Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

Summary

Cultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with ¹²⁵I-factor Xa as the ligand.

Keywords

Extrinsic pathway inhibitor - Tissue factor - Factor VIIa - Factor Xa - Human endothelium

• References

• 1 Sanders NL, Bajaj SP, Zivelin A, Rapaport SI. Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component. Blood 1985; 66: 204-212
• 2 Rao LV M, Rapaport SI. Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation. Blood 1987; 69: 645-651
• 3 Broze Jr GJ, Miletich JP. Characterization of the inhibition of tissue factor in serum. Blood 1987; 69: 150-155
• 4 Broze Jr GJ, Warren LA, Novotny WF, Higuchi DA, Girard JJ, Miletich JP. The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: Insights into its possible mechanism of action. Blood 1988; 71: 335-343
• 5 Wun TC, Kretzmer KK, Girard TJ, Miletich JP, Broze Jr GJ. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three Kunitz- type inhibitory domains. J Biol Chem 1988; 263: 6001-6004
• 6 Broze Jr GJ, Miletich JP. Isolation of the tissue factor inhibitor produced by Hep G2 hepatoma cells. Proc Natl Acad Sci USA 1987; 84: 1886-1890
• 7 Bajaj MS, Rana SV, Wysolmerski RB, Bajaj SP. Inhibitor of the factor VIIa-tissue factor complex is reduced in patients with disseminated intravascular coagulation but not in patients with severe hepatocellular disease. J Clin Invest 1987; 79: 1874-1878
• 8 Rana SV, Reimers HJ, Pathikonda MS, Bajaj SP. Expression of tissue factor and factor VIIa/tissue factor inhibitor activity in endotoxin or phorbol stimulated U937 monocytelike cells. Blood 1988; 71: 256-262
• 9 Balconi G, Dejana E. Cultivation of endothelial cells: Limitations and perspectives. Med Biology 1986; 64: 231-245
• 10 Rao LV M, Bajaj SP. Purification of human factor VII utilizing O-(Diethylaminoethyl)-Sephadex and Sulfopropyl Sephadex chromatography. Anal Biochem 1984; 136: 357-361
• 11 Bajaj SP, Rapaport SI, Prodanos C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem 1981; 11: 397-412
• 12 Usharani P, Wam-Cramer BJ, Kasper CK, Bajaj SP. Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. J Clin Invest 1985; 75: 76-83
• 13 Rao LV M, Rapaport SI. Affinity purification of human brain tissue factor utilizing factor VII bound to immobilized anti-factor VII. Anal Biochem 1987; 165: 365-370
• 14 Wam-Cramer BJ, Maki SL, Zivelin A, Rapaport SI. Partial purification and characterization of extrinsic pathway inhibitor (the factor Xa-dependent plasma inhibitor of factor VIIa/tissue factor). Thromb Res 1987; 48: 11-22
• 15 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-2756
• 16 Almus FE, Rao LV M, Rapaport SI. Decreased inducibility of tissue factor activity on human umbilical vein endothelial cells cultured with endothelial cell growth factor and heparin. Thromb Res 1988; 50: 339-344
• 17 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 18 Jaffe EA. Cell biology of endothelial cells. Hum Pathol 1987; 18: 234-239
• 19 Warr TA, Wam-Cramer BJ, Rao LV M, Rapaport SI. Physiologic variation in extrinsic pathway inhibitor. Arteriosclerosis 1988; 8: 681a
• 20 Brox JH, Østerud B, Bjorklid E, Fenton II JW. Production and availability of thromboplastin in endothelial cells. The effects of thrombin, endotoxin and platelets Br J Haematol 1984; 57: 239-246
• 21 Gaidai KS. Thromboplastin synthesis in endothelial cells. Haemostasis 1984; 14: 378-385
• 22 Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest 1987; 79: 124-130