Contrasting Effects of Platelet GPVI Deletion versus Syk Inhibition on Mouse Jugular Vein Puncture Wound Structure

Platelet glycoprotein (GP)VI is a transmembrane protein that was originally characterized as a collagen receptor supporting platelet adhesion and activation through its association with the Fc receptor g-chain (FcRg). The FcRg subunit contains immunoreceptor tyrosine-based activation motifs (ITAMs) that recruit and activate Syk (spleen tyrosine kinase), a key player in intracellular signaling pathways. Absence or dysfunction of GPVI produces a mild bleeding defect in humans similar to the impaired hemostasis reported in the murine knockout. Here, we have taken an ultrastructure approach to examine the impact of ligand binding to GPVI versus the downstream pharmacologic inhibition of the GPVI-dependent signaling pathway. Clots were generated for analysis following a puncture wound in the mouse external jugular vein. Images were obtained using mice genetically missing GPVI and mice pretreated with the Syk inhibitor, BI 1002494. Our study was designed to test the hypothesis that the predominant contribution of GPVI to hemostasis is mediated by a Syk-dependent signaling cascade. If true, clot structure observed with a Syk inhibitor versus the GPVI knockout would be similar. If the extracellular domains of the protein had a Syk-independent platelet adhesion role, then significant comparative differences in thrombus structure would be expected. Our results clearly indicate an important, Syk-independent role of the GPVI extracellular domain in the adherence of platelets within the intravascular crown of a growing venous clot, a site distant from exposed collagen-rich adventitia. In striking contrast, the adventitial proximal role of GPVI was Syk-dependent with the GPVI knockout and Syk inhibitor giving the same, limited structural outcome of collagen-proximal platelet cytosol loss and a thinned extravascular cap. Consistent with the lesser role of Syk-dependent processes on thrombus structure, Syk inhibitor had no detectable effect on jugular puncture wound bleeding times while the knockout had a statistically significant, but modest effect on bleeding time. Based on this contrast, we suggest that Syk inhibition may be the more selective approach to modulating the role of GPVI in occlusive clotting.