No abstract

Clot-associated prothrombinase and thrombin activities may contribute to thrombus extension after thrombolytic and anticoagulant treatment. We studied prothrombin activation after adding human purified prothrombin to human clot. By using two different drugs with an exclusive direct anti-activated factor X activity (DX9065a) or anti-activated factor II activity (r-hirudin), we tried to determine whether clot-bound thrombin and prothrombinase could be inhibited in our experimental system when human purified prothrombin was added. Standard clots were prepared from platelet-poor human plasma after addition of calcium. We measured clot-bound thrombin or free thrombin using a direct simple chromogenic assay. In parallel, prothrombin fragment 1+2 measurement was used to monitor prothrombin activation. For this, two protocols were used. We introduced the direct inhibitors before starting the activation process (protocol A) or at the time of the activation process (protocol B). We found a direct correlation between thrombin generation and prothrombin fragment 1+2 with an increase of thrombin activity on clots and in the incubation mixtures when clots were incubated in human purified pothrombin alone. Two protocols were used: in the first, clots were pre-incubated in presence of drugs before adding prothrombin; and in the second, clots were incubated in the presence of prothrombin and drugs. Prothrombin activation was not inhibited when clots were incubated with r-hirudin and consequently thrombin generation still occurred. However, added r-hirudin blocks thrombin activity on the clots and in the incubation mixture, but does not prevent prothrombin activation, as shown by the increase of prothrombin fragment 1+2. In contrast, DX9065a did not suppress clot-bound thrombin. However, DX9065a blocks prothrombin activation whichever protocol was used. The results show that hirudin is a poor inhibitor of thrombin generation in contrast to DX9065a. On the other hand, DX9065a cannot inhibit thrombin bound to clot in contrast to hirudin.

No abstract

The procoagulant activity of a thrombus is essentially due to clot-associated factor IIa and factor Xa activities.The aim of this review is to underline that specific antithrombin and anti-Xa drugs, such as r-hirudin and DX 9065a, respectively, are complementary, and could be used in combination in clinical trials in patients with acute arterial thrombosis such as coronary syndromes. After standardization of the in-vitro techniques for clot-bound thrombin and clot-associated factor Xa, we have studied the anticoagulant effect of unfractionated heparin and a low-molecular heparin in an in-vitro model. We have confirmed the inability of heparins to inhibit clot-bound thrombin and clot-associated factor Xa. We have compared r-hirudin, a direct thrombin inhibitor, with DX9065a, a direct factor Xa inhibitor. We have observed that r-hirudin inhibited clot-bound thrombin but not clot-bound factor Xa. After r-hirudin treatment interruption, a hypercoagulation rebound has been reported and it could be in relation with the persistence of factor Xa activity in the clot. We have demonstrated that DX9065a inhibits clot-bound factor Xa but does not inhibit clot-bound factor IIa. The complementary effect of DX9065a and r-hirudin is demonstrated in this experimental model. It is likely that several other combinations of drugs may also exhibit an increase of the antithrombotic activity which could be of interest in clinical implication for the treatment of several groups of patients at high risk of arterial thrombosis.

No abstract