Two Independent Binding Sites on Monolayers of Human Endothelial Cells Are Responsible for Interaction with Coagulation Factor VII and Factor VIIa

 

PDF Download Buy Article Permissions and Reprints

Summary

The interaction of radiolabeled factor VII (FVII) and factor VIIa (FVIIa) with endotoxin-stimulated endothelial cells (EC), known to express tissue factor (TF), and unstimulated EC was studied. FVII/FVIIa binding to EC-monolayers was saturable within 4.5-6 h, reversible, temperature and calcium dependent on both, endotoxin-stimulated and on unstimulated EC. Upon 2 h of incubation on EC, FVII was partially converted to FVIIa in the absence of protease inhibitors. The affinity of this binding was K _d = 45.4 ± 18.7 nM with a calculated number of binding sites B _max = 3.75 ± 0.31 × 10⁶ molecules/cell. In addition to unlabeled FVII and FVIIa, other vitamin K-dependent proteins reduced binding of [¹²⁵I]-FVII/FVIIa to about 60-70%, and this type of common binding site for vitamin K-dependent proteins revealed a K _d = 32.2 ± 5.6 nM and a B _max = 3.03 ± 0.14 × 10⁶ molecules/cell. Moreover, in the presence of 1 μM prothrombin to suppress common binding sites, only on endotoxin-stimulated EC additional inhibition of FVII/FVIIa binding was achieved by anti-TF antibodies. The characteristics of the FVII/FVIIa-TF interaction with a K _d = 17.2 ± 5.2 nM and a B _max = 342,000 ± 1,100 binding sites/cell revealed a similar saturation kinetics in radioligand binding and in functional factor X activation within 90-120 min. These data indicate the presence of at least two independent binding sites for FVII/FVIIa on stimulated EC of which about 10% are TF specific. The existence of binding sites common for vitamin K-dependent proteins on both types of EC may improve the availability of FVII/FVIIa once EC become stimulated and express TF on their surface.

