**1. Introduction**

220 Perioperative Considerations in Cardiac Surgery

based on high clinical suspicion. The most important principles of therapy include: discontinuation of any form of heparin therapy, treatment with indirect inhibitors of factor Xa or direct thrombin inhibitors and reversal of vitamin K antagonists if they were used previously. Additionally, transfusion of platelets should be avoided since it will only

Heparin -induced thrombocytopenia (HIT) is a rare highly under-diagnosed but life

 HIT is a clinico-pathological syndrome requiring multiple laboratory tests to make the final diagnosis. Most often treatment must be initiated before final diagnosis is

 The most important principles of therapy include: discontinuation of any form of heparin, avoidance of platelet transfusion, treatment with indirect inhibitors of factor Xa or direct thrombin inhibitors and reversal of vitamin K antagonists if they were used

Management of a HIT-positive patient undergoing cardiac surgery with the use of CPB

[1] Warkentin TE. Agents for the treatment of Heparin-Induced Thrombocytopenia.

[2] Ortel TL. Heparin Induced Thrombocytopenia: when low –platelet count is a mandate for anticoagulation. Hematology Am Soc Hematol Educ Program. 2009; 225-32. [3] Levy JH, Winkler AM. Haparin-Induced Thrombocytopenia and cardiac surgery. Curr

[4] Wąsowicz M, Vegas A, Borger MA, Harwood S. Bivalirudin anticoagulation for cardiopulmonary bypass in a patient with Heparin-Induced Thrombocytopenia. Can J

[5] Sniecinski R, Hursting MJ, Paidas MJ, Levy JH. Etiology and assessment of hypercoagulability with lessons from Heparin-Induced Thrombocytopenia. Anesth

precipitate their activation and aggravate the clinical symptoms.

threatening complication of heparin therapy.

presents a challenge for the anesthesiologist.

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Opin Anaesthesiol 2010; 23: 74-79

Anesth 2005; 52: 1093-98

Anal 2011; 112: 46-58.

**7. Key points** 

established

previously.

**8. References** 

*Overview of diverse functional aspects* 

Human platelets (PLTs) are anucleate, discoid small cells (2 - 4 µm by 0.5 µm) that normally circulate at concentrations of 150 - 400 x 109/L for a maximum of 10 days. They are primed to undergo explosive activation following damage from the vessel wall and play a central role in both primary hemostasis and arterial thrombosis, including adhesion, aggregation, and coagulation but also chemotaxis, inflammation, and proliferation.

The normal vascular endothelium produces potent PLT inhibitors such as nitric oxide, prostacyclin and natural ADPase (CD39). However, once sub-endothelial components like collagen, fibronectin, laminin, or von Willebrand factor (vWF) become exposed, PLTs undergo a highly regulated set of functional responses including adhesion, followed by spreading, release reactions (degranulation), induction of pro-coagulant activity, microparticle formation, and clot retraction. These activities result in rapid formation of a vascular (white) plug, which then is stabilized by the activation of soluble plasma components resulting in the formation of fibrin and the inclusion of erythrocytes and leukocytes. This red plug initiates the healing process during which a part of fibrin is degraded again by fibrinolysis. Under physiological conditions thrombus formation is strongly limited to the region of the damaged vessel wall by inhibitory mechanisms of intact endothelial cells and the coagulation cascade.

PLTs are enriched in surface glycoprotein (GP) receptors that mediate interactions among PLTs themselves, with the sub-endothelium and with white blood cells. PLTs also contain specific granules for the storage of calcium (Ca++), adenine/guanine nucleotides, and serotonin (dense bodies) and the storage of coagulation factors (e.g. vWF, FV), multimerin, thrombospondin-1, fibrinogen, IgE, growth factors (e.g. PDGF, TGF-β, ECGF, EGF, VEGF, bFGF, IGF-I), and cytokines (e.g. PF4, RANTES) (α granules). Serotonin (5 hydroxytryptamine) acts predominantly as a local vasoconstrictor but has also proinflammatory properties by stimulation of monocytes and attraction of T lymphocytes (IL-16). Contents of α granules mediate e.g. host defense, recruitment and activation of leukocytes as well as regulation of tissue repair by mitogenic effects on smooth muscle cells, macrophages, monocytes, and fibroblasts. Other important pro-inflammatory mediators are present in the cytosol (IL-1β, CD40L) and are generated from mRNA (relict from megacaryocytes) and are released upon PLT activation. CD40L stabilizes aggregation by interference with GP IIb-IIIa and stimulates endothelial cells to express ICAM-1, VCAM-1, E-selectin, and the vitronectin receptor, thus modulates leukocyte-endothelium and PLTendothelium interactions.

