**Mechanisms Involved in Diabetes-Associated Platelet Hyperactivation**

Voahanginirina Randriamboavonjy

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60539

## **1. Introduction**

Diabetes mellitus is a multi-factorial disease caused by a combination of genetic and environ‐ mental factors. Although insulin resistance and dysregulation of glucose and lipid homeostasis are the primary hallmarks of the disease, it is now well accepted that the morbidity and mortality associated with diabetes mostly result from micro- and macro-vascular complica‐ tions [1]. An early step in the pathogenesis of the vascular complications of diabetes is the development of endothelial dysfunction which is characterised by a decrease in nitric oxide (NO) bioavailability, prostacyclin production and a general reduction in the anti-thrombo‐ genic properties of vascular wall [2]. Diabetes is also characterised by an alteration of platelet function. Indeed, platelets from patients with type 1 or type 2 diabetes are hyperreactive and demonstrate increased adhesiveness as well as exaggerated aggregation and thrombus formation. Several mechanisms have been reported to mediate the hyperreactivity of platelets from diabetic patients including morphological changes such as increased mean platelet volume and accelerated platelet turnover, biochemical changes such as increased reactive oxygen species (ROS) production, increased synthesis of thromboxane A2 (TXA2) and thrombin and a dysregulated Ca2+ homeostasis. Platelets from diabetic patients also demon‐ strate increased surface expression of adhesion proteins such as P-selectin and the αIIbβ3 integrin and reduced membrane fluidity. These changes characteristic of the "diabetic platelet" have been mostly attributed to the metabolic dysregulation associated to the insulin resistance and dyslipidemia. However, given that platelet hyperreactivity has also been found in patients with type 1 diabetes mellitus it is suggested that hyperglycemia alone can account for at least part of the altered platelet response in patients with diabetes mellitus. Oxidative stress which characterizes both types of diabetes has also been shown to be an important factor mediating the phenotypic changes of diabetic platelets. In this chapter I will first give an overview of the

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physiological platelet activation, and then discuss the role of factors such as insulin resistance, hyperglycemia, dyslipidemia on platelet function. Next, I will describe the intracellular mechanisms underlying platelet hyperreactivity in diabetes. Finally, the impact of diabetes on the responsiveness to anti-platelet therapy will be discussed.

## **2. Physiological platelet activation**

Platelets are anucleated cells generated from megakaryocytes, and after their release into the blood, circulate for approximately 10 days. The main role of platelets is to maintain hemostasis. Under normal conditions, platelet adhesion to the vascular wall is inhibited due to the antithrombotic nature of the endothelial cell surface and the permanent release of anti-platelet factors by the endothelium [3]. Following vascular injury, especially under the influence of high shear stress, platelets tether and adhere to the exposed subendothelial collagen via the von Willebrand factor (vWF)-mediated binding to platelet glycoprotein Ib/V/IX complex. The initial interaction is subsequently strengthened by the interaction of collagen to its receptor glycoprotein VI (GPVI) and the integrin α2β1. The ligation of these receptors activates Src family tyrosine kinases (SFKs) which lead to the phosphorylation and activation of the phospholipase Cγ2 (PLC γ2). The latter hydrolyses membrane Phosphatidylinositol-4, 5 bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3) which respectively activates the protein kinase C (PKC) and stimulates the release of Ca2+ from the intracellular stores. PKC binds to and phosphorylates the cytoplasmic tail of the β3 subunit of the αIIbβ3 integrin complex, which then recruits talin and kindlin-3. This in turns causes a conformational change in the extracellular domain of the integrin and enables the binding of circulating fibrinogen (inside-out signalling). Fibrinogen links activated αIIbβ3 integrins of neighbouring platelets and initiates platelet aggregation. After its binding, fibrinogen initiates the so-called "outside-in" signalling of the integrin. This step includes the activation of SFKs which promotes the tyrosine phosphorylation of β3 integrin and the binding of cytoskeletal proteins leading to the activation of the phosphatidylinositide 3-kinases (PI3K) and platelet adhesion. Ca2+ and DAG also act together to activate the calcium- and diacylglycerol-regulated guanosine exchange factor (CalDAGGEF), a guanosine exchange factor important for the activation of the small GTPase Rap1. The latter activates mitogen-activated protein kinases (MAPKs) which are known to be upstream of the phospholipase A2 (PLA2)/cyclooxygenase (COX) signalling cascade resulting into the production of thromboxane A2. PKC and MAPK act in concert to stimulate the release of different second mediators from platelet granules. These agonists; including adenosine diphosphate (ADP), serotonin and the formed throm‐ boxane A2 bind to their respective G protein–coupled receptors (GPCRs). Through the activation of G protein-mediated signalling pathways, they can further increase their own formation and/or release, thus acting as positive-feedback amplifying platelet responses by recruiting additional platelets and promoting aggregation. (for review see references [4, 5]) (figure 1). In addition to stimulating degranulation, the increase in platelet [Ca2+)i also leads to the activation of calpains, a family of Ca2+-dependent neutral cysteine proteases. While some calpains are expressed only in specific tissues, platelets are known to express at least two isoforms of this enzyme i.e µ-calpain (calpain 1) and m-calpain (calpain 2). The µ- and mcalpain were initially named for the Ca2+ concentration (micromolar versus millimolar) required for their activation in vitro [6]. However it is now clear that additional mechanisms such as phosphorylation also regulate their proteolytic activity [7]. Several reports have highlighted the importance of µ- and m-calpain in platelet activation. Indeed, once activated, calpains induce the limited proteolysis of a number of proteins implicated in cytoskeletal rearrangement, degranulation and aggregation. Proteins identified to-date that are targeted by calpain include spectrin, adducin and talin as well as platelet-endothelial cell adhesion molecule-1 (PECAM-1), the myosin light chain kinase and N-ethylmaleimide-sensitive-factor attachment receptor proteins such as N-ethylmaleimide sensitive factor attachment protein-23 and vesicle-associated membrane protein-3. Furthermore, µ-calpain modulates αIIbβ3 integrin-mediated outside-in signaling and platelet spreading by cleaving the β3 subunit of the αIIbβ3 integrin [8]. In the final stages of platelet activation, phosphatidylserine, which is normally sequestered in the inner leaflet of the plasma membrane, is relocated to the outer leaflet leading to the shedding of microparticles and giving platelets a procoagulant surface. After stimulation, Ca2+ is removed from the cytosol and sequestred into the intracellular stores and/or extruded into the extracellular space by the action of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) and the plasma membrane Ca2+-ATPase (PMCA), respectively. Forma‐ tion of platelet plug or primary hemostasis is associated with the activation of the coagulation cascade which results into fibrin deposition and linking (secondary hemostasis) and the formation of a red clot. After the clot has been formed, platelets rearrange and contract their intracellular actin/myosin cytoskeleton. Given that the intracellular actin network is connected to the internal part of the integrin αIIbβ3, platelet contractile force on the fibrin network will lead to "clot retraction". Finally, the fibrin is slowly dissolved by the fibrinolytic enzyme, plasmin, and the platelets are cleared by phagocytosis and wound healing will take place.

physiological platelet activation, and then discuss the role of factors such as insulin resistance, hyperglycemia, dyslipidemia on platelet function. Next, I will describe the intracellular mechanisms underlying platelet hyperreactivity in diabetes. Finally, the impact of diabetes on

Platelets are anucleated cells generated from megakaryocytes, and after their release into the blood, circulate for approximately 10 days. The main role of platelets is to maintain hemostasis. Under normal conditions, platelet adhesion to the vascular wall is inhibited due to the antithrombotic nature of the endothelial cell surface and the permanent release of anti-platelet factors by the endothelium [3]. Following vascular injury, especially under the influence of high shear stress, platelets tether and adhere to the exposed subendothelial collagen via the von Willebrand factor (vWF)-mediated binding to platelet glycoprotein Ib/V/IX complex. The initial interaction is subsequently strengthened by the interaction of collagen to its receptor glycoprotein VI (GPVI) and the integrin α2β1. The ligation of these receptors activates Src family tyrosine kinases (SFKs) which lead to the phosphorylation and activation of the phospholipase Cγ2 (PLC γ2). The latter hydrolyses membrane Phosphatidylinositol-4, 5 bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3) which respectively activates the protein kinase C (PKC) and stimulates the release of Ca2+ from the intracellular stores. PKC binds to and phosphorylates the cytoplasmic tail of the β3 subunit of the αIIbβ3 integrin complex, which then recruits talin and kindlin-3. This in turns causes a conformational change in the extracellular domain of the integrin and enables the binding of circulating fibrinogen (inside-out signalling). Fibrinogen links activated αIIbβ3 integrins of neighbouring platelets and initiates platelet aggregation. After its binding, fibrinogen initiates the so-called "outside-in" signalling of the integrin. This step includes the activation of SFKs which promotes the tyrosine phosphorylation of β3 integrin and the binding of cytoskeletal proteins leading to the activation of the phosphatidylinositide 3-kinases (PI3K) and platelet adhesion. Ca2+ and DAG also act together to activate the calcium- and diacylglycerol-regulated guanosine exchange factor (CalDAGGEF), a guanosine exchange factor important for the activation of the small GTPase Rap1. The latter activates mitogen-activated protein kinases (MAPKs) which are known to be upstream of the phospholipase A2 (PLA2)/cyclooxygenase (COX) signalling cascade resulting into the production of thromboxane A2. PKC and MAPK act in concert to stimulate the release of different second mediators from platelet granules. These agonists; including adenosine diphosphate (ADP), serotonin and the formed throm‐ boxane A2 bind to their respective G protein–coupled receptors (GPCRs). Through the activation of G protein-mediated signalling pathways, they can further increase their own formation and/or release, thus acting as positive-feedback amplifying platelet responses by recruiting additional platelets and promoting aggregation. (for review see references [4, 5])

(figure 1). In addition to stimulating degranulation, the increase in platelet [Ca2+)i

the activation of calpains, a family of Ca2+-dependent neutral cysteine proteases. While some calpains are expressed only in specific tissues, platelets are known to express at least two

also leads to

the responsiveness to anti-platelet therapy will be discussed.

**2. Physiological platelet activation**

178 The Non-Thrombotic Role of Platelets in Health and Disease

Platelets store quite high concentrations of chemokines, cytokines, growth factors and vasoactive substances. The latter are sequestered into 2 major types of granules; α-granules and dense granules. On the one hand, alpha-granules are known to contain growth factors including vascular endothelial growth factor, platelet-derived growth factor, endostatin, transforming growth factor-β; chemokines such as platelet factor 4 (CXCL4) and CCL5 and adhesion molecules such as P-selectin. Dense granules, on the other hand, store mainly small molecules (e.g ADP and serotonin) and ions (such as Ca2+ and Mg2+). Upon activation, platelets release soluble proteins contained in their granules and redistribute some α-granules contents to the membrane (e.g P-selectin, CD40 ligand). Given the variety of proteins released upon platelet activation, it is clear that platelets may affect the vascular wall in different ways. Indeed, beyond their role in hemostasis, platelets are involved in angiogenesis as well as in vascular inflammation.

### **3. Effect of insulin resistance on platelet function**

Insulin exerts an inhibitory effect on platelets but the intracellular mechanisms remain not fully characterized and whether the effects are mediated by the insulin receptor is unclear.

**Figure 1. Platelet major signalling pathways.** Collagen binding either to GPVI or α2β1 integrin or vWF binding to Gp1b-IX-V leads to the activation of Src family kinases and tyrosine phosphorylation of the phospholipase Cγ2. PLCγ2 cleaves phosphatidylinositol (4, 5)-bisphosphate PIP2 to generate inositol (1, 4, 5)-trisphosphate (IP3) and diacylglycer‐ ol (DAG). IP3 and DAG are responsible for the mobilization of calcium from intracellular stores leading to calpain acti‐ vation and the activation of isoforms of protein kinase C (PKC), respectively. Ca2+ and DAG activate the Ca2+ and DAG-regulated guanosine exchange factor (CalDAGGEF) which in turn activate the small GTPase Rap1. The latter ac‐ tivates the mitogen-activated kinase (MAPK) which leads to thromboxane A2 generation, granule secretion and integ‐ rin activation. The released factors potentiate platelet signaling via the activation of G protein-coupled receptors/ phospholipase Cβ pathway. The binding of fibrinogen to activated integrin initiates platelet spreading by activating the Phosphoinositide 3-kinase (PI3K) pathway.

Indeed, there is no evidence of the expression of insulin receptor on platelets [9] and it is speculated that the effects are rather mediated by the activation of insulin-like growth factor (IGF) receptor [10, 11]. Insulin decreases thrombin-induced increase in Ca2+ and attenuates agonist-induced platelet aggregation [12]. One of the mechanisms described to mediate the anti-platelet effect of insulin is the activation of the AMP-activated protein kinase (AMPK) and Akt by the PI3K [13]. Insulin can also inhibit Ca2+ mobilization by activating the inhibitory Gprotein Gi [14]. However, given that insulin can also stimulate the release of ADP [15], it is assumed that whether insulin activates or inhibits platelets may depend on its concentration. Although the insulin effect in platelets has been initially believed to involve the activation of nitric oxide synthase (NOS), there are contradictory reports on the expression and function of NOS in human platelets [13, 16]. Insulin resistance refers to the loss of response to insulin stimulation. The molecular mechanisms of insulin resistance are complex but it has been shown that the faculty of insulin to inhibit platelet activation is lost in diabetic patients [17] and the inhibitory effect of insulin on the interaction of platelet with collagen and other agonists is blunted by insulin resistance in obese subjects [18]. Moreover, in non diabetic, obese women, there is a direct correlation between platelet reactivity assessed by thromboxane A2 generation and insulin resistance [19].

