Vav1 and Vav3 Have Critical but Redundant Roles in Mediating Platelet Activation by Collagen

Abstract

Vav family proteins are guanine nucleotide exchange factors for the Rho/Rac family of small GTP-binding proteins. In addition, they have domains that mediate protein-protein interactions, including one Src homology 2 (SH2) and two Src homology 3 (SH3) domains. Vav1, Vav2, and Vav3 play a crucial role in the regulation of phospholipase C␥ (PLC␥) isoforms by immuno-tyrosinebased activation motif (ITAM)-coupled receptors, including the T-and B-cell antigen receptors. We have reported in platelets, however, that Vav1 and Vav2 are not required for activation of PLC␥2 in response to stimulation of the ITAM-coupled collagen receptor glycoprotein VI (GPVI). Here we report that Vav3 is tyrosinephosphorylated upon activation of GPVI but that Vav3-deficient platelets also exhibit a normal response upon activation of the ITAM receptor. In sharp contrast, platelets deficient in both Vav1 and Vav3 show a marked inhibition of aggregation and spreading upon activation of GPVI, which is associated with a reduction in tyrosine phosphorylation of PLC␥2. The phenotype of Vav1/ 2/3 triple-deficient platelets is similar to that of Vav1/3 double-deficient cells. These results demonstrate that Vav3 and Vav1 play crucial but redundant roles in the activation of PLC␥2 by GPVI. This is the first time that absolute redundancy between two protein isoforms has been observed with respect to the regulation of PLC␥2 in platelets.Collagen is the most thrombogenic component of the subendothelial matrix, inducing powerful platelet activation through the GPVI 1 -FcR␥-chain receptor complex. GPVI signals through sequential activation of Src and Syk family tyrosine kinases (1, 2). The Src kinases Fyn and Lyn stimulate tyrosine phosphorylation of two conserved tyrosines in the FcR␥-chain immunotyrosine-based activation motif (ITAM) (2-4). This leads to engagement of Syk via its two SH2 domains and its subsequent activation. Syk orchestrates a downstream signaling cascade that is regulated through the interaction of several adapter proteins, including LAT, Gads, and SLP-76, and leads to activation of effector enzymes, including phosphatidylinositol (PI) 3-kinases, Tec kinases, and phospholipase C␥ (PLC␥). The functional role of many of the proteins in this cascade, including GPVI (5), FcR␥ chain (1), Syk (1), LAT (6), Gads (7), SLP-76 (8), Btk (9), Tec (9), and PLC␥2 (10, 11), has been shown by the impairment or abolition of response in platelets from genetically deficient mice. In sharp contrast, platelets from mice deficient in Vav1 and Vav2 show minimal functional impairment in responses to collagen or GPVI-specific agonists such as convulxin and CRP (12), despite their role in signaling by other ITAM receptors, including the B-cell and T-cell antigen receptors. The Vav family of GTP exchange factors consists of three members (13-16), which share a common structural arrangement. The amino terminus contains a calponin homology domain and an acidic region, which contains regulatory tyrosine phosphorylation sites. This is followed by Dbl homology, plec...

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…Human and mouse platelet-rich plasma and washed platelets were isolated from whole blood as previously described [32, 33]. Consent for human blood was obtained from each donor, and platelet preparation was carried out with ethical approval.…”

“…For platelet stimulation and subsequent detection of protein tyrosine phosphorylation, platelets were pre-treated with 10 µM lotrafiban, 2 units/mL apyrase and 10 µM indomethacin, and then stimulated and subjected to lysis and western blotting as described [33]. For co-immunoprecipitation experiments, 1 × 10 9 washed human platelets were lysed in 1% Brij97 lysis buffer [15] and pre-cleared with 20 µL protein G sepharose beads for 60 minutes at 4°C.…”

“…Mouse or human platelet-rich plasma was diluted to 2 × 10 7 platelets/mL in modified Tyrode’s buffer [33]. Mouse platelets were pre-incubated with 5 μg/mL of rat anti-mouse GPVI clone JAQ3 [36] for 10 minutes at 37°C and then allowed to spread on glass-bottom dishes coated with 10 µg/mL collagen for 45 minutes at 37°C.…”

“…Non-adhered platelets were removed by gentle washing with modified Tyrode’s buffer [33]. The relevant antibodies and the membrane dye DiOC6 were incubated for 10 minutes at 37°C with the spread platelets at a final concentration of 10 ng/mL for JAQ1, 5 ng/mL for CD9, 50 ng/mL for IgG controls and 0.2 µM for DiOC6.…”

Preferential protection of protein interaction network hubs in yeast: Evolved functionality of genetic redundancy

“…Notably, although evidence suggests that a rapid loss of functional overlap indeed describes the fate of most duplicated genes, this hypothesis is also violated by numerous well documented examples (13,14). In one such case, recent knockdown experiments in Caenorhabditis elegans have revealed duplicate genes that have been conserved in a functionally redundant state for Ͼ80 million years of evolution (15).…”