Activation of Prothrombin by Factor Xa Bound to the Membrane Surface of Human Umbilical Vein Endothelial Cells: Its Catalytic Efficiency Is Similar to That of Prothrombinase Complex on Platelets
Upon incubation of human prothrombin with factor Xa bound to human umbilical vein endothelial cells (HUVEC) (0.5–0.6 fmol factor Xa/105 cells), three bonds at Arg273 Thr274 and Arg322−Ile323 were cleaved, yielding and releasing fragment 1–2 and a degraded form of α-thrombin, but not meizothrombin, into the fluid phase. The apparent Km for prothrombin and the Vmax were 0.25± 0.07 μM and 210± 40 fmol thrombin/min/105 cells, respectively. For the maximally bound factor Xa, the calculated catalytic efficiency (kcat=6–7 s−1) was similar to those reported for the prothrombinase complex formed on the phospholipid vesicles and natural membrane surfaces. The prothrombin derivatives lacking the 10-carboxygulutamic acid (Gla) residues-containing region were not activated by the cell-bound factor Xa. The activation rate of prothrombins with Gla residues variously modified to γ-methyleneglutamic acids was reduced in accordance with the number of modified residues. For the inhibition of prothrombin activation, intact fragment 1 was needed; the Gla-domain alone did not affect the reaction. Binding of monoclonal antibodies to the region of 1–48 or the kringle 1 region of prothrombin also interfered with the prothrombin activation. Prothrombin activation on the surface of HUVEC appeared to proceed via formation of a cellular prothrombinase complex composed of phospholipids of HUVEC membrane, endogenous factor Va, factor Xa, and prothrombin. The Gla-domain and kringle 1 regions are indispensable for the molecule to serve as an effective substrate for the cell-bound factor Xa.