## **4. Effect of hyperglycemia and AGEs**

Indeed, there is no evidence of the expression of insulin receptor on platelets [9] and it is speculated that the effects are rather mediated by the activation of insulin-like growth factor (IGF) receptor [10, 11]. Insulin decreases thrombin-induced increase in Ca2+ and attenuates agonist-induced platelet aggregation [12]. One of the mechanisms described to mediate the anti-platelet effect of insulin is the activation of the AMP-activated protein kinase (AMPK) and Akt by the PI3K [13]. Insulin can also inhibit Ca2+ mobilization by activating the inhibitory Gprotein Gi [14]. However, given that insulin can also stimulate the release of ADP [15], it is assumed that whether insulin activates or inhibits platelets may depend on its concentration. Although the insulin effect in platelets has been initially believed to involve the activation of nitric oxide synthase (NOS), there are contradictory reports on the expression and function of NOS in human platelets [13, 16]. Insulin resistance refers to the loss of response to insulin stimulation. The molecular mechanisms of insulin resistance are complex but it has been

**Figure 1. Platelet major signalling pathways.** Collagen binding either to GPVI or α2β1 integrin or vWF binding to Gp1b-IX-V leads to the activation of Src family kinases and tyrosine phosphorylation of the phospholipase Cγ2. PLCγ2 cleaves phosphatidylinositol (4, 5)-bisphosphate PIP2 to generate inositol (1, 4, 5)-trisphosphate (IP3) and diacylglycer‐ ol (DAG). IP3 and DAG are responsible for the mobilization of calcium from intracellular stores leading to calpain acti‐ vation and the activation of isoforms of protein kinase C (PKC), respectively. Ca2+ and DAG activate the Ca2+ and DAG-regulated guanosine exchange factor (CalDAGGEF) which in turn activate the small GTPase Rap1. The latter ac‐ tivates the mitogen-activated kinase (MAPK) which leads to thromboxane A2 generation, granule secretion and integ‐ rin activation. The released factors potentiate platelet signaling via the activation of G protein-coupled receptors/ phospholipase Cβ pathway. The binding of fibrinogen to activated integrin initiates platelet spreading by activating

GpIb-IX-V GPVI

FcR SFK (Fyn, Lyn)

**PLC 2**

IP3

Ca2+

Calpain Rap1

DAG

CalDAGGEF1

PKC

PAR1 PAR4

Thrombin

**Gq G12,13 Gi**

**PLC**

vWF

IIb3

SFK (cSrc)

PI3K p190-RhoGAP

Platelet spreading

2 1

P2Y1

Fibrinogen

the Phosphoinositide 3-kinase (PI3K) pathway.

Activation

ADP

P2Y12

180 The Non-Thrombotic Role of Platelets in Health and Disease

cAMP

**Gq Gi**

Collagen

Granulesecretion Integrin activation

> GPCR-mediated Signal amplification

TP

TXA2

MAPKs

RIAM

TP

TXA2 synthesis

Despite the fact that diabetes is characterized by chronic hyperglycemia, there is evidence that acute hyperglycemia can directly affect platelet reactivity. Indeed, a prospective randomised double blind controlled study has shown that 24h euglycaemic treatment significantly increased the plasma levels of the platelet and endothelial activation markers soluble P-selectin and vWF [20]. Moreover, it has been shown that challenging healthy non-diabetic subjects with 24h hyperglycemia-hyperinsulinemia altered the insulin signaling pathway [21]. Indeed, the glycogen synthase kinase 3β (GSKβ3) and the tyrosine phosphatase SHP2 as well as tissue factor were upregulated on mRNA and protein levels while mRNA for the syntaxin 4-binding protein was downregulated. High glucose can also increase calcium influx in platelets by enhancing a PI3K-dependent transient receptor potential channel canonical type 6 [22]. The latter is known to be significantly highly expressed in diabetic platelets. Hyperglycemia is able to increase the expression and/or activity of PKC [23, 24], a central kinase in the regulation of platelet activity. Another consequence of hyperglycemia in platelets is the induction of mitochondrial dysfunction. One of the recent mechanisms linking hyperglycemia and mitochondrial dysfunction is the activation of aldose reductase and subsequent ROS produc‐ tion which result to p53 phosphorylation [25] or the stimulation of the PLCγ2/PKC/p38α MAPK pathway and the increase in TXA2 production [26]. More recent findings showed that the hyperglycemia-induced platelet activation could be attributed to the downregulation of different micro-RNA (miR) such as miR-223 and miR-146a. Indeed, low platelet and plasma miR-223 and miR-146a expression has been associated with an increased risk for ischemic stroke in patients with diabetes mellitus [27]. Many of the deleterious effects of glucose have been attributed to its metabolite methylgyloxal (MG), a highly reactive dicarbonyl metabolite that is generated endogenously by the nonenzymatic degradation of the glycolytic intermedi‐ ates, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate [28]. It is known that plasma levels of MG in diabetic individuals are enhanced. Study investigating the effect of MG on platelets showed that acute application of MG to platelets increases intracellular Ca2+ levels and activates classical PKCs at the same time as inhibiting PI3K/Akt and the β3-integrin outside-in signalling. Moreover, *in vivo*, MG increases thrombus size but reduces its stability in mice [29]. Although most of the effects of MG have been attributed to the formation of advanced glycation end-products (AGEs) and the subsequent activation of the AGE receptor; RAGE, this study has highlighted a direct effect of MG on platelets which may contribute to the diabetes-associated platelet hyperaggregability. Several studies have shown that AGEs can activate platelets and that platelets express RAGE. The expression of surface markers such as P-selectin (CD62) and CD63 (a lysosomal glycoprotein) has been shown to be significantly increased by AGE stimulation [30] suggesting AGE-induced platelet degranulation. However, this study failed to report the detailed intracellular mechanism involved. Studies performed in mice could show that AGEs induce a prothrombotic phenotype via interaction with platelet glycoprotein IV (CD36) [31]. The serum- and glucocorticoid-inducible kinase 1 (SGK1) has been also suggested to mediate AGE-induced platelet hyperactivation [32]. Indeed, SGK1 expression is known to be regulated by hyperglycaemia and AGEs. In platelets, SGK1 increases store-operated calcium entry (SOCE) and thereby regulates several Ca2+-dependent platelet functions such as degranulation, integrin αIIbβ3 activation, phosphatidylserine exposure, aggregation and thrombus formation.

### **5. Effect of dyslipidemia**

The fact that glycemic control alone has proven insufficient to reduce thrombotic complica‐ tions in diabetic patients suggests that other factors may contribute to platelet hyperreactiv‐ ity in diabetes [33]. One feature of diabetes is the presence of dyslipidemia which is characterized by high plasma triglyceride concentration, reduced high density lipoprotein (HDL) concentration, and increased concentration of low density lipoprotein (LDL)[34]. There is evidence that dyslipidemia contributes to the diabetes-associated platelet hyperac‐ tivation. By binding to a pertussis sensitive G-protein coupled receptor on platelets, LDL induces an increase in cytosolic [Ca2+]i , IP3 formation and activation of PKC [35]. Howev‐ er, the pro-thrombotic properties of LDL seem to be rather associated to its oxidation. Indeed, oxidized-LDL can directly interact with platelets specific receptors such as the lectinlike oxidized LDL receptor-1 [36] or the CD36 [37, 38]. The latter involves the activation of the MAPK c-Jun N-terminal kinase (JNK)2 and its upstream activator MKK4. Not only are platelets activated by ox-LDL but activated platelets are also known to be able to form ox-LDL via platelet gp91phox (NOX2)-dependent ROS generation [39] suggesting the contribu‐ tion of platelets to circulating ox-LDL. The formed ox-LDL has been demonstrated to be either uptaken by monocytes [39] or amplify platelet activation [40]. On the molecular levels, LDL activates the platelet arachidonic acid signalling cascade, i.e phosphorylation of p38 MAPK and cytosolic phospholipase A2, leading to increased TXA2 formation [41]. Interestingly, there is less information on the direct effects of triglycerides on platelets. However, the link between hyperlipidemia and platelet hyperactivation is supported by the fact that lipid-lowering agents possess anti-thrombotic properties [42, 43].

#### **6. Dysregulation of calcium signalling**

One of the characteristics of platelets from patients with type 2 diabetes is the alteration of the intracellular Ca2+ homeostasis. Different mechanisms have been reported to be respon‐ sible for this abnormality. One of the mechanisms leading to the latter phenomenon is the reduction in platelet Ca2+-ATPase activity in diabetic subjects [44, 45]. Moreover, the function of the Na+ /Ca2+ exchanger is known to be significantly altered in platelets from diabetic

patients [46]. Another mechanism contributing to the enhanced resting cytosolic calcium in platelets from diabetic patients is the increased passive Ca2+ leakage rate from the intracel‐ lular stores [47]. Given that most of the intracellular signalling in platelets is regulated by calcium, it is more than expected that a dysregulation in calcium homeostasis would affect platelet function in many ways. One of the consequences of the increased [Ca2+]i in platelet is the activation of calpains. Although calpain-mediated proteolysis is involved in physio‐ logical platelet activation, type 2 diabetes has been shown to be associated with the overactivation of calpain in platelets [45] leading to marked changes in the platelet proteome [48]. In platelets from diabetic patients, the integrin-linked kinase and septin-5 were found to be new calpain substrates and their cleavage was shown to be involved in the en‐ hanced platelet adhesion and spreading as well as enhanced α-granule secretion, respective‐ ly. Moreover, calpain was able to cleave the chemokine RANTES into a variant with an enhanced chemotactic activity. The *in vivo* relevance of calpain in inducing the hyperreac‐ tivity of platelets from diabetic patients was supported by the finding that treatment of diabetic mice with calpain inhibitor preserved the platelet proteome, and reversed the diabetes-associated platelet hyperactivation [48].

#### **7. Increased apoptosis**

increased by AGE stimulation [30] suggesting AGE-induced platelet degranulation. However, this study failed to report the detailed intracellular mechanism involved. Studies performed in mice could show that AGEs induce a prothrombotic phenotype via interaction with platelet glycoprotein IV (CD36) [31]. The serum- and glucocorticoid-inducible kinase 1 (SGK1) has been also suggested to mediate AGE-induced platelet hyperactivation [32]. Indeed, SGK1 expression is known to be regulated by hyperglycaemia and AGEs. In platelets, SGK1 increases store-operated calcium entry (SOCE) and thereby regulates several Ca2+-dependent platelet functions such as degranulation, integrin αIIbβ3 activation, phosphatidylserine exposure,

The fact that glycemic control alone has proven insufficient to reduce thrombotic complica‐ tions in diabetic patients suggests that other factors may contribute to platelet hyperreactiv‐ ity in diabetes [33]. One feature of diabetes is the presence of dyslipidemia which is characterized by high plasma triglyceride concentration, reduced high density lipoprotein (HDL) concentration, and increased concentration of low density lipoprotein (LDL)[34]. There is evidence that dyslipidemia contributes to the diabetes-associated platelet hyperac‐ tivation. By binding to a pertussis sensitive G-protein coupled receptor on platelets, LDL

er, the pro-thrombotic properties of LDL seem to be rather associated to its oxidation. Indeed, oxidized-LDL can directly interact with platelets specific receptors such as the lectinlike oxidized LDL receptor-1 [36] or the CD36 [37, 38]. The latter involves the activation of the MAPK c-Jun N-terminal kinase (JNK)2 and its upstream activator MKK4. Not only are platelets activated by ox-LDL but activated platelets are also known to be able to form ox-LDL via platelet gp91phox (NOX2)-dependent ROS generation [39] suggesting the contribu‐ tion of platelets to circulating ox-LDL. The formed ox-LDL has been demonstrated to be either uptaken by monocytes [39] or amplify platelet activation [40]. On the molecular levels, LDL activates the platelet arachidonic acid signalling cascade, i.e phosphorylation of p38 MAPK and cytosolic phospholipase A2, leading to increased TXA2 formation [41]. Interestingly, there is less information on the direct effects of triglycerides on platelets. However, the link between hyperlipidemia and platelet hyperactivation is supported by

the fact that lipid-lowering agents possess anti-thrombotic properties [42, 43].

One of the characteristics of platelets from patients with type 2 diabetes is the alteration of the intracellular Ca2+ homeostasis. Different mechanisms have been reported to be respon‐ sible for this abnormality. One of the mechanisms leading to the latter phenomenon is the reduction in platelet Ca2+-ATPase activity in diabetic subjects [44, 45]. Moreover, the function

/Ca2+ exchanger is known to be significantly altered in platelets from diabetic

, IP3 formation and activation of PKC [35]. Howev‐

aggregation and thrombus formation.

182 The Non-Thrombotic Role of Platelets in Health and Disease

induces an increase in cytosolic [Ca2+]i

**6. Dysregulation of calcium signalling**

of the Na+

**5. Effect of dyslipidemia**

Although being anucleated, there is evidence demonstrating that platelets possess the necessary machinery to undergo apoptosis [49]. Among other mechanisms, calpain seems to play an important role in platelet apoptosis [50, 51]. Indeed, although caspase is activated during platelet apoptosis, this seems to be downstream of calpain activation. The increased calpain activation in platelets from diabetic patients described above suggests that diabetic platelets may be more prone to apoptosis. Several factors have been reported to induce platelet apoptosis including the diabetes-associated oxidative stress which is an important stimulus for inducing mitochondrial damage [52]. Mitochondria not only are the target of oxidative stress but are also able to generate ROS therefore amplifying the reaction to oxidative stress [52, 53]. Platelets from patients with type 2 diabetes demonstrate an increased ATP content but decreased mitochondrial membrane potential [54] supporting the alteration of mitochon‐ drial function. Another mechanism involved in platelet apoptosis is the development of endoplasmic reticulum (ER) stress. It has been shown that diabetes mellitus was associated with the production of hyperreactive platelets expressing an altered protein disulfide isomer‐ ase and 78-kDa glucose-regulated protein [55]. Moreover, homocysteine, which levels are known to be significantly increased in diabetic patients, has been shown to stimulate ER stressmediated platelet apoptosis by activating the caspase pathway [56]. One of the consequences of platelet activation and apoptosis is the generation of intact membrane vesicles known as microparticles. The formation of platelet-derived microparticles (PMPs) is known to be Ca2+ and calpain-dependent. Although PMPs are involved in hemostasis due to their procoagulant properties, elevated levels of PMPs in blood from diabetic patients has been suggested to participate in the increased vascular complications in diabetes [57, 58].

## **8. Increased mean platelet volume**

Platelet reactivity and size have been shown to directly correlate. Indeed, young and large platelets exhibit higher activity than old and small ones. The mean platelet volume (MPV) is an indicator of the average size of platelets which has been largely used to investigate the relationship between platelet size and activity. There is evidence that MPV is significantly increased in diabetic patients and that it directly correlates with glycemic control [59-61].

