Increased thrombin responsiveness in platelets from mice lacking glycoprotein V

Abstract

A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V ؊͞؊ platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard-Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover, in vitro analysis demonstrated that GP V ؊͞؊ platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V ؊͞؊ mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors. P latelet thrombosis and hemostasis are complex reactions that depend on adhesive interactions mediated by specific receptors. A major platelet complex is glycoprotein (GP) Ib-IX-V. The initial adhesion of platelets is primarily mediated by binding of platelet membrane GP Ib-IX-V to von Willebrand factor (vWf) found on damaged vessel walls (1). After adhesion, binding of other agonists such as thrombin, ADP, and collagen induce signaling events that ultimately activate the receptor function of ␣⌱⌱b␤3 for soluble fibrinogen, leading to platelet aggregation (2). Although platelet aggregates are required for normal hemostasis, they can in addition cause arterial thrombosis in atherosclerotic arteries, e.g., acute myocardial infarction and stroke, inducing ischemic complications of cardiovascular disease (3, 4).The importance of the GP Ib-IX-V complex in normal platelet function is underscored by the study of Bernard-Soulier syndrome (BSS), an inherited bleeding disorder characterized by large platelets that are defective in adhesion to damaged vessel walls (1). This genetic disorder is caused by a lack of functional GP Ib-IX-V and has been linked to defects in either GP Ib or GP IX (5). The activities mapped to the GP Ib subunit of the GP Ib-IX-V complex include vWf (6) and thrombin binding (7, 8) on the extracellular domain and actin-binding protein (9-11) and 14-3-3 (12-15) binding on the cytoplasmic domain.Several studies indicate functional activities for the GP V subunit of the GP Ib-IX-V complex. In one example, GP V has been shown to be cleaved by thrombin from the platelet surface during thrombin-mediated platelet stimulation (16), but the role of GP V cleavage in this thrombin-induced platelet response is unresolved (17). In another example, the signa...

Platelet receptors and signaling in the dynamics of thrombus formation

“…61,63,69 Apart from GP Ibα, GP V may also be involved in this scenario, since it is a prominent α-thrombin substrate and GP V -/-mice display enhanced platelet sensitivity to low doses of thrombin leading to in vivo increased thrombogenesis and embolus formation. 70,71 It has been suggested that proteolysis of GP V by α-thrombin reveals the ability of GP Ibα to act as a thrombin ligand and induce stimulatory responses. 72 Despite recognizing that an intact GP Ib/IX/V complex may be required for optimal thrombin responsiveness, there is little doubt that PARs are sufficient to activate platelets and account for most, if not all, of the thrombin-induced signaling.…”

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

“…Precipitated, GTP-bound Rap1B was separated by SDS-PAGE on 10 -20% acrylamide gradient gels, transferred to nitrocellulose, and identified by immunoblotting with a specific polyclonal antibody. Intracellular Ca 2ϩ concentration was evaluated using Fura-2-loaded platelets and calculated as described under "Experi- GPIb-IX-V on the platelet surface, and this interaction may contribute to platelet activation (33)(34)(35)(36)(37)(38). We therefore examined the role of GPIb-IX-V in thrombin-induced activation of Rap1B.…”

“…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).…”

Identification of a DNA-binding site and transcriptional target for the EWS–WT1(+KTS) oncoprotein

“…These two genes share the same genomic structure and are adjacent to each other, consistent with a tandem gene duplication event. GP5 encodes a subunit of the GP1b-V-IX receptor that binds von Willebrand factor and is important in platelet aggregation (Roth et al 1996;Gurney et al 2002), and it may act to negatively regulate the response of the activated receptor (Ramakrishnan et al 1999). We have shown that LRRC15 also encodes a transmembrane glycoprotein, expressed at the leading edge of migrating cells.…”