Regulation of platelet contractility by agonists present across a thrombus

The generation of cell traction forces plays an important role in tissue morphogenesis. Platelets self-assemble into a layered thrombus structure, with a tightly packed core close to the vascular injury site and a looser shell toward the vessel lumen. The mechanical and proteolytic stability of the thrombus core might be linked to increased platelet contractility, reinforced by reciprocal interactions with the local microenvironment. How this spatially regulates platelet contractility is thus a clinically important question. The aim of this in vitro study was to clarify how platelet agonists that are present at different locations in a thrombus coregulate actomyosin contractility. In single-platelet contractile force measurements, thrombin led to ∼50% higher traction forces after an hour than adenosine diphosphate or collagen-related peptide. Stimulation of protease-activated receptor 1 (PAR1) in combination with PAR4 maximized platelet contractility without changing adhesion signaling. Only thrombin induced the persistent phosphorylation of Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) and myosin regulatory light chain 9 (MYL9) in adherent platelets, consistent with PAR>Gα13>RhoA signaling. Thrombin further induced elevated protein expression of MYL9 and led to splicing of ROCK1 pre-mRNA. Antiplatelet drugs counteracted clot contraction in vitro in a thrombin concentration-dependent manner, more potently at low thrombin concentrations. Our findings demonstrate a mechanism in which thrombin maximizes sustained platelet contractility. They further delineate the intimate spatiotemporal and functional relationship between thrombin and platelets during stabilization of the hemostatic plug. Our results also caution against the combined use of antiplatelet and anticoagulant treatments, which might increase bleeding diathesis due to defective clot contraction.