## **9. Hyporesponsiveness to anti-platelet therapy**

One feature of platelets from diabetic patients is their hyporesponsiveness to anti-platelet therapy. Indeed, there is evidence that anti-platelet therapy is less effective in diabetic patients when compared with patients without diabetes [62]. One example is the so-called "aspirin resistance" in which diabetic patients are refractory to the anti-platelet effect of aspirin [63]. Aspirin or salicylic acid acetylates and irreversibly inhibits cyclooxygenase thereby inhibiting the TXA2 formation. Aspirin has been also shown to activate the NO/cGMP pathway. Although aspirin resistance is seen in the majority of diabetic patients, the exact molecular mechanism is still unclear. One of the mechanisms proposed to mediate aspirin-resistance is the increased glycation of platelet proteins which may alter the acetylation process [64]. Some *in vitro* studies have also shown a direct link between hyperglycemia and aspirin resistance. Certainly, high glucose can acutely reduce the antiaggregating effect of aspirin by inhibiting the aspirin-induced activation of the NO/cGMP/PKG pathway without affecting the aspirininduced inhibition of TXA2 synthesis [65]. Given that acute stimulation of platelets with other monosaccharides such as fructose and galactose was shown to lead to a similar alteration of the aspirin effect on platelets and that lactic acid also impaired the inhibition of platelet aggregation with aspirin, it has been suggested that lactic acid might be the mediator of the glucose-induced inhibition of the aspirin effect in platelets [66]. Interestingly, the platelet resistance seems to be specific to aspirin since hyperglycemia-induced platelet hyperactivation in type 2 diabetes could be reversed by a nitric oxide-donating agent [67]. More recently, non-HDL cholesterol has been reported to be an independent risk factor for aspirin resistance in patients with type 2 diabetes [68].

Diabetes is also known to be associated to a reduced responsiveness of platelets to the P2Y12 ADP receptor antagonist clopidogrel [69, 70]. Although not directly investigated in diabetic patients, upregulation of ADP receptor levels, increase in ADP exposure or accelerated platelet turnover may contribute to clopidogrel resistance.

#### **10. Conclusions**

The fact that the diabetic milieu can affect platelet function in several ways explains the failure of glycemic control alone to reduce the risk of atherothrombotic events in diabetic patients. Indeed, the increased platelet hyperreactivity is the result of complicated inter-regulated mechanisms. Moreover, given that diabetic platelets are resistant to most anti-platelet therapy, there is a need of new therapeutical strategies to improve platelet function in diabetes. Certainly, management of both glycemia and dyslipidemia would improve the effects of antiplatelet therapy. Moreover, the facts that calpain plays a key role in platelet activation and that calpain activity is elevated in diabetic platelets, makes it tempting to suggest the Ca2+-activated proteases as a promising therapeutic target to prevent thrombotic complications in diabetic patients.

## **Author details**

**8. Increased mean platelet volume**

184 The Non-Thrombotic Role of Platelets in Health and Disease

patients with type 2 diabetes [68].

**10. Conclusions**

turnover may contribute to clopidogrel resistance.

**9. Hyporesponsiveness to anti-platelet therapy**

Platelet reactivity and size have been shown to directly correlate. Indeed, young and large platelets exhibit higher activity than old and small ones. The mean platelet volume (MPV) is an indicator of the average size of platelets which has been largely used to investigate the relationship between platelet size and activity. There is evidence that MPV is significantly increased in diabetic patients and that it directly correlates with glycemic control [59-61].

One feature of platelets from diabetic patients is their hyporesponsiveness to anti-platelet therapy. Indeed, there is evidence that anti-platelet therapy is less effective in diabetic patients when compared with patients without diabetes [62]. One example is the so-called "aspirin resistance" in which diabetic patients are refractory to the anti-platelet effect of aspirin [63]. Aspirin or salicylic acid acetylates and irreversibly inhibits cyclooxygenase thereby inhibiting the TXA2 formation. Aspirin has been also shown to activate the NO/cGMP pathway. Although aspirin resistance is seen in the majority of diabetic patients, the exact molecular mechanism is still unclear. One of the mechanisms proposed to mediate aspirin-resistance is the increased glycation of platelet proteins which may alter the acetylation process [64]. Some *in vitro* studies have also shown a direct link between hyperglycemia and aspirin resistance. Certainly, high glucose can acutely reduce the antiaggregating effect of aspirin by inhibiting the aspirin-induced activation of the NO/cGMP/PKG pathway without affecting the aspirininduced inhibition of TXA2 synthesis [65]. Given that acute stimulation of platelets with other monosaccharides such as fructose and galactose was shown to lead to a similar alteration of the aspirin effect on platelets and that lactic acid also impaired the inhibition of platelet aggregation with aspirin, it has been suggested that lactic acid might be the mediator of the glucose-induced inhibition of the aspirin effect in platelets [66]. Interestingly, the platelet resistance seems to be specific to aspirin since hyperglycemia-induced platelet hyperactivation in type 2 diabetes could be reversed by a nitric oxide-donating agent [67]. More recently, non-HDL cholesterol has been reported to be an independent risk factor for aspirin resistance in

Diabetes is also known to be associated to a reduced responsiveness of platelets to the P2Y12 ADP receptor antagonist clopidogrel [69, 70]. Although not directly investigated in diabetic patients, upregulation of ADP receptor levels, increase in ADP exposure or accelerated platelet

The fact that the diabetic milieu can affect platelet function in several ways explains the failure of glycemic control alone to reduce the risk of atherothrombotic events in diabetic patients. Voahanginirina Randriamboavonjy\*

Address all correspondence to: voahangy@vrc.uni-frankfurt.de

Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany

## **References**


dylinositol 3-kinase-dependent pathway. Arterioscler Thromb Vasc Biol 2008;28:746-51.

[23] Assert R, Scherk G, Bumbure A, et al. Regulation of protein kinase C by short term hyperglycaemia in human platelets in vivo and in vitro. Diabetologia 2001;44:188-95.

[9] Rauchfuss S, Geiger J, Walter U, et al. Insulin inhibition of platelet-endothelial inter‐ action is mediated by insulin effects on endothelial cells without direct effects on pla‐

[10] Blair TA, Moore SF, Williams CM, et al. Phosphoinositide 3-kinases p110alpha and p110beta have differential roles in insulin-like growth factor-1-mediated Akt phos‐ phorylation and platelet priming. Arterioscler Thromb Vasc Biol 2014;34:1681-8.

[11] Hers I. Insulin-like growth factor-1 potentiates platelet activation via the IRS/

[12] Ishida M, Ishida T, Ono N, et al. Effects of insulin on calcium metabolism and plate‐

[13] Fleming I, Schulz C, Fichtlscherer B, et al. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human plate‐

[14] Ferreira IA, Eybrechts KL, Mocking AI, et al. IRS-1 mediates inhibition of Ca2+ mobi‐ lization by insulin via the inhibitory G-protein Gi. J Biol Chem 2004;279:3254-64.

[15] Randriamboavonjy V, Schrader J, Busse R, et al. Insulin induces the release of vasodi‐ lator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent path‐

[16] Bohmer A, Gambaryan S, Tsikas D. Human blood platelets lack nitric oxide synthase

[17] Ferreira IA, Mocking AI, Feijge MA, et al. Platelet inhibition by insulin is absent in

[18] Westerbacka J, Yki-Jarvinen H, Turpeinen A, et al. Inhibition of platelet-collagen in‐ teraction: an in vivo action of insulin abolished by insulin resistance in obesity. Arte‐

[19] Basili S, Pacini G, Guagnano MT, et al. Insulin resistance as a determinant of platelet

[20] Kotzailias N, Graninger M, Knechtelsdorfer M, et al. Acute effects of hyperglycaemia on plasma concentration of soluble P-selectin and von Willebrand factor in healthy volunteers -a prospective randomised double blind controlled study. Thromb Res

[21] Rao AK, Freishtat RJ, Jalagadugula G, et al. Alterations in insulin-signaling and coag‐ ulation pathways in platelets during hyperglycemia-hyperinsulinemia in healthy

[22] Liu D, Maier A, Scholze A, et al. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphati‐

type 2 diabetes mellitus. Arterioscler Thromb Vasc Biol 2006;26:417-22.

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PI3Kalpha pathway. Blood 2007;110:4243-52.

let aggregation. Hypertension 1996;28:209-12.

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[50] Wolf BB, Goldstein JC, Stennicke HR, et al. Calpain functions in a caspase-independ‐ ent manner to promote apoptosis-like events during platelet activation. Blood 1999;94:1683-92.

[36] Chen M, Kakutani M, Naruko T, et al. Activation-dependent surface expression of LOX-1 in human platelets. Biochem Biophys Res Commun 2001;282:153-8.

[37] Chen K, Febbraio M, Li W, et al. A specific CD36-dependent signaling pathway is re‐ quired for platelet activation by oxidized low-density lipoprotein. Circ Res

[38] Podrez EA, Byzova TV, Febbraio M, et al. Platelet CD36 links hyperlipidemia, oxi‐

[39] Carnevale R, Pignatelli P, Lenti L, et al. LDL are oxidatively modified by platelets via GP91(phox) and accumulate in human monocytes. FASEB J 2007;21:927-34.

[40] Carnevale R, Bartimoccia S, Nocella C, et al. LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism. Atherosclerosis

[41] Colas R, Sassolas A, Guichardant M, et al. LDL from obese patients with the metabol‐ ic syndrome show increased lipid peroxidation and activate platelets. Diabetologia

[42] Tehrani S, Mobarrez F, Antovic A, et al. Atorvastatin has antithrombotic effects in patients with type 1 diabetes and dyslipidemia. Thromb Res 2010;126:e225-e231. [43] Tsai NW, Lin TK, Chang WN, et al. Statin pre-treatment is associated with lower pla‐ telet activity and favorable outcome in patients with acute non-cardio-embolic ische‐

[44] Rosado JA, Saavedra FR, Redondo PC, et al. Reduced plasma membrane Ca2+-AT‐ Pase function in platelets from patients with non-insulin-dependent diabetes melli‐

[45] Randriamboavonjy V, Pistrosch F, Bolck B, et al. Platelet sarcoplasmic endoplasmic reticulum Ca2+-ATPase and mu-calpain activity are altered in type 2 diabetes melli‐

[46] Li Y, Woo V, Bose R. Platelet hyperactivity and abnormal Ca2+ homeostasis in diabe‐

[47] Zbidi H, Jardin I, Bartegi A, et al. Ca2+ leakage rate from agonist-sensitive intracellu‐ lar pools is altered in platelets from patients with type 2 diabetes. Platelets

[48] Randriamboavonjy V, Isaak J, Elgheznawy A, et al. Calpain inhibition stabilizes the

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## **Chapter 10**

## **Platelets in Alzheimer's Disease**

Barbara Plagg and Christian Humpel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60535

## **1. Introduction**

[65] Russo I, Viretto M, Barale C, et al. High glucose inhibits the aspirin-induced activa‐ tion of the nitric oxide/cGMP/cGMP-dependent protein kinase pathway and does not affect the aspirin-induced inhibition of thromboxane synthesis in human platelets.

[66] Kobzar G, Mardla V, Samel N. Short-term exposure of platelets to glucose impairs in‐ hibition of platelet aggregation by cyclooxygenase inhibitors. Platelets

[67] Gresele P, Marzotti S, Guglielmini G, et al. Hyperglycemia-induced platelet activa‐ tion in type 2 diabetes is resistant to aspirin but not to a nitric oxide-donating agent.

[68] Kim JD, Park CY, Ahn KJ, et al. Non-HDL cholesterol is an independent risk factor for aspirin resistance in obese patients with type 2 diabetes. Atherosclerosis

[69] Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, et al. Platelet function profiles in pa‐ tients with type 2 diabetes and coronary artery disease on combined aspirin and clo‐

[70] Geisler T, Anders N, Paterok M, et al. Platelet response to clopidogrel is attenuated in diabetic patients undergoing coronary stent implantation. Diabetes Care

Diabetes 2012;61:2913-21.

190 The Non-Thrombotic Role of Platelets in Health and Disease

Diabetes Care 2010;33:1262-8.

pidogrel treatment. Diabetes 2005;54:2430-5.

2011;22:338-44.

2014;234:146-51.

2007;30:372-4.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that gradually leads to severe cognitive impairment. The neuropathological hallmarks of AD include beta-amyloid plaques, tau neurofibrillary tangles, inflammation and glial responses, vascular dysfunction, synapse loss and cholinergic neurodegeneration. Currently, the diagnosis of possible or probable AD is based on a time consuming combination of clinical and psychological testing, imaging, and the analysis of three well-established biomarkers (beta-amyloid(42), total tau and phospho-tau-181) in cerebrospinal fluid. The search for biomarkers in blood is of high importance to avoid invasive lumbar puncture and to allow fast and easy analysis of a high number of patients. Biomarkers have been screened in blood of AD patients in plasma/serum, peripheral blood mononuclear cells (PBMCs), monocytes or also in platelets.

Platelets are interesting targets to study AD because they share some properties with neurons: they contain the neurotransmitter serotonin and the amyloid-precursor protein (APP), which produces the beta-amyloid, which aggregates in the brain of AD patients. Moreover, platelets are an easily accessible source of human cells. This review focuses on changes in the platelets of AD patients and will summarize (1) platelet activation including mean platelet volume, membrane fluidity and coated platelets, (2) serotonin metabolism, (3) APP isoforms and processing enzymes, including secretases, (4) oxidative stress and radicals, including nitric oxide metabolism and mitochondrial pathologies, including cytochrome-c oxidase and monoamino-oxidase-B and (5) enzymatic activity, such as glycogen synthase kinase-3 or phospholipase A2. In spite of all efforts, the discrepant results so far have prevented the establishment of a valid platelet-derived AD biomarker. With this survey we provide a detailed review about the major current findings on the potential use of platelet markers in AD.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by progressive deterioration in cognition leading to premature death. The neuropathological hallmarks of AD include beta-amyloid (Aβ) plaques, neurofibrillary tau tangles, inflammation

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and glial responses, synapse loss and cholinergic neurodegeneration among others. The worldwide prevalence of dementia was estimated as high as 24.2 million by 2005 and is expected to quadruplicate by 2050 [1, 2]. North America and Western Europe show the highest prevalence rate of dementia for persons >60 years [3]. Most dementia cases (70%) are attributed to AD, placing a considerable burden on society [2]. The increasing rate of AD cases not only stresses the establishment of efficient therapeutic intervention but also the identification of economic and reliable biomarkers. In both domains great efforts and smaller progress have been made in the last decades, while the desired breakthrough has yet to be awaited.

