Platelets, antiplatelet agents and platelet function testing

Antiplatelet drugs are the cornerstone in the prevention of coronary thrombosis during and after PCI. This section contains a brief overview of platelet function and mode of action; and describes the available antiplatelet medications and their limitations.

1.6.1. Platelets and thrombosis

Platelets are anucleate cells which are produced in the bone marrow and circulate for approximately 10 days. They have a vital role in rapid haemostasis following injury,

their primary function being the initiation of thrombosis at the site of arterial damage by the processes of adhesion, activation and aggregation.

1.6.1.1. Platelet adhesion

Platelets do not adhere to healthy endothelial cells, which have several antiplatelet properties: they carry a negative charge, and they secrete the antiplatelet agents nitric oxide, prostacyclin and ADP-ase. However, where the endothelial layer is interrupted in spontaneous plaque rupture or angioplasty, platelets rapidly bind to exposed subendothelial collagen. As described by Ruggeri and Ware in 1993, in the presence of the high sheer forces found in fast flowing blood in small vessels and around arterial stenoses, this attachment is largely mediated by von Willebrand factor (vWF). This glycoprotein, the largest known soluble globular protein, is generated in endothelial cells and released into both the blood and the subendothelium. It is also produced by megakaryocytes, the cells which give rise to platelets, and is stored in platelet α- granules. vWF changes shape from a globular structure to an extended chain conformation both on binding to collagen and under conditions of shear stress above a critical value (Siedlecki et al, 1996). This conformational change allows the binding of platelets via platelet membrane glycoprotein receptors such as GP 1bα. Platelet adhesion triggers activation of the platelet.

1.6.1.2. Platelet activation

Activation is a complex collection of different molecular processes and conformational changes which must occur before the aggregation of platelets into a stable platelet plug. Several platelet activating agents are known including collagen and vWF/thrombin complex in subendothelial tissue, and the soluble agonists ADP, thrombin and thromboxane A2 (TXA2), each with its specific platelet membrane receptor (see below). Binding of an agonist rapidly causes a marked rise in platelet intracellular calcium, triggering a number of events. The platelet loses its cytoskeletal structure to change shape from a disc to a globule with multiple arm-like extensions and blebs. The blebs are shed as microvesicles and the arms increase the surface area available for platelet-platelet binding. Highly pro-coagulant factors are released from α- and dense granules into the immediate vicinity; and the surface of the platelet become highly pro-coagulant due to the redistribution of phospholipid molecules. Platelet activation also leads to a conformational change in the IIbIIIa (Integrin αIIbβ3) complex in the platelet membrane (Seiss, 1989), the final event required for agonist-mediated aggregation.

Activation is propagated by several mechanisms, including the binding of the adenine nucleotide ADP (released from platelet granules) to the purinergic P2Y1 and P2Y12 G protein-coupled platelet receptors.

1.6.1.3. Platelet aggregation

Activated platelets form stable clumps by the cross linking of cell membrane IIbIIIa molecules. IIbIIIa is part of the integrin family, large proteins which span the cell membrane. Integrins have 2 main functions: to mediate cell attachment to surrounding tissue, and as cell signalling intermediaries (they have also been studied for their potential roles in invasion of tissue by metastatic cancer cells, the functioning of viruses and allergic disorders such as asthma). IIbIIIa is the most abundant platelet glycoprotein, with approximately 80 000 molecules per platelet (Scarborough et al, 1999).

Recent work has shown that platelets are also capable of forming clumps without activation, independent of soluble agonists and IIbIIIa, in response to high shear stress. Nesbitt, Jackson et al (2009) showed that discoid platelets could form stable clumps, via membrane tethers, in injured non-stenosed mouse vessels. These initially formed downstream from the site of vascular injury and had sufficient strength to occlude vessels. Secondary, agonist-dependent consolidation of the platelet clump then occurred at the site of tissue injury in the areas of lower shear created around the developing thrombus.

1.6.2. Platelet agonist receptors and inhibitors used in PCI

Identification of the various cell receptors involved in platelet function has led to the development of drugs designed to block these receptors in order to reduce arterial thrombosis in patients with cardiovascular disease. A detailed review of contemporary antiplatelet therapy has been provided by White, 2011.

