The Rac GTPases, members of the Ras related family of Rho GTPases, have been shown to be involved in the regulation of cell spreading, adhesion and actin cytoskeleton reorganization in hematopoietic cells (
Roberts, A.W., et al.;
Gu Y., et al.). Adhesion of platelets to vascular matrix containing fibrinogen or collagen is the first and a critical step that leads to platelet aggregation. The possibility that individual Rac GTPases may play a role in platelet activation was examined in this study. The Rac2 gene targeted (Rac2−/−) mice were used to investigate the contribution of Rac2 GTPase in platelet adhesion and aggregation. All experiments were conducted using an equal number (1–2 x 108/ml) of washed platelets from WT and Rac2−/− mice. Firstly, the role of Rac2 in platelet adhesion was examined by quantifying adhesion of platelets to fibrinogen coated wells. Platelets from Rac2−/−mice exhibited a 35% less adhesion to fibrinogen than platelets from WT mice. Secondly, binding of platelets to soluble fibrinogen was assessed by stimulating the platelets with ADP, collagen or phospholipase C (PLC) in the presence of Oregon Green conjugated fibrinogen followed by flow cytometry analysis. ADP-, PLC- and collagen-induced fibrinogen binding to platelets from Rac2−/−mice, as compared to platelets from WT mice, was decreased by 65%, 40% and 35%, respectively. Thirdly, platelet aggregation was monitored by an optical density method using a Chronolog Aggregometer. Agonists that induce aggregation responses via specific receptors for example thrombin, collagen and U46619 (a stable analog of thromboxane A2) as well as agonists that bypass receptors and directly activate protein kinase C by generating diacylglycerol (e.g. PLC from Clostridium perfringens) or by mimicking diacylglycerol (e.g. phorbol 12-myristate 13-acetate, PMA) were used in this study. At threshold concentrations, thrombin (0.04 U/ml), collagen (4 μg/ml), U46619 (0.5 μM) and PLC (0.04 U/ml) all elicited a slower onset of aggregation, as depicted by a prolonged shape change phase, in platelets from Rac2−/−than platelets from WT mice. Aggregation responses induced by thrombin, collagen and U46619 were all diminished in platelets from Rac2−/−mice as compared to platelets from WT mice. Moreover, platelets from Rac2−/−, but not from WT, mice failed to exhibit irreversible aggregation even when challenged with higher concentrations of U46619 (10 μM) or collagen (8 μg/ml). Platelet aggregation responses induced by PLC and PMA, agents that bypass receptors, were also decreased in platelets from Rac2−/−mice than in platelets from WT mice. These data lead us to suggest that Rac2 plays a critical role not only in platelet adhesion to fibrinogen, a step necessary for subsequent platelet aggregation, but also in sustenance and perpetuation of platelet aggregation. Furthermore, Rac2 appears to be involved in regulation of agonist-receptor mediated as well as direct, receptor-independent, activation of platelets.
2005, The American Society of Hematology