RhoA/ROCK signaling contributes to sex differences in the activation of human platelets

• Sex differences in reactivity of human platelets were investigated.
• Although similar basal levels, female platelets exhibited higher levels of GPIIb/IIIa activation upon ADP stimulation.
• Enhanced activity of the RhoA/ROCK signaling pathway might lead to greater sensitivity of female platelets to ADP.

ABSTRACTStudies of sex-dependent differences in platelet aggregation and glycoprotein (GP)IIb/IIIa activation have demonstrated that platelets from females are more sensitive to agonists than those from males. To date, there is little understanding of these differences at a molecular level. Here, sex differences in reactivity of platelets from 86 women and 86 men were investigated. Platelet degranulation (CD62P expression) and activation of GPIIb/IIIa (PAC-1 binding), with and without ADP, were assessed. Extent of shape change (ESC) in response to ADP was measured. Basal CD62P and PAC-1 expression did not differ between the sexes. In response to ADP activation, mean PAC-1 binding in platelets from female donors was 17.9 ± 3.5% vs. 14.0 ± 4.1% in platelets from male donors, and ESC was significantly greater in platelets from females (p < 0.05). Evaluation of basal expression of signaling molecules along the ADP receptor pathway leading to GPIIb/IIIa activation and subsequent RhoA/ROCK signaling via GPIIb/IIIa ‘outside-in’ signaling showed that platelets from females produce 3-fold greater levels of phosphorylated protein kinase C (PKC) substrates. There was a 2.5-fold greater level of activated RhoA, and platelet sub-fractionation analysis demonstrated 2.7-fold more RhoA in the membrane fraction of female vs. male platelets. Similarly, there was a 2.8-fold increase in levels of phosphorylated myosin light chain (MLC) in platelets from females vs. males. The increased signaling activity in platelets from females mirrors their greater sensitivity to agonists. These findings further our understanding of the molecular differences between platelets from males and females.