Abstract
BackgroundThe current model of fibrinolysis accepts fibrin as the only accelerator of tissue plasminogen activator (tPA),-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, under non-reducing conditions, non-covalently associated FXa fragments of 33kDa and 13kDa are generated. Contrary to fibrinolysis dogma, generation of Xa33/13 exposes C-terminal lysine binding sites for plasminogen and tPA, thus converting FXa into a fibrinolysis accelerator. Here we investigated whether Xa33/13 is produced in plasma when FXa has irreversibly associated with its inhibitor, antithrombin (AT), and if the product acquires fibrinolysis accelerator function.Methods/resultsWestern blot analysis of clotting plasma revealed the appearance of Xa33 antigen correlating to the appearance of Xa13-AT. Purified Xa-AT was cleaved approximately 40-fold faster by plasmin compared to FXa. The resulting species has slightly different cleavage sites as determined by N-terminal sequencing, but predicted exposure of new C-terminal lysine. Immobilized Xa33/13-AT bound 125I-plasminogen. To follow purified plasminogen activation by tPA, a chromogenic assay demonstrated Xa-AT enhanced tPA activity several fold over FXa. An excess of Xa-AT in plasma to overcome the endogenous FXa and AT significantly enhanced the rate of fibrinolysis.ConclusionThese data demonstrate a novel function for the Xa-AT complex and support the concept for auxiliary cofactors in fibrinolysis.Heart and Stroke Foundation of British Columbia & Yukon BackgroundThe current model of fibrinolysis accepts fibrin as the only accelerator of tissue plasminogen activator (tPA),-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, under non-reducing conditions, non-covalently associated FXa fragments of 33kDa and 13kDa are generated. Contrary to fibrinolysis dogma, generation of Xa33/13 exposes C-terminal lysine binding sites for plasminogen and tPA, thus converting FXa into a fibrinolysis accelerator. Here we investigated whether Xa33/13 is produced in plasma when FXa has irreversibly associated with its inhibitor, antithrombin (AT), and if the product acquires fibrinolysis accelerator function. The current model of fibrinolysis accepts fibrin as the only accelerator of tissue plasminogen activator (tPA),-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, under non-reducing conditions, non-covalently associated FXa fragments of 33kDa and 13kDa are generated. Contrary to fibrinolysis dogma, generation of Xa33/13 exposes C-terminal lysine binding sites for plasminogen and tPA, thus converting FXa into a fibrinolysis accelerator. Here we investigated whether Xa33/13 is produced in plasma when FXa has irreversibly associated with its inhibitor, antithrombin (AT), and if the product acquires fibrinolysis accelerator function. Methods/resultsWestern blot analysis of clotting plasma revealed the appearance of Xa33 antigen correlating to the appearance of Xa13-AT. Purified Xa-AT was cleaved approximately 40-fold faster by plasmin compared to FXa. The resulting species has slightly different cleavage sites as determined by N-terminal sequencing, but predicted exposure of new C-terminal lysine. Immobilized Xa33/13-AT bound 125I-plasminogen. To follow purified plasminogen activation by tPA, a chromogenic assay demonstrated Xa-AT enhanced tPA activity several fold over FXa. An excess of Xa-AT in plasma to overcome the endogenous FXa and AT significantly enhanced the rate of fibrinolysis. Western blot analysis of clotting plasma revealed the appearance of Xa33 antigen correlating to the appearance of Xa13-AT. Purified Xa-AT was cleaved approximately 40-fold faster by plasmin compared to FXa. The resulting species has slightly different cleavage sites as determined by N-terminal sequencing, but predicted exposure of new C-terminal lysine. Immobilized Xa33/13-AT bound 125I-plasminogen. To follow purified plasminogen activation by tPA, a chromogenic assay demonstrated Xa-AT enhanced tPA activity several fold over FXa. An excess of Xa-AT in plasma to overcome the endogenous FXa and AT significantly enhanced the rate of fibrinolysis. ConclusionThese data demonstrate a novel function for the Xa-AT complex and support the concept for auxiliary cofactors in fibrinolysis.Heart and Stroke Foundation of British Columbia & Yukon These data demonstrate a novel function for the Xa-AT complex and support the concept for auxiliary cofactors in fibrinolysis.
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