**The Traditional Role of Platelets in Hemostasis**

Douglass A. Drelich and Paul F. Bray

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60595

## **1. Introduction**

[48] S. Patel-Hett *et al.*, The spectrin-based membrane skeleton stabilizes mouse megakar‐ yocyte membrane systems and is essential for proplatelet and platelet formation.

[49] A. Sui et al., Tmod3 participates in platelet formation and sizing in mouse fetal liver

*B*lood 118, 1641 (Aug 11, 2011).

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

FASEB J 28, 278.9 (2014).

Hemostatic balance is central to health maintenance. Hemostasis must be initiated rapidly to prevent excessive blood loss. However, it must be tightly controlled to prevent over exuberant thrombus formation with resultant pathologic occlusion of arterial or venous vascular beds. Platelets are central to this hemostatic balance via primary hemostasis, support of coagulation, and even anti-fibrinolytic effects. Quantitative and qualitative platelet disorders have classi‐ cally focused on hemorrhagic and thrombotic diseases, the severity of which can range from mild to life-threatening. Recent advances have demonstrated that platelets have functions beyond their traditional hemostatic role such as supporting vascular integrity, angiogenesis, immune function, tumor metastases, etc. These "non-traditional" functions of platelet will be discussed in other chapters. In this chapter we present a brief review of the traditional roles of platelets in hemostasis and thrombosis.

## **2. Structure**

Platelets have many unique structural features that facilitate their contributions to thrombus formation. The cell membrane of platelets consists of a phospholipid bilayer embedded with cholesterol, glycoproteins, and glycolipids. Platelet membranes are asymmetrically organized. Negatively charged phospholipids in resting platelets are preferentially present on the inner leaflet, most notably phosphatidylserine.[1] The platelet membrane is rich in a variety of glycoproteins (GPs) that bind agonists to activate platelets and that serve primarily adhesive functions (Table 1). Transmembrane glycoproteins may distribute preferentially to cholesterolrich microdomains, called "lipid rafts."[2]

© 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.


**Table 1.** Platelet Surface Adhesion Proteins

Resting platelets in circulation have a stable discoid shape that is maintained by a circumfer‐ ential coil of microtubules and a membrane cytoskeleton composed of actin, spectrin and other proteins.[3] The platelet plasma membrane is contiguous with the open canalicular system (OCS), a complex series of connecting tunnels that are open to the extracellular space. In addition to supplying membrane surface area to the spreading platelet, the canalicular system provides a potential route for the release of granule contents critical for delivery of the numerous vasoactive elements present in platelet granules. This also serves as a storage site for glycoproteins that are receptors for adhesive molecules. While the OCS is contiguous with the extracellular space, the dense tubular system is a closed channel network analogous to the sarcoplasmic reticulum as a site where calcium can be sequestered. Release of calcium from this system is a critical step in platelet activation.

Platelets have three different types of granules (Table 2). Dense granules contain adenine nucleotides (e.g., ADP and ATP), calcium, bioactive amines (e.g., serotonin and histamine) and polyphosphates. α-granules are rich in larger adhesive proteins.


**Table 2.** Platelet Granule Contents important for hemostasis

## **3. Thrombopoiesis**

**Platelet Surface Receptors for Adhesive Proteins**

GPIIb-IIIa αIIbβ<sup>3</sup> CD61 (β subunit), CD41 Fibrinogen (and several others)

Resting platelets in circulation have a stable discoid shape that is maintained by a circumfer‐ ential coil of microtubules and a membrane cytoskeleton composed of actin, spectrin and other proteins.[3] The platelet plasma membrane is contiguous with the open canalicular system (OCS), a complex series of connecting tunnels that are open to the extracellular space. In addition to supplying membrane surface area to the spreading platelet, the canalicular system provides a potential route for the release of granule contents critical for delivery of the numerous vasoactive elements present in platelet granules. This also serves as a storage site for glycoproteins that are receptors for adhesive molecules. While the OCS is contiguous with the extracellular space, the dense tubular system is a closed channel network analogous to the sarcoplasmic reticulum as a site where calcium can be sequestered. Release of calcium from

Platelets have three different types of granules (Table 2). Dense granules contain adenine nucleotides (e.g., ADP and ATP), calcium, bioactive amines (e.g., serotonin and histamine) and

**GP designation Integrin designation Other names Primary ligands**

GPIb-V-IX n/a CD42 Von Willebrand Factor

GPIa-IIa α2β<sup>1</sup> VLA-2 Collagen

GPIc-IIa α5β<sup>1</sup> VLA-5 Fibronectin

GPIV n/a GPIIIB, CD36 Collagen

**Table 1.** Platelet Surface Adhesion Proteins

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

this system is a critical step in platelet activation.

**Table 2.** Platelet Granule Contents important for hemostasis

**Dense granules**

Platelet factor 4 (PF4) von Willebrand Factor

Fibrinogen Fibronectin Factor V Factor XI Protein S PAI-1

ADP ATP Calcium Serotonin **α-granules**

polyphosphates. α-granules are rich in larger adhesive proteins.

In the healthy state, platelets have an average lifespan of 8-9 days. This requires an active production mechanism. Bone marrow megakaryocytes produce approximately 1011 platelets daily. Each individual magakaryocyte can produce between 1000 and 3000 platelets.[4] Most of the molecules present in the mature platelet are produced by the megakaryocyte, but some such as fibrinogen and immunogloulin, are endocytosed from the surrounding plasma milieu. Megakaryocytes produce platelets by extending long projections. Cytoplasm in the developing platelets largely resembles that of the megakaryocyte. However, certain contents, particularly granules, appear to be moved into the developing proplatelets by an active transport mecha‐ nism.[5]

Several cytokines effect the development of platelets. IL-3, GM-CSF, and stem cell factor all appear important in maintaining the health and proliferation of megakaryocytes. However, the key regulator of platelet formation is thrombopoietin (TPO). TPO is a 50-70 kDa protein that has homology to erythropoietin.[6] TPO interacts with its key receptor c-Mpl, leading to dimerization initiating a signal transduction cascade through JAK, STAT, and MAPK path‐ ways. TPO is made in the liver and to a lesser extent the kidney.

## **4. Platelet-mediated hemostasis**

During both normal in vivo hemostasis and pathologic thrombus formation, numerous physiologic responses occur simultaneously, such as vasoconstriction, platelet plug formation and coagulation. Platelet thrombus formation itself involves a set of unique molecular responses and signaling pathways that also occur simultaneously. From a discussion point of view, this complexity makes it convenient to arbitrarily compartmentalize these processes.

#### **4.1. Tethering and firm attachment**

Platelet plug formation is initiated by exposure to a break in the endothelial lining of blood vessels. This has two important sequelae. The first is the loss of a variety of inhibitors of platelet function. The intact endothelium produces nitric oxide and prostacyclin both of which are inhibitors of platelet function, and the loss of endothelium leads to the loss of CD39 which in its intact state breaks down adenosine diphosphate (ADP), an activator of platelets. Exposure of subendothelial elements also allows the initial recruitment of platelets from the circulation via interactions between adhesive glycoproteins on the platelet surface and subendothelial proteins. [7]

Von Willebrand Factor (VWF) is critical for platelet-mediated thrombus formation in vessels with high shear rates and high shear stress. VWF is a multimeric protein that ranges in molecular weight from 0.5 daltons (dimers) to greater than 20 million daltons (multimers).[8] The hemostatic efficacy of VWF is directly proportional to its size with the largest molecules being the most prothrombotic. Subendothelial VWF is derived from plasma VWF that binds collagen after vessel injury and the abluminal secretion from endothelial cells. VWF circulates as a globular protein but undergoes conformational changes when exposed to high shear stress conditions. This unfolding exposes binding domains that allow the large von Willebrand multimers to form a bridge between subendothelial collagen and circulating platelets. The von Willebrand protein contains multiple functional domains including binding domains for both collagen and platelet GPIbα.

The initial binding of VWF to the platelets is mediated by interaction between the A1 domain of VWF with the GPIbα subunit of the GPIb-V-IX complex.[9] GPIbα has an N-terminal segment comprised of two β-loops flanking a leucine-rich repeat segment. GPIX is a small, single chain polypeptide. The exact contribution of this peptide to the function of the complex is not well understood. This bridging mediates a rapid but reversible platelet adhesion that allows for rolling of platelets along the damaged endothelium. Occupation of this complex by VWF also leads to platelet signaling responses, including rearrangement of the cytoskeleton, increase in intracellular calcium, and granule release. The reduced platelet velocity mediated by the VWF-GPIbα interaction, coupled with activation of integrin α2β1 enables stable, irreversible interactions to form between collagen and platelet integrin α2β1.

#### **4.2. Activation**

Once platelets are captured from the circulation, activation steps lead to numerous changes in the platelets. These include conformational changes, rapid calcium influx, degranulation, thromboxane production, etc. The changes are induced by numerous agonists interacting with specific receptors on the platelet plasma membrane.

With increasingly sophisticated technologies for assessing platelet function and thrombus formation in vivo and under flow condition, there is an enhanced appreciation of the hetero‐ geneity of platelets in a developing thrombus. Thus, there appears to be diverse microenvir‐ onments such that regions near the vessel wall may contain degranulated and irreversibly activated platelets, while the luminal region may have minimally activated and reversible adhered platelets that may or may not undergo thrombus stabilization.[10, 14]

As noted above, exposure of subendothelial collagen begins the initial tethering process. Once platelets are engaged in rolling on this matrix they have the opportunity to interact with GPVI, which is the major platelet collagen signaling receptor.[15] GPVI is a type 1 transmembrane protein belonging to the Ig superfamily. It associates with an Fc receptor γ-chain which serves as the signal transducing unit. Engagement of repetitive motifs on collagen by multiple GPVI molecules leads to crosslinking of GPVI dimers and phosphorylation of the FcRγ chain immunoreceptor tyrosine-based activation motifs (ITAMs). This initiates a Syk-dependent signaling cascade finally resulting in activation of phospholipase Cγ2 (PLCγ2) and phosphoi‐ nositide-3 kinase (PI3K) that generates inositol-1,4,5-trisphosphate (IP3). IP3 induces calcium mobilization, degranulation and integrin αIIbβ3 activation. Activated αIIbβ3 binds fibrinogen and VWF, leading to platelet aggregation.