AD is classified into clinically indistinguishable early onset genetic AD (onset < 60 years) and late onset sporadic AD (> 60 years). Early onset AD accounts for approx. 1-5% of all cases and is associated with a more rapid progression of the neurodegenerative process and autosomal dominantly inherited mutations with a high penetrance. Sporadic AD accounts for >95% of all dementia cases and turned out to be a more heterogeneous disease. Currently, the diagnosis of possible or probable AD according to the NINCDS-ADRDA criteria is based on a time consuming combination of psychological testing, imaging, and the analysis of three wellestablished biomarkers (beta-amyloid(42), total tau and phospho-tau-181) in cerebrospinal fluid (CSF).

Biochemical markers mirror the physiological changes and can be objectively measured and evaluated as an indicator of pathological changes. A good biomarker must be sufficiently sensitive to detect early changes and specific to differentiate AD from clinically similar conditions. Besides high diagnostic and prognostic accuracy, biomarkers should allow global reproducibility with non-invasive and easy-to-perform tests. Within the search for peripheral AD-specific biomarkers, anucleated blood platelets have shown to be a promising target, since they represent the principal component of human blood affected by (early) biochemical alterations during AD. Moreover, platelets are an easily accessible source of human tissue and contain proteins found in neuronal cells.

In this chapter we aim to review changes in AD occurring in platelets to understand whether they may have a potential as putative biomarkers for diagnosing AD. We will focus on (1) platelet activation including mean platelet volume, membrane fluidity and coated platelets, (2) serotonin metabolism, (3) APP isoforms and processing enzymes, including secretases, (4) oxidative stress and radicals, including nitric oxide metabolism and mitochondrial patholo‐ gies, including cytochrome-c oxidase and monoamino-oxidase-B activity and (5) enzymatic activity, such as glycogen synthase kinase-3 or phospholipase A2. In spite of all efforts made, the discrepant results so far have prevented the establishment of a valid platelet-derived AD biomarker. With this survey we provide a detailed review about the major current findings on the potential use of platelet markers in AD.

## **2. Platelet activation**

Vascular risk factors were traditionally considered to distinguish between vascular dementia and AD. However, in the last decade a series of studies revealed that vascular events are involved in the development of AD and the arbitrary classification into vascular dementia and AD is very much outdated [4-7]. Several studies reported vascular alterations such as an increased number of fragmented vessels, atrophic string vessels, changed vessel diameters, altered capillary membrane and collagen accumulation in the basement membrane of AD patients [8, 9]. Besides, deposits of Aβ in cerebral vessels (cerebral amyloid angiopathy (CAA)) induce severe damage of the vessel wall and alter the cerebral blood flow promoting the progression of AD [10]. Moreover, it became clear that AD associated alterations are not solely limited to the brain and occur in vessels and blood cells of the peripheral system [11].

Activation and aggregation of platelets are important steps for haemostasis at sites of vascular injury, while uncontrolled activation can trigger thrombotic vessel occlusion at sides of atherosclerotic plaque rupture [12] and lead to chronic inflammatory reactions. In AD, activated platelets are strongly linked to vascular processes and are proposed to be the missing link for the association between atherosclerotic events and AD [13]. Adhesion and activation of platelets on the vascular wall progressively lead to vascular inflammation and atheroscle‐ rosis, thus playing a key role in the development of AD-associated conditions. Increased platelet activation has been identified in the late 90s, due to damaged cerebral endothelial cells or membrane abnormalities in the AD brain [14].

Moreover, increased platelet activation, measured by GPIIb-IIIa complex activation or Pselectin expression is significantly higher in AD patients with fast cognitive decline compared to slow cognitive decline [13]. Furthermore it is known, that peripheral Aβ peptides contribute to platelet adhesion and activation in the initiation of thrombus formation [15, 16]. Recently it has been shown that activated platelets aggregate at sites of vascular Aβ promoting CAA by inducing platelet thrombus formation leading to vessel occlusion at vascular Aβ plaques [17, 18]. Because platelets are major players in blood flow alterations and vascular diseases, it was suggested that they do not only mirror AD related changes, but promote actively the progres‐ sion of AD. In light of these findings it is conceivable that increased platelet activity could induce the progression of AD by contributing to peripheral vascular damage and endothelial senescence. Uncontrolled activation of platelets in AD-subjects may result in chronic inflam‐ mation mediating endothelial cell stress, which, in turn, may trigger platelet activation [19]. Alternatively, systemic inflammation in AD patients may result in platelet activation creating a vicious, self-amplifying circle [20]. Thus, it is conceivable that activated platelets contribute at different sites to the progression of AD.


**Table 1.** Platelet activation in AD

and glial responses, synapse loss and cholinergic neurodegeneration among others. The worldwide prevalence of dementia was estimated as high as 24.2 million by 2005 and is expected to quadruplicate by 2050 [1, 2]. North America and Western Europe show the highest prevalence rate of dementia for persons >60 years [3]. Most dementia cases (70%) are attributed to AD, placing a considerable burden on society [2]. The increasing rate of AD cases not only stresses the establishment of efficient therapeutic intervention but also the identification of economic and reliable biomarkers. In both domains great efforts and smaller progress have

been made in the last decades, while the desired breakthrough has yet to be awaited.

fluid (CSF).

contain proteins found in neuronal cells.

192 The Non-Thrombotic Role of Platelets in Health and Disease

the potential use of platelet markers in AD.

**2. Platelet activation**

AD is classified into clinically indistinguishable early onset genetic AD (onset < 60 years) and late onset sporadic AD (> 60 years). Early onset AD accounts for approx. 1-5% of all cases and is associated with a more rapid progression of the neurodegenerative process and autosomal dominantly inherited mutations with a high penetrance. Sporadic AD accounts for >95% of all dementia cases and turned out to be a more heterogeneous disease. Currently, the diagnosis of possible or probable AD according to the NINCDS-ADRDA criteria is based on a time consuming combination of psychological testing, imaging, and the analysis of three wellestablished biomarkers (beta-amyloid(42), total tau and phospho-tau-181) in cerebrospinal

Biochemical markers mirror the physiological changes and can be objectively measured and evaluated as an indicator of pathological changes. A good biomarker must be sufficiently sensitive to detect early changes and specific to differentiate AD from clinically similar conditions. Besides high diagnostic and prognostic accuracy, biomarkers should allow global reproducibility with non-invasive and easy-to-perform tests. Within the search for peripheral AD-specific biomarkers, anucleated blood platelets have shown to be a promising target, since they represent the principal component of human blood affected by (early) biochemical alterations during AD. Moreover, platelets are an easily accessible source of human tissue and

In this chapter we aim to review changes in AD occurring in platelets to understand whether they may have a potential as putative biomarkers for diagnosing AD. We will focus on (1) platelet activation including mean platelet volume, membrane fluidity and coated platelets, (2) serotonin metabolism, (3) APP isoforms and processing enzymes, including secretases, (4) oxidative stress and radicals, including nitric oxide metabolism and mitochondrial patholo‐ gies, including cytochrome-c oxidase and monoamino-oxidase-B activity and (5) enzymatic activity, such as glycogen synthase kinase-3 or phospholipase A2. In spite of all efforts made, the discrepant results so far have prevented the establishment of a valid platelet-derived AD biomarker. With this survey we provide a detailed review about the major current findings on

Vascular risk factors were traditionally considered to distinguish between vascular dementia and AD. However, in the last decade a series of studies revealed that vascular events are

#### **2.1. Effects on platelet volume**

The platelet volume is a marker of platelet activation and thus involved in the pathophysiology of multiple pro-inflammatory diseases. Along with platelet activation, platelet volume values are considered indicators for vascular events linked to cerebral vascular dementia. In AD, both increased [21, 22] and decreased [23, 24] mean platelet volume has been reported. These heterogeneous results point out the importance of establishing methodological consensus in the isolation and processing of platelets, since they are very sensitive to cellular damage.


**Table 2.** Platelet volume in AD

#### **2.2. Effects on membrane fluidity**

Discrepant results have been published concerning platelet membrane fluidity: while de‐ creased fluidity of cell membranes from platelets is associated with normal ageing, increased internal membrane fluidity is one of the first alterations to be reported in platelets [25, 26]. Increased platelet membrane fluidity becomes apparent by a decrease in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) in labelled membranes. It was proposed, that this apparent abnormality of membranes in AD patients is due to an increase in internal membranes [26]. However, no significant differences in either cholesterol or phospholipid compared to healthy subjects has been observed [26], while an increase of intracellular platelet membranes in AD patients has been reported [27]. Other findings suggest furthermore that demented patients with increased platelet membrane fluidity display an earlier onset and a more rapid deterioration of cognitive symptoms compared to other demented subjects [25]. In light of these findings, platelet membrane fluidity was proposed as a biological risk factor for AD, since it is associated with significant contributions to the risk of developing AD [28, 29]. However, others failed to demonstrate differences in platelet membrane fluidity between AD patients and healthy controls, concluding that platelet membrane fluidity cannot be considered as antemortem biomarker for AD [30-32]. An increase in DPH fluorescence anisotropy has been reported [33-36] and as anisotropy is inversely related to membrane fluidity, these results indicate in contrast to the previously mentioned studies a decrease of the external and internal membrane fluidity in AD [33]. It was suggested that the short life span of platelets makes them less susceptible for long-term modifications. And finally, a reduced fluidity in the platelet inner mitochondrial membrane has been reported, which was associated with oxidative damage [37], while others correlated membrane fluidity with alterations of APP fragments [38].


**Table 3.** Platelet membrane fluidity in AD

#### **2.3. Coated platelets**

**2.1. Effects on platelet volume**

194 The Non-Thrombotic Role of Platelets in Health and Disease

**Table 2.** Platelet volume in AD

**2.2. Effects on membrane fluidity**

The platelet volume is a marker of platelet activation and thus involved in the pathophysiology of multiple pro-inflammatory diseases. Along with platelet activation, platelet volume values are considered indicators for vascular events linked to cerebral vascular dementia. In AD, both increased [21, 22] and decreased [23, 24] mean platelet volume has been reported. These heterogeneous results point out the importance of establishing methodological consensus in the isolation and processing of platelets, since they are very sensitive to cellular damage.

**References Effect on platelet volume**

Yesil et al., 2012 ↑ in AD

Yagi et al., 1984 ↑ in vascular dementia compared to AD

Wang et al., 2013 ↓ in AD and MCI; associated with MMSE

Discrepant results have been published concerning platelet membrane fluidity: while de‐ creased fluidity of cell membranes from platelets is associated with normal ageing, increased internal membrane fluidity is one of the first alterations to be reported in platelets [25, 26]. Increased platelet membrane fluidity becomes apparent by a decrease in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) in labelled membranes. It was proposed, that this apparent abnormality of membranes in AD patients is due to an increase in internal membranes [26]. However, no significant differences in either cholesterol or phospholipid compared to healthy subjects has been observed [26], while an increase of intracellular platelet membranes in AD patients has been reported [27]. Other findings suggest furthermore that demented patients with increased platelet membrane fluidity display an earlier onset and a more rapid deterioration of cognitive symptoms compared to other demented subjects [25]. In light of these findings, platelet membrane fluidity was proposed as a biological risk factor for AD, since it is associated with significant contributions to the risk of developing AD [28, 29]. However, others failed to demonstrate differences in platelet membrane fluidity between AD patients and healthy controls, concluding that platelet membrane fluidity cannot be considered as antemortem biomarker for AD [30-32]. An increase in DPH fluorescence anisotropy has been reported [33-36] and as anisotropy is inversely related to membrane fluidity, these results indicate in contrast to the previously mentioned studies a decrease of the external and internal membrane fluidity in AD [33]. It was suggested that the short life span of platelets makes them less susceptible for long-term modifications. And finally, a reduced fluidity in the platelet inner mitochondrial membrane has been reported, which was associated with oxidative damage [37], while others correlated membrane fluidity with alterations of APP fragments [38].

Liang et al., 2013 ↓ in vascular dementia and AD

Coated-platelets (PLTs) are a subset of platelets produced upon dual-agonist stimulation with collagen und thrombin retaining several procoagulant α-granule proteins on their surface [39]. PLTs are important for the coagulation cascade because of their ability to generate thrombin at sites of vascular damage. Effectively, it was suggested that increased levels of PLTs may be related to prothrombotic conditions [40], while decreased levels of PLTs were linked to an increased risk for haemorrhage [41]. It was also demonstrated that coated-platelet levels are elevated in amnestic mild cognitive impairment (MCI) and correlate with the progression of AD [40, 42-45]. Additionally, the same group showed that MCIs with elevated coated-platelet levels are more likely to develop AD, while they found no significant alterations in patients with frontotemporal lobe dementia [44, 45]. So far, coated platelets seem to be an interesting target, which needs to be reproduced internationally by other research groups.

## **3. Serotonin**

Several studies report abnormalities in serotonin (5-HT) concentration as well as alterations in its uptake during AD, possibly linked to psychobehavioral problems such as e.g. depression [46-48]. Platelets accumulate high levels of serotonin in dense granules and release it upon activation [49]. Early studies failed to demonstrate altered serotonin processing in platelets [50, 51], while in the early 90s heterogeneous results regarding 5-HT Km (Michaelis-Menten constant) and maximal velocity (Vmax) binding affinity were published [52, 53, 54]. Compared to controls, patients with AD demonstrated in different studies significantly lower platelet serotonin concentrations [55-58]. However, an increased 5-HT concentration in low-density platelet populations was recently reported [59]. Likewise, heterogeneous results have been reported for serotonin uptake: both increased and decreased serotonin uptake was found in AD platelets [52, 60]. In light of these data and despite great efforts, so far platelet-derived serotonin has not been established as a reliable biomarker for AD.


Vmax = maximum number of 5-HT uptake sites; Km = Michaelis-Menten constant; EOAD = Early Onset AD

**Table 4.** Platelet 5-HT in AD

## **4. Amyloid Precursor protein (APP) and secretases**

#### **4.1. APP isoforms and ratios**

APP is an integral membrane protein with a large extracellular domain and a shorter, intra‐ cellular C-terminal tail. Three major APP isoforms (770, 751 and 695 kDa) have been described. APP 751 und APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI), while APP 695 lacks this domain. The APP isoforms are cut by different enzymes (secretases) into smaller peptides, whereas sequential cleavage by β-secretase (BACE1) and γ-secretase (ADAM-10) generates the neurotoxic Aβ fragments. Conversely, cleavage by α-secretase precludes the formation of amyloid fragments by processing APP within the Aβ domain.

