Abstract
BACKGROUND: A signaling pathway is difficult, if not impossible, to elucidate in platelets using only in vivo studies. Likewise, the physiological significance of signaling information obtained exclusively from in vitro observations is unknown. Therefore, both in vitro and in vivo experiments are required to establish the physiological significance of a signaling pathway. OBJECTIVE: To evaluate the physiological significance of signaling data obtained from botrocetin (bt)/von Willebrand factor (VWF)-stimulated washed platelets. METHOD: Stable thrombus formation in response to FeCl(3)-induced injury of the mouse carotid artery was used to evaluate the physiological significance of signaling data obtained from bt/VWF-stimulated washed platelets. RESULTS: Syk, PLCgamma2, Galphaq and P2Y12, but not LAT, were found either to be required for or to affect stable thrombus formation. Prior in vitro studies had demonstrated that LAT is not required for bt/VWF-induced platelet aggregation in the presence of exogenous fibrinogen. These data provide the first demonstration of the in vivo role for these signaling molecules in GPIb-dependent/initiated signal transduction and are consistent with the signaling pathway deduced from in vitro studies of bt/VWF-stimulated washed platelets using metabolic inhibitors and knockout mice. CONCLUSION: The broad agreement between the in vitro and the in vivo results establish that bt/VWF stimulation of washed platelets can provide physiologically significant glycoprotein Ib-dependent/initiated signaling data.