The exposure of subendothelial collagen also exposes extravascular tissue factor, initiating coagulation and thrombin generation. This cascade is enhanced by PS exposure on activated

platelet and endothelial cell membranes. Thrombin is a potent activator of platelets. Human platelets express two thrombin-activated G protein coupled receptors (GPCRs), PAR1 and PAR4.[16, 17] PAR activation occurs when a protease, such as thrombin, binds and cleaves the amino-terminus of the receptor. Binding of the new amino-terminus to the second extracellular loop of the PAR induces conformational changes in transmembrane domains enabling activation of G proteins.[18, 20] PAR1 and PAR4 activation lead to activation of Gαq, which activates PLCβ. PLCβ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and IP3, leading to PKC activation and increased calcium mobilization, respectively.[21] In platelets, these pathways work in concert to activate the integrin αIIbβ3 resulting in aggregation. PAR1 has a higher affinity for thrombin, and calcium transiently rises sharply after PAR1 activation followed by a relatively fast return to baseline levels. In contrast, PAR4 induces a more gradual and sustained rise in calcium and accounts for the majority of intracellular calcium flux.[22, 23] These platelet PAR1 and PAR4 kinetic signaling differences are reminiscent of the initiation and propagation phases of coagulation, where there is a burst of thrombin generation (quickly shut off by tissue factor pathway inhibitor [TFPI]) followed by a sustained and quantitatively greater production of thrombin by the intrinsic pathway.


**Table 3.** Important Platelet Receptors and Agonists

collagen after vessel injury and the abluminal secretion from endothelial cells. VWF circulates as a globular protein but undergoes conformational changes when exposed to high shear stress conditions. This unfolding exposes binding domains that allow the large von Willebrand multimers to form a bridge between subendothelial collagen and circulating platelets. The von Willebrand protein contains multiple functional domains including binding domains for both

The initial binding of VWF to the platelets is mediated by interaction between the A1 domain of VWF with the GPIbα subunit of the GPIb-V-IX complex.[9] GPIbα has an N-terminal segment comprised of two β-loops flanking a leucine-rich repeat segment. GPIX is a small, single chain polypeptide. The exact contribution of this peptide to the function of the complex is not well understood. This bridging mediates a rapid but reversible platelet adhesion that allows for rolling of platelets along the damaged endothelium. Occupation of this complex by VWF also leads to platelet signaling responses, including rearrangement of the cytoskeleton, increase in intracellular calcium, and granule release. The reduced platelet velocity mediated by the VWF-GPIbα interaction, coupled with activation of integrin α2β1 enables stable,

Once platelets are captured from the circulation, activation steps lead to numerous changes in the platelets. These include conformational changes, rapid calcium influx, degranulation, thromboxane production, etc. The changes are induced by numerous agonists interacting with

With increasingly sophisticated technologies for assessing platelet function and thrombus formation in vivo and under flow condition, there is an enhanced appreciation of the hetero‐ geneity of platelets in a developing thrombus. Thus, there appears to be diverse microenvir‐ onments such that regions near the vessel wall may contain degranulated and irreversibly activated platelets, while the luminal region may have minimally activated and reversible

As noted above, exposure of subendothelial collagen begins the initial tethering process. Once platelets are engaged in rolling on this matrix they have the opportunity to interact with GPVI, which is the major platelet collagen signaling receptor.[15] GPVI is a type 1 transmembrane protein belonging to the Ig superfamily. It associates with an Fc receptor γ-chain which serves as the signal transducing unit. Engagement of repetitive motifs on collagen by multiple GPVI molecules leads to crosslinking of GPVI dimers and phosphorylation of the FcRγ chain immunoreceptor tyrosine-based activation motifs (ITAMs). This initiates a Syk-dependent signaling cascade finally resulting in activation of phospholipase Cγ2 (PLCγ2) and phosphoi‐ nositide-3 kinase (PI3K) that generates inositol-1,4,5-trisphosphate (IP3). IP3 induces calcium mobilization, degranulation and integrin αIIbβ3 activation. Activated αIIbβ3 binds fibrinogen

The exposure of subendothelial collagen also exposes extravascular tissue factor, initiating coagulation and thrombin generation. This cascade is enhanced by PS exposure on activated

irreversible interactions to form between collagen and platelet integrin α2β1.

adhered platelets that may or may not undergo thrombus stabilization.[10, 14]

specific receptors on the platelet plasma membrane.

and VWF, leading to platelet aggregation.

collagen and platelet GPIbα.

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

**4.2. Activation**

There are two important amplification pathways in platelet activation.[24] The first is through the release of ADP from dense granule secretion. ADP is a potent platelet agonist that, when added to in vitro platelets, leads to TXA2 production, phosphorylation of a number of proteins, increased cytosolic Ca++, shape change, aggregation, and secretion. This pathway is required for maximal platelet aggregation induced by other agonists. Platelets have two ADP receptors, P2Y1 and P2Y12, and both are GPCRs. P2Y12 activates Gαi, which promotes aggregation by inhibiting cyclic AMP (cAMP) formation. P2Y12 mediated activation of protein kinase A leads to VASP phorphorylation. P2Y12 is inhibited by the thienopyridines, commonly used antiplatelet agents that have benefit in the management of ischemic vascular disease. P2Y1 appears to be necessary, but not sufficient to induce full platelet aggregation. Platelets from P2Y1 knockout mice cannot change shape or aggregate to ADP but cAMP is still decreased in those platelets due to its effect on P2Y12. P2Y1 activates Gαq with subsequent calcium mobilization.

The second feedback amplification pathway involves the metabolism of arachidonic acid (AA) to thromboxane A2 (TXA2).[24] A number of agonists stimulate the release of arachidonic acid from the stores in the plasma membrane, in particular phosphatidylcholine and phosphati‐ dylethanolamine. Phospholipase A2 (PLA2) is the most important enzyme in the release of AA from those phospholipids. PLA2 can be activated by rising cytosolic calcium levels though there also appear to be calcium independent mechanisms. Released AA is then metabolized by cycloxegenase 1 (COX-1) to Prostaglandin G2 which subsequently is converted to Prosta‐ glandin H2. Thromboxane synthase then produces TXA2. Aspirin irreversibly acetylates COX and also has benefit in preventing arterial ischemic syndromes. TXA2 diffuses out of platelets and binds to prostanoid GPCR family receptors, notably TPα and TPβ, which also activate platelets via Gq.

Epinephrine activates platelets through adrenergic α2a GPCRs that couple with Gαi family members to inhibit adenylyl cyclase leading to decreased cAMP and increased intracellular calcium concentration. It appears that epinephrine synergizes with other agonists, particularly ADP. It is unclear if epinephrine can lead to full aggregation by itself in vitro, although there are reports of families with mild bleeding disorders due to defects in epinephrine-induced platelet aggregation.

#### **4.3. Shape change**

The most dramatic observable change to platelets as they undergo activation is the change from their discoid form to a spread form with many filopodia. Agonists, such as thrombin and TXA2, activate GPCRs coupled to Gα12/13, which signal through RhoA –ROCK and myosin to reorganize the actin cytoskeleton and produce shape change.[24] Platelets contain large amounts of actin in both the globular (G-actin) and multimeric filamentous (F-actin) forms. Following activation; the proportion of F-actin increases from 40-50% to 70-80%. In an organized process, actin filaments from the resting platelet are cleaved into smaller fragments. These then form the beginnings of new, longer actin filaments. This process is regulated, in part, by increase phosphatidylinositol-4,5-biphosphate (PIP2). Simultaneous to the changes in actin, myosin is phosphorylated by myosin light chain kinase activated by the calciumcalmodulin complex. This leads to association with F-actin as well as binding the complex to the membrane via interaction with the GPIb-IX complex. In resting platelets, filamin acts to stabilize the actin framework underlying the membrane and limits the movement of the GPIb. Increasing cytoplasmic Ca++ concentrations activate calpain cleaving the actin binding protein leading to release from the GPIb complex. The outcome of this complex series of reactions is the centralization of actin into thick, fibrous masses associated with phosphorylated myosin filaments.

#### **4.4. Degranulation**

The above-mentioned agonists all induce platelet exocytosis of granules.[25] **S**oluble **N**ethylmaleimide-sensitive factor **a**ttachment protein **re**ceptors or SNAREs mediate this delivery.[26] This includes t-SNAREs (target receptors), v-SNAREs (vesicle associated membrane receptors), and soluble components such as N-ethylmaleimide-sensitive fusion proteins (NSF) and NSF attachment protein. Reorganization of the cytoskeleton in conjunction with the SNARE machinery facilitates exocytosis of these granules, which contain a large variety of mediators important to the hemostatic and other roles of platelets.

**Dense Granules**. There are approximately three to eight dense granules per platelet. These are 20 to 30 nm in size and are electron dense due to the high calcium content. Dense granules also have high concentrations of serotonin, ADP, and ATP. ADP is an important platelet activator and this concentration of ADP in the dense granules and its delivery to developing thrombi by degranulation is an important amplification step in activating other platelets localized by adhesive molecules.

