**2. The role of platelets in atherogenesis**

#### *Role of the arterial plaque*

224 Perioperative Considerations in Cardiac Surgery

*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,

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.

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

prostacyclin, prostaglandin E1, and theophylline.

*Secretion* 

In contrast to stable angina pectoris, which is generally caused by a reduced oxygen supply to the myocardium due to coronary vasoconstriction, acute coronary syndromes (ACS) arise from an acute plaque rupture within an epicardial coronary artery with subsequent PLT aggregation and thrombus formation. Reperfusion strategies aim to dissolve the thrombotic plaque either through administration of fibrinolytic agents or direct coronary interventions. Paradoxically, despite a sufficiently reestablished blood flow, reperfusion injury occurs and myocardial dysfunction can progress. This is mainly triggered by activated PLTs released from the plaque area or the circulation itself (cardiovascular risk factors are per se associated with an increased basal activity of circulating PLTs [9,10]). Activated PLTs in turn promote inflammatory reactions within the ischemic myocardium, thus plug growing. Consequently, up to 50% of all patients with successful revascularization and normal epicardial blood flow following interventional therapy do not have adequate tissue reperfusion [11,12].

Following a modern concept of atherogenesis, apart from endothelial dysfunction (characterized by decreased vasodilatation upon stimulation with acetylcholine and increased pro-coagulant inflammatory activities) lipid deposition on the intima is one of the first pathological events in the genesis of an arterial plaque. The lipid-rich nucleus is separated from blood flow by a fibrous cap and rich in free cholesterol crystals, cholesteryl esters, oxidized LDL and monocytes/macrophages. The latter undergo phagocytosis of fatty acids and oxidized LDL and differ to foam cells. Additionally, there are particularly heavy PLT deposits and high amounts of tissue factor, which favors thrombin formation. Thrombin, in turn, activates additional PLTs and supports their aggregation to already adhering PLTs (recruitment), which, in parallel, stimulate the migration of smooth muscle cells and fibroblasts by a PDGF-dependent mechanism. After intima proliferation and monocyte migration increased shear forces (high blood flow or tension) and the liberation of proteolytic enzymes (plasminogen activator, metalloproteinases) can promote plaque rupture with all known sequelae. PLTs are not just involved in thrombotic complications by formation of vascular occlusions but also trigger plaque progression and promote myocardial malperfusion by participation in recurrent vasoconstrictions and local/systemic inflammatory reactions.

#### *PLT-mediated microembolization*

The regeneration of the afflicted myocardial area largely depends on the integrity and recovery of the microcirculation distal to the stenosis. Importantly, there is an increased embolization of thrombotic material from the arterial plaque lesion during plaque growing and particularly in the reperfusion phase after coronary interventions. This may promote intermittent coronary vasospasms distal to the stenosis through the release of serotonin and TxA2 resulting in inadequate perfusion, myocardial ischemia, tissue damage, unstable angina or non-ST-segment elevation myocardial infarction. On the other hand, the contact of intact endothelial cells with activated PLTs has the potential to modify chemotactic, proteolytic and adhesive properties inducing increased surface expression of endothelial adhesion receptors (VCAM-1, ICAM-1, vitronectin receptor), which participate in the recruitment of PLTs to the inflamed endothelium. Additionally, the elevated release of endothelial pro-inflammatory substances (MIP-1, IL-6, IL-8) supports chemotaxis, adhesion, and transmigration of monocytes.

#### *PLT-mediated inflammation*

The exposure of sub-endothelial compounds is not required for PLT adhesion in acute inflammatory processes such as ischemia/reperfusion. E.g., p selectin expression of inflamed endothelial cells has been demonstrated to mediate PLT rolling through GP Ib indicating that the vWF receptor mediates both PLT adhesion to the sub-endothelial matrix and to "intact" endothelial cells. Additionally, endothelial adhesion receptors that bridge PLTs via fibrinogen are up-regulated in response to endothelial inflammation (e.g. by IL-1β or CD40L of activated PLTs or thrombin). In this manner, PLTs (if activated or resting) adhere to the vessel wall and promote the recruitment of neutrophils and monocytes by the release of a variety of pro-inflammatory mediators and growth hormones. Furthermore, adhering PLTs can induce up-regulation of NF-κB in endothelial cells leading to further inflammatory changes in the vessel wall. High doses of ASA (≥ 900 mg/d) can influence the NF-κB activation, thus promote the stillstand of atherosclerotic plaque progression [13]. Consequently, cardiac patients with elevated systemic CRP levels benefit especially from antiplatelet therapy with ASA [14]. Another strategy to limit reperfusion injury uses monoclonal antibodies to adhesion receptors such as p selectin (present on endothelial cells and PLTs), CD11/CD18 (present on leukocytes), and the vitronectin receptor (present on endothelial cells). Abciximab not only blocks GP IIb-IIIa on PLTs, but also the vitronectin receptor on endothelial cells explaining its favorable effect on myocardial perfusion, microcirculation, and recovery of left ventricular function [14-17].