Platelets are of particular interest in AD research, because they contain high levels of APP [61-63]. In contrast to neuronal tissues where isoform 695 lacking the KPI domain is the most abundant one, platelets express mainly APP770 whereas APP695 is marginally present [64]. Platelets contain α-, β-, γ- secretase activities and generate different APP fragments: sAPPα, sAPPβ, the amyloidogenic fragment (C99) and Aβ peptides [65, 66]. Platelet APP is mainly processes by the α-secretase pathway releasing soluble APP (sAPP) [66] and predominantly Aβ(40). Both APP and Aβ are stored in α-granules of platelets and become released upon activation by agents like the physiological agonists thrombin and collagen. A recent study reports significant up-regulation of platelet APP isoforms compared to controls, and a correlation between APP mRNA levels and cognitive impairment [67]. The same group found significant up-regulation of platelet mRNA expression level of total APP and APP containing a KPI domain in patients with AD and frontotemporal lobe dementia compared to controls [68]. We have recently shown that platelet-secreted APPβ in MCI and AD is significantly increased when measured with ELISA compared to control subjects, while no changes in sAPPα are seen [69].


**Table 5.** Platelet APP expression in AD

51], while in the early 90s heterogeneous results regarding 5-HT Km (Michaelis-Menten constant) and maximal velocity (Vmax) binding affinity were published [52, 53, 54]. Compared to controls, patients with AD demonstrated in different studies significantly lower platelet serotonin concentrations [55-58]. However, an increased 5-HT concentration in low-density platelet populations was recently reported [59]. Likewise, heterogeneous results have been reported for serotonin uptake: both increased and decreased serotonin uptake was found in AD platelets [52, 60]. In light of these data and despite great efforts, so far platelet-derived

Andersson et al., 1991 unchanged maximum number of binding sites (Bmax) and binding affinity

↓ Km and Vmax

females tested)

↓ ability to accumulate 5-HT

↑ affinity of binding of 5-HT to the platelet membrane in AD (only

↓ 5-HT concentrations in non-psychotic female and psychotic male

↓ 5-HT concentrations in the late phase of AD compared to other

↑ Vmax 5-HT-uptake in mild and moderate AD Trend ↓ Vmax 5-HT-uptake in severe AD

AD patients compared to controls

phases and controls

APP is an integral membrane protein with a large extracellular domain and a shorter, intra‐ cellular C-terminal tail. Three major APP isoforms (770, 751 and 695 kDa) have been described.

Prokselj et al., 2014 ↓ 5-HT concentrations in AD compared to AD controls

Vmax = maximum number of 5-HT uptake sites; Km = Michaelis-Menten constant; EOAD = Early Onset AD

serotonin has not been established as a reliable biomarker for AD.

**References Effects on serotonin**

196 The Non-Thrombotic Role of Platelets in Health and Disease

Kumar et al., 1995a ↓ 5-HT concentration

Koren et al., 1993 ↓ [3H]-5-HT uptake

Milovanovic et al., 2014 ↑ in low-density platelet populations

**4. Amyloid Precursor protein (APP) and secretases**

Inestrosa et al., 1993

Kumar et al., 1995b

Arora et al., 1991

Mimica et al., 2008

Muck-Seler et al., 2009

**Table 4.** Platelet 5-HT in AD

**4.1. APP isoforms and ratios**

Tukiainen et al., 1981 unchanged 5-HT uptake in

Several studies showed that the platelet APP ratio (defined as the ratio between the upper 130kDa and the lower 106-110kDa isoforms) is significantly lower in AD patients compared to controls and patients with other forms of dementia [70-77]. It seems that the alteration of platelet APP isoforms is an early event in AD and the ratio shows to be a consistent predictor for the conversion from MCI to AD. In fact, MCI subjects converting to AD showed signifi‐ cantly decreased APP ratios at baseline compared to other dementia forms and stable MCI subjects [78-80]. Furthermore, the APP ratio positively correlates with cognitive decline, i.e. the lower the ratio, the more severe the disease [38, 73]. Furthermore, it has been shown that carriers of the APOE4 allele are associated with a larger reduction in the APP ratio [81]. Moreover, administration of acetylcholine esterase inhibitors [82, 83] increases the ratio of APP forms in AD suggesting a possible effect of these drugs on APP trafficking in platelets. The proposed cut-off scores are around 0.56 with a sensitivity of 88% and specificity of 89% [80, 84]. Thus, the APP ratio has been proposed as a potential biomarker in prodromal AD-stages and a reliable indicator for the disease progress. Despite the promising homogeneous findings the validity of APP ratio as a useful supportive biomarker for AD diagnosis is not yet inter‐ nationally established. Methodological problems including lack of sensitivity and diversity of the used antibodies and different isolation procedures of platelets may account for this problem.


**Table 6.** Platelet APP ratio in AD

#### **4.2. Enzymatic activity: BACE1 and ADAM-10**

APP is cleaved into secreted (soluble) APP (sAPP), smaller intracellular fragments and the Aβ peptides (40, 42 or 43 amino acids) by three secretases (α, β, γ). The α-secretase leads to the non-amyloidogenic pathway, while cleavage of APP by β -secretase (BACE1) and γ-secretase (ADAM-10) generates the toxic Aβ fragments. So far, several authors report increased BACE1 in AD by Western blotting or ELISA analysis [69, 73, 85, 86]. However, using a novel ELISA system a significant decrease of BACE1 N-terminal and C-terminal fragments has been reported in AD [87]. ADAM-10 is the major constitutive α-secretase for APP processing [88, 89]. To date, platelet ADAM10 (a disintegrin and metalloprotease) has been reported to be significantly reduced in platelets in AD [85, 90, 91] while others failed to detect changes in alpha- and beta-secretase activities in AD [92]. Moreover, reduction in ADAM-10 levels correlates with the progression and seems to be stage-dependent [91].


**Table 7.** Platelet secretase activity in AD

proposed cut-off scores are around 0.56 with a sensitivity of 88% and specificity of 89% [80, 84]. Thus, the APP ratio has been proposed as a potential biomarker in prodromal AD-stages and a reliable indicator for the disease progress. Despite the promising homogeneous findings the validity of APP ratio as a useful supportive biomarker for AD diagnosis is not yet inter‐ nationally established. Methodological problems including lack of sensitivity and diversity of the used antibodies and different isolation procedures of platelets may account for this

Stroke);

mutations

Borroni et al., 2002, 2003, 2004 ↓ in MCIs converting to AD and early stages of probable AD Liu et al., 2005 ↓ in AD; increases with galantamine treatment for 12 weeks

APP is cleaved into secreted (soluble) APP (sAPP), smaller intracellular fragments and the Aβ peptides (40, 42 or 43 amino acids) by three secretases (α, β, γ). The α-secretase leads to the non-amyloidogenic pathway, while cleavage of APP by β -secretase (BACE1) and γ-secretase (ADAM-10) generates the toxic Aβ fragments. So far, several authors report increased BACE1 in AD by Western blotting or ELISA analysis [69, 73, 85, 86]. However, using a novel ELISA system a significant decrease of BACE1 N-terminal and C-terminal fragments has been reported in AD [87]. ADAM-10 is the major constitutive α-secretase for APP processing [88, 89]. To date, platelet ADAM10 (a disintegrin and metalloprotease) has been reported to be significantly reduced in platelets in AD [85, 90, 91] while others failed to detect changes in alpha- and beta-secretase activities in AD [92]. Moreover, reduction in ADAM-10 levels

Zainaghi et al., 2007, 2012 ↓ in MCI converting to dementia upon follow-up

↓ in AD and elderly patients with Down Syndrom; ratio

↓ specific in AD compared to PD and HS (Hemorrhagic

unchanged in cognitively normal young adults carrying PS-1

correlated with the severity of the disease

**References Effects on APP ratio**

198 The Non-Thrombotic Role of Platelets in Health and Disease

Tang et al., 2006 ↓ in AD

**4.2. Enzymatic activity: BACE1 and ADAM-10**

Rosenberg et al., 1997 ↓ in AD compared to controls

Padovani et al., 2001, 2002 ↓ in MCI and mild and very mild AD

Srisawat et al., 2013 ↓ in Thai patients with AD

correlates with the progression and seems to be stage-dependent [91].

problem.

Di Luca et al., 1996, 1998

Baskin et al., 2000, 2001

**Table 6.** Platelet APP ratio in AD

#### **4.3. Activation of platelets by beta-amyloid**

It is well known that different heterogeneous amyloidogenic peptides (Aβ(1-40), Aβ(1-42), Aβ(25-36)) as well as aggregated Aβ can induce platelet aggregation [15, 16]. Platelets store and release preferentially the 40 amino acid Aβ fragment in their granules upon stimulation with physiological agonists like thrombin, collagen or calcium ionophores [93, 61, 94, 95]. Once released, Aβ peptides trigger platelet activation, initiating a vicious feedback loop of platelet activation and Aβ release. Further, apoptotic stimuli significantly increase platelet Aβ(40) but not Aβ(42) suggesting that this pathway determines altered APP processing [96, 97]. Recently it was shown that Aβ induces platelet activation independent of known physiological agonists [16]. It was furthermore suggested that platelets modulate aggregation of soluble Aβ into fibrillar Aβ and facilitate platelet adhesion at vascular Aβ accumulations, contributing to the full occlusion of the affected vessel [18]. At this point it seems therefore likely that platelets and platelet-derived Aβ may contribute to a significant degree to the amyloid burden in the vascular walls promoting CAA in AD patients [96].

## **5. Oxidative stress, radicals and mitochondrial pathologies**

Excessive chronic oxidative stress and production of radicals in the AD brain has been considered to promote cellular degeneration. Especially, nitric oxide (NO) and peroxynitrite (ONOO) are very reactive toxic radicals (ROS). It becomes more and more clear that a dysre‐ gulation of mitochondria and the involvement of cytochrome C-oxidase may play a role in this process. Vascular damage and endothelial dysfunction may play a role in AD and platelets serve as a source of oxidative stress. Mitochondria are the major sites responsible for more than 90 % of the ROS generation. In AD, mitochondrial DNA of cortical neurons was reported to induce excessive oxidative damage and increased DNA mutations [98-100]. Moreover, abnormal mitochondrial size and decreased mitochondrial number in AD and MCI are likely to increase ROS generation and oxidative damage [99, 101]. In platelets reduced Complex IV and Complex III activity has been repeatedly associated with AD [102-105, 32]. Subsequently, Aβ was shown to interact directly with mitochondria and inhibit platelet Complex IV activity inducing oxidative stress [106].

#### **5.1. Nitric oxide and peroxynitrite**

An increase in nitric oxide synthase (NOS) has been associated with normal aging and with AD [107, 108, 33]. Similarly to NO, peroxynitrite (ONOO) was found to be increased in AD patients and both NO and ONOO are linked to reduced Na+/K+-ATPase activity in platelet membranes of AD patients [108]. Additionally, carriers of the epsilon 4 allele of apolipoprotein E (APOE) show higher NOS compared to non-carriers [109]. In contrast, others found signif‐ icantly lower NO concentrations in platelets of AD patients and a generally higher platelet aggregation rate. Since NO is known to inhibit platelet aggregation, NO might be responsible for the aggregation of platelets in the observed AD cohort [110].


**Table 8.** Platelet nitric oxide and peroxynitrite concentrations in AD

#### **5.2. Cytochrome c oxidase**

Cytochrome C oxidase (COX) is an enzyme located in mitochondria and may play a role in the production of radicals. In most studies, reduced platelet COX activity has been found in AD [111-113, 102, 103, 32, 104] but also in cognitively normal individuals with a maternal history of AD [114]. However, another study did not find any differences in COX activity in AD [115]. Recently, reduced mitochondrial COX activity has been found, which correlates to decreased mitochondrial membrane potential, resulting in higher lipid peroxides, superoxide radicals and protein carbonyls [111]. It has been proposed, that reduced COX activity causes higher tissue vulnerability and reduced oxygen availability [104].

#### **5.3. Monoamino-oxidase B**

Monoamino-oxidase-B (MAO-B) is an important enzyme located in the mitochondria and plays a role in metabolic processes of serotonin. Studies on platelet MAO-B activity yielded inconsistent results up to date: increased MAO-B activity was reported by several groups [116-126], while few researchers could not find any abnormalities [127,128]. The decline of mini-mental state examination scores (MMSE) preceded the elevation of MAO-B activity


**Table 9.** Platelet COX activity in AD

abnormal mitochondrial size and decreased mitochondrial number in AD and MCI are likely to increase ROS generation and oxidative damage [99, 101]. In platelets reduced Complex IV and Complex III activity has been repeatedly associated with AD [102-105, 32]. Subsequently, Aβ was shown to interact directly with mitochondria and inhibit platelet Complex IV activity

An increase in nitric oxide synthase (NOS) has been associated with normal aging and with AD [107, 108, 33]. Similarly to NO, peroxynitrite (ONOO) was found to be increased in AD patients and both NO and ONOO are linked to reduced Na+/K+-ATPase activity in platelet membranes of AD patients [108]. Additionally, carriers of the epsilon 4 allele of apolipoprotein E (APOE) show higher NOS compared to non-carriers [109]. In contrast, others found signif‐ icantly lower NO concentrations in platelets of AD patients and a generally higher platelet aggregation rate. Since NO is known to inhibit platelet aggregation, NO might be responsible

for the aggregation of platelets in the observed AD cohort [110].

**References Effects on nitric oxide**

**Table 8.** Platelet nitric oxide and peroxynitrite concentrations in AD

tissue vulnerability and reduced oxygen availability [104].

**5.2. Cytochrome c oxidase**

**5.3. Monoamino-oxidase B**

Kawamoto et al., 2005 ↑ NO and peroxynitrite in AD

Vignini et al., 2007, 2013; ↑ NO and ONOO(-) peroxynitrite in AD Marcourakis et al., 2008 ↑ NOS activity in APOE epsilon 4 carriers

Yu and Jia, 2009 ↓ NO and eNOS (endothelial nitric oxide synthase)

Cytochrome C oxidase (COX) is an enzyme located in mitochondria and may play a role in the production of radicals. In most studies, reduced platelet COX activity has been found in AD [111-113, 102, 103, 32, 104] but also in cognitively normal individuals with a maternal history of AD [114]. However, another study did not find any differences in COX activity in AD [115]. Recently, reduced mitochondrial COX activity has been found, which correlates to decreased mitochondrial membrane potential, resulting in higher lipid peroxides, superoxide radicals and protein carbonyls [111]. It has been proposed, that reduced COX activity causes higher

Monoamino-oxidase-B (MAO-B) is an important enzyme located in the mitochondria and plays a role in metabolic processes of serotonin. Studies on platelet MAO-B activity yielded inconsistent results up to date: increased MAO-B activity was reported by several groups [116-126], while few researchers could not find any abnormalities [127,128]. The decline of mini-mental state examination scores (MMSE) preceded the elevation of MAO-B activity

inducing oxidative stress [106].