**α-Granules**. There are 50 to 80 α–granules per platelet. They are much larger than dense granules at approximately 200 nm in diameter. Upon platelet activation, α granules fuse with the plasma membrane, releasing their cargo, substantively increasing the total platelet membrane surface area. α-granule membranes are rich in important adhesive integral membrane proteins, like GPIb-IX and αIIbβ3, which enhance adhesive properties. α-granule cargo includes adhesive proteins and coagulation factors like fibrinogen and VWF, represent‐ ing an important amplification feature of platelet thrombus growth. Fibrinogen is present in concentrations greater than that of plasma. Notably, megakaryocytes do not appear to synthesize fibrinogen, and it is endocytosed via αIIbβ3. Patients lacking αIIbβ3 also lack α-granule fibrinogen. α-granule VWF has high molecular weight, which is the most efficient form for hemostasis. Platelets α-granules also contribute substantial amounts of coagulation Factor V and Factor XI, as well as thrombospondin-1 which is important for platelet activation via signaling through CD47. α-granules contain a number of antifibrinolytic molecules including α2-antiplasmin and plasminogen activator inhibitor (PAI-1).

Proteomic studies indicate α-granules contain more than 300 different soluble proteins.[27] Many of the non-hemostatic and systemic effects of circulating platelets are mediated by these molecules, and include chemokines (e.g., PF4, β-TG, MCP-, RANTES and others), antimicrobial proteins (thymosin-β4 and thrombocidins), immune modulators (complement, factor H, IgG), growth factors (PDGF, TGF β and others) and pro-angiogenic (VGF, FGF) and anti-angiogenic (endostatin, angiostatin) factors.

#### **4.5. Aggregation**

dylethanolamine. Phospholipase A2 (PLA2) is the most important enzyme in the release of AA from those phospholipids. PLA2 can be activated by rising cytosolic calcium levels though there also appear to be calcium independent mechanisms. Released AA is then metabolized by cycloxegenase 1 (COX-1) to Prostaglandin G2 which subsequently is converted to Prosta‐ glandin H2. Thromboxane synthase then produces TXA2. Aspirin irreversibly acetylates COX and also has benefit in preventing arterial ischemic syndromes. TXA2 diffuses out of platelets and binds to prostanoid GPCR family receptors, notably TPα and TPβ, which also activate

Epinephrine activates platelets through adrenergic α2a GPCRs that couple with Gαi family members to inhibit adenylyl cyclase leading to decreased cAMP and increased intracellular calcium concentration. It appears that epinephrine synergizes with other agonists, particularly ADP. It is unclear if epinephrine can lead to full aggregation by itself in vitro, although there are reports of families with mild bleeding disorders due to defects in epinephrine-induced

The most dramatic observable change to platelets as they undergo activation is the change from their discoid form to a spread form with many filopodia. Agonists, such as thrombin and TXA2, activate GPCRs coupled to Gα12/13, which signal through RhoA –ROCK and myosin to reorganize the actin cytoskeleton and produce shape change.[24] Platelets contain large amounts of actin in both the globular (G-actin) and multimeric filamentous (F-actin) forms. Following activation; the proportion of F-actin increases from 40-50% to 70-80%. In an organized process, actin filaments from the resting platelet are cleaved into smaller fragments. These then form the beginnings of new, longer actin filaments. This process is regulated, in part, by increase phosphatidylinositol-4,5-biphosphate (PIP2). Simultaneous to the changes in actin, myosin is phosphorylated by myosin light chain kinase activated by the calciumcalmodulin complex. This leads to association with F-actin as well as binding the complex to the membrane via interaction with the GPIb-IX complex. In resting platelets, filamin acts to stabilize the actin framework underlying the membrane and limits the movement of the GPIb. Increasing cytoplasmic Ca++ concentrations activate calpain cleaving the actin binding protein leading to release from the GPIb complex. The outcome of this complex series of reactions is the centralization of actin into thick, fibrous masses associated with phosphorylated myosin

The above-mentioned agonists all induce platelet exocytosis of granules.[25] **S**oluble **N**ethylmaleimide-sensitive factor **a**ttachment protein **re**ceptors or SNAREs mediate this delivery.[26] This includes t-SNAREs (target receptors), v-SNAREs (vesicle associated membrane receptors), and soluble components such as N-ethylmaleimide-sensitive fusion proteins (NSF) and NSF attachment protein. Reorganization of the cytoskeleton in conjunction with the SNARE machinery facilitates exocytosis of these granules, which contain a large

variety of mediators important to the hemostatic and other roles of platelets.

platelets via Gq.

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

platelet aggregation.

**4.3. Shape change**

filaments.

**4.4. Degranulation**

Platelets contain ≈80,000 αIIbβ3 (GPIIb-IIIa) complexes, the most abundant plasma membrane GP.[28] In the resting platelet, αIIbβ3 exists primarily in a low affinity conformation that is not able to bind its major ligands, which are fibrinogen, VWF, fibronectin and thrombospondin-1. The final common pathway of platelet activation leads to integrin activation to a high affinity state.[29] This is referred to as inside-out signaling. The high affinity conformation binds fibrinogen (or other adhesive ligands), and the bound fibrinogen serves as a bridge to other platelets, resulting in an expanding platelet aggregate. The importance of αIIbβ3 in platelet function and normal hemostasis is underscored by the moderately severe bleeding seen in patients with Glanzmann thrombasthenia, an inherited disorder caused by absent or dysfunc‐ tional αIIbβ3.

αIIbβ3 is the prototypic member of the integrin family of heterodimeric integral membrane adhesion receptors. This receptor consists of 18 α subunits that associate noncovalently with 8 β subunits. αIIb is expressed only in megakaryocytes and platelets, and localizes to the plasma membrane, OCS, and α-granules. β3 has a broad tissue distribution. Platelets also express the αvβ3 vitronectin receptor in low abundance. Crystalization of the extracellular domain of αvβ3 and the head domain of αIIbβ3 have provided detailed structural information about these integrins.[30, 32]

Talin is an abundant cytoskeletal protein that links integrins to the actin cytoskeleton. The agonist-induced rise in intracellular calcium results in binding of the talin head domain to the cytoplasmic domain of integrin β3. This interaction leads to an unclasping of the intracellular and transmembrane components of the αIIb and β3 molecules, causing spreading of the two proteins and exposure of the ligand binding site. The precise molecular details by which talin is enabled to bind β3 are unclear, but efficient integrin activation likely involves (1) the guanine nucleotide exchange factor CalDAG-GEFI, (2) activation of the small GTPase Rap1, (3) kindlin-3 binding to the β3 cytoplasmic tail, and (4) calpain cleavage of talin.

## **5. Role in coagulation**

Platelets contribute substantially to thrombin generation, which further induces additional platelet activation. In addition, platelet thrombus stabilization requires local fibrin generation that depends on thrombin generation. When platelets are stimulated by strong agonists, the negatively charged phospholipids on the inner leaflet of the platelet plasma membrane are "flipped" to the outer leaflet. This reorganization may be mediated by the calcium activated scramblase TMEM16F.[33] Translocation of negatively charged phospholipids forms a stage upon which coagulation reactions occur. The formation of the "tenase" complex that converts Factor X to activated Factor X requires phospholipid. The development of the prothrombinase complex also requires negatively charged phospholipid as the surface upon which the complex assembles.

Activation of platelets by strong agonists also leads to the development and shedding of platelet microparticles. These have a high density of negatively charged phospholipids and are thus able to support the formation of the "tenase" and prothrombinase complex as noted above. They also contain coagulation Factor Va with which to support the formation of thrombin as well as supplying arachidonic acid which can contribute to further formation of TXA2.

Platelets α-granule release also provides coagulation factors V, XI and XIII. Factor V may be particularly important as platelet Factor V is modified in a manner rendering it more resistant to cleavage by activated protein C.

## **6. Platelets in pathologic thrombosis**

Pathologic studies show that venous thrombi are platelet-poor, while arterial thrombi are platelet-rich. In addition, although anti-platelet therapy is known to have benefit in preventing recurrent venous thrombi, the benefits appear to be greater for myocardial infarction and stroke. These pathologic and clinical observations are consistent with the known effect of shear stress on platelet thrombus formation. The effects of higher shear stress are clear for VWF. VWF adopts a folder globular structure under a low shear environment, obscuring the domains that mediate binding to platelets. In contrast, the mechanical effects induced by high shear unfolds VWF and exposes the GPIbα A1-binding domain of VWF. In addition, high shear rates are able to activate platelets directly.[34] In summary, platelets make a modest contribution to venous thrombosis and a more substantive contribution to arterial thrombosis. However, the fundamental molecular mechanisms of platelet thrombus formation appear to be similar in health and disease.

## **Author details**

express the αvβ3 vitronectin receptor in low abundance. Crystalization of the extracellular domain of αvβ3 and the head domain of αIIbβ3 have provided detailed structural information

Talin is an abundant cytoskeletal protein that links integrins to the actin cytoskeleton. The agonist-induced rise in intracellular calcium results in binding of the talin head domain to the cytoplasmic domain of integrin β3. This interaction leads to an unclasping of the intracellular and transmembrane components of the αIIb and β3 molecules, causing spreading of the two proteins and exposure of the ligand binding site. The precise molecular details by which talin is enabled to bind β3 are unclear, but efficient integrin activation likely involves (1) the guanine nucleotide exchange factor CalDAG-GEFI, (2) activation of the small GTPase Rap1, (3)

Platelets contribute substantially to thrombin generation, which further induces additional platelet activation. In addition, platelet thrombus stabilization requires local fibrin generation that depends on thrombin generation. When platelets are stimulated by strong agonists, the negatively charged phospholipids on the inner leaflet of the platelet plasma membrane are "flipped" to the outer leaflet. This reorganization may be mediated by the calcium activated scramblase TMEM16F.[33] Translocation of negatively charged phospholipids forms a stage upon which coagulation reactions occur. The formation of the "tenase" complex that converts Factor X to activated Factor X requires phospholipid. The development of the prothrombinase complex also requires negatively charged phospholipid as the surface upon which the complex

Activation of platelets by strong agonists also leads to the development and shedding of platelet microparticles. These have a high density of negatively charged phospholipids and are thus able to support the formation of the "tenase" and prothrombinase complex as noted above. They also contain coagulation Factor Va with which to support the formation of thrombin as well as supplying arachidonic acid which can contribute to further formation of

Platelets α-granule release also provides coagulation factors V, XI and XIII. Factor V may be particularly important as platelet Factor V is modified in a manner rendering it more resistant

Pathologic studies show that venous thrombi are platelet-poor, while arterial thrombi are platelet-rich. In addition, although anti-platelet therapy is known to have benefit in preventing recurrent venous thrombi, the benefits appear to be greater for myocardial infarction and

kindlin-3 binding to the β3 cytoplasmic tail, and (4) calpain cleavage of talin.

about these integrins.[30, 32]

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

**5. Role in coagulation**

assembles.

