Platelet thrombin receptor antagonism and atherothrombosis

Abstract

Clinical manifestations of atherothrombotic disease, such as acute coronary syndromes, cerebrovascular events, and peripheral arterial disease, are major causes of mortality and morbidity worldwide. Platelet activation and aggregation are ultimately responsible for the progression and clinical presentations of atherothrombotic disease. The current standard of care, dual oral antiplatelet therapy with aspirin and the P2Y(12) adenosine diphosphate (ADP) receptor inhibitor clopidogrel, has been shown to improve outcomes in patients with atherothrombotic disease. However, aspirin and P2Y(12) inhibitors target the thromboxane A(2) and the ADP P2Y(12) platelet activation pathways and minimally affect other pathways, while agonists such as thrombin, considered to be the most potent platelet activator, continue to stimulate platelet activation and thrombosis. This may help explain why patients continue to experience recurrent ischaemic events despite receiving such therapy. Furthermore, aspirin and P2Y(12) receptor antagonists are associated with bleeding risk, as the pathways they inhibit are critical for haemostasis. The challenge remains to develop therapies that more effectively inhibit platelet activation without increasing bleeding complications. The inhibition of the protease-activated receptor-1 (PAR-1) for thrombin has been shown to inhibit thrombin-mediated platelet activation without increasing bleeding in pre-clinical models and small-scale clinical trials. PAR-1 inhibition in fact does not interfere with thrombin-dependent fibrin generation and coagulation, which are essential for haemostasis. Thus PAR-1 antagonism coupled with existing dual oral antiplatelet therapy may potentially offer more comprehensive platelet inhibition without the liability of increased bleeding.

Figure 1

Sites of action of current and emerging antithrombotic drugs and antiplatelet agents. Adapted with permission. (Copyright © 2007 American Heart Association. All rights reserved.) Platelet adherence to the endothelium occurs at sites of vascular injury through the binding of GP receptors to exposed extracellular matrix proteins (collagen and vWF). Platelet activation occurs via complex intracellular signalling processes and causes the production and release of multiple agonists, including TXA₂ and ADP, and local production of thrombin. These factors bind to their respective G protein-coupled receptors, mediating paracrine and autocrine platelet activation. Further, they potentiate each other's actions (P2Y₁₂ signalling modulates thrombin generation). The major platelet integrin GPIIb/IIIa mediates the final common step of platelet activation by undergoing a conformational shape change and binding fibrinogen and vWF leading to platelet aggregation. The net result of these interactions is thrombus formation mediated by platelet/platelet interactions with fibrin. Current and emerging therapies inhibiting platelet receptors, integrins, and proteins involved in platelet activation include the thromboxane inhibitors, the ADP receptor antagonists, the GPIIb/IIIa inhibitors, and the novel PAR antagonists and adhesion antagonists. TP, thromboxane receptor; 5-HT2A, 5-hydroxy tryptamine 2A receptor. Reversible-acting agents are indicated by brackets.

Figure 2

Intracellular signalling pathways and phenotypic effects mediated by PAR-1 activation by thrombin in platelets. Reproduced with permission. (Copyright © 2005 International Society on Thrombosis and Haemostasis. All rights reserved.) Members of the Gα12/13, Gαq, and Gαi/z families activate PAR-1 signalling pathways. First, the α-subunits of G12 and 13 bind RhoGEFs (which activate small G proteins) and drive Rho-mediated cytoskeletal responses leading to changes in platelet morphology. Second, Gαq activates phospholipase Cβ and triggers phosphoinositide hydrolysis, calcium mobilization, and protein kinase C activation. This then activates pathways leading to granule secretion, as PAR-1–Gαq-coupled ADP release is especially important for thrombin-mediated platelet activation. In addition, calcium-regulated kinases and phosphatases, GEFs, MAP kinases, and other proteins that mediate platelet aggregation are activated. Gαi promotes platelet responses by inhibiting adenylyl cyclase. Gβγ subunits can activate phosphoinositide-3 kinase, other lipid-modifying enzymes, protein kinases, MAP kinase cascades, and channels. Phosphoinositide-3 kinase modifies the inner leaflet of the plasma membrane to provide molecular docking sites. Finally, PAR-1 also promotes growth factor shedding and activation of receptor tyrosine kinases involved in cell growth and differentiation. GEF, guanine nucleotide exchange factors; MAP, mitogen-activated protein; IP3, inositol triphosphate 3; DAG, diacylglycerol; WASP, Wiskott–Aldrich syndrome protein; SRE, serum response element; MLC, myosin light chain.

Figure 3

Role of thrombin-mediated PAR-1 signalling in haemostasis vs. thrombosis: rationale for PAR-1 antagonist. In addition to activating PARs on platelets, thrombin facilitates fibrin formation and protein C activation. Available data in mice and non-human primate models indicate that PAR antagonists have the potential to block platelet activation by thrombin while sparing these other functions of thrombin, including coagulation, that are absolutely necessary for haemostasis. Murine studies suggest that fibrin(ogen) is relatively more important than thrombin-induced platelet activation for haemostasis, and this is supported by high bleeding risk associated with the use of anticoagulants in man. Thus, inhibition of thrombin-induced platelet activation might provide for a larger therapeutic index than can be achieved with coagulation inhibitors for the prevention or treatment of thrombosis.

Figure 4

PAR-1 antagonists in clinical development. Reproduced with permission. (Copyright © 2006 and 2008. The American Chemical Society. All rights reserved.)^, Chemical structure of PAR-1 antagonist SCH 530348 and presumed structure of E-5555.