PAR4, but Not PAR1, Signals Human Platelet Aggregation via Ca2+ Mobilization and Synergistic P2Y12 Receptor Activation

Abstract

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y 12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1-and PAR4-mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y 12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y 12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

“…Calcium Mobilization-Changes in intracellular calcium concentrations were measured as described previously (21). Briefly, washed isolated human platelets were incubated with 2.5 M Fura2-AM at 37°C for 30 min and washed.…”

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

“…These studies were approved by the Thomas Jefferson University Institutional Review Board, and informed consent was obtained from all donors before blood draw. Washed platelets were prepared as previously described ( 21,38 ). The fi nal platelet concentration of washed platelets in Tyrodes buffer was adjusted to a physiological concentration of 3 × 10 8 platelets/ml.…”

Protease-activated receptors in cancer: A systematic review

“…Human platelets express two functional thrombin receptors, PAR1 and PAR4 (63)(64)(65). Thrombin acts through PAR1 and PAR4 on human platelets to signal activation responses, such as calcium mobilization, release of procoagulant molecules (e.g., P-selectin) from α-granules, release of small molecules (e.g., ADP) from dense granules, activation of glycoprotein IIbIIIa/integrin α IIb β 3 (GPIIbIIIa) and aggregation (65)(66)(67)(68)(69)(70)(71)(72). Thrombin activates PAR1 at concentrations 10-fold less than PAR4, but activation of PAR4 provides a longer stimulus (61,73).…”