TXA2.

to cleavage by activated protein C.

**6. Platelets in pathologic thrombosis**

Douglass A. Drelich and Paul F. Bray\*

\*Address all correspondence to: paul.bray@jefferson.edu

The Division of Hematology and The Cardeza Foundation for Hematologic Research, De‐ partment of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Phila‐ delphia, PA, USA

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Suppl 1:24-27


**The Role of Platelets in Inflammation, Infection and Immunity**

## **Chapter 3**

## **Role of Platelets in Inflammation**

Mònica Arman, Holly Payne, Tatyana Ponomaryov and Alexander Brill

Additional information is available at the end of the chapter

http://dx.doi.org/10. 5772/60536

## **1. Introduction**

#### **1. 1. Inflammation**

Inflammation is a complex of responses of the innate immune system to pathological stimuli such as microbes, pathogens or damage-associated molecular patterns (DAMPs). Local inflammation includes the following classical symptoms: *dolor* (pain), *calor* (heat), *rubor* (redness), *tumor* (swelling) and *functio laesa* (loss of function). Systemic inflammation occurs in different contexts like massive trauma, chronic disease, or as a response to an infection, in which case it is designated as sepsis. Clinical responses during systemic inflammation (systemic inflammatory response syndrome, SIRS) include altered body temperature, elevated pulse rate, elevated respiratory rate, abnormal white blood cell count and other symptoms. [1] The inflammatory response includes (but is not limited to) recruitment of immune cells, such as neutrophils and monocytes, by the vessel wall, followed by their extravasation to tissues. Although inflammation involves multiple mechanisms beyond this process (e. g. , involving complement and kinin systems as well as changes in vascular tonus), we will concentrate herein on the platelet role in vascular endothelial activation and interactions with leukocytes, with a special focus on *in vivo* data.

## **2. Platelet-derived mediators regulating inflammation**

Platelets have multiple roles beyond hemostasis and thrombosis and were described as inflammatory cells several decades ago. [2] Platelets contain a number of inflammatory peptide and protein mediators, some of which they retain the capability of synthesizing *de novo*, whereas others are stored and secreted from granules (dense granules, α-granules or lyso‐

© 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.

somes). [3] The release of these cytokines and chemokines, as well as eicosanoids, upon activation enables platelets to recruit leukocytes to the site of inflammation or injury. The table below lists some of the platelet-derived inflammatory mediators:


**Table 1.** Inflammatory mediators synthesized by and stored in platelets

Platelet α-granules contain large proteins, many of which are involved in regulation of the inflammatory response. [4, 5] Among them, Platelet Factor 4 (PF4) is the most abundant protein secreted by activated platelets (accounting for ~25% of α-granule content). [6] It functions as a chemoattractant for monocytes. PF4 accelerates atherogenesis by causing vascular inflam‐ mation and promoting retention of lipoproteins in the vascular wall, which contributes to atherosclerosis. PF4 prevents full interaction of LDL with its receptor, causing lipoproteins to be retained on the cell surface rather than being catabolized. [7]

Platelet-originating thromboxane A2 (TxA2), which is made *de novo* from arachidonic acid upon activation, induces platelet activation and aggregation. [8] This may form a positive feedback loop facilitating further release of stored cytokines.

somes). [3] The release of these cytokines and chemokines, as well as eicosanoids, upon activation enables platelets to recruit leukocytes to the site of inflammation or injury. The table

below lists some of the platelet-derived inflammatory mediators:

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

**Table 1.** Inflammatory mediators synthesized by and stored in platelets

be retained on the cell surface rather than being catabolized. [7]

Platelet α-granules contain large proteins, many of which are involved in regulation of the inflammatory response. [4, 5] Among them, Platelet Factor 4 (PF4) is the most abundant protein secreted by activated platelets (accounting for ~25% of α-granule content). [6] It functions as a chemoattractant for monocytes. PF4 accelerates atherogenesis by causing vascular inflam‐ mation and promoting retention of lipoproteins in the vascular wall, which contributes to atherosclerosis. PF4 prevents full interaction of LDL with its receptor, causing lipoproteins to

**Molecule Family Location** IL-1β Cytokine Synthesized Thromboxane A2 Eicosanoid Synthesized PF4/CXCL4 Chemokine α- granules β-thromboglobulin (CXCL7/ NAP-2) Chemokine α- granules RANTES (CCL5) Chemokine α- granules CD40L Cytokine α- granules PDGF Growth factor α- granules TGF-β Growth factor α- granules TNF-α Cytokine α- granules IL-1α Cytokine α- granules GRO-α (CXCL1) Cytokine α- granules ENA-78 (CXCL5) Cytokine α- granules SDF-1 (CXCL12) Cytokine α- granules MIP-1α (CCL3) Chemokine α- granules MCP-3 (CCL7) Chemokine α- granules NAP-2 (CXCL7) Chemokine α- granules TARC (CCL17) Chemokine α- granules Interleukin-8 (CXCL8) Chemokine α- granules Polyphosphates Phosphates Dense granules ATP Nucleotide Dense granules Serotonin Monoamine Dense granules Glutamate Amino Acid Dense granules Platelet-derived IL-1α has been shown to mediate cerebral inflammation *in vivo*. [9] IL-1α secreted from platelets promotes expression of the adhesion molecules ICAM-1 and VCAM-1 on endothelial cells. It also accelerates transendothelial migration of neutrophils and contrib‐ utes to chronic inflammatory diseases, such as multiple sclerosis. [9] Platelet IL-1α and IL-1β have proinflammatory roles in rheumatoid arthritis; it has been shown that platelet depletion attenuates the disease in mice. [10]

Besides storage, platelets can also synthesize biologically active proteins. For example, thrombin activation results in synthesis of pro-IL-1β. [11] Interestingly, synthesis of pro-IL-1β was inhibited by neutralization of the beta-3 integrin, which implies that direct antiplatelet therapy could have an anti-inflammatory effect. Platelets contain the splicing machinery allowing for cytokine mRNA maturation. [12] IL-1β potentiates its own synthesis in platelets by an autocrine loop, and its production by activated platelets occurs *in vivo*, where it accumulates in thrombus in the ferric chloride-treated carotid artery. [13] This represents a link connecting sterile thrombotic process with formation of proinflammatory milieu. IL-1β from platelets causes both up-regulation of endothelial adhesion receptors and release of proinflammatory IL-6 and IL-8 from endothelial cells. IL-1β is also responsible for activation of NF-κB in endothelial cells, which is required for transcription of inflammatory genes MCP-1 and ICAM-1. [7]

Platelet derived growth factor (PDGF) is able to chemoattract monocytes and eosinophils. [4] The chemokine RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) recruits monocytes to the inflamed endothelium in a P-selectin-dependent fashion. [14] RANTES plays a role in many inflammatory disorders including asthma, atherosclerosis and delayed-type hypersensitivity reactions. [15]

Among other platelet-derived chemokines, Macrophage Inflammatory Protein (MIP)-1α induces leukocyte chemotaxis *in vivo*. [8, 16] MIP-1α is a chemoattractant for monocytes, macrophages, T-cells and neutrophils and is involved in transendothelial migration at sites of inflammation. [17] MIP-1α is required for a normal inflammatory response to certain types of viruses. MIP-1α-null mice develop a reduced inflammatory response to influenza virus and coxsackievirus-induced myocarditis. [17]

Platelets store large amounts of the pro-inflammatory molecule CD40 ligand (CD40L). Interaction of CD40L with CD40 on endothelial cells and macrophages causes release of IL-8 and MCP-1, which attract neutrophils and monocytes. [18] Similarly to IL-1β, CD40L induces adhesion receptor expression on endothelium and release of chemokines thus mediating leukocyte recruitment.

Platelets contain Polyphosphate (polyP) in their dense granules. [19] Proinflammatory and procoagulant functions of polyP have recently been demonstrated. [20] PolyP, released upon platelet stimulation, binds to factor XII activating the FXII-driven contact activation system. The resulting release of the inflammatory mediator bradykinin culminates in the accumulation of neutrophils and increased vascular permeability through binding its receptor BR2. Target‐ ing polyP, for example, with phosphatases may be of potential therapeutic benefit for treating such diseases as rheumatoid arthritis and atherosclerosis. [20] However, the role of polyP in activating the contact phase system has recently been questioned. [21]

Thus, platelets store and release a substantial repertoire of inflammatory mediators. These molecules may contribute to multiple inflammation-related diseases, which make platelets important players in the field of inflammation.

