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

Abstract

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, Arg²⁸⁶-Thr²⁸⁷, and Arg³²²-IIe³²³ were cleaved, yielding and releasing fragment 1-2 and a degraded form of α-thrombin, but not meizothrombin, into the fluid phase. The apparent K_m for prothrombin and the V_max 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 (k_cat=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 γ-carboxyglutamic 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.