Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin.
Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.
D000990:Antithrombin III / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives; D001777:Blood Coagulation; D003619:Dansyl Compounds; D004789:Enzyme Activation; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D006493:Heparin; D006801:Humans; D007020:Hypoprothrombinemias / Q000097:blood / Q000235:genetics*; D007700:Kinetics; D010974:Platelet Aggregation; D011516:Prothrombin / Q000235:genetics* / Q000302:isolation & purification; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D003619:Dansyl Compounds; C040593:prothrombin Metz; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D000990:Antithrombin III; D011516:Prothrombin; D006493:Heparin; C040592:thrombin Metz; D001120:Arginine; D013917:Thrombin