## **3. Platelet-endothelium interactions**

Under physiological (non-inflammatory) conditions, production of platelet inhibitors (such as prostacyclin and nitric oxide) by endothelial cells limits platelet interaction with intact endothelium. Adhesion of activated platelets to intact Human Umbilical Vein Endothelial Cells (HUVEC) was demonstrated *in vitro*. [22] Mechanisms of this involved αIIbβ3 integrin (glyco‐ protein (GP) IIb/IIIa) on platelets, ICAM-1 and αvβ3 integrin on the endothelium and von Willebrand Factor (VWF), fibrinogen and fibronectin as bridging molecules. Platelet GPIbα, which is constitutively expressed and does not require activation, was reported to be a receptor to endothelial P-selectin. [23] There is a report demonstrating that platelets contain P-selectin glycoprotein ligand (PSGL)-1 (although 25-100-fold fewer than leukocytes). [24] Blocking PSGL-1 down-regulates the number of rolling and captured platelets on stimulated venule endothelium suggesting that this route can also be implicated in platelet-vessel wall interaction under inflammatory conditions. Integrin αvβ3, a vitronectin receptor on the endothelial cells, was shown to participate in platelet recruitment to stimulated endothelium. [25]

*In vivo*, platelets do not spontaneously interact with intact endothelium in murine mesenteric venules. [26] Stimulation of murine vessels with Weibel-Palade body secretagogues calcium ionophore or histamine results in rapid platelet adhesion followed by rolling, peaking 1 min after stimulation. This "stop-and-go" platelet translocation on stimulated endothelium was absent in VWF-null mice. Cleavage of GPIbα from platelet surface also prevented platelet binding to the vessel wall. Therefore, interaction of platelets with activated endothelium *in vivo* is mediated by binding of platelet GPIbα to endothelium-expressed VWF. [26]

Another pathway of platelet binding to the vascular wall involves the glycoprotein VI (GPVI), a major platelet receptor for collagen. This route is most important in platelet interactions with atherosclerotic plaques. Inhibition of GPVI by infusion of GPVI-Fc, a dimeric soluble form of GPVI fused with human Fc fragment, to ApoE-/- mice decreased transient platelet interactions with atherosclerotic artery wall by about half. [27] Long-term administration of a GPVIblocking antibody also improved endothelial function and prevented propagation of athero‐ sclerosis. [27] GPVI binds activated endothelium through vitronectin and improves cardiac function in a mouse model of heart ischemia-reperfusion by reducing inflammation in the infarcted myocardium. [28] Infusion of soluble GPVI-Fc in either ischemia or reperfusion phase substantially decreased infarct size.

In a model of cerebral ischemia-reperfusion, platelet tumbling on and adhesion to the brain vascular endothelium has been demonstrated. [29] This was specific to veins and no plateletvessel wall interactions were observed in arteries of different diameter. This interaction was almost entirely dependent on P-selectin as administration of anti-P-selectin antibody abolished it. Neutralization of αIIbβ3 had certain inhibitory effect too, though less manifested than blocking P-selectin. Rolling and adhesion of platelets was reported also during reperfusion period after retinal ischemia. This process was dependent on endothelial P-selectin, and the time course of P-selectin *de novo* synthesis in the endothelium corresponded to the kinetics of platelet-endothelial interactions. [30]

of neutrophils and increased vascular permeability through binding its receptor BR2. Target‐ ing polyP, for example, with phosphatases may be of potential therapeutic benefit for treating such diseases as rheumatoid arthritis and atherosclerosis. [20] However, the role of polyP in

Thus, platelets store and release a substantial repertoire of inflammatory mediators. These molecules may contribute to multiple inflammation-related diseases, which make platelets

Under physiological (non-inflammatory) conditions, production of platelet inhibitors (such as prostacyclin and nitric oxide) by endothelial cells limits platelet interaction with intact endothelium. Adhesion of activated platelets to intact Human Umbilical Vein Endothelial Cells (HUVEC) was demonstrated *in vitro*. [22] Mechanisms of this involved αIIbβ3 integrin (glyco‐ protein (GP) IIb/IIIa) on platelets, ICAM-1 and αvβ3 integrin on the endothelium and von Willebrand Factor (VWF), fibrinogen and fibronectin as bridging molecules. Platelet GPIbα, which is constitutively expressed and does not require activation, was reported to be a receptor to endothelial P-selectin. [23] There is a report demonstrating that platelets contain P-selectin glycoprotein ligand (PSGL)-1 (although 25-100-fold fewer than leukocytes). [24] Blocking PSGL-1 down-regulates the number of rolling and captured platelets on stimulated venule endothelium suggesting that this route can also be implicated in platelet-vessel wall interaction under inflammatory conditions. Integrin αvβ3, a vitronectin receptor on the endothelial cells,

was shown to participate in platelet recruitment to stimulated endothelium. [25]

*vivo* is mediated by binding of platelet GPIbα to endothelium-expressed VWF. [26]

*In vivo*, platelets do not spontaneously interact with intact endothelium in murine mesenteric venules. [26] Stimulation of murine vessels with Weibel-Palade body secretagogues calcium ionophore or histamine results in rapid platelet adhesion followed by rolling, peaking 1 min after stimulation. This "stop-and-go" platelet translocation on stimulated endothelium was absent in VWF-null mice. Cleavage of GPIbα from platelet surface also prevented platelet binding to the vessel wall. Therefore, interaction of platelets with activated endothelium *in*

Another pathway of platelet binding to the vascular wall involves the glycoprotein VI (GPVI), a major platelet receptor for collagen. This route is most important in platelet interactions with atherosclerotic plaques. Inhibition of GPVI by infusion of GPVI-Fc, a dimeric soluble form of GPVI fused with human Fc fragment, to ApoE-/- mice decreased transient platelet interactions with atherosclerotic artery wall by about half. [27] Long-term administration of a GPVIblocking antibody also improved endothelial function and prevented propagation of athero‐ sclerosis. [27] GPVI binds activated endothelium through vitronectin and improves cardiac function in a mouse model of heart ischemia-reperfusion by reducing inflammation in the infarcted myocardium. [28] Infusion of soluble GPVI-Fc in either ischemia or reperfusion phase

activating the contact phase system has recently been questioned. [21]

important players in the field of inflammation.

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

**3. Platelet-endothelium interactions**

substantially decreased infarct size.

Platelet adhesion to endothelium of atherosclerotic plaques can also be mediated by the glycoprotein αIIb. [31] Platelet adhesion to the atherosclerotic plaque in apoE-/- αIIb-double deficient mice was virtually abolished as compared with apoE-/- αIIb+/+ controls. Formation of atherosclerotic lesions was reduced in the absence of αIIb. Platelet-vessel wall interactions through αIIb are also implicated in the pathogenesis of such thromboinflammatory disease as ischemic stroke as shown in a model of cerebral ischemia-reperfusion in mice. The exact mechanism of αIIb involvement in interactions with inflamed but non-denuded endothelium remains to be clarified. It is known that ischemia promotes fibrinogen deposition on the vessel wall, which leads to platelets recruitment. [32] Local hypoxia and pro-inflammatory shift (like generation of reactive oxygen species (ROS)) result in VWF expression on the endothelium mediating platelet accrual. [33, 34] Thus, two major ligands for αIIbβ3, fibrinogen and VWF, can appear on the endothelial surface during inflammation and recruit platelets via this integrin.

Platelet accumulation in lung and cerebral vasculature was described in a murine model of malaria[35] as well as in patients. [36] Platelets might damage endothelium and support leukocyte accumulation in the brain vessels thus promoting cerebral inflammation as a part of malaria pathogenesis. [37] Platelet depletion protects mice from disease progression. [38] The role of platelets in malaria may be complex depending on the stage of the disease: platelets could attenuate parasite growth at the early stages whereas at later stages platelets support disease-related inflammation. [39]

Activated platelets can be found in circulation in patients with various inflammatory diseases, such as sepsis, cerebrovascular ischemia and diabetes. [40-42] Besides posing a danger for excessive thrombosis, circulating activated platelets confer a proinflammatory signal. Acti‐ vated platelets infused into mice stimulate release of Weibel-Palade body constituents and form complexes with leukocytes, leading to elevated recruitment of leukocytes to the vessel wall. [43] Thrombin-activated platelets accumulate at the atherosclerotic carotid artery wall. This process is dramatically inhibited when platelets lack CD40L. [44]

Interaction of platelets with endothelium mediates accumulation of monocytes and deposition of proinflammatory cytokines (e.g. , RANTES) at the vessel wall (Figure 1). It was directly demonstrated in mice using repeated infusions of activated platelets or bone marrow trans‐ plantation techniques, that platelets promote the development of larger atherosclerotic plaques. [45, 46] This effect is predominantly mediated by platelet P-selectin.

Thus, platelets bind to the activated/inflamed vascular wall by a set of receptors including Pselectin, glycoproteins Ibα, αIIb and VI as well as CD40L. Activated platelets are able to induce a pro-inflammatory shift in the endothelium. Platelet-mediated endothelial activation plays a role in the development of various diseases that have an inflammatory component in their pathogenesis.

**Figure 1.** Platelet cross-talk with endothelium and leukocytes.

## **4. Platelet-leukocyte interactions**

Under physiological conditions, platelets and leukocytes do not bind to each other. Such interaction becomes possible in prothrombotic or proinflammatory state with increased number of blood platelet-leukocyte aggregates (PLA) (Figure 1) observed in such diseases as diabetes mellitus, stroke and others. [47-49]

Binding of platelets to leukocytes can mediate recruitment of the latter to the vessel wall and render leukocytes more prothrombotic promoting synthesis of tissue factor by monocytes. [50] This interaction starts by binding of P-selectin on activated platelets to PSGL-1 on leukocytes initiating a signaling cascade inside leukocytes, which leads to activation of integrins, in particular, Mac-1 and LFA-1 on leukocyte membrane. [51-53] Mac1 can bind platelet receptor GPIbα directly or αIIbβ3 through fibrinogen bound to the integrin on platelets. [54, 55] Plateletleukocyte binding is an active process as pre-activation of leukocytes potentiates this interac‐ tion whereas tyrosine kinase inhibitors down-regulate it. Full activation of the integrins triggers outside-in signaling regulating multiple leukocyte functions such as transmigration, production of ROS and phagocytosis. [56] Platelet-mediated activation of Mac-1 can lead to sequestration and activation of coagulation Factor X resulting in thrombin generation. [57] This phenomenon suggests that platelet-leukocyte interaction triggers also the coagulation cascade.