**5.1. Nitric oxide and peroxynitrite**

200 The Non-Thrombotic Role of Platelets in Health and Disease

[116-119]. It has been suggested that MAO-B activity might be an indicator for severity and clinical progress in AD [57]. In another study, non-psychotic AD patients showed significantly higher platelet MAO-B activity, suggesting that MAO-B activity can differentiate between psychotic and non-psychotic subtypes of AD [56].


**Table 10.** Platelet MAO-B activity in AD

## **6. Changes of other biological systems**

## **6.1. GSK3β activity**

Glycogen synthase kinase 3-beta (GSK3β) is involved in the regulation of glycogen synthesis by phosphorylating and inactivating the glycogen synthase and in the regulation of intracel‐ lular signalling pathways [129-131]. Moreover, in neural cell tissues GSK3β is the most important Tau kinase and has been linked to synaptic plasticity and neural injury [132, 133]. Therefore, deregulation of GSK3β has a significant impact on the formation of neurofibrillary tangles. GSK3β is also expressed in platelets and might be involved in platelet activation [134]. A substantial higher expression of GSK3β has been shown in AD and MCI platelets as compared to healthy controls, which also correlated with worse memory performance [135].

#### **6.2. Phospholipase activity**

It is known that AD is associated with chronic inflammatory responses and platelets express inflammatory mediators such as chemokines, interleukins, adhesive proteins and contain enzymes such as phospholipase-A2 (PLA2) [20]. Phospholipases are important platelet enzymes involved in the metabolism of membrane phospholipids and inflammatory synthesis. PLA2 and phospholipase C (PLC) are altered in peripheral blood cells and significantly decreased PLC activity in platelets of AD patients has been reported [136], suggesting aberrant phospholipase metabolism. Further, decreased PLA2 activity was found in human platelets [137, 138], which correlates with the degree of cognitive impairment and is modulated by cognitive training in healthy elders [139]. In contrast, an increased platelet PLA2 activity in individuals with AD has also been shown [140]. Thus, again the reports on these enzyme activities are very controversial and do not allow to establish a concluding interpretation.


**Table 11.** Platelet PLA2 and PLC activity in AD

#### **6.3. Overview of other platelet components found to be altered in AD**

Several other biological systems have been studied in platelets of AD patients, such as e.g. decreased plasma antioxidant power levels [107], decreased cathepsin D [141], increased platelet glutamine synthetase-like protein level in MCI [112], increased NA,K-ATPase activities in AD [107], unchanged Vitamin E and cholesterol content between AD and controls [32] or increased phenolsulphotransferase activity in demented patients [142] or decreased platelet peripheral-type benzodiazepine binding [143]. However, we do not claim to provide

a complete overview of all markers. Anyhow, none of these markers has been found to be a suitable reproducible marker for AD.

## **7. Platelets as biomarkers**

**6. Changes of other biological systems**

202 The Non-Thrombotic Role of Platelets in Health and Disease

Glycogen synthase kinase 3-beta (GSK3β) is involved in the regulation of glycogen synthesis by phosphorylating and inactivating the glycogen synthase and in the regulation of intracel‐ lular signalling pathways [129-131]. Moreover, in neural cell tissues GSK3β is the most important Tau kinase and has been linked to synaptic plasticity and neural injury [132, 133]. Therefore, deregulation of GSK3β has a significant impact on the formation of neurofibrillary tangles. GSK3β is also expressed in platelets and might be involved in platelet activation [134]. A substantial higher expression of GSK3β has been shown in AD and MCI platelets as compared to healthy controls, which also correlated with worse memory performance [135].

It is known that AD is associated with chronic inflammatory responses and platelets express inflammatory mediators such as chemokines, interleukins, adhesive proteins and contain enzymes such as phospholipase-A2 (PLA2) [20]. Phospholipases are important platelet enzymes involved in the metabolism of membrane phospholipids and inflammatory synthesis. PLA2 and phospholipase C (PLC) are altered in peripheral blood cells and significantly decreased PLC activity in platelets of AD patients has been reported [136], suggesting aberrant phospholipase metabolism. Further, decreased PLA2 activity was found in human platelets [137, 138], which correlates with the degree of cognitive impairment and is modulated by cognitive training in healthy elders [139]. In contrast, an increased platelet PLA2 activity in individuals with AD has also been shown [140]. Thus, again the reports on these enzyme activities are very controversial and do not allow to establish a concluding interpretation.

**References Effects on PLA2 and PLC activity**

**6.3. Overview of other platelet components found to be altered in AD**

Several other biological systems have been studied in platelets of AD patients, such as e.g. decreased plasma antioxidant power levels [107], decreased cathepsin D [141], increased platelet glutamine synthetase-like protein level in MCI [112], increased NA,K-ATPase activities in AD [107], unchanged Vitamin E and cholesterol content between AD and controls [32] or increased phenolsulphotransferase activity in demented patients [142] or decreased platelet peripheral-type benzodiazepine binding [143]. However, we do not claim to provide

Krzystanek et al., 2007 ↑ PLA2 in AD

Matushima et al., 1995, 1998 ↓ PLC in AD

**Table 11.** Platelet PLA2 and PLC activity in AD

Gattaz et al., 1995, 1996, 2004 ↓ PLA2 in AD and MCI

**6.1. GSK3β activity**

**6.2. Phospholipase activity**

Biomarkers must objectively reflect physiological processes linked to AD and should be globally reproducible with easy-to-perform tests [144]. Moreover, besides being sensitive enough to differentiate AD from clinically similar diseases, a good biomarker should be able to detect early disease-associated changes. In AD, abnormal metabolism of APP, hyperphos‐ phorylation of Tau, induction of oxidative stress and inflammatory cascades result in patho‐ logical changes in liquid fluids. Currently, the laboratory diagnosis of AD is based on the combination of three CSF biomarkers yielding a sensitivity of >95% and specificity of >85% [145, 146]. However, considering the invasiveness of lumbar puncture and the growing incidence rate of AD, the need for biomarkers in more accessible body fluids is necessary. Blood measurements are minimally invasive and less time-consuming compared to CSF. The establishment of new markers in plasma/serum has proven to be difficult because changes mirror a broad spectrum of physiological processes not necessarily related to AD [144]. Moreover, changes in plasma/serum are very small and heterogeneous, thus making the search for reliable and sensitive biomarkers challenging.

Within the search for peripheral biomarkers in AD, blood platelets have been of great interest during the last decades. These anuclear blood cells share several homeostatic functions with neurons such as accumulation and release of neurotransmitters like serotonin, and expression of receptors and enzymes [147]. Moreover, human platelets have shown to be the most important source of more than 90% of the circulating APP [148, 149]. On grounds of these findings, platelets can be considered a valid peripheral model for the analysis of metabolic pathways linked to AD. Despite great efforts, to date no specific platelet biomarker has been successfully established, while some platelet components have shown to be of greater validity than others.

Overall, the so far published studies underline the need to establish concurrent methods in order to yield comparable results and avoid methodological diversity among institutes. Platelets contain several types of surface receptors responding to external stimuli with activation or inhibitory actions [150]. In response to physiological and non-physiological stimuli, platelet granules and their contents are released, thus requiring careful handling during isolation and experimentation in order to avoid activation. Effectively, different platelet isolation and processing procedures may account to a significant extent for the observed interrater differences between institutes. Moreover, different phases of the disease, comorbidities or medications need to be taken into consideration when evaluating platelet status in AD patients. Additionally, further analysis and classification of platelets with respect to their different features (e.g. density fraction) might be helpful to generate more significant data. Besides a homogeneous patient group, the selection of healthy, age-matched controls is a known challenge in AD-research and might additionally explain varying data. Therefore, the so far obtained results need to be replicated in large and homogeneous cohorts according to the same methodological protocol by independent researchers. To date, the use of platelet biomarkers in the diagnostic routine of AD can yet not be recommended and is in urgent need of final evaluation.

## **8. Conclusions**

In conclusion, the search for platelet biomarker so far has highlighted some candidates worth further investigation, while the breakthrough in identifying one satisfyingly specific and sensitive marker has yet to be awaited. Overall, among the studied platelet components and according to the available data, the platelet APP ratio and COX activity seem the most promising candidates for establishing new peripheral biomarkers for AD.

## **Acknowledgements**

This study has been supported by the Austrian Science Funds (P24734-B24).

## **Author details**

Barbara Plagg and Christian Humpel\*

\*Address all correspondence to: christian.humpel@i-med.ac.at

Laboratory of Psychiatry and Exp. Alzheimer's Research, Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Austria

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**Platelets in Tissue Regeneration and Wound Repair**

## **Chapter 11**

## **Platelets in Tissue Regeneration**

Ronaldo J. F. C. do Amaral and Alex Balduino

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61184

## **1. Introduction**

Platelets are especially well known for their thrombotic role. However, besides their role on stopping bleeding, platelets contribute for several mechanisms and steps in wound healing and tissue repair, such as inflammation, angiogenesis, cells proliferation, and differentiation. The potential of platelets to be used therapeutically to assist in wound repair led researchers around the world to look at platelet-based products and their capacity to promote tissue regeneration, *in vitro* and *in vivo*. In this chapter, we will discuss the main growth factors present in platelet granules that affect tissue regeneration. In addition, we will consider how platelet-derived products, such as those obtained from platelet-rich plasma (PRP), can be used to enhance tissue regeneration. We will review the applications of this knowledge in clinical trials, and *in vivo* models, as well as discussing the capacity for platelet products to substitute for classical components of media for *in vitro* cell culture.

## **2. Tissue repair related growth factors in platelets granules**

Approximately, one trillion platelets circulate in the bloodstream of a human adult (4 liters of blood at 3X108 platelets / ml). Platelets have a lifespan of approximately 10 days. They are synthesized by megakaryocytic cells in the bone marrow of long bones and approximately 10% are replenished daily. "Old" and damaged platelets are cleared from the blood by phagocytes in the liver and the spleen [1]. While in the circulation, platelets survey the vasculature for evidence of damage. If damage is perceived, they participate in hemostatic events. When platelets are activated upon vascular injury, they change their discoid form to a more spherical morphology with pseudopods, and they release their granular content [2]. Those are not only related to the coagulation process and hemostasis, but also to tissue repair. The granules are of 3 different types: α-granules, dense granules, and lysosymes. α-granules

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

are the most abundant granule-type in human platelets (50-80 per platelet) and contain a diverse protein repertoire [3] including a variety of molecules with biological activity (Table 1). These molecules are released following platelet activation and play important functional roles at sites of vascular damage.


**Table 1.** Main bioactive molecules present in platelets alpha granules Adapted from [4].

Many platelet-derived bioactive proteins play critical roles in inflammation, angiogenesis and wound healing. For example, TGF-β1, the most abundant isoform of TGF-β present in platelets, has an important role in all wound healing phases. It coordinates multiple pathophysiological events including the initial recruitment of inflammatory cells to a site of injury, angiogenesis, re-epitheliazation following damage, and the induction of extracellu‐ lar matrix production by fibroblasts [5]. PDGF is a chemoattractant molecule for fibroblasts and smooth muscle cells, as well as an inducer of proliferation of mesenchymal cells [6]. FGF-2, the main FGF isoform present in platelets, promotes angiogenesis by supporting endothelial cell growth [7]. It is also a potent fibroblast mitogen and induces hyaluronic acid synthesis to facilitate a scarless wound healing [8]. EGF enables mesenchymal cells prolifer‐ ation, chemotaxis and cytoprotection [9]. VEGF is a pro-angiogenic biomolecule that stimulates blood vessel formation [10], and expression of adhesion proteins that enhance leukocyte adhesion [11]. In fact, a total of more than 300 bioactive agents have been identi‐ fied that are released from activated platelets [12]. These agents differ in their origin with some components being synthesized in the parent megakaryocyte while others are scav‐ enged from plasma and concentrated in platelet granules [13].

are the most abundant granule-type in human platelets (50-80 per platelet) and contain a diverse protein repertoire [3] including a variety of molecules with biological activity (Table 1). These molecules are released following platelet activation and play important functional

> Fibrinogen Fibronectin Vitronectin Trombospondin-1

Factor V Factor IX Protein S Anti-thrombin

Plasminogen

α-2 antiplasmin

Metaloprotease-4 α-1-antitripsin

PF-4 (*platelet factor 4*) Endostatins β-tromboglobulins

Many platelet-derived bioactive proteins play critical roles in inflammation, angiogenesis and wound healing. For example, TGF-β1, the most abundant isoform of TGF-β present in platelets, has an important role in all wound healing phases. It coordinates multiple pathophysiological events including the initial recruitment of inflammatory cells to a site of injury, angiogenesis, re-epitheliazation following damage, and the induction of extracellu‐ lar matrix production by fibroblasts [5]. PDGF is a chemoattractant molecule for fibroblasts and smooth muscle cells, as well as an inducer of proliferation of mesenchymal cells [6]. FGF-2, the main FGF isoform present in platelets, promotes angiogenesis by supporting

CD40-L P-selectin

**Table 1.** Main bioactive molecules present in platelets alpha granules Adapted from [4].

Plasminogen activatior inhibitor

TIMP-4(*tissue inhibitor of metalloprotease-4)*

TGF-β (transforming growth factor beta) PDGF (platelet-derived growth factor) FGF (fibroblast growth factor) EGF (epidermal growth fator)

VEGF (vascular endothelial growth factor)

roles at sites of vascular damage.

222 The Non-Thrombotic Role of Platelets in Health and Disease

GROWTH FACTORS

ADHESION PROTEINS

COAGULATION FACTORS

FIBRINOLYTIC FATORS

PROTEASES AND ANTIPROTEASES

MEMBRANE GLYCOPROTEINS

OTHER PROTEINS

**CATEGORY MOLECULE**

Dense granules contain factors related to platelet activation, such as Ca2+ and ADP, serotonin, histamine, dopamine, and catecolamines. Local release of these components, in response to platelet activation or thrombotic events, results in altered recruitment of inflammatory cell types and altered vascular permeability [14]. Finally, lysosomes contain hydrolytic enzymes and catalases [15]. The nature of the contents of the platelet granules are summarised in MItrugno et al (2015) in this publication.

