Prostacyclin reverses platelet stress fibre formation causing platelet aggregate instability

Abstract

Abstract Prostacyclin (PGI 2 ) modulates platelet activation to regulate haemostasis. Evidence has emerged to suggest that thrombi are dynamic structures with distinct areas of differing platelet activation. It was hypothesised that PGI 2 could reverse platelet spreading by actin cytoskeletal modulation, leading to reduced capability of platelet aggregates to withstand a high shear environment. Our data demonstrates that post-flow of PGI 2 over activated and spread platelets on fibrinogen, identified a significant reduction in platelet surface area under high shear. Exploration of the molecular mechanisms underpinning this effect revealed that PGI 2 reversed stress fibre formation in adherent platelets, reduced platelet spreading, whilst simultaneously promoting actin nodule formation. The effects of PGI 2 on stress fibres were mimicked by the adenylyl cyclase activator forskolin and prevented by inhibitors of protein kinase A (PKA). Stress fibre formation is a RhoA dependent process and we found that treatment of adherent platelets with PGI 2 caused inhibitory phosphorylation of RhoA, reduced RhoA GTP-loading and reversal of myosin light chain phosphorylation. Phospho-RhoA was localised in actin nodules with PKA type II and a number of other phosphorylated PKA substrates. This study demonstrates that PGI 2 can reverse key platelet functions after their initial activation and identifies a novel mechanism for controlling thrombosis.