*In vivo*, interactions between platelets and leukocytes occur in various thrombo-inflammatory conditions. For example, blood stasis in the carotid artery induces P-selectin-dependent accumulation of leukocytes surrounded by platelets in the vicinity of the vessel wall. [58] Platelet depletion almost completely abrogates leukocyte recruitment suggesting that devel‐ opment of the inflammatory response in this model is platelet-dependent. Platelet P-selectin is implicated in recruiting leukocytes not only to the inflammation site but also in pure thrombosis. Thrombi in ferric chloride-challenged carotid arteries of P-selectin-deficient mice contained less leukocytes than in control animals. [59] This confirms the central role of Pselectin in platelet interactions with leukocytes.

Formation of platelet-leukocyte rosettes *in vivo* depends on platelet activation. It has been reported that plasma level of circulating PLA more specifically reflects platelet activation than platelet P-selectin expression. [60] Resting platelets infused into mice do not associate with leukocytes. [61] Intravenous injection of collagen together with αIIbβ3 antagonist to prevent formation of platelet aggregates, results in rapid development of platelet-leukocyte conjugates. These conjugates roll on the vascular wall both in C57BL/6 and aged ApoE-deficient mice prone to atherosclerosis. In both cases, this rolling was mediated by P-selectin. Binding of activated platelets to leukocytes supported leukocyte recruitment by the endothelium through VCAM-1, and elevated leukocyte interactions with vessel wall in inflammation and atherosclerosis. [45] Infusion of activated P-selectin positive but not P-selectin deficient platelets elevated monocyte binding to atherosclerotic endothelium in mice. Formation of PLA resulted in deposition of chemokines, such as RANTES and PF4, on the endothelium thus supporting development of atherosclerosis. Besides leukocytes, activated platelets mediate lymphocyte homing in peripheral lymph nodes. [62] Again, all these phenomena were dependent on platelet surface P-selectin.

Cell activation in leukocyte-platelet interaction is bi-directional, i. e. , not only platelets activate leukocytes but also vice versa. [56] In particular, various leukocyte-derived molecules can activate platelets and promote platelet-mediated fibrin deposition. Activated platelets stimulate neutrophils to release their chromatin designated as Neutrophil Extracellular Traps (NETs), [63] at least in part by presenting High Mobility Group Box 1 (HMGB1). [64] NETs can recruit and activate platelets. [65] Histones, an integral part of NETs, directly activate platelets and induce platelet aggregation. [66]

In conclusion, platelets interact with both endothelium and leukocytes mediating accumula‐ tion of the latter at the inflammatory site, thus supporting the central step in the inflammatory response.

## **5. Platelet Toll-like receptors**

a pro-inflammatory shift in the endothelium. Platelet-mediated endothelial activation plays a role in the development of various diseases that have an inflammatory component in their

Under physiological conditions, platelets and leukocytes do not bind to each other. Such interaction becomes possible in prothrombotic or proinflammatory state with increased number of blood platelet-leukocyte aggregates (PLA) (Figure 1) observed in such diseases as

Binding of platelets to leukocytes can mediate recruitment of the latter to the vessel wall and render leukocytes more prothrombotic promoting synthesis of tissue factor by monocytes. [50] This interaction starts by binding of P-selectin on activated platelets to PSGL-1 on leukocytes initiating a signaling cascade inside leukocytes, which leads to activation of integrins, in particular, Mac-1 and LFA-1 on leukocyte membrane. [51-53] Mac1 can bind platelet receptor GPIbα directly or αIIbβ3 through fibrinogen bound to the integrin on platelets. [54, 55] Plateletleukocyte binding is an active process as pre-activation of leukocytes potentiates this interac‐ tion whereas tyrosine kinase inhibitors down-regulate it. Full activation of the integrins triggers outside-in signaling regulating multiple leukocyte functions such as transmigration, production of ROS and phagocytosis. [56] Platelet-mediated activation of Mac-1 can lead to sequestration and activation of coagulation Factor X resulting in thrombin generation. [57]

pathogenesis.

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

**Figure 1.** Platelet cross-talk with endothelium and leukocytes.

**4. Platelet-leukocyte interactions**

diabetes mellitus, stroke and others. [47-49]

Toll-like receptors (TLRs) are a family of innate immunity pattern-recognition receptors that trigger inflammation in response to microbial products or products of inflamed tissues. TLRs function as front-line sensors of infection, as they recognize conserved structures in pathogens designated as pathogen-associated molecular patterns (PAMPs). [67] TLRs can also sense DAMPs, released by activated or necrotic host cells and upregulated following tissue damage. [68] Human and murine platelets express TLR2, TLR4, TLR7 and TLR9. [69-76] TLR6 has been detected in human platelets. [69] Expression of TLR1 has been reported in one[69] but not in another[77] study.

**Platelet TLR2**. Pam3CSK4, a synthetic agonist of the TLR2/1 complex, triggers platelet activation including integrin αIIbβ<sup>3</sup> transition to an active state, aggregation, alpha- and dense granule release and CD40L expression. [78-80] These responses are inhibited in TLR2-deficient murine platelets and in human platelets by pretreatment with TLR2-blocking antibody. [79] Pam3CSK4 also triggers formation of platelet–neutrophil aggregates (PNA). [79, 80]

In periodontitis, a chronic inflammatory disease of the supportive dental tissues, the gramnegative periodontopathogens directly induce TLR2- and TLR4-dependent surface expression of CD40L in human platelets. [81] *In vivo* challenge with live *Porphyromonas gingivalis* induced formation of PNA in wild-type but not TLR2-deficient mice. [79] *Ex vivo* experiments showed that platelet TLR2 mediated formation of PNA and enhanced phagocytosis of periodonto‐ pathogens. [81]

Human cytomegalovirus (HCMV), a widespread pathogen that correlates with various diseases including atherosclerosis, binds TLR2-positive platelet subpopulation. This results in platelet degranulation, release of proinflammatory CD40L and IL-1β and proangiogenic vascular endothelial–derived growth factor (VEGF). Murine CMV activates wild-type but not TLR2-deficient mouse platelets. HCMV-activated platelets bind to and activate neutrophils, supporting their adhesion and transmigration through endothelial monolayers. [82] In an *in vivo* model, CMV increased the number of PLA and plasma VEGF levels and demonstrated a trend to enhance neutrophil extravasation in a TLR2-dependent fashion. [82]

**Platelet TLR4***.* Platelet activation with thrombin causes increase of TLR4 surface expression in one [83] but not another [72] study. Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, is a natural ligand for TLR4. *In vitro*, some studies have reported no [77, 84] or even inhibitory effect[85] of LPS on platelet aggregation whereas others have shown that LPS can potentiate platelet aggregation induced by low doses of other agonists. [86, 87] *In vivo,* intravenous injection of LPS in mice induces formation of platelet aggregates mainly in lung and liver microvasculature. [88, 89] Platelet TLR4 mediated microvascular LPSinduced thrombosis in murine cremaster muscle venules. [84, 90]

LPS present on platelets and leukocytes from patients with hemolytic uremic syndrome (HUS) caused PLA formation. [91] LPS binds human platelets through a complex of TLR4 and CD62, leading to platelet activation. The specificity of LPS binding to platelet TLR4 was confirmed *in vivo* using TLR4 deficient mice. [92]

Platelet TLR4 contributes to LPS-induced thrombocytopenia *in vivo*. [71, 72] In one study, LPS induced thrombocytopenia in wild-type but not TLR4-deficient mice. [71] TLR4-positive but not TLR4-negative platelets accumulated in the murine lungs in response to LPS in a neutro‐ phil-dependent fashion. In another study, LPS produced thrombocytopenia and increased serum levels of TNFα in LPS-sensitive mice but not in mice carrying mutated TLR4. [72] LPSinduced TNFα production in LPS-sensitive mice was reduced by platelet depletion and could be restored by platelet transfusion. [72]

In mouse endotoxemia, TLR4-positive but not TLR4-negative platelets accumulate in the lungs. [71] LPS induces platelet binding to sequestered neutrophils primarily in the liver sinusoids and pulmonary capillaries leading to formation of NETs, [93] which are able to trap and kill microbes. [63]

**Platelet TLR7**. A recent study showed that platelet TLR7 mediates platelet activation in response to the single stranded RNA encephalomyocarditis virus (EMCV). [74] This interac‐ tion led to platelet granule release, P-selectin surface expression, and increase in PNA, both in mouse and human blood, but did not induce platelet aggregation. There were, however, implications to the host immune response and survival. TLR7 expressed on platelets is implicated in protection against EMCV-induced mortality. Transfusion of TLR7-positive platelets into TLR7-null mice prolonged survival after infection with EMCV whereas transfu‐ sion of platelets lacking TLR7 into wild-type mice did not affect the survival rate. [74]

**Platelet TLR9**. In platelets, TLR9 is located in intracellular compartments. [75] TLR9 responds to carboxy(alkylpyrrole) protein adducts, an altered-self ligand generated in oxidative stress, in both human and mouse platelets. This interaction results in aggregation *in vitro* and thrombosis *in vivo*. [76] Physiological platelet agonists synergize with TLR9 ligands by increasing TLR9 expression on the platelet membrane. [76]

## **6. Platelets in sepsis**

function as front-line sensors of infection, as they recognize conserved structures in pathogens designated as pathogen-associated molecular patterns (PAMPs). [67] TLRs can also sense DAMPs, released by activated or necrotic host cells and upregulated following tissue damage. [68] Human and murine platelets express TLR2, TLR4, TLR7 and TLR9. [69-76] TLR6 has been detected in human platelets. [69] Expression of TLR1 has been reported in one[69] but not in

**Platelet TLR2**. Pam3CSK4, a synthetic agonist of the TLR2/1 complex, triggers platelet activation including integrin αIIbβ<sup>3</sup> transition to an active state, aggregation, alpha- and dense granule release and CD40L expression. [78-80] These responses are inhibited in TLR2-deficient murine platelets and in human platelets by pretreatment with TLR2-blocking antibody. [79]

