Abstract
Procoagulant activities associated with human clots may contribute to thrombus extension. We investigate the inhibition of clot-associated factor Xa and thrombin activities by purified human antithrombin either alone or as combination with a low molecular weight heparin (enoxaparin) as compared with unfractionated heparin (UFH). The standard clots were prepared by recalcification of frozen platelet-poor human plasma. Clot-associated thrombin was measured on the clot after clot incubation in recalcified buffer or recalcified prothrombin solution. The enzymatic reaction was measured using a specific substrate for thrombin (CBS 3447). The thrombin concentration was determined both on the clots and in the reaction mixtures. In parallel, prothrombin fragment 1.2 and thrombin-antithrombin complexes (TAT) were measured using enzyme-linked immunosorbent assay methods. We demonstrated that in the presence of purified human prothrombin and antithrombin (AT), a partial inhibition of clot associated thrombin activity correlated with an increase of TAT complexes. However, antithrombin was unable to inhibit thrombin generation induced by the clot-associated factor Xa. Enoxaparin (low molecular weight heparin) and UFH did not enhance clot-bound thrombin inhibition induced by AT. We conclude that clot-bound thrombin is accessible to human antithrombin alone. AT is also able to inhibit thrombin generated by factor Xa-associated clot. However, neither a low molecular weight heparin or UFH enhanced the effect of AT alone.
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