Control of coagulation by the RhoA pathway and the exchange factor Arhgef1

Abstract

Activation of the thrombin receptors, called PAR, leads to a remodeling of the actin cytoskeleton depending on the small G proteins of the Rho family. The main one, RhoA, controls platelet contractility. The Arhgef1 nucleotide exchange factor, responsible for the activation of RhoA, is not well known. Our goal is to identify the role of Arhgef1 in platelet activity and thrombin generation in response to thrombin-activated PAR receptors. Thrombin generation, platelet activation and aggregation are measured by fluorimetry, flow cytometry, and turbidimetry in the blood of Arhgef1+/+ and Arhgef1−/− mice. The formation of a thrombus in the carotid artery in response to the local application of FeCl3, is followed by Echo-Doppler. In response to thrombin, aggregation of washed platelets and intracellular calcium are similar in Arhgef1−/− and Arhgef1+/+ mice. However, the secretion of alpha granules, exposure of phosphatidylserine and release of microparticles associated with platelet activation are decreased compared to Arhgef1+/+ mice. The generation of whole blood thrombin was significantly reduced (25%) in Arhgef1−/− mice compared to Arhgef1+/+. These changes result in an increase in the time of an occlusive thrombus occurrence induced by FeCl3 in the carotid artery. Our results show the involvement of the platelet RhoA pathway in the self-amplification of thrombin generation by a mechanism dependent on Arhgef1 and PAR receptors. Changes in the distribution of membrane phospholipids suggest that this pathway could participate in the deleterious effects of thrombin in atherothrombosis.