Kinetics of Thrombocyte and Thrombocyte-Microparticle Recruitment in Laser Induced Arterial Thrombus Formation in Zebrafish.

We have been using zebrafish as a genetic model to study hemostasis and thrombosis. In this pursuit, we studied the initial events in thrombus formation in a laser induced arterial thrombus formation. First, we identified in zebrafish blood microparticles that have membrane proteins similar to those found in thrombocytes. We have then shown that in Weinstein transgenic line (where endothelial cells are labeled with GFP using fli-1 promoter) thrombocytes were also labeled with the GFP. In this line, we found two populations of GFP positive thrombocytes one that is more intense and larger in size than the other and others were of intermediate intensities and sizes. In Weinstein line as well as in Handin transgenic line which carries exclusively GFP labeled thrombocytes (driven under GpIIb promoter), we found GFP labeled microparticles. The GFP microparticles in both lines were similar in numbers suggesting that thrombocytes are generating more microparticles. They ranged in size between 0.2 to 0.8 microns. Thrombin and collagen treatment of thrombocytes increased the generation of microparticles. We also found that the microparticles agglutinated in a vWF dependent fashion.

In Lin transgenic line (where mostly red cells are labeled with GFP using GATA-1 promoter), we found a small percentage of thrombocytes were also labeled with GFP (corresponding to less intense GFP thrombocytes in Weinstein line). By using the microparticles from Lin and Weinstein lines, we found that the agglutinates contained, thrombocyte microparticles, and to a larger extent red cell microparticles. By labeling the thrombocyte microparticles with DiI-C₁₈ (a dye that selectively labels approximately 10% of thrombocytes at defined concentration), we found that microparticles accumulated first at the site of injury. Intravenous pan-caspase inhibitor (z-VAD-FMK) injections in zebrafish resulted in significant reduction of microparticles and prolongation of time to adherence in laser induced thrombosis assay.

In Weinstein line we noted that less intense GFP thrombocytes were more intensely labeled with DiI-C₁₈. We defined DiI-C₁₈ +ve thrombocytes as young thrombocytes and found that expression of GPIb, and GPIIb/IIIa on native thrombocytes and P-selectin, annexin V and calcium levels after thrombocyte activation were higher in young thrombocytes compared to mature DiI-C₁₈ -ve thrombocytes. We also found that in an aggregation reaction, young and mature thrombocytes formed independent clusters with a preference for formation of young clusters first. By using dye labeling methods as well as above transgenic lines we showed that on laser induced arterial injury young thrombocytes initiated arterial thrombus formation.

To identify novel differences between the two populations of thrombocytes, we performed microarray analysis on thrombocyte RNA using a control red cell RNA and found approximately 100 five-fold upregulated genes in thrombocytes compared to red cells. These included genes such as xfo-6. We are currently studying the gene expression differences between the two thrombocyte populations.

In summary, we found that microparticles adhere first to sub-endothelial matrix followed by young thrombocyte clustering and later by mature and young thrombocytes clusters in growing thrombus. The knowledge on the differences between the thrombocyte populations and their microparticles as well as their recruitment into thrombus, might provide insight into thrombus formation in mammals and may suggest novel antithrombotic targets.