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Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.

The activation of factor XI by meizothrombin was investigated using recombinant meizothrombin (R155A meizothrombin) that is resistant to autocatalytic removal of fragment 1. Meizothrombin was capable of activating factor XI at an activation rate similar to that of thrombin. Dextran sulphate and heparin, known cofactors of thrombin-mediated factor XI activation, did not stimulate the activation of factor XI by meizothrombin. However, the activation of factor XI by meizothrombin was markedly enhanced by vesicles containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas PC/PS or PC/PE vesicles only had a minor effect on the activation. Thrombin-mediated factor XI activation was not influenced by phospholipids. The effect of PC/PS/PE and PC/PS vesicles was studied in a factor XI dependent clot lysis assay. In this assay, factor XI inhibits clot lysis by a feedback loop in the intrinsic pathway via thrombin-mediated factor XI activation. Removal of endogenous phospholipids in plasma by centrifugation resulted in an increased clot lysis, which could be restored to the pre-centrifugation level by the addition of PC/PS/PE vesicles, but not by PC/PS vesicles. When clot lysis was initiated by factor IXa in the presence of a factor XIa blocking antibody, there was no difference in inhibitory effect of PC/PS/PE or PC/PS vesicles. These data suggested that the differences in clot lysis inhibition observed between PC/PS/PE and PC/PS vesicles were caused by factor XI activation by meizothrombin. Meizothrombin-mediated factor XI activation may therefore play an important role in the antifibrinolytic feedback loop in the intrinsic pathway.

Fibrin-bound thrombin is protected from inactivation by antithrombin III, while its coagulant potential is retained. In the presence of heparin, ternary complexes between thrombin, fibrin and heparin are formed. In these complexes the coagulant activity of thrombin is retained, whereas the anticoagulant activity of fibrin-bound heparin is neutralized. The limited effectiveness of heparin in the prevention of both venous thrombosis and coronary reocclusion is probably related to the protective effect of fibrin on the inactivation of thrombin by anti-thrombin III. Recently, it has been shown that factor XI can be activated by thrombin, resulting in the generation of additional thrombin via the intrinsic pathway. This additional thrombin is capable of stabilizing the clot by protecting it from fibrinolysis. We studied the effect of heparin on the activation of factor XI by fibrin-bound thrombin. First, we used fibrin monomers coupled to Sepharose to which thrombin and unfractionated heparin (UFH) were bound. Factor XI activation by thrombin was the same in the presence of fibrin-Sepharose or control-Sepharose. The addition of heparin (0.1 U/ml) resulted in a 91 and 15-fold enhancement in the presence of control-Sepharose and fibrin-Sepharose, respectively. Next, we added complexes of heparin, thrombin and fibrin monomer to factor XII and XI double-deficient plasma in the presence or absence of a reconstituting amount of factor XI. In the presence of factor XI, additional fibrin formation was observed indicating that factor XI activation by thrombin in complex with fibrin and heparin can take place in plasma. We then studied the effect of other heparin-like anticoagulants on the thrombin-mediated factor XI activation. UFH enhanced thrombin-mediated factor XI activation 68-fold, LMWH (low molecular weight heparin, Fragmin) 12-fold, danaparoid (Orgaran) 3-fold, while the pentasaccharide ORG 31540 did not result in an enhancement. Binding studies of these anticoagulants to fibrin-Sepharose showed that LMWH bound with approximately the same affinity as UFH, while danaparoid and the pentasaccharide did not bind to fibrin. We conclude that fibrin-bound thrombin is capable of factor XI activation. Furthermore, heparin bound in a complex with fibrin can act as a cofactor for this activation. This factor XI activation capacity may play a role in the limited effectiveness of heparin. Provided that thrombin-mediated factor XI activation plays an important role in vivo, danaparoid and especially the pentasaccharide may be better anticoagulants than UFH and LMWH.

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes.(ABSTRACT TRUNCATED AT 250 WORDS)