Platelet activation, with concomitant release of granular contents, happens in parallel with coagulation or thrombosis. The natural participation of platelets and in hemostasis and tissue repair has led to the development of products that could help in those processes.

Due to the variety of possible uses and number of studies, PRP is perhaps the main platelet based product investigated for tissue regeneration purposes. PRP is a platelet concentrate in a small volume of plasma obtained after a centrifugal spin of whole blood to remove red cells and while cells. Regular platelet concentration in peripheral blood is 150-350 x 106 per milliliter. In the context of tissue engineering and wound repair, the term PRP refers to a platelet concentration in plasma above this regular range, that can be injected into a wound site to affect or accelerate repair. The clinical use of PRP, mainly in the cases of bone and soft tissue regeneration, presents a platelet concentration of at least 109 per milliliter, which is around 5 times higher than physiological levels [16]. For peri-implant bone regeneration, for example, the recommended platelet concentration is approximately 109 per milliliter. In lower concen‐ trations the effect is suboptimal and in higher it is inhibitory [17].

The therapeutic action of platelet concentrates derives from the release of factors involved in tissue repair upon platelet activation. The clot that is formed during that activation may also play a role of a temporary extracellular matrix which will allow cells proliferation and differentiation [16]. In that case, an elevated platelet concentration would be expected to generate an elevated local concentration of released bioactive factors. However the correlation between the platelet concentration and the concentration of released bioactive agents may not be exact, due to variations between blood donors [18], or between platelet preparation methods [19]. Moreover, some growth factors that act in tissue repair are also present in plasma. Such growth factors include HGF and IGF-1. Consequently, the concentrations of these factors at sites of wounds may only be slightly altered according to the platelet concentration [20]. Generally, it is considered that a platelet concentration 5 times higher than in peripheral blood can lead to an enhanced local concentration of growth factors that varies from 3 to 5 times in excess of normal pathophysiological levels [4]. Thus, by serving as a reservoir of concentrated growth factors involved in cell proliferation and differentiation, platelet concentrates can contribute to tissue growth and repair. In a similar manner, platelet-derived bioactive products may find use in cell culture protocols.

## **3. How platelet products can improve cell culture**

Since the beginning of cell culture techniques, many innovations were designed to optimize the process of cell expansion *in vitro*, analyzing their differentiation capacity, as well as their response to chemicals and promising pharmaceutical molecules. With time, the field im‐ proved. Plastics, glasses, bioreactors and engineering technology evolved a lot, at the same time that the biological field also evolved.

Methods to grow cells *in vitro* try to reproduce appropriately all physiological conditions that are observed *in vivo*, and try to mimic it *in vitro*. The culture medium is the source of soluble factors that will enable cell growth and survival. Classical cell culture media are comprised of a basal balanced salt solution such as MEM (Eagle's minimal essential medium), DMEM (Dulbecco's modified Eagle's medium), IMDM (Iscove's modified DMEM), RPMI (Roswell Park Memorial Institute medium), Medium-199, HamF12 and McCoy's medium. Although they provide inorganic salts, amino acids, vitamins and glucose, a protein-rich supplement is required to provide growth factors. Traditionally, Fetal Bovine Serum (FBS) is added to the basal medium [21] as the main source of growth factors to stimulate cell proliferation; FBS contains transport proteins carrying hormones (e.g. transcortin), minerals, trace elements (e.g. transferrin), and lipids (e.g. lipoproteins). In addition, FBS contain attachment and spreading factors, acting as germination points for cell attachment; and stabilizing and detoxifying factors needed to maintain pH or to inhibit proteases either directly, such as α-antitrypsin or α2 macroglobulin, or indirectly, by acting as an unspecific sink for proteases and other (toxic) molecules [22]. FBS is obtained by cardiac puncture of bovine fetuses without anaesthesia. Jochems strongly discussed the ethical issues on the use of FBS. The use of cell culture is strongly recommended as an alternative to animal experimentation. However, the require‐ ment for FBS, obtained from animal sources, end up making the concept of cell culture as an alternative to animal experimentation somewhat unethical [23].

Besides the ethical issues, scientific issues are also pointed on the use of FBS. Firstly, lot-to-lot variations make it necessary to test samples before purchase, as its molecular composition of FBS may vary [24]. FBS might interfere with cells genotype and phenotype, influencing experimental outcome. For example, it can promote cell proliferation in fibroblasts, whilst inhibiting it in epithelial cells [25]. It can be contaminated with viruses, bacteria, mycoplasmas, yeast, fungi, immunoglobulins, endotoxins, and possibly prions [26], contraindicating it for use for cells that would be further transplanted into humans. FBS is not totally chemically defined, as many substances present in it have not yet been characterized [27], some don't have their function fully elucidated, and others may even be toxic [28].

Serum, obtained from clotted whole blood, is known to be more suitable to cell culture than plasma from the same organism; despite the difficulty in obtaining it in large quantities. This is likely to be due to the release of proteins and growth factors from activated platelets during the clotting process [29], [30].Therefore, PRP, platelet lysates and other platelet-derived products can substitute FBS in cell culture. As the platelets are present in an elevated concen‐ tration, the growth factors important for cell culture are also more concentrated, as already discussed in this chapter. Finally, platelets can be easily obtained from human sources and therefore better mimic the effects of serum in human cells. Thinking of cell therapy, it can be used for growing cells that would be later transplanted into humans, especially when in an autologous approach.

## **4. How platelets can be used to improve tissue repair**

contribute to tissue growth and repair. In a similar manner, platelet-derived bioactive products

Since the beginning of cell culture techniques, many innovations were designed to optimize the process of cell expansion *in vitro*, analyzing their differentiation capacity, as well as their response to chemicals and promising pharmaceutical molecules. With time, the field im‐ proved. Plastics, glasses, bioreactors and engineering technology evolved a lot, at the same

Methods to grow cells *in vitro* try to reproduce appropriately all physiological conditions that are observed *in vivo*, and try to mimic it *in vitro*. The culture medium is the source of soluble factors that will enable cell growth and survival. Classical cell culture media are comprised of a basal balanced salt solution such as MEM (Eagle's minimal essential medium), DMEM (Dulbecco's modified Eagle's medium), IMDM (Iscove's modified DMEM), RPMI (Roswell Park Memorial Institute medium), Medium-199, HamF12 and McCoy's medium. Although they provide inorganic salts, amino acids, vitamins and glucose, a protein-rich supplement is required to provide growth factors. Traditionally, Fetal Bovine Serum (FBS) is added to the basal medium [21] as the main source of growth factors to stimulate cell proliferation; FBS contains transport proteins carrying hormones (e.g. transcortin), minerals, trace elements (e.g. transferrin), and lipids (e.g. lipoproteins). In addition, FBS contain attachment and spreading factors, acting as germination points for cell attachment; and stabilizing and detoxifying factors needed to maintain pH or to inhibit proteases either directly, such as α-antitrypsin or α2 macroglobulin, or indirectly, by acting as an unspecific sink for proteases and other (toxic) molecules [22]. FBS is obtained by cardiac puncture of bovine fetuses without anaesthesia. Jochems strongly discussed the ethical issues on the use of FBS. The use of cell culture is strongly recommended as an alternative to animal experimentation. However, the require‐ ment for FBS, obtained from animal sources, end up making the concept of cell culture as an

Besides the ethical issues, scientific issues are also pointed on the use of FBS. Firstly, lot-to-lot variations make it necessary to test samples before purchase, as its molecular composition of FBS may vary [24]. FBS might interfere with cells genotype and phenotype, influencing experimental outcome. For example, it can promote cell proliferation in fibroblasts, whilst inhibiting it in epithelial cells [25]. It can be contaminated with viruses, bacteria, mycoplasmas, yeast, fungi, immunoglobulins, endotoxins, and possibly prions [26], contraindicating it for use for cells that would be further transplanted into humans. FBS is not totally chemically defined, as many substances present in it have not yet been characterized [27], some don't have

Serum, obtained from clotted whole blood, is known to be more suitable to cell culture than plasma from the same organism; despite the difficulty in obtaining it in large quantities. This is likely to be due to the release of proteins and growth factors from activated platelets during

may find use in cell culture protocols.

224 The Non-Thrombotic Role of Platelets in Health and Disease

time that the biological field also evolved.

**3. How platelet products can improve cell culture**

alternative to animal experimentation somewhat unethical [23].

their function fully elucidated, and others may even be toxic [28].

Platelet concentrates, with the concentration of 5 x1010 platelets / unit are usually used for the treatment and prevention of severe hemorrhage [31]. The use of blood products for wound closure and stimulation of repair, such as fibrin glues, was first described in the 1970s [32]. The platelet gel emerged in the 1990s as a cheaper and autologous alternative compared to fibrin glues [33]. In 1987, PRP emerged as a product of autologous transfusion after open heart surgery, to prevent the need for a homologous product [34]. In 1998, Marx et al. described the use of platelets as an accelerator of tissue repair/regeneration, in that case bone formation in bone grafts for maxillofacial surgery [35].

Since then, most of the studies have shown an increase in bone repair, musculoskeletal tissues (muscles, tendons, and cartilage) and other "soft" tissues when platelet concentrates are used [36]. In the case of bone repair in maxillofacial surgery, the use of a platelet product efficacy and safety have been proven. In that case, only 9 out of 1,287 maxillae and mandible short implants (<8,5mm) from 661 patients between 2001 and 2008 had been lost. All implants had been embedded in liquid PRGF (plasma-rich in growth fators). Briefly, PRGF is obtained by centrifugation of whole blood collected in 9ml tubes containing sodium citrate at 580 x g for 8 minutes. Next, the 1ml fraction above red cell fraction is collected and activated with calcium chloride [37]. Although platelet products lack osteoinductive factors as BMPs, they can enhance bone formation. When PRP is added to human autogenous bone grafts, the bone density is higher, higher the proportion of mature bone and lesser osteoclast resorption, compared to size and age-matched grafts without PRP after 4 months of surgery [36]. Human PRP with the presence of peripheral blood mononucleated cells had its angiogenic properties proven in a nude animal model of critical size calvarial defect. Moreover, when it has been used synergistically with BMP-2, the effect on bone healing was augmented, as observed by histology, bone mineral density and bone mineral content after 8 weeks of implantation [38]. As said before, the induction of bone regeneration is more effective when PRP is used with approximately 1 million platelets per microliter. This was shown in a study where femurs of New Zealand white rabbits receiving an titanium implant where treated or not with autolo‐ gous PRP. Lower concentrations than 1 million platelets per microliter resulted in suboptimal peri-implant bone formation, whereas higher concentrations caused an inhibitory effect [17]. In addition, mesenchymal cells treated with PRP are also able to promote better repair and bone maturation in mandibular bone defects models, being pointed as an alternative to autogenous grafts. This has been shown when bone defects of canine mandible were filled with autologous PRP gel, autologous PRP gel with bone-marrow MSCs or autogenous particulate cancellous bone and marrow (PCBM). Briefly, PRP was obtained by 50mL blood collection in heparin, followed by two centrifugation steps which resulted in platelet concen‐ tration 438% above baseline. PRP activation was performed by adding thrombin/calcium solution. Increased bone formation and neovascularization was observed in the PRP plus MSC group. [39].

In muscle injuries, some factors present in PRP such as IGF-1 and bFGF can accelerate tissue repair. In contrast, TGF-β may lead to a fibrotic repair, increasing the possibility of the recurrence of new lesions [40]. Although mice with muscular injuries have demonstrated functional improvement when treated with high-frequency ultrasound-treated PRP, in order to lyse platelets and release of growth factor [41]. A preliminary study on muscle strain injuries in professional sportsman showed significant increase in the recovery time from injury when treated with autologous conditioned serum (ACS). ACS was obtained by blood collection without anti-coagulants followed by incubation and centrifugation for the retrieving of the serum [42]. Nonetheless, the action of PRP in muscle injuries still requires further investigation [43]. The first double-blinded, randomized, placebo controlled PRP clinical study on acute muscle injury, did not confirm the benefits for the use of PRP to enable the return to sports activities by athletes. In this case, PRP was prepared using a commercially available system (Arthrex double syringe ACP system) according to the manufacturer's instructions, and apparently was not activated prior to injection [44]. However, the methodology used in this work has been questioned, due to delayed administration and low dosage of PRP injections. The authors replied that there is no consensus on time of PRP injections, as well as that their PRP preparation method was in accordance with the literature [45].

Animal studies [46], [47] and human trials in tendon injuries show positive results through the use of PRP [48]–[50]. Although clinical trials with appropriate methodologies have not yet proven the effectiveness of PRP in this type of injury [51], localized platelet delivery can induce mobilization of circulating cells to sites of rat tendon injuries with concomitant in‐ crease in collagen synthesis [52]. *In vitro*, platelets can induce proliferation, collagen synthe‐ sis [53] and release of angiogenic factors in human tenocytes [54].A systematic review stated there were strong evidence against PRP injection for chronic lateral epicondylar tendinop‐ athy. In a total of 6 studies, 5 showed no significant benefit at the final follow-up, while 1 presented benefits for PRP injections compared to corticosteroid injection [55]. However, the results presented in that review have been questioned [56]. Surprisingly, another systematic review, selecting 9 studies, concluded there was limited but evolving evidence to support PRP injections in lateral epicondylitis, suggesting that further studies regarding the prepara‐ tion of PRP as well as the timing of the interventions are needed [57].