• References

• 1 Colucci M, Balconi G, Lorenzet R, Pietra A, Locati D, Donati MB, Semeraro N. Cultured human endothelial cells generate tissue factor in response to endotoxin. J Clin Invest 1983; 71: 1893-1896
  CrossrefPubMedGoogle Scholar
• 2 Brox JH, Osterud B, Björklid E, Fenton JW. Production and availability of thromboplastin in endothelial cells: the effect of thrombin, endotoxin and platelets. Br J Haematol 1984; 57: 239-246
  CrossrefPubMedGoogle Scholar
• 3 Rivers RPA, Hathaway WE, Weston WL. The endotoxin-induced coagulant activity of human monocytes. Br J Haematol 1975; 30: 311-316
  CrossrefPubMedGoogle Scholar
• 4 Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 1986; 163: 740-745
  CrossrefPubMedGoogle Scholar
• 5 Schorer AE, Kaplan ME, Rao GHR, Moldow CF. Interleukin-1 stimulates endothelial cell tissue factor production and expression by a prostaglandin-independent mechanism. Thromb Haemostas 1986; 56: 256-259
  PubMedGoogle Scholar
• 6 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
  CrossrefPubMedGoogle Scholar
• 7 Warr TA, Rao LVM, Rapaport SI. Disseminated intravascular coagulation in rabbits induced by administration of endotoxin or tissue factor: effect of anti-tissue factor antibodies and measurement of plasma extrinsic pathway inhibitor activity. Blood 1990; 75: 1481-1489
  PubMedGoogle Scholar
• 8 Edgington TS, Mackman N, Brand K, Ruf W. The structural biology of expression and function of tissue factor. Thromb Haemostas 1991; 66: 67-79
  PubMedGoogle Scholar
• 9 Bach RR. Initiation of coagulation by tissue factor. CRC C R Biochem 1988; 23: 339-368
  PubMedGoogle Scholar
• 10 Scarpati EM, Sadler JE. Regulation of endothelial cell coagulant properties. J Biol Chem 1989; 264: 20705-20713
  PubMedGoogle Scholar
• 11 Bach R, Gentry R, Nemerson Y. Factor VII binding to tissue factor in reconstituted phospholipid vesicles: Induction of cooperativity by phosphatidylserine. Biochemistry 1986; 25: 4007-4020
  CrossrefPubMedGoogle Scholar
• 12 Fair DS, MacDonald MJ. Cooperative interaction between factor VII and cell surface-expressed tissue factor. J Biol Chem 1987; 262: 11692-11698
  PubMedGoogle Scholar
• 13 Sakai T, Lund-Hansen T, Paborsky L, Pedersen AH, Kisiel W. Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation. J Biol Chem 1989; 264: 9980-9988
  PubMedGoogle Scholar
• 14 Broze GJ. Binding of human factor VII and VIIa to monocytes. J Clin Invest 1982; 70: 526-535
  CrossrefPubMedGoogle Scholar
• 15 Osterud B, Björklid E. Human factor VII associated with endotoxin-stimulated monocytes in whole blood. Biochem Biophys Res Commun 1982; 108: 620-626
  CrossrefPubMedGoogle Scholar
• 16 Ruf W, Edgington TS. Two sites in the tissue factor extra-cellular domain mediate the recognition of the ligand factor VIIa. Proc Natl Acad Sci 1991; 88: 8430-8434
  CrossrefPubMedGoogle Scholar
• 17 Fraker PJ, Speck Jr JC. Protein and cell membrane iodination with sparingly soluble chloramine, 1,3,4,6-tetrachloro-3a,6a-diphenyl glycouril. Biochem Biophys Res Commun 1978; 80: 849-857
  CrossrefPubMedGoogle Scholar
• 18 Preissner KT, Wassmuth R, Müller-Berghaus G. Physicochemical characterization of human S-protein and its function in the blood coagulation system. Biochem J 1985; 231: 349-355
  CrossrefPubMedGoogle Scholar
• 19 Preissner KT, Delvos U, Müller-Berghaus G. Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism. Biochemistry 1987; 26: 2521-2528
  CrossrefPubMedGoogle Scholar
• 20 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
  CrossrefPubMedGoogle Scholar
• 21 Tanaka H, Janssen B, Preissner KT, Müller-Berghaus G. Purification of glycosylated apoprotein of tissue factor from human brain and inhibition of its procoagulant activity by a specific antibody. Thromb Res 1985; 40: 745-756
  CrossrefPubMedGoogle Scholar
• 22 Born VJJ, Ram IE, Alderkamp GHJ, Reinaldapoot HH, Bertina RM. Application of factor VII-sepharose affinity chromatography in purification of human tissue factor apoprotein. Thromb Res 1986; 42: 635-643
  CrossrefPubMedGoogle Scholar
• 23 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-2756
  CrossrefPubMedGoogle Scholar
• 24 Klagsbrun M, Sullivan R, Smith S, Rybka R, Shing Y. Purification of endothelial cell growth factors by heparin affinity chromatography. Methods Enzymol 1987; 147: 95-105
  PubMedGoogle Scholar
• 25 Wroblewski F, LaDue JS. Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med 1955; 90: 210-215
  CrossrefPubMedGoogle Scholar
• 26 Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672
  CrossrefPubMedGoogle Scholar
• 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
  CrossrefPubMedGoogle Scholar
• 28 Merill CR, Switzer RC, Van Keuren ML. Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and highly sensitive silver stain. Proc Natl Acad Sci USA 1979; 76: 4335-4339
  CrossrefPubMedGoogle Scholar
• 29 Ploplis VA, Edgington TS, Fair DS. Initiation of the extrinsic pathway of coagulation. J Biol Chem 1987; 262: 9503-9508
  PubMedGoogle Scholar
• 30 Sakai T, Kisiel W. Binding of human factor X and Xa to HepG2 and J82 human tumor cell lines. Evidence that FXa binds to tumor cells independent of factor Va. J Biol Chem 1990; 265: 9105-9113
  PubMedGoogle Scholar
• 31 Heimark RL, Schwartz SM. Binding of coagulation factor IX and X to the endothelial cell surface. Biochem Biophys Res Commun 1983; 111: 723-731
  CrossrefPubMedGoogle Scholar
• 32 Rodgers GM, Shuman MA. Characterisation of the interaction between factor Xa and bovine aortic endothelial cells. Biochim Biophys Acta 1985; 844: 320-329
  CrossrefPubMedGoogle Scholar
• 33 Stern MS, Nawroth PP, Kisiel W, Vehar G, Esmon CT. The binding of factor IXa to cultured bovine endothelial cells. J Biol Chem 1985; 260: 6717-6722
  PubMedGoogle Scholar
• 34 Ruf W, Rehemtulla A, Edgington TS. Phospholipid-independent and dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem 1991; 265: 2158-2166
  PubMedGoogle Scholar
• 35 Toomey JR, Smith KJ, Stafford DW. Localization of the human tissue factor recognition determinant of human factor VIIa. J Biol Chem 1991; 266: 19198-19202
  PubMedGoogle Scholar
• 36 Ruf W, Kalnik MW, Lund-Hansen T, Edgington TS. Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions. J Biol Chem 1991; 266: 15719-15725
  PubMedGoogle Scholar
• 37 Sakai T, Lund-Hansen T, Thim L, Kisiel W. The ϒ-carboxyglutamic acid domain of human factor VIIa is essential for its interaction with cell surface tissue factor. J Biol Chem 1990; 265: 1890-1894
  PubMedGoogle Scholar
• 38 Rodgers GM, Broze Jr GJ, Shuman MA. The number of receptors for factor VII correlates with the ability of cultured cells to initiate coagulation. Blood 1984; 63: 434-438
  PubMedGoogle Scholar
• 39 Nemerson Y, Repke D. Tissue factor accelerates the activation of coagulation factor VII: the role of a bifunctional coagulation cofactor. Thromb Res 1985; 40: 351-358
  CrossrefPubMedGoogle Scholar
• 40 Rao LVM, Rapaport SI. Activation of factor VII bound to tissue factor: A key early step in the tissue factor pathway of coagulation. Proc Natl Acad Sci USA 1988; 85: 6687-6691
  CrossrefPubMedGoogle Scholar
• 41 Broze 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: Insight into its possible mechanism of action. Blood 1988; 71: 335-343
  PubMedGoogle Scholar
• 42 Warn-Cramer BJ, Almus FE, Rapaport SI. Studies on the factor Xa-dependent inhibitor of factor VIIa/tissue factor (extrinsic pathway inhibitor) from cell supernates of cultured human umbilical vein endothelial cells. Thromb Haemostas 1989; 61: 101-105
  PubMedGoogle Scholar