Activated Thrombin-Activatable Fibrinolysis Inhibitor (Tafia) Attenuates Fibrin-Dependent Plasmin Generation On Thrombin-Activated Platelets

Background Thrombin-activated platelets can promote fibrinolysis by binding plasminogen in a fibrinogen-dependent manner and enhancing its activation by tissue-type plasminogen activator (t-PA). Whether t-PA also binds to activated platelets and the mechanism for regulation of platelet-dependent fibrinolysis remain unknown. Objectives Determine the mechanism of plasminogen and t-PA binding on thrombin-activated platelets and its regulation by activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Methods Plasminogen and t-PA binding with or without TAFIa treatment was quantified using flow cytometry. Plasmin generation on platelets was quantified using a plasmin-specific substrate. Mass spectrometry analyses identified fibrinogen as a potential target of TAFIa. Thrombus formation was studied in mice lacking fibrinogen (Fg(-/-)) using intravital microscopy. Results Plasminogen and t-PA bind to platelets activated by thrombin but not by other agonists, including protease-activated receptor agonists (PAR-AP). Plasminogen binds via its kringle domains because epsilon-aminocaproic acid eliminates binding, whereas t-PA binds via its finger and kringle domains. Plasminogen binding is fibrinogen-dependent because it is abolished on (a) Fg(-/-)platelets, and (b) thrombi in Fg(-/-)mice. Binding requires thrombin-mediated fibrinogen modification because addition of batroxobin to PAR-AP activated platelets has no effect on plasminogen binding but induces t-PA binding. TAFIa reduces plasminogen and t-PA binding to thrombin-activated platelets and attenuates plasmin generation in a concentration-dependent manner. Mass spectrometry identified K556 on the fibrinogen alpha-chain as a potential thrombin cleavage site that generates a TAFIa sensitive C-terminal lysine residue. Conclusion These findings provide novel mechanistic insights into how platelets activated by thrombin at sites of vascular injury can influence fibrinolysis.