Due to specific binding between sub-endothelial agents and specific GP receptors, PLTs begin to slow down and transiently adhere or roll along the damaged area of the vessel wall. Under conditions of high shear, as found in the arterial circulation, the initial PLT-subendothelium interactions are exclusively mediated by vWF present in bridges between subendothelial collagen and GP Ib (V-IX) of the adhering PLTs. The following steps of activation via various signal transduction pathways (outside-in signaling) and the elevation of cytoplasmatic Ca++ levels are then mediated by other receptor-ligand interactions. When the cytoplasmatic Ca++ concentration exceeds a certain threshold, cytoskeletal changes occur, which mediate shape change from discoid to sphere, pseudopod formation, and conformational change of the fibrinogen receptor GP IIb-IIIa (receptor activation). Only the activated GP IIb-IIIa complex is able to bind soluble plasma fibrinogen (and vWF under high shear conditions) leading to further spreading of the stimulated PLTs along the site of injury and ultimate aggregation characterized by fibrinogen bridges between the activated GP IIb-IIIa complexes on adjacent PLTs. Simultaneous release and surface exposure of granule components (e.g. ADP and serotonin from dense bodies, vWF and p selectin from αgranules) and cyclooxygenase (COX)-related thromboxane A2 (TxA2) formation/expression result in further recruitment, activation and aggregation of other PLTs near to the growing plug. At least, internal, anionic, negatively charged phospholipids are exposed by transbilayer flip flop of the inner membrane leaflet and pro-coagulant microvesicles are generated. The exposure of anionic phospholipids, mainly phosphatidylserine, provides a surface upon which PLTs can support thrombin generation by accelerating the tenase and prothrombinase reactions of the plasmatic coagulation pathway. Thrombin, the key enzyme of the coagulation cascade and the most potent PLT agonist, interacts with two binding sites on PLTs, a) the region of GP Ib (high affinity) and b) a specific epitope of the thrombin receptor (moderate affinity). Binding of thrombin leads to cleavage of the extracellular domain (protease-activated receptor PAR), whereby the generated free polypeptide (SFLLRN-x) can directly activate further thrombin receptors (thrombin-receptor activating peptide TRAP). Human PLTs express two kinds of PARs activated either by lower (PAR-1) or higher concentrations of thrombin (PAR-4). Receptor activation by thrombin results in further activation of GP IIb-IIIa, TxA2 formation, and secretion of granular components such as ADP that promotes recruitment and activation of adjacent PLTs into the vicinity of the growing plug and the inclusion of leukocytes. Thrombin also converts soluble fibrinogen into insoluble fibrin, which is cross-linked by the thrombin-activated FXIII to confer stability of the otherwise fragile plug/thrombus. At last, the activated PLTs rearrange their intracellular actin/myosin cytoskeleton, which leads to clot retraction. The latter is inhibited by blockade of GP IIb-IIIa receptors representing central links between the contractile elements.

#### *Adhesion*

As described, adhesion is mediated by GPs of the PLT surface that recognize specific structural components of the extracellular matrix such as collagen and elastic fibrils embedded in a gel of proteoglycans and water. This first contact *(contact phase)* between PLTs and the sub-endothelium is mediated by interaction between GP Ib-V-IX (vWF receptor belonging to the leucine-rich GPs) with collagen-bound vWF. Thus, the main task of GP Ib-V-IX is the adhesion of circulating PLTs to immobilized vWF despite high shear

E-selectin, and the vitronectin receptor, thus modulates leukocyte-endothelium and PLT-