In periodontitis, a chronic inflammatory disease of the supportive dental tissues, the gramnegative periodontopathogens directly induce TLR2- and TLR4-dependent surface expression of CD40L in human platelets. [81] *In vivo* challenge with live *Porphyromonas gingivalis* induced formation of PNA in wild-type but not TLR2-deficient mice. [79] *Ex vivo* experiments showed that platelet TLR2 mediated formation of PNA and enhanced phagocytosis of periodonto‐

Human cytomegalovirus (HCMV), a widespread pathogen that correlates with various diseases including atherosclerosis, binds TLR2-positive platelet subpopulation. This results in platelet degranulation, release of proinflammatory CD40L and IL-1β and proangiogenic vascular endothelial–derived growth factor (VEGF). Murine CMV activates wild-type but not TLR2-deficient mouse platelets. HCMV-activated platelets bind to and activate neutrophils, supporting their adhesion and transmigration through endothelial monolayers. [82] In an *in vivo* model, CMV increased the number of PLA and plasma VEGF levels and demonstrated a

**Platelet TLR4***.* Platelet activation with thrombin causes increase of TLR4 surface expression in one [83] but not another [72] study. Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, is a natural ligand for TLR4. *In vitro*, some studies have reported no [77, 84] or even inhibitory effect[85] of LPS on platelet aggregation whereas others have shown that LPS can potentiate platelet aggregation induced by low doses of other agonists. [86, 87] *In vivo,* intravenous injection of LPS in mice induces formation of platelet aggregates mainly in lung and liver microvasculature. [88, 89] Platelet TLR4 mediated microvascular LPS-

LPS present on platelets and leukocytes from patients with hemolytic uremic syndrome (HUS) caused PLA formation. [91] LPS binds human platelets through a complex of TLR4 and CD62, leading to platelet activation. The specificity of LPS binding to platelet TLR4 was confirmed

Platelet TLR4 contributes to LPS-induced thrombocytopenia *in vivo*. [71, 72] In one study, LPS induced thrombocytopenia in wild-type but not TLR4-deficient mice. [71] TLR4-positive but not TLR4-negative platelets accumulated in the murine lungs in response to LPS in a neutro‐ phil-dependent fashion. In another study, LPS produced thrombocytopenia and increased

trend to enhance neutrophil extravasation in a TLR2-dependent fashion. [82]

induced thrombosis in murine cremaster muscle venules. [84, 90]

*in vivo* using TLR4 deficient mice. [92]

Pam3CSK4 also triggers formation of platelet–neutrophil aggregates (PNA). [79, 80]

another[77] study.

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

pathogens. [81]

Sepsis is an uncontrolled systemic reaction to an infection. It can progress into severe sepsis with multiple organ dysfunction and cognitive impairment. Septic shock, in which patients suffer vascular collapse and often are irresponsive to fluid resuscitation and vasopressor therapy, is often the terminal event of severe sepsis. [1] Sepsis is a complex process presenting with multiple pathogenic features, such as dysregulation of the immune and coagulation systems, thrombosis, disruption of endothelial barrier function, increased vascular permea‐ bility, microvascular sequestration, tissue damage, etc. [94] This complexity is likely to be responsible for the failure to find new treatments for sepsis, [95] and for the lack of good animal models. [96, 97]

Platelets are both cellular effectors and cellular targets in the pathophysiology of sepsis. Regardless of the initiating events in sepsis, platelets play an important role in the development of multiple organ failure via their haemostatic and thrombotic potential, resulting in throm‐ botic microangiopathy and disseminated intravascular coagulation. [98, 99, 100] Evidence for an important role of platelets is provided by clinical studies and animal model data demon‐ strating beneficial effect of antiplatelet agents in sepsis (reviewed in [101]).

Sepsis is frequently accompanied by thrombocytopenia, which is closely associated with disease severity and mortality rate. [98, 99] Multiple mechanisms may contribute to thrombo‐ cytopenia in sepsis: disseminated intravascular coagulation with peripheral consumption and destruction of platelets, impaired thrombopoiesis, direct activation by bacteria or their products, phagocytosis, etc. [102-104] Thrombocytopenia is also detected after injection of LPS in mice (a common model for sepsis) [88, 89] through a TLR4-dependent mechanism. [71, 72]

Alterations in circulating platelets occur in septic patients. CD62P expression was elevated in septic platelets in some studies[105, 106] but not in the others[107, 108] Other platelet activation markers found in sepsis include membrane expression of thrombospondin (TSP)[109, 110] and CD63, [106] elevated soluble CD40L level[108] and an increase in beta-thromboglobulin and the beta-thromboglobulin-to-PF4 ratio. [111] Increased VEGF release by agonist-stimulated platelets from septic samples has been reported. [107] Moreover, triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1), secreted upon platelet activation, is found in the plasma of patients with sepsis in levels that correlate with disseminated intravascular coagulation. [112, 113]. Animal studies suggest that TLT-1 dampens inflammation and augments platelet aggregation, reducing local hemorrhage. [112] Furthermore, soluble TLT-1 increases platelet adherence to the endothelium[114] and is involved in the regulation of inflammation in the course of sepsis by suppressing leukocyte activation and affecting plateletneutrophil crosstalk. [115]

An *in vitro* study showed that platelets from septic patients are hyper-adhesive to cultured endothelium. [110] Alterations in platelet aggregation [107, 116] and increase in PLA level, which both might contribute to inflammation and vascular injury, have also been found in sepsis. [105, 109]

Changes in platelet transcriptome have also been reported in sepsis. [117] Expression of spliced tissue factor mRNA in platelets from septic subjects was associated with tissue factordependent procoagulant activity. [118] This may be one of the mechanisms by which platelets contribute to microvascular thrombosis in sepsis. [119, 118]

It is likely that different platelet-activating pathways cooperate during sepsis. Platelets are activated by some (but not all) bacteria or their products, and by NETs. [65, 103, 104] This could have beneficial roles in fighting infection (i. e. , pathogen capture within thrombus, pathogen killing, etc). However, uncontrolled thrombus formation in response to bacteria or NETs could have detrimental effects in sepsis. Other processes, such as imbalance between plasma level of high molecular weight VWF and its cleavage protease ADAMTS-13, imbalanced coagula‐ tion, systemic endothelial activation, and leukocyte activation, might contribute to potentiat‐ ing platelet activation in sepsis. [99, 100]

## **7. Platelets and neurovascular inflammation**

The central nervous system (CNS) is an immune-privileged site, separated from blood by the blood brain barrier (BBB). Under pathological conditions, BBB may be disrupted. This lets cells from blood into the cerebral tissue and facilitates innate and adaptive immune responses in the CNS. [120, 121]

Platelets are present in the inflamed CNS microvasculature in mice and are capable of activating brain endothelial cells via IL-1α release. Platelets, as inflammatory cells, participate in neural diseases associated with pathogen-induced and sterile inflammation. [122-124]

cytopenia in sepsis: disseminated intravascular coagulation with peripheral consumption and destruction of platelets, impaired thrombopoiesis, direct activation by bacteria or their products, phagocytosis, etc. [102-104] Thrombocytopenia is also detected after injection of LPS in mice (a common model for sepsis) [88, 89] through a TLR4-dependent mechanism. [71, 72]

Alterations in circulating platelets occur in septic patients. CD62P expression was elevated in septic platelets in some studies[105, 106] but not in the others[107, 108] Other platelet activation markers found in sepsis include membrane expression of thrombospondin (TSP)[109, 110] and CD63, [106] elevated soluble CD40L level[108] and an increase in beta-thromboglobulin and the beta-thromboglobulin-to-PF4 ratio. [111] Increased VEGF release by agonist-stimulated platelets from septic samples has been reported. [107] Moreover, triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1), secreted upon platelet activation, is found in the plasma of patients with sepsis in levels that correlate with disseminated intravascular coagulation. [112, 113]. Animal studies suggest that TLT-1 dampens inflammation and augments platelet aggregation, reducing local hemorrhage. [112] Furthermore, soluble TLT-1 increases platelet adherence to the endothelium[114] and is involved in the regulation of inflammation in the course of sepsis by suppressing leukocyte activation and affecting platelet-

An *in vitro* study showed that platelets from septic patients are hyper-adhesive to cultured endothelium. [110] Alterations in platelet aggregation [107, 116] and increase in PLA level, which both might contribute to inflammation and vascular injury, have also been found in

Changes in platelet transcriptome have also been reported in sepsis. [117] Expression of spliced tissue factor mRNA in platelets from septic subjects was associated with tissue factordependent procoagulant activity. [118] This may be one of the mechanisms by which platelets

It is likely that different platelet-activating pathways cooperate during sepsis. Platelets are activated by some (but not all) bacteria or their products, and by NETs. [65, 103, 104] This could have beneficial roles in fighting infection (i. e. , pathogen capture within thrombus, pathogen killing, etc). However, uncontrolled thrombus formation in response to bacteria or NETs could have detrimental effects in sepsis. Other processes, such as imbalance between plasma level of high molecular weight VWF and its cleavage protease ADAMTS-13, imbalanced coagula‐ tion, systemic endothelial activation, and leukocyte activation, might contribute to potentiat‐

The central nervous system (CNS) is an immune-privileged site, separated from blood by the blood brain barrier (BBB). Under pathological conditions, BBB may be disrupted. This lets cells from blood into the cerebral tissue and facilitates innate and adaptive immune responses in

contribute to microvascular thrombosis in sepsis. [119, 118]

**7. Platelets and neurovascular inflammation**

ing platelet activation in sepsis. [99, 100]

neutrophil crosstalk. [115]

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

sepsis. [105, 109]

the CNS. [120, 121]

Sterile neurovascular inflammation accompanies such neural disorders as stroke, multiple sclerosis, and Alzheimer's disease (summarized in [125]). Ischemic stroke elicits a strong inflammatory response. [126] Inhibition of platelet adhesion to the injured vessel wall by blocking surface receptors GPIbα or GPVI protected mice from ischemic injury, implying that platelets are involved in stroke-related cerebral inflammation. [127] The lack of ADAMTS13, an enzyme cleaving VWF rendering it less proadhesive, promoted brain damage whereas infusion of ADAMTS13 ameliorated the defect, [128] further suggesting that platelet adhesion is an important pathogenetic step in ischemic stroke.

