Rho Kinase Regulates Platelet Adhesion and Aggregation on Collagen under Flow

Abstract number: P0301

Calaminus¹ SDJ, Auger¹ JM, McCarty¹ OJT, Wakelam² MJO, Watson¹ SP

¹¹Centre for Cardiovascular Sciences, The Medical School, University of Birmingham, Birmingham, UK ²²CRUK Centre for Cancer Studies, The Medical School, University of Birmingham, Birmingham, UK

Rho kinase has been reported to play an important role in sustaining platelet adhesion to von Willebrand Factor (VWF) at intermediate and high shear rates, although it is not required for the initial tethering event (Schoenwaelder et al. 2002, J. Biol. Chem 277, 14738–46). In this study, we have asked whether Rho kinase also contributes to platelet adhesion to collagen under shear conditions, a process that is dependant on the capture of platelets by vWF.

Method:  The interaction of platelets with collagen has been investigated under static and shear conditions in real-time. Experiments were performed in the presence of the inhibitors of Rho kinase, Y27632 and HA-1077, and the inhibitor of myosin II, blebbistatin, since myosin light chain kinase is regulated by Rho kinase. For the shear experiments, whole blood was labeled with DiOC₆, and incubated with the inhibitors Y27632 or HA-1077. In separate experiments, washed platelets were incubated with inhibitors and DiOC₆, and then reconstituted with red blood cells (50% v/v).

Results:  None of the inhibitors had an effect on adhesion or formation of filopodia or lamellipodia on collagen under static conditions. In contrast, platelet adhesion to collagen under shear was increased by approximately 2-fold as determined by measurement of surface area in the presence of Y27632 (20 mM) or HA-1077 (20 mM) in both whole blood and reconstituted washed platelets after 3 minutes of flow at 1000 s^–1. This was accompanied by ~30% reduction in aggregate size. Blebbistatin had no effect on the degree of surface coverage in reconstituted washed platelets, although it reduced aggregate size.

Conclusion:  This study demonstrates a role for Rho kinase in regulating adhesion and aggregate formation on collagen at high shear, which is partially mediated through regulation of myosin light chain phosphorylation.