Tissue Factor Mediated Activation of Prothrombin Complex Concentrates and its Inhibition by Antithrombin Drugs.

Surface enhanced laser desorption/ionization (SELDI) is a new technique used to investigate proteins, their up/down regulation during pathogenesis (biomarkers), cleavage products and protein/protein interactions. SELDI has been used to profile biomarkers in such diseases as cancer and AIDS. More recently this technique has also been used to investigate drug protein, receptor ligand and other interactions. SELDI can be used to monitor the activation and/or inhibition of enzymes that regulate important processes, such as coagulation and fibrinolysis. This application can be important in the development of new drugs used to treat thrombosis. Plasma samples obtained from patients treated with activated and non-activated forms of prothrombin complexes have provided specific biomarker profiles which can be further analyzed to understand the sites and the mechanism of the prohemostatic and activation profiles of the factor concentrates. This study was designed to determine if prothrombin complex (konyne) could be activated using tissue factor (TF) and if this activation could be differentially inhibited by anti-IIa drugs. This was accomplished by setting up the following reactions mixtures: TF alone; konyne alone; TF and konyne; TF, konyne and one of the anti-IIa drugs (argatroban, hirudin or angiomax). Saline was used to maintain the proteins at the same concentration in all reaction mixtures. TF was consistently added first followed by the test drug or saline and finally by konyne. The mixtures were incubated at 37°C for 15 minutes and then spotted on a strong anion exchange chip (SAX2). The chips were read using a PBSII system (Ciphergen, Freemont, CA). When konyne was added to TF a high intensity peak at 50 kDa was evident showing that konyne was activated by TF. It is speculated that the peak at 50 kDa may represent factor Xa. When argatroban was added to the reaction mixture at concentrations of 10 and 1 μg/ml, the peak at 50 kDa was not present. This indicates that argatroban is capable of inhibiting the formation of factor Xa thus it may interfere in the TF/VIIa mediated conversion of factor X to Xa. However, the addition of angiomax at the same concentrations did not cause the elimination of this peak suggesting that the mechanism of action of angiomax is somewhat different from argatroban. Hirudin showed some inhibition of the 50 kDa peak at 10 μg/ml. This suggests that hirudin is not as efficient in preventing the formation of the peak at 50 kDa. The inhibition of this peak may prove to be a specific mechanism of action of argatroban and to a lesser extent hirudin in the control of thrombogensis. Since prothrombin complex concentrates such as konyne are composed of purified factor II, VII, IX and X these provide a novel experimental setting to understand the TF mediated activation of prothrombin complexes and can be used in the understanding of the TF mediated activation process and thus inhibition by antithrombin/anti-Xa drugs. Other prothrombin complex concentrates such as proplex and FEIBA can also be used.