Due to specific binding between sub-endothelial agents and specific GP receptors, PLTs begin to slow down and transiently adhere or roll along the damaged area of the vessel wall. Under conditions of high shear, as found in the arterial circulation, the initial PLT-subendothelium interactions are exclusively mediated by vWF present in bridges between subendothelial collagen and GP Ib (V-IX) of the adhering PLTs. The following steps of activation via various signal transduction pathways (outside-in signaling) and the elevation of cytoplasmatic Ca++ levels are then mediated by other receptor-ligand interactions. When the cytoplasmatic Ca++ concentration exceeds a certain threshold, cytoskeletal changes occur, which mediate shape change from discoid to sphere, pseudopod formation, and conformational change of the fibrinogen receptor GP IIb-IIIa (receptor activation). Only the activated GP IIb-IIIa complex is able to bind soluble plasma fibrinogen (and vWF under high shear conditions) leading to further spreading of the stimulated PLTs along the site of injury and ultimate aggregation characterized by fibrinogen bridges between the activated GP IIb-IIIa complexes on adjacent PLTs. Simultaneous release and surface exposure of granule components (e.g. ADP and serotonin from dense bodies, vWF and p selectin from αgranules) and cyclooxygenase (COX)-related thromboxane A2 (TxA2) formation/expression result in further recruitment, activation and aggregation of other PLTs near to the growing plug. At least, internal, anionic, negatively charged phospholipids are exposed by transbilayer flip flop of the inner membrane leaflet and pro-coagulant microvesicles are generated. The exposure of anionic phospholipids, mainly phosphatidylserine, provides a surface upon which PLTs can support thrombin generation by accelerating the tenase and prothrombinase reactions of the plasmatic coagulation pathway. Thrombin, the key enzyme of the coagulation cascade and the most potent PLT agonist, interacts with two binding sites on PLTs, a) the region of GP Ib (high affinity) and b) a specific epitope of the thrombin receptor (moderate affinity). Binding of thrombin leads to cleavage of the extracellular domain (protease-activated receptor PAR), whereby the generated free polypeptide (SFLLRN-x) can directly activate further thrombin receptors (thrombin-receptor activating peptide TRAP). Human PLTs express two kinds of PARs activated either by lower (PAR-1) or higher concentrations of thrombin (PAR-4). Receptor activation by thrombin results in further activation of GP IIb-IIIa, TxA2 formation, and secretion of granular components such as ADP that promotes recruitment and activation of adjacent PLTs into the vicinity of the growing plug and the inclusion of leukocytes. Thrombin also converts soluble fibrinogen into insoluble fibrin, which is cross-linked by the thrombin-activated FXIII to confer stability of the otherwise fragile plug/thrombus. At last, the activated PLTs rearrange their intracellular actin/myosin cytoskeleton, which leads to clot retraction. The latter is inhibited by blockade of GP IIb-IIIa receptors representing central links between the contractile

As described, adhesion is mediated by GPs of the PLT surface that recognize specific structural components of the extracellular matrix such as collagen and elastic fibrils embedded in a gel of proteoglycans and water. This first contact *(contact phase)* between PLTs and the sub-endothelium is mediated by interaction between GP Ib-V-IX (vWF receptor belonging to the leucine-rich GPs) with collagen-bound vWF. Thus, the main task of GP Ib-V-IX is the adhesion of circulating PLTs to immobilized vWF despite high shear

endothelium interactions.

elements. *Adhesion* 

forces. GP Ib-V-IX consists of four subunits: the central part is GP V surrounded by GP Ib<sup>α</sup> and GP Ibβ, which are covalently linked to each other by disulfide bridges and noncovalently bound to GP IX. GP Ibα possesses binding sites for vWF and thrombin. In contrast to GP IIb-IIIa, whose surface expression increases after thrombin activation, the surface density of GP Ib-V-IX is reduced by receptor internalization after PLT activation. Recent evidence suggests that, apart from GP Ib-V-IX, another PLT membrane receptor for collagen, GP VI, is strictly required for the initial PLT tethering following vascular injury. Both receptors appear to act in concert to recruit PLTs to the sub-endothelium in-vivo. GP VI mediates the activation (opening) of other adhesive receptors (GP IIb-IIIa, GP Ia-IIa) by shifting them from a low to a high affinity binding state required for stable PLT arrest. GP VI belongs to the immunoglobulin superfamily and forms complexes with the FcR ʏ-chain at the cell surface. In the absence of GP VI, PLTs completely failed to adhere and aggregate on the damaged vessel wall. Further immunoglobulin adhesion receptors, PECAM (PLTendothelial cell adhesion molecule)-1 and ICAM (intracellular adhesion molecule)-2 probably mediate adhesion to leukocytes and PLT related inflammation, but their global role for PLT function is mostly unknown.

The contact phase is stabilized via further adhesion to collagen, fibronectin, laminin, and thrombospondin *(stabilization phase)*. Binding of collagen leads to the formation of pseudopods (shape change) and peptide as well as GP IIb-IIIa activation via tyrosine phosphorylation *(activation phase)*. Starting from released arachidonic acid (AA) the adhering PLTs form TxA2, which slows down the blood flow due to its vasoconstricting activity. Additionally TxA2 induces the release of soluble granule components like ADP *(secretion phase)* leading to the recruitment of still resting PLTs that then become activated and aggregate with already adhering PLTs through activated GP IIb-IIIa. By spreading of the PLT aggregate over the complete sub-endothelium, the vessel wall lesion is separated from blood flow, and blood loss is kept as low as possible.

#### *Aggregation*

Aggregation depends on three conditions: shear force, Ca++, and fibrinogen. The latter are stored in PLT granules and are released in high concentrations during PLT activation. While the *primary aggregation phase* is reversible and characterized by "loose links" via fibrinogen bridges, the *secondary aggregation phase* is irreversible and induced by the degranulation process. GP IIb-IIIa, a β3 integrin, plays a central role in aggregation. 60,000 to 100,000 GP IIb-IIIa receptors can be detected per PLT. 70% are bound to the surface, and 30% are only released from intracellular stores (open canalicular system, α-granules) upon PLT activation. Circulating PLTs carry resting, not activated GP IIb-IIIa complexes (low affinity functional state) that only can bind immobilized fibrinogen. The binding sites for soluble fibrinogen become accessible after conformational change during activation, which strongly depends on Ca++ (high affinity functional state). The binding of fibrinogen to the activated receptor induces a further conformational change (ligand-occupied functional state) with exposure of cryptic epitopes (LIBS = ligand-induced binding site) and transmembrane signal transduction (post-occupancy) events.

