Down Regulation of Thrombin Procoagulant Activity by Prothrombin Activation Fragment 1.2.

Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Under physiological condition, prothrombin activation catalyzed by the fully assembled prothrombinase complex via an ordered, sequential reaction with primary cleavage at Arg320-lle321 followed by the second cleavage at Arg271-Thr272, forming fragment 1.2 and thrombin. The fragment 1.2 contains the amino-terminal gla domain and the two kringle domains. It has been shown that the kringle 2 domain in fragment 2 can bind to the exosite II of thrombin and inhibit allosterically its activity by inducing conformational changes at the active site. Nascent thrombin that is generated on platelet surface will remains non-covalenty bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate thrombin activity, we tested the effect of anionic phospholipid-bound fragment 1.2 (0–10 μM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on esterase activity towards S2238 (D-Phe-Pip-Arg-pNA.2HCI) suggesting a competitive inhibition of fibrinogen binding. We also labeled thrombin at the active site with fluorescein-FPRCK (5-fluorescein-D-Phe-Pro-Arg chloromethyl-ketone) and monitored fluorescence changes following fragment 1.2 binding in a spectrofluorometer. Anionic phospholipid-bound fragment 1.2 induced changes in the active site similar to fragment 2 with half maximal effect at ~8 μM. We tested the effect of fragment 1.2 on fluorescein-FPR thrombin binding to platelets. Fragment 1.2 inhibited thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that membrane-bound prothrombin fragment 1.2 may play an important role in hemostasis by down regulating thrombin procoagulant activity because of its interaction with exosite 2.