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/10(5) cells), three bonds at Arg273-Thr274, Arg286-Thr287, and Arg322-Ile323 were cleaved, yielding and releasing fragment 1-2 and a degraded form of alpha-thrombin, but not meizothrombin, into the fluid phase. The apparent Km for prothrombin and the Vmax were 0.25 +/- 0.07 microM and 210 +/- 40 fmol thrombin/min/10(5) 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 gamma-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 gamma-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.
Sugo
T
T
Institute of Hematology, Jichi Medical School, Tochigi.
Nakamikawa
C
C
Tanabe
S
S
Matsuda
M
M
D000595:Amino Acid Sequence; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D011516:Prothrombin / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*; D014471:Umbilical Veins
D010446:Peptide Fragments; D011516:Prothrombin; D013917:Thrombin; D015951:Factor Xa