Interestingly, limiting platelet aggregation with αIIbβ3 inhibitors did not protect from stroke in mice[127] and humans. [129] Altogether there are several candidates on the platelet surface or inside platelet granules, including GPIb, GPVI, and VWF that could be potential targets for stroke treatment through reducing thrombo-inflammation without inducing bleeding complications[126, 130-133]

Neuronal loss is accompanied by BBB breakdown and vascular inflammation in age-related **Alzheimer's disease** (AD). [134] Platelet function in AD is altered, and platelet activation state (determined by plasma soluble GPVI levels) is considered a potential biomarker for the disease progression. [135-138] Platelets contain substantial amounts of amyloid precursor protein[139] and various forms of tau protein that could have diagnostic value as biomarkers and/or play a role in disease pathogenesis. [140]

**Multiple sclerosis** (MS) is a devastating T-cell mediated autoimmune neuroinflammatory disease. [141] High levels of platelet activation markers (surface expression of P-selectin) and increased plasma content of platelet-derived microparticles (PMP) were detected in MS patients. [142] Chronic lesions of MS patients contain tissue factor, as has been demonstrat‐ ed by proteomics approach, [143] and elevated levels of platelet-specific αIIb and β<sup>3</sup> transcripts were detected by microarray. [144] These findings are in concert with platelet presence in human MS lesions and in the murine brain in experimental autoimmune encephalomyelitis (EAE), rodent model of MS. [145] Platelet depletion as well as blocking GPIbα or αIIbβ3 by antibody Fab fragments in the inflammatory rather than immunization phase of the disease resulted in decreased EAE severity. Intravital microscopy revealed that platelets directly contributed to leukocyte rolling and adhesion to endothelium of the inflamed postcapillary venules via GPIb-Mac-1 interaction. [145]

Platelet activation in neuroinflammation may result from direct recognition of specific structures of damaged tissue. For instance, massive platelet activation and degranulation was induced upon systemic administration in mice of sialated glycosphingolipids (gangliosides), components of astroglial and neuronal lipid rafts of BBB. The cerebral gangliosides GT1b and GQ1b are specifically recognized by platelets with P-selectin playing the central role. [146]

The pathogenesis of **migraine**, the third most frequent disease worldwide, [147] involves sterile inflammation and hypersensitization of pain pathways. [148] Spontaneous platelet activation and aggregation in migraine patients have been known for years [149, 150] and expression of platelet receptors to fibrinogen and serotonin are altered in migraine patients. [151] PLA accumulating in the blood of patients with migraine[152] may link severe headaches and stroke. [152, 153] Preliminary observations suggest that antiplatelet therapies may be effective to reduce the severity of migraine. [154]

## **8. Platelets in allergic inflammation**

Allergic diseases include a variety of conditions (atopic dermatitis, asthma, etc) that are caused by immune responses to environmental antigens. The hallmarks of allergy are the activation of TH2 lymphocytes and the production of allergen-specific IgE antibodies, with the latter causing excessive activation of mast cells, eosinophils and basophils. This may become fatal when hypersensitivity results in systemic response designated as anaphylaxis. In chronic allergic inflammation, large numbers of immune cells accumulate at the affected site, causing substantial tissue damage. [155] The link between platelet activation and allergy has been studied for many years. [156, 157]

Independent studies report elevated plasma levels of platelet activation blood markers (βthromboglobulin (β-TG), PF4, P-selectin, and PMPs) in patients with atopic dermatitis (AD) and psoriasis. [158-160] as well as in a mouse model of AD. [161] Plasma β-TG and PF4 may be markers for the severity of AD and psoriasis. [158]

In AD, it is possible that platelets contribute to an itch–scratch–hemorrhage cycle via release of pruritogenic factors such as histamine, 5-HT, acid proteases, IL-1β, TGF-β, PAF, and prostaglandin E2. [157]

While studies on platelet activation markers in asthmatic patients are inconclusive likely due to differences in experimental design, [162-167] the role of platelets in lung allergic inflamma‐ tion has been established in mouse models. [168-171] There is a significant association between activation of platelets and eosinophils in the airways of individuals with asthma. [164] Moreover, circulating PLA are detected in the blood of allergen-challenged asthmatic patients and mice. [168] Platelets are essential for leukocyte recruitment to human and murine lungs in allergic inflammation [168, 170] and to the skin in chronic hapten-induced dermatitis, another mouse model of AD. [172] PLA circulate in the blood of asthmatic patients and in allergen-challenged mice. [165, 168] In all cases, the role of platelets was P-selectin-dependent.

Platelets express functional low (FcεRII) and high affinity (FcεRI) receptors for IgE at low level [173-175] Murine platelets can chemotactically respond to the sensitizing allergen via FcεR *in vitro* and *in vivo*, with platelet influx preceding the influx of leukocytes. [171] Upon engagement of IgE receptors, platelets release a variety of biologically active media‐ tors[175, 176] including RANTES, a potent eosinophil chemoattractant. [177] IgE is stored in platelet α-granules and released upon activation, which may potentially amplify the allergic response. [178]

Multiple products released by activated platelets are able to exacerbate the allergic response, e. g. , thromboxanes, histamine, and serotonin. [179, 180, 181] PAF is a potent mediator of allergic inflammation that is both released by and activates immune and inflammatory cells, including platelets [55, 182, 183]. In mice, platelets, and not mast cells, are the main source of serotonin released during allergic inflammatory response. [184] Besides allergic mediators platelets also contain substances limiting inflammation, for example, lipoxins, produced during platelet-leukocyte interactions. [185-188]

In asthma, platelets have been found to actively participate in most of its main features, including bronchial hyperresponsiveness, bronchoconstriction, airway inflammation and airway remodelling. [169, 189]

In conclusion, delineation of platelet contribution to the allergic response may be beneficial in developing more effective therapies, [190] as well as diagnostic and prognostic tools to evaluate efficacy of treatment of various allergic diseases. [191]

## **9. Concluding remarks**

activation and aggregation in migraine patients have been known for years [149, 150] and expression of platelet receptors to fibrinogen and serotonin are altered in migraine patients. [151] PLA accumulating in the blood of patients with migraine[152] may link severe headaches and stroke. [152, 153] Preliminary observations suggest that antiplatelet therapies may be

Allergic diseases include a variety of conditions (atopic dermatitis, asthma, etc) that are caused by immune responses to environmental antigens. The hallmarks of allergy are the activation of TH2 lymphocytes and the production of allergen-specific IgE antibodies, with the latter causing excessive activation of mast cells, eosinophils and basophils. This may become fatal when hypersensitivity results in systemic response designated as anaphylaxis. In chronic allergic inflammation, large numbers of immune cells accumulate at the affected site, causing substantial tissue damage. [155] The link between platelet activation and allergy has been

Independent studies report elevated plasma levels of platelet activation blood markers (βthromboglobulin (β-TG), PF4, P-selectin, and PMPs) in patients with atopic dermatitis (AD) and psoriasis. [158-160] as well as in a mouse model of AD. [161] Plasma β-TG and PF4 may

In AD, it is possible that platelets contribute to an itch–scratch–hemorrhage cycle via release of pruritogenic factors such as histamine, 5-HT, acid proteases, IL-1β, TGF-β, PAF, and

While studies on platelet activation markers in asthmatic patients are inconclusive likely due to differences in experimental design, [162-167] the role of platelets in lung allergic inflamma‐ tion has been established in mouse models. [168-171] There is a significant association between activation of platelets and eosinophils in the airways of individuals with asthma. [164] Moreover, circulating PLA are detected in the blood of allergen-challenged asthmatic patients and mice. [168] Platelets are essential for leukocyte recruitment to human and murine lungs in allergic inflammation [168, 170] and to the skin in chronic hapten-induced dermatitis, another mouse model of AD. [172] PLA circulate in the blood of asthmatic patients and in allergen-challenged mice. [165, 168] In all cases, the role of platelets was P-selectin-dependent.

Platelets express functional low (FcεRII) and high affinity (FcεRI) receptors for IgE at low level [173-175] Murine platelets can chemotactically respond to the sensitizing allergen via FcεR *in vitro* and *in vivo*, with platelet influx preceding the influx of leukocytes. [171] Upon engagement of IgE receptors, platelets release a variety of biologically active media‐ tors[175, 176] including RANTES, a potent eosinophil chemoattractant. [177] IgE is stored in platelet α-granules and released upon activation, which may potentially amplify the

effective to reduce the severity of migraine. [154]

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

**8. Platelets in allergic inflammation**

studied for many years. [156, 157]

prostaglandin E2. [157]

allergic response. [178]

be markers for the severity of AD and psoriasis. [158]

Platelets are important players in the development of inflammation. They store multiple inflammatory molecules that, upon release, chemoattract key innate immune cells leukocytes and stimulate endothelium. Platelets interact with leukocytes and support their interaction with vessel wall and egression to tissues. Platelets play a pivotal role in various inflammationrelated diseases and targeting platelets could be a promising approach to manipulate the inflammatory response.

## **Author details**

Mònica Arman, Holly Payne, Tatyana Ponomaryov and Alexander Brill\*

\*Address all correspondence to: a. brill@bham. ac. uk

Centre for Cardiovascular Sciences, Institute of Biomedical Research, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

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## **Chapter 4**
