A thrombin receptor function for platelet glycoprotein Ib–IX unmasked by cleavage of glycoprotein V

Abstract

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V ؊͞؊ platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib-IX-V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y 12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (G q-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib-IX-V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib-IX via thrombin acting as a ligand, resulting in platelet activation.G lycoprotein (GP) Ib-IX-V is a major complex on the platelet surface, second only to ␣⌱⌱b␤3. This complex consists of several subunits: GP Ib␣, GP Ib␤, GP IX, and GP V in the ratio of 2:2:2:1. Absence of GP Ib-IX-V results in a severe bleeding disorder known as Bernard Soulier syndrome characterized by giant platelets and impaired von Willebrand factor (vWf) binding (1). GP Ib␣ is a receptor for vWf, and the GP Ib-IX-V complex is critical for platelet adhesion under arterial shear conditions (2). A role for GP Ib-IX-V in platelet activation has been proposed on the basis of observations that the signaling molecule 14-3-3 (3, 4) is associated with the complex, and that phosphorylation of pp72 syk occurs upon vWf binding to GP Ib␣ (5). In fact, Zaffran et al. (6) recently showed that in heterologous Chinese hamster ovary (CHO) cells expressing both ␣⌱⌱b␤3 and GP Ib-IX, inside-out activation of ␣⌱⌱b␤3 could occur upon vWf adhesion.The GP Ib␣ subunit also has a thrombin binding site on the extracellular domain that overlaps the vWf binding domain (7). Additionally, the complex has a platelet-specific thrombin substrate, GP V, that is cleaved very early during thrombin-induced platelet aggregation (8). Platelets from Bernard Soulier syndrome patients show an impaired response to thrombin (9), and antibodies that block thrombin binding to GP Ib␣ also partially inhibit platelet responses to thrombin (9). More recently, thrombin binding to GP Ib␣ has been shown to enhance platelet procoagulant activity (10). However, the physiological significance of this interaction ha...

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…A prediction from this model is that part of the platelet-bound thrombin should be found in lipid rafts, along with the other components of this enzyme-cofactor-substrate complex. Recently, cleavage of GPV by thrombin has been shown to play a signaling role in platelet activation (57). Whether the cleavage of GPV also facilitates the exposure of the FXI-binding site in the GPIb-IX-V complex remains to be tested.…”

“…Access to the active site is restricted by two surface loops, the 50-insertion loop (Leu 45 -Asn 57 , thrombin B chain numbering system) and the autolysis loop (Leu 144 -Gly 155 ), that are situated on the northern and southern rims of the active-site cleft, respectively. Enzyme specificity is further defined by two ligand-binding sites (exosites) that are characterized by a high density of solvent-exposed basic residues.…”

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).…”

TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

“…These data imply that different signaling pathways could be involved in the activation induced by thrombin or TRAP, a concept that seems to be accepted in additional literature. 2,30 It may be argued that the differences noted in our activation studies could be related to the difficulty in achieving equivalent concentrations with the two activating agents. It is worth mentioning that concentrations of thrombin or TRAP that induced similar aggregating responses (ie, 0.1 U/ml of thrombin and 30 mol/L of TRAP) caused equivalent levels of secretion and of phosphorylation of those proteins present in whole platelet lysates, but still induced differential intensity of phosphorylation of proteins associated with the insoluble cytoskeletal fractions.…”

“…Both receptors seem to act simultaneously although independently. 2,30 However, the ultrastructural features observed with TRAP do not seem to be physiological. Our present study further supports these concepts.…”