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

J Biochem. 1995 Feb;117(2):244-50. doi: 10.1093/jb/117.2.244.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Division
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / metabolism*
  • Enzyme Activation
  • Factor Xa / metabolism*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Peptide Fragments / pharmacology
  • Prothrombin / metabolism*
  • Substrate Specificity
  • Thrombin / metabolism*
  • Umbilical Veins

Substances

  • Peptide Fragments
  • Prothrombin
  • Thrombin
  • Factor Xa