The protein kinase C inhibitor RO318220 potentiates thrombin-stimulated platelet-supported prothrombinase activity

Abstract

AbstractProthrombinase activity was tested on thrombin- and SFLLRN-activated platelets treated with RO318220, a potent inhibitor of protein kinase C. RO318220 completely inhibited platelet dense and α-granule secretion at a concentration of 20 μM but had no effect on prothrombinase activity in the presence of excess factor Va (20 nM). This indicates that protein kinase C activity and agonist-initiated secretion are not necessary for the development of a procoagulant surface. Treatment with 75 to 150 μM RO318220 potentiated platelet-supported thrombin generation up to 280% of control platelets with no change in Kd appFXa. Treated with increasing concentrations of RO318220, an increasing proportion of thrombin-stimulated platelets bound annexin V with decreasing binding sites per platelet. A lower mean forward scatter (FSC-H) of platelets treated with RO318220 suggested platelet vesiculation as a result of RO318220 treatment; however, 100 μM calpeptin pretreatment eliminated the decrease in FSC-H without affecting either the increase in platelets positive for annexin V binding, the decrease in binding sites per platelet, or the 3-fold increase in prothrombinase activity. Thus, RO318220 appears to increase prothrombinase activity by increasing platelet responsiveness to thrombin rather than by inducing platelet vesiculation. This suggests that RO318220 inhibits a signaling molecule within a negative regulatory pathway that governs platelet procoagulant surface changes. (Blood. 2003;102:2472-2481)