1.6.2.1. IIbIIIa inhibition

Platelet activation leads to a conformational change in the IIbIIIa molecule in the platelet cell membrane and allows permanent cross-linking of platelets and the formation of a platelet plug. Activated platelet IIbIIIa can bind to multiple soluble ligands, such as fibrinogen, fibronectin and vWF. Intravenously administered IIbIIIa inhibiting drugs have been in routine clinical use for many years to treat coronary thrombosis (Nurden et al, 2009). Intravenous IIbIIIa inhibitors are still used during PCI, particularly when coronary thrombus is present or when flow appears poor, although less than before. An unexpected event in the evolution of antiplatelet therapy was that in all 5 trials of oral IIbIIIa agents, increased mortality and increased bleeding rates and,

paradoxically, increased thrombotic events. Chew et al presented these results in a 2001 meta-analysis. These findings were never fully explained but use of oral IIbIIIa inhibitors ceased and attention was turned to different antiplatelet agents affecting alternative platelet receptors.

1.6.2.2. Thromboxane A2 and aspirin

TXA2 is produced by platelets from arachidonic acid by the cyclooxygenase (COX-1) pathway, involved in prostaglandin synthesis (Vane, 1971). Platelets have 2 variants of TXA2 receptor, TPα and TPβ. Both are G-protein linked receptors which trigger an increase in intracellular calcium levels and further platelet activation. TXA2 is also widely produced by other mechanisms, such as the COX-2 pathway, by several other cell types. Roth and Majerus in 1975 suggested that aspirin exerted its antiplatelet effect by the rapid and permanent acetylation and inactivation of platelet cyclooxygenase. Aspirin has been shown to improve cardiovascular outcomes in secondary prevention in several studies e.g. in ISIS-2, 1988, the death rate 34 days post MI was 9.4% on aspirin and 12.0% on placebo, risk ratio 0.78. Aspirin‟s role in primary prevention is less clear, when bleeding may outweigh the benefit.

1.6.2.3. Adenosine diphosphate (ADP) and P2Y₁₂ receptor inhibitors

ADP is released by damaged endothelial cells and erythrocytes, but is also excreted (in much larger amounts) by activated platelets, causing a self-propagating cascade of platelet activation leading to the formation of a platelet plug at the site of an endothelial breach. It is thought that the binding of ADP to P2Y1 is responsible for the change in shape of activating platelets via changes in intracellular calcium level. The binding of ADP to P2Y12 inhibits adenylate cyclase, a transmembranous G protein- coupled enzyme catalysing the conversion of ATP to cAMP. The inhibition of cAMP production reduces the activity of protein kinases, which can no longer phosphorylate vasodilator stimulated phosphoprotein (VASP). VASP phosphorylation is required for GP IIb/IIIa receptor inhibition; reduced cAMP therefore results in activation of IIb/IIIa receptors and promotion of platelet aggregation (Jin and Kunapuli, 1998). P2Y1 is widely expressed throughout the body and so has not been a focus of antiplatelet research in humans. P2Y12 is expressed only in platelets and the brain; several P2Y12 inhibitors have been developed which are in clinical use, including the thienopyridines clopidogrel and prasugrel; and ticagrelor, a nucleoside analogue (White, 2011).

1.6.2.4. Thrombin and thrombin inhibitors

Thrombin is an essential component in thrombus formation. It is the final protease in the coagulation cascade, cleaving fibrinogen to form a fibrin clot. Antithrombin is the

primary natural inhibitor of blood coagulation proteases, which only exerts its effect when in contact with heparin-like glyocosaminoglycans with which it forms a ternary complex (Li, 2004) – the basis of heparin as an anticoagulant. However, thrombin also has a direct effect on platelets unrelated to its interaction with fibrinogen (Davey and Luscher, 1967), and is a potent platelet agonist.

Two human platelet thrombin receptors have now been demonstrated: the protease activated receptors (PAR) 1 and 4 (Kahn 1998). PARs are G protein-coupled receptors, very widespread in all organs, which have complex interactions with multiple agonists and antagonists. Vorapaxar and atopaxar are novel oral drugs in development, both targeting PAR1, for potential use in the treatment of ACS. A major clinical trial in which vorapaxar was added to standard therapy after NSTE-ACS was halted early due to excess bleeding without a reduction in the composite primary endpoint. Intracranial haemorrhage rates were 1.1% in the vorapaxar group vs. 0.2% in the placebo group (Tricoci et al, 2012). Atopaxar has been tested in Phase II clinical trials with promising results (Wiviott, 2011).