Exploring potential anticoagulant drug formulations using thrombin generation test

Abstract

Many anticoagulant drugs inhibiting proteins of the coagulation cascade have been developed. The main targets of anticoagulant drugs are thrombin and factor Xa; inhibiting these factors delays thrombus growth, thus preventing thrombosis while increasing bleeding risk. A balance between thrombosis and bleeding is ensured in the 'therapeutic window' of the anticoagulant drug concentration range. Novel anticoagulant drugs and combinations thereof are being developed. We rank coagulation factors as potential anticoagulant drug targets in combination with thrombin inhibitors, aptamer HD1 and bivalirudin, providing a background for several promising dual target treatment strategies. The thrombin generation test was used to assess the whole coagulation cascade in normal and factor-deficient human blood plasma. Potential therapeutic windows were estimated for coagulation factors, ranking them as targets for anticoagulant drugs. Thrombin and factor Xa have been revealed as the most promising targets, which fully agrees with the current drug development strategy. Inhibitors of factors Va and VIIa are expected to have narrow therapeutic windows. Inhibitors of factors VIIIa and IXa are expected to have a moderate anticoagulant effect. Factors XI and XII are poor targets for anticoagulant drugs. Compared with plasma that is deficient in factor II, the thrombin inhibitors bivalirudin and aptamer HD1 had increased activity. Both inhibitors were tested in deficient plasma providing a model of potential drug combination. The most promising combinations were anti-thrombin with anti-V/Va and also anti-thrombin with anti-IX/IXa. Each combination had an incremental dose-effect dependence that is promising from the standpoint of the therapeutic window.

Keywords: Anticoagulant; Bivalirudin; Coagulation cascade; DNA aptamer; Thrombin.

Fig. 1

The coagulation cascade is a sophisticated regulatory network controlling the formation of fibrin fiber. Tissue factor triggers coagulation under vessel damage, whereas factor XII induces supplementary contact activation intrinsic pathway. HK – high molecular weight kininogen, PL – phospholipid surface, TM – thrombomodulin, APC – activated protein C, TFPI – tissue factor pathway inhibitor. The scheme is derived from a diagram from Enzyme Research Laboratories .

Fig. 2

Thrombin generation in normal and deficient plasma. A – TGT parameters; B – thrombin generation in normal, factor II-, factor V-, and factor VII-deficient plasma; C – thrombin generation in normal, factor VIII-, factor IX-, and factor X-deficient plasma; D – thrombin generation in normal, factor XI-, and factor XII-deficient plasma.

Fig. 3

Time to peak and peak thrombin concentration in plasma with different factor content: A – factor II, B – factor V, C – factor VII, and D – factor X deficient plasma (no peak – time to peak exceeds 240 min).

Fig. 4

Time to peak and peak thrombin concentration in plasma with different factor content: A – factor VIII-, B – factor IX-, C – factor XI-, and D – factor XII-deficient plasma.

Fig. 5

Thrombin generation in normal plasma in the presence of thrombin inhibitors: A, B – bivalirudin; C, D – aptamer HD1.

Fig. 6

Thrombin generation inhibited with bivalirudin and aptamer HD1 with different factor contents: A – factor II-, B – factor V-, C – factor VII-, and D – factor X-deficient plasma (no peak – time to peak exceeds 240 min).

Fig. 7

Thrombin generation inhibited with bivalirudin and aptamer HD1 with different factor content: A – factor VIII-, B – factor IX-, C – factor XI-, and D – factor XII-deficient plasma.