Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Abstract

To cite this article: Lindahl TL, Macwan AS, Ramstr€ om S. Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets. J Thromb Haemost 2016; 14:1639-41.See also French SL, Arthur JF, Lee H, Nesbitt WS, Andrews RK, Gardiner EE, Hamilton JR. Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood. This issue, pp 1642-54.Thrombin is the most important enzyme in coagulation and also a potent activator of platelets. It activates platelets via N-terminal cleavage of protease-activated receptors (PARs) 1 and 4. Although structurally similar, both PARs have distinct and complementary roles; for example, PAR1 is more sensitive than PAR4 to low concentrations of thrombin [1], whereas PAR4 gives rise to a more prolonged calcium mobilization after activation compared with a transient 'spike' by PAR1 [2]. Moreover, PAR4 is more important for clot lysis resistance [3]. After activation, a subpopulation of the platelets become procoagulant by allowing the formation of coagulation factor complexes on their surface. They do not participate in aggregation but facilitate generation of even more thrombin and, consequently, amplification of the response [4]. Experimental data suggest that collagen exposed during vessel damage in combination with thrombin is the most potent trigger for formation of procoagulant platelets [5], but the signaling processes regulating the formation of the aggregatory and procoagulant platelet subpopulations are still not known in detail.The article by Hamilton and co-workers in this issue of JTH [6] shows for the first time that activation of PAR4 is crucial for the thrombin-induced activation of platelets in an in vitro thrombus model; that is, platelets adhered on a collagen surface in a flow chamber system. Two tools were essential for this study, a rabbit polyclonal antibody directed towards the cleavage site for thrombin at the N-terminal of PAR4 and a fluorescence resonance energy transfer-based thrombin activity sensor linked to an anti-CD41a antibody, to prevent washing out by the flowing blood, which was invented by Welsh et al. [7]. The anti-PAR4 antibody only attenuated the peak of the thrombin-induced cytosolic calcium but abolished the sustained elevation of cytosolic calcium; thus PAR4 was responsible for the sustained elevation, in accordance with previous studies [8][9][10][11][12]. Presumably this sustained elevation is essential for the creation of the negatively charged surface and the procoagulant activity, which is supported by the known very strong effect in this direction by the calcium ionophore A23187 (see Fig. 4G in [6] and [13]).The relative importance of PAR1 and PAR4 for different aspects of platelet activation is far from clear; published studies have shown various, even contradicting, results. To be able to answer questions regarding the relative importance of PAR1 and PAR4 in the platelet procoagulant response, we need to st...

Supramaximal calcium signaling triggers procoagulant platelet formation

“…22,25,37 Many other signaling pathways have been proposed to regulate platelet procoagulant activity to some extent, and these may also be reinterpreted in terms of this model. These signaling pathways include other plasma membrane channels, such as TRPC6, 23,38 Clchannels, 20 and aquaporins 39 ; plasma membrane receptors, including protease-activated receptor 1, 40,41 protease-activated receptor 4, 42,43 P2Y 12 , 44 and integrin a IIb b 3 [45][46][47] ; signaling platforms such as lipid rafts 18,48 and diverse intracellular signaling proteins such as Rac1 49 ; different protein kinase C isoforms 7,50,51 ; and nuclear receptors. 52 We suggest that although many of these pathways are likely to regulate the initial [Ca 21 ]cyt signal, it may be useful to reassess the contribution of these pathways because they may also reveal targets to selectively inhibit platelet procoagulant activity.…”

Protease-Activated Receptor PAR-4: An Inducible Switch between Thrombosis and Vascular Inflammation?

“…By virtue of its slower on/off kinetics, PAR-4 ensures a sustained calcium signalling response and, according to a recent study, is more important than PAR-1 for the procoagulant effect of thrombin on platelets during prolonged thrombus formation. 144,145 PAR-4 has thus emerged as a promising candidate target for new anti-thrombotic therapies. This approach could be particularly suited in patients in whom complete thrombin inhibition or selective PAR-1 blockade leads to excessive bleeding or other undesired effects, as well as in patients with myocardial damage or diabetes who display heightened platelet PAR-4 reactivity.…”

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)-1H-indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ-Thrombin

“…13 Unfortunately, tool compounds to probe the role of PAR4 in hemostasis and thrombosis have been lacking and the field has relied almost exclusively on PAR4 antibodies. 14 Additionally, thrombin activates PARs through cleavage of the extracellular domain of the receptor, revealing an encrypted tethered ligand (TL) that binds intramolecularly to activate the receptor.…”