The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion

Abstract

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collageninduced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VI null platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VI null or FcR-␥ null animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VI null platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils. IntroductionPlatelet membrane receptors interact with surface-bound adhesive ligands and, as such, become essential for hemostasis and thrombosis. 1 There are numerous unique receptors interacting with different adhesive ligands suggesting that a large opportunity exists for functional redundancy in platelet adhesion. However, an emerging theme of platelet biology is the relevance of different membrane receptors in different areas of the vasculature. 2,3 A specific example is the exclusive role for the platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor in areas of the vascular system where flow rates and high shear occur, such as in small arteries and arterioles. 4 Thus, defining the physiologic relevance of an individual receptor and its ligand is an important aspect for understanding participation of the platelet in hemostasis and thrombosis.Among adhesive ligands of the extravascular matrix, collagen is a significant component with a number of known collagen receptors on the platelet surface. 5,6 One of the more recently characterized collagen receptors is GP VI. 7 The molecular cloning of GP VI revealed it to be a member of the immunoglobulin superfamily of type I transmembrane proteins. [8][9][10] The surface expression of GP VI requires the concomitant expression of the ␥-subunit of the FcR receptor (FcR-␥) and their association is functionally relevant as collagen binding to GP VI results in platelet signaling via the immunoreceptor tyrosine-based activation motif (ITAM) located in the FcR-␥ subunit. 8,[11][12][13][14] As with many of the platelet receptors, the in vivo relevance of GP VI was established prior to its description and recognition as a p...

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…A significant defect in PAR4 peptide-induced secretion was observed, but this was only a 20% decrease (Figure 3K); since there was no PAR4 aggregation defect (Figure 3E), a role for Tspan9 in PAR4-induced platelet activation appears unlikely. To test whether a Tspan9 collagen aggregation phenotype could be revealed at a 50% reduced surface expression level of GPVI, which could make a role for Tspan9 in regulating GPVI more critical, Tspan9-deficient mice were crossed with GPVI-deficient mice [28]. This allowed the effect of Tspan9 deficiency to be investigated on a GPVI-heterozygous background.…”

“…Heterozygous breeding pairs were used to generate Tspan9 knockout mice with litter-matched controls, and mice were aged at least 8 weeks before use in experiments. GPVI-deficient mice [28] were kindly provided by Prof Jerry Ware (University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA). Mouse experiments at the Biomedical Services Unit, University of Birmingham, UK, were performed in accordance with the Animals (Scientific Procedures) Act 1986.…”

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

“…[39][40][41] For in vitro studies, we used CRP as the GPVI-selective agonist because this peptide contains 10 GPO repeat sequences and serves as a potent activator of GPVImediated signaling and does not recognize integrin ␣2␤1 . 42 In this way, we can differentiate GPVI-mediated platelet responses from integrin ␣2␤1-mediated platelet responses. Signaling through GPVI/FcR␥-chain ITAM-dependent pathway is thought to be counterbalanced by proteins bearing ITIM motifs such as those contained in members of the Ig-ITIM superfamily.…”

Platelets at the vascular interface

“…And even though GPVI has adapted to meet the accelerated temporal needs of hemostasis[30], the ITAM signaling response is still slower compared to the GPCR response. Consistently, the mild bleeding phenotype of patients [31,32] or mice[33,34] lacking GPVI suggests that this receptor has a minor role in classical hemostasis when compared to the major GPCRs[15]. Surprisingly, GPVI was shown to be more important for thrombus stabilization than for thrombus initiation[35], suggesting the existence of ligands other than collagen.…”