ALBERT

Description: Human prothrombinase assembled on synthetic membranes composed of phosphatidylcholine and phosphatidylserine (PCPS) catalyzes thrombin formation almost exclusively by sequential cleavage of prothrombin at Arg320 to yield the protease meizothrombin (mIIa) as an intermediate which is then further cleaved at Arg271. This cleavage pathway arises because Arg320 in intact prothrombin is cleaved ∼30-fold faster than Arg271. When prothrombin lacks γ-carboxyglutamic acid modifications (desGlaII), product formation is modestly decreased but bond cleavage largely occurs in the opposite order, yielding the zymogen, prethrombin 2 (P2), as an intermediate. This results from a reduction in the rate of cleavage at Arg320 in intact prothrombin and a partly compensating gain of function in cleavage at Arg271. Thus, membrane binding and/or other interactions mediated by γ-carboxyglutamic acids in the substrate play a major role in modulating the pathway for cleavage and whether the intermediate is a zymogen or protease. We now extend these approaches to evaluate prothrombinase function on activated platelets and human umbilical vein endothelial cells (HUVECs) at the physiologic concentration of prothrombin. Prothrombin activation was detected by measuring the appearance of product(s) with proteolytic activity and by analysis of the cleavage process by SDS-PAGE and western blot analysis quantitatively imaged by infrared fluorescence. This approach was validated by documenting equivalence in the progress curves obtained by western blot imaging or following staining of total protein in a purified system with PCPS membranes. Prothrombin cleavage was assessed using human platelets (108/ml), activated with thrombin and studied with saturating concentrations of Va and a limiting concentration of Xa. The pattern of prothrombin cleavage, intermediate accumulation and product formation was clearly distinct from that observed with PCPS membranes, indicating substantial flux towards thrombin formation via initial cleavage at Arg271 followed by cleavage at Arg320 producing the zymogen P2 as an intermediate. Transient formation of mIIa from initial cleavage at Arg320 was undetectable. Thus, prothrombinase assembled on thrombin activated platelets cleaves prothrombin in a way that is reminiscent of the cleavage of desGlaII rather than fully carboxylated prothrombin seen with PCPS membranes. In contrast, equivalent studies with prothrombinase assembled on thrombin activated HUVECs produced cleavage patterns consistent with significant flux towards thrombin formation via initial cleavage of prothrombin at Arg320 yielding mIIa as an intermediate. The use of desGlaII with HUVECs yielded lower rates and cleavage patterns similar to those obtained with fully carboxylated prothrombin in the platelet reactions. Our results document that the cleavage pathway for thrombin formation is dependent on cell type. Because mIIa is a protease with a different spectrum of activities from thrombin, its formation restricted to the vessel wall suggests an important regulatory role for the modulation of the pathway of prothrombin cleavage. Such bimodal regulation of the pathway for prothrombin cleavage on HUVECs and platelets suggests differential roles of prothrombinase assembled on platelets versus endothelium in regulating the hemostatic response to vascular injury.