Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy.

We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo.

Glycoprotein Ib-V-IX, a receptor for von Willebrand factor, couples physically and functionally to the Fc receptor gamma-chain, Fyn, and Lyn to activate human platelets.

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin alpha(IIb)beta(3). We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (triangle upA1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor gamma-chain through interaction with Syk SH2 domains. GP Ib physically associates with the gamma-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cgamma2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated gamma-chain, phosphorylation of PLCgamma2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR gamma-chain and members of the Src family kinases, leading to phosphorylation of the gamma-chain, recruitment, and activation of Syk. Phosphorylation of PLCgamma2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.