Thrombin binding to GPIbα induces platelet aggregation and fibrin clot retraction supported by resting αIIbβ3 interaction with polymerized fibrin

Abstract

SummaryWe have investigated the mechanisms leading to platelet aggregation following thrombin interaction with the glycoprotein (GP) Ib-IX-V complex. We show that platelets desensitized for the two thrombin receptors, PAR-1 and PAR-4, are still able to aggregate in response to thrombin and that this aggregation can be inhibited by a monoclonal antibody (VM16d) that blocks thrombin binding to GPIbα, or by pretreatment of platelets with Mocarhagin, a protease that specifically cleaves GPIbα. The thrombin/GPIbα-initiated signaling cascade induces platelet shape change through activation of the Rho kinase p160ROCK, independent of calcium mobilization, transient MEK-1 phosphorylation as well as the cleavage of talin through a calcium-independent mechanism. This signaling cascade does not induce the exposure of high affinity αIIbβ3 integrin receptors, nor does it lead to µ-calpain cleavage of filamin or the integrin cytoplasmic tail. In contrast, we provide evidence that binding of thrombin to GPIbα induces fibrin binding to resting αIIbβ3 leading to fibrin-dependent platelet aggregation and clot retraction, that can be selectively inhibited by αIIbβ3 antagonists such as RGDS, the dodecapeptide or lamifiban, as well as by the fibrin polymerization inhibitor GPRP-amide.

Plasmin-mediated Activation of Platelets Occurs by Cleavage of Protease-activated Receptor 4

“…Platelet Desensitization-Platelet desensitization was performed according to the method of Dubois et al (19), where washed platelets were incubated for 45 min at 37°C without stirring in the absence or presence of either 50 M SFLLRN, 500 M AYPGKF, or 1 unit/ml plasmin. Immediately after incubation, platelets were assayed for their responses to various agonists.…”

“…If plasmin proteolysis proceeded at a slow rate relative to thrombin, then it could explain why a relatively short incubation time failed to desensitize further PAR stimulation. Therefore, in an effort to facilitate a greater extent of desensitization, platelets were incubated with plasmin for a prolonged period using a recently published protocol (19). Upon further stimulation with 1 unit/ml thrombin, a complete loss of both aggregation and calcium release occurred relative to control treatment (Fig.…”

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

“…Human platelets, prepared as described elsewere, 16 were incubated with 1 NIH U/mL of thrombin and/or 1 mM of ionophore A23187 (Sigma, St. Louis, MO, USA), and/or 10 mg/mL of collagen (Stago, Asnière, France), for 15 min at 37°C without stirring. Platelets were subsequently pelleted by centrifugation, first at 1500 g for 15 min and then, after the residual platelets had been discarded, by centrifugation at 12000 g for 2 min.…”

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

“…This represents a high affinity receptor for thrombin, and a mounting body of evidence indicates that it may contribute to the activation of platelets (32)(33)(34)(35)(36)(37). It has been known for years that platelets from patients affected by the Bernard-Soulier syndrome, which lack GPIb-IX-V, show impaired response to thrombin (38).…”