Fibrin as a thrombin sink: a factor retaining a thrombus in the stillness phase

ABSTRACT: The process of nonocclusive thrombus formation is well known, but the mechanism keeping the thrombus silent at the end stage remains unclear. The aim of this work was to evaluate the role of fibrin in limiting further growth of a thrombotic remnant. Intravital microscopy showed that attachment of platelets to a fibrin-rich thrombus stopped after partial thrombus disaggregation, indicating that the thrombus activation potential is lost, a stage we named the stillness phase. Histological analyses showed that 80% of the internal cross-section area of the thrombus remnant is bordered by fibrin, whereas 20% of the superficial thrombus area was covered only by a few platelet layers, suggesting a role of fibrin in limiting platelet recruitment. This result was confirmed in a flow-based assay in which fibrin-rich thrombi recruited circulating platelets inefficiently as compared with fibrin-poor thrombi. Moreover, we found that in vitro, lysis of fibrin with recombinant tissue plasminogen activator (rtPA) released active thrombin. This observation was confirmed in vivo because treating a thrombus with rtPA to promote fibrin breakdown during the stillness phase resulted in the release of thrombin, leading to an unexpected regrowth of the thrombus. This finding was further supported by the dynamics of thrombus formation in FgaEK mice, which displayed repeated cycles of thrombus growth and detachment after vessel injury, with an inability to reach the stillness phase, accompanied by the continuous release of active thrombin. Altogether, these findings identify a novel role of fibrin in maintaining an end-stage thrombotic remnant in an inactive state.