#### *Activation*

Upon changes in biochemical pathways several soluble PLT agonists (e.g. ADP, thrombin, TxA2) are formed, which bind to specific G protein-linked receptors. Via signal transduction pathways, each agonist amplifies the activation step through the formation of *second*  *messengers*. One of these messengers, phospholipase C, forms inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG), whereby IP3 enhances the intracellular Ca++ concentration and DAG activates proteinkinase C, which in turn phosphorylates a series of further signal proteins that control the degranulation process and the activation of GP IIb-IIIa. Cytoplasmatic Ca++ activates phospholipase A2, which leads to the liberation of AA from phospholipids of the cell membrane. Aspirin-sensitive COX-1 and thromboxane synthetase then form TxA2, which has vasoconstricting activity and stimulates the secretion of granule components after interaction with specific TxA2 receptors. Two TxA2 receptors can be distinguished on the PLTs surface (TPα and TPβ), of which TPα is most important. COX-1 inhibition results in reduced secretion and inhibition of secondary aggregation. Receptors that directly inhibit PLTs stimulate adenylate cyclase (increased formation of cAMT) via GS proteins and are activated by PLT antagonists like adenosine, β-adrenergic substances, prostacyclin, prostaglandin E1, and theophylline.

PLTs are presently the only cells that express ADP specific receptors (P2Y1, P2Y12, P2X1). Like other activation receptors, ADP receptors are linked to G-proteins. Due to their key role in the pathogenesis of arterial thrombosis, they are of particular pharmacological interest. The P2Y1 receptor is linked to the initiation of shape change, mediation of Ca++ mobilization and activation of phospholipase C. Activation of P2Y12 inhibits cAMP formation via inhibitory Gproteins and is predominantly responsible for TxA2 formation, p selectin surface expression and conformational changes of GP IIb-IIIa (receptor activation), thus sustained PLT aggregation. All of these mechanisms are affected by thienopyridines. Like P2Y1, P2X1 mediates Ca++ influx and shape change but seems not to be influenced by thienopyridines.

#### *Secretion*

During adhesion, PLTs begin to release stored components from the granules in the order dense bodies, α-granules, and lysosomes. Dependent on ATP and Ca++ the degranulation process initiates the secondary, irreversible phase of aggregation and reinforces the activation/recruitment of further circulating PLTs as well as fibrin formation resulting in thrombus consolidation. As described above, the interaction of released ADP (from PLTs, damaged vessel wall cells, endothelial cells, red blood cells) with its specific purinergic receptors plays a central role in this process. Released serotonin reinforces vasoconstriction and thus slows down the blood flow. Released α-granule contents attract leukocytes and fibroblasts (β-TG, PF4), promote mitogenic and proliferative effects in fibroblasts and smooth muscle cells (growth factors like PDGF), or exhibit pro-inflammatory activity (IL-1). P selectin is found in both PLTs (α-granules) and endothelial cells (Weibel-Palade bodies) and is expressed on cell surface only after cellular activation. P selectin is the decisive receptor for PLT adhesion to leukocytes and triggers inflammatory reactions but also plays a central part in vascular repair processes. Interestingly, p selectin is significantly increased in all states of coronary artery disease: stable angina (showing also increased TxA2 formation and fibrinogen binding due to increased GP Ib and GP IIb-IIIa expression [1-3]), unstable angina (showing also increased LIBS expression [4]), and acute myocardial infarction (AMI). Here, increased p selectin levels are indicative for an increased thrombotic re-occlusion risk [5]. When coronary stenting is combined with dual antiplatelet therapy, p selectin expression and GP IIb-IIIa activation are as low as after conventional coronary angioplasty [6-8]. Besides p selectin, thrombin promotes chemotaxis of monocytes and mitogenesis in lymphocytes and mesenchymal cells (smooth muscle cells, fibroblasts). In addition, released coagulation factors (vWF, fibrinogen, FV, PAI-1) fulfill pro-coagulant or anti-fibrinolytic tasks. The pro-coagulant activity can be reinforced by microparticle formation, small membrane vesicles extruded from activated PLTs that exhibit a high binding affinity for FVa and FVIIIa, thus facilitating the formation of the tenase and prothrombinase complexes. Lysosomal enzymes like collagenease or elastase degradate surrounding fibrils and induce changes in atherosclerotic plaques.