Cartilage, as an avascular tissue, and so injuries are usually critical and difficult to repair. Consequently, there is a need for new regenerative methods to address the specific demands imposed by cartilaginous injuries [58]. In 2003, PRGF was first used in a case of cartilaginous avulsion in a football player, causing an accelerated and rapid repair, which enabled the athlete's earlier return to sport activities [59]. Intra-articular PRP injections in patients with chronic cartilage degeneration also demonstrated positive results evaluated by clinical score methods as IKDC and EQ-VAS. In those studies, PRP was prepared by two-centrifugation steps which increased platelet concentration of 600% comparing to whole blood counting, and was activated by calcium solution prior to injection [60], [61]. On the other hand, *in vitro* analyses on chondrocytes proliferation and chondrogenic induction generated controversial results in the literature: In general, PRP induced chondrocyte proliferation [62]. Regarding chondrogenic induction, PRP appeared as an inducer [63], [64], while contradictory results showing the promotion of fibrogenic phenotype have also been observed [65], [66]. These antitheses may be related to different methodologies for PRP production, and therefore requires a better assessment of the PRP effect on chondrogenic cells.

with autologous PRP gel, autologous PRP gel with bone-marrow MSCs or autogenous particulate cancellous bone and marrow (PCBM). Briefly, PRP was obtained by 50mL blood collection in heparin, followed by two centrifugation steps which resulted in platelet concen‐ tration 438% above baseline. PRP activation was performed by adding thrombin/calcium solution. Increased bone formation and neovascularization was observed in the PRP plus MSC

In muscle injuries, some factors present in PRP such as IGF-1 and bFGF can accelerate tissue repair. In contrast, TGF-β may lead to a fibrotic repair, increasing the possibility of the recurrence of new lesions [40]. Although mice with muscular injuries have demonstrated functional improvement when treated with high-frequency ultrasound-treated PRP, in order to lyse platelets and release of growth factor [41]. A preliminary study on muscle strain injuries in professional sportsman showed significant increase in the recovery time from injury when treated with autologous conditioned serum (ACS). ACS was obtained by blood collection without anti-coagulants followed by incubation and centrifugation for the retrieving of the serum [42]. Nonetheless, the action of PRP in muscle injuries still requires further investigation [43]. The first double-blinded, randomized, placebo controlled PRP clinical study on acute muscle injury, did not confirm the benefits for the use of PRP to enable the return to sports activities by athletes. In this case, PRP was prepared using a commercially available system (Arthrex double syringe ACP system) according to the manufacturer's instructions, and apparently was not activated prior to injection [44]. However, the methodology used in this work has been questioned, due to delayed administration and low dosage of PRP injections. The authors replied that there is no consensus on time of PRP injections, as well as that their

Animal studies [46], [47] and human trials in tendon injuries show positive results through the use of PRP [48]–[50]. Although clinical trials with appropriate methodologies have not yet proven the effectiveness of PRP in this type of injury [51], localized platelet delivery can induce mobilization of circulating cells to sites of rat tendon injuries with concomitant in‐ crease in collagen synthesis [52]. *In vitro*, platelets can induce proliferation, collagen synthe‐ sis [53] and release of angiogenic factors in human tenocytes [54].A systematic review stated there were strong evidence against PRP injection for chronic lateral epicondylar tendinop‐ athy. In a total of 6 studies, 5 showed no significant benefit at the final follow-up, while 1 presented benefits for PRP injections compared to corticosteroid injection [55]. However, the results presented in that review have been questioned [56]. Surprisingly, another systematic review, selecting 9 studies, concluded there was limited but evolving evidence to support PRP injections in lateral epicondylitis, suggesting that further studies regarding the prepara‐

Cartilage, as an avascular tissue, and so injuries are usually critical and difficult to repair. Consequently, there is a need for new regenerative methods to address the specific demands imposed by cartilaginous injuries [58]. In 2003, PRGF was first used in a case of cartilaginous avulsion in a football player, causing an accelerated and rapid repair, which enabled the athlete's earlier return to sport activities [59]. Intra-articular PRP injections in patients with chronic cartilage degeneration also demonstrated positive results evaluated by clinical score

PRP preparation method was in accordance with the literature [45].

tion of PRP as well as the timing of the interventions are needed [57].

group. [39].

226 The Non-Thrombotic Role of Platelets in Health and Disease

PRP has also shown to induce mesenchymal stem cells (MSCs) proliferation. Regarding induction of osteogenesis on MSCs, mouse MSCs were treated with activated by thrombin/ calcium solution human PRP or washed platelets (WPLT), where the platelets had been suspended in phosphate saline rather than plasma, with equal platelets concentration, 4 times above the baseline. Interestingly, both stimulated cells proliferation in earlier time points, while WPLT induced higher proliferation than PRP in later time points. Alternatively, ALP activity and collagen type I expression, those indicatives of osteogenic differentiation, were increased in PRP rather than WPLT [67]. In others studies, PRP gel could induce osteocalcin and collagen type 1 expression in rat MSC [68], as well as activated PRP, with platelet con‐ centration 4 times higher the baseline, induced greater human MSC mineralization [69]. Interestingly, when the growth factors present in PRP are released in a controlled manner, through the association of PRP with alginate hydrogel, human MSCs alkaline phosphatase activity is induced [70]. As for chondrogenic induction *in vitro*, mRNA levels for aggrecan, Sox-9, and Runx2 were increased in buffered, i.e. inactivated, PRP treated human MSCs [71]. Subchondral bone MSCs cultured in a 3D model also showed chondrogenic potential induced by PRP (activated by freezing and thawing process), but not osteogenic or adipogenic [72]. Moreover, PRP associated with MSCs was able to induce chondrogenesis *in vitro* and *in vivo* in full-thickness rabbit articular cartilage injury model [73]. Recently, a systematic review selected 27 articles analyzing the role of PRP on MSCs in vitro proliferation and differentiation, in comparison to FBS. It has been seen that PRP stimulates cells proliferation, preserves their immunomodulatory capacity and may delay the acquirement of a senescent phenotype. The majority of the studies also showed that PRP maintains cells adipogenic, osteogenic and chondrogenic differentiation capacity, in fewer cases enhanced it, while in rare cases dimin‐ ished the adipogenic differentiation capacity [74].

Platelets have been shown to play an important role in the repair of many different tissues such as skin [75], nervous tissue [76], corneal [77], myocardial [78], and vascular [79]. In addition, platelet-derived products demonstrate distinct antimicrobial effects [80], and contribute to orthopedic repair [81] and plastic surgery applications [82]. It is worth noting the potential for therapeutic effects in sports medicine, as the need for elite athletes to recover quickly from injuries and achieve their regular level of efficiency is huge. PRP use is currently allowed by the global anti-doping agency [83].

There have been no reports of serious health problems arising after the therapeutic use of PRP so far, but despite evidence demonstrating its positive effects, especially in repair of muscu‐ loskeletal injuries, there are few published clinical studies, and even smaller the number of works with sufficient methodological quality to ensure evidence-based decision-making use of PRP [43]. Likewise, there is still a need for basic studies to better understand some still open issues, such as the optimal concentration of platelets, the harm or the benefits of the presence of leukocytes, a possible combination of recombinant proteins with PRP [15] and analysis of its action at the cellular level.

## **5. The need of standardization**

Due to the variety of protocols, many classifications are used to characterize platelet prepara‐ tions for tissue engineering purposes. Many groups refer generically to platelets used in tissue engineering applications as Platelet-Rich Plasma (PRP). However, this can cause confusion, since many biomedical researchers use the PRP abbreviation to refer to a simple enrichment by centrifugation of whole blood to remove red blood cells and leukocytes. Nonetheless, tissue engineers have adopted the term PRP to refer to platelet concentrates that can be re-injected to a site of tissue injury with or without modification or activation. Moreover, additional acronyms are used to sub classify the platelet preparations used in tissue injury-studies. Among these classifications, we can find: PDWHF (platelet derived wound healing factors) [84], L- PRP (leukocyte and platelet - rich plasma) [85], PRFM (plasma rich fibrin matrix) [86], PRGF (plasma rich in growth factors) [87], among others.

Likewise, the PRP gel preparation used in tissue engineering applications cannot be referred to as a " fibrin glue", as the clot formed in the PRP activation contain the same components and at similar concentrations to that found in a native blood clot and consists primarily of fibrin, fibronectin and vitronectin, besides the bioactive molecules. The fibrin glue is only considered a concentrate of fibrinogen, which is polymerized by thrombin and calcium [16]. This platelet gel was originally used as a substitute to fibrin glue in oral and maxillofacial surgery [33]. It was also used in cutaneous chronic wounds [88], including diabetic ulcers [89], degenerative disorders of the knee [90], gynecologic, cardiac, and general surgical procedures [91].

Various commercial systems for the preparation of autologous platelet products for local injection into wound sites have been developed. Among the main ones are: SmartPrep ® 2APC +TM (Harvest Technologies), Biomet GPS III ® (Biomet Manufacturing Corp.), Arthrex ACP ® (Arthrex Inc), Cascade ® platelet - rich fibrin matrix (Musculoskeletal Trasnplant Founda‐ tion), Cascade ® platelet - rich plasma therapy (Musculoskeletal Trasnplant Foundation), BTI plasma rich in growth factors (PRGF) (Biotechnology Institute) and Magellan ® Autologous Platelet Separator System (Arteriocyte) [92]. Despite such variety, the basic formulation of PRP consists of primarily autologous blood collection in the presence of anticoagulants. After collection, the blood is centrifuged once or twice in order to separate the plasma with platelets from erythrocytes and leukocytes and concentrate them. After the second centrifugation, part of the plasma is used to resuspend the platelet concentrate, formulating the final PRP. The portion of the plasma that is not used in this ressupension is commonly described as plateletpoor plasma (PPP). The release of growth factors contained in platelets granules occurs following activation of the PRP with exogenous thrombin, collagen, or calcium chloride, forming a clot. Some methodologies also may use freeze-thawing cycles or sonication by ultrasound treatment in order to disrupt platelets membrane and release of the growth factors. In other cases, platelets may not be activated at all. Calcium chloride is important to enable fibrin polymerization and thrombin generation by the endogenous coagulation cascade. The final product, i.e. the supernatant liquid without the clot, is actually a serum derived from PRP [4]. Both commercial and non-commercial forms may vary on the speed and number of centrifu‐ gation steps to concentrate the platelets, the usage or not of anticoagulant, the type of antico‐ agulant, the presence of leukocytes, which may release matrix metalloproteinase and reactive oxygen species that can increase tissue damage, and the substance that will induce platelet activation. In the end, these variations generate diversity in types, concentration and speed of the release of growth factors, which may explain different results among papers [93]. In order to normalize and standardize platelets products for tissue engineering purposes, mainly by the platelet concentration, activation (whether it occurs or not an how), and presence of white blood cells, some classification systems have been proposed [83], [94].In summary, platelets act as reservoirs of growth factors and bioactive agents. The localized application of concen‐ trated preparations of these reagents from autologous platelet donations appears to facilitate and accelerate wound healing and tissue repair. Some controversy exists as to the effectiveness of this treatment. However, significant variations in the platelet preparations, mainly platelet concentration and activation, may explain some or all of the variations.

## **Author details**

loskeletal injuries, there are few published clinical studies, and even smaller the number of works with sufficient methodological quality to ensure evidence-based decision-making use of PRP [43]. Likewise, there is still a need for basic studies to better understand some still open issues, such as the optimal concentration of platelets, the harm or the benefits of the presence of leukocytes, a possible combination of recombinant proteins with PRP [15] and analysis of

Due to the variety of protocols, many classifications are used to characterize platelet prepara‐ tions for tissue engineering purposes. Many groups refer generically to platelets used in tissue engineering applications as Platelet-Rich Plasma (PRP). However, this can cause confusion, since many biomedical researchers use the PRP abbreviation to refer to a simple enrichment by centrifugation of whole blood to remove red blood cells and leukocytes. Nonetheless, tissue engineers have adopted the term PRP to refer to platelet concentrates that can be re-injected to a site of tissue injury with or without modification or activation. Moreover, additional acronyms are used to sub classify the platelet preparations used in tissue injury-studies. Among these classifications, we can find: PDWHF (platelet derived wound healing factors) [84], L- PRP (leukocyte and platelet - rich plasma) [85], PRFM (plasma rich fibrin matrix) [86],

Likewise, the PRP gel preparation used in tissue engineering applications cannot be referred to as a " fibrin glue", as the clot formed in the PRP activation contain the same components and at similar concentrations to that found in a native blood clot and consists primarily of fibrin, fibronectin and vitronectin, besides the bioactive molecules. The fibrin glue is only considered a concentrate of fibrinogen, which is polymerized by thrombin and calcium [16]. This platelet gel was originally used as a substitute to fibrin glue in oral and maxillofacial surgery [33]. It was also used in cutaneous chronic wounds [88], including diabetic ulcers [89], degenerative disorders of the knee [90], gynecologic, cardiac, and general surgical

Various commercial systems for the preparation of autologous platelet products for local injection into wound sites have been developed. Among the main ones are: SmartPrep ® 2APC +TM (Harvest Technologies), Biomet GPS III ® (Biomet Manufacturing Corp.), Arthrex ACP ® (Arthrex Inc), Cascade ® platelet - rich fibrin matrix (Musculoskeletal Trasnplant Founda‐ tion), Cascade ® platelet - rich plasma therapy (Musculoskeletal Trasnplant Foundation), BTI plasma rich in growth factors (PRGF) (Biotechnology Institute) and Magellan ® Autologous Platelet Separator System (Arteriocyte) [92]. Despite such variety, the basic formulation of PRP consists of primarily autologous blood collection in the presence of anticoagulants. After collection, the blood is centrifuged once or twice in order to separate the plasma with platelets from erythrocytes and leukocytes and concentrate them. After the second centrifugation, part of the plasma is used to resuspend the platelet concentrate, formulating the final PRP. The portion of the plasma that is not used in this ressupension is commonly described as platelet-

its action at the cellular level.

procedures [91].

**5. The need of standardization**

228 The Non-Thrombotic Role of Platelets in Health and Disease

PRGF (plasma rich in growth factors) [87], among others.

Ronaldo J. F. C. do Amaral1,2 and Alex Balduino2,3\*


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## *Edited by Steve Kerrigan and Niamh Moran*

Platelets play a key role in thrombosis and haemostasis. However recent evidence clearly demonstrates that the functional role of platelets extends to many other processes in the body. With an internationally recognised list of contributing authors, The Non-Thrombotic Role of Platelets in Health and Disease, is a unique and definitive source of state-of-the-art knowledge about the additional role of platelets outside thrombosis and haemostasis. The intended audience for The Non-Thrombotic Role of Platelets in Health and Disease includes platelet biologists, microbiologists, immunologists, haematologists, oncologists, respiratory physicians, cardiologists, neurobiologists, tissue engineers, as well as students and fellows in these areas.

The Non-Thrombotic Role of Platelets in Health and Disease

The Non-Thrombotic Role of

Platelets in Health and Disease

*Edited by Steve Kerrigan and Niamh Moran*

Photo by Ugreen / iStock