Pharmacologic modulation of thrombin generation associated with human clots by human purified antithrombin alone or in the presence of low molecular weight heparin or unfractionated heparin

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.