Download PDF
Thromb Haemost 1994; 72(03): 465-474
DOI: 10.1055/s-0038-1648890
Original Article
Schattauer GmbH Stuttgart

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

Independent of Protein S and Distinct from Known Ligands
Neelesh Bangalore
The Plasma Derivatives Laboratory, American Red Cross Holland Laboratory, Rockville, Maryland, USA
,
William N Drohan
The Plasma Derivatives Laboratory, American Red Cross Holland Laboratory, Rockville, Maryland, USA
,
Carolyn L Orthner
The Plasma Derivatives Laboratory, American Red Cross Holland Laboratory, Rockville, Maryland, USA
› Author Affiliations
Further Information

Publication History

Received 03 February 1994

Accepted after resubmission 26 May 1994

Publication Date:
25 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Activated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

 

  • References

  • 1 Stenflo J. A new vitamin K-dependent protein. J Biol Chem 1976; 251: 355-363
    PubMedGoogle Scholar
  • 2 Kisiel W. Human plasma protein C. Isolation, characterization, and mechanism of activation by thrombin. J Clin Invest 1979; 64: 761-769
    CrossrefPubMedGoogle Scholar
  • 3 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-2252
    CrossrefPubMedGoogle Scholar
  • 4 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
    PubMedGoogle Scholar
  • 5 Walker FJ, Sexton PW, Esmon CT. The inactivation of blood coagulation by APC through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-342
    CrossrefPubMedGoogle Scholar
  • 6 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-489
    PubMedGoogle Scholar
  • 7 Taylor Jr FB, Lockhart MS. Whole blood clot lysis: In vitro modulation by activated protein C. Thromb Res 1985; 37: 639-649
    CrossrefPubMedGoogle Scholar
  • 8 DeFouw NJ, Haverkate F, Bertina RM, Koopman J, van Wijngaarden A, van Hinsberg VWM. The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro. Blood 1986; 67: 1189-1192
    PubMedGoogle Scholar
  • 9 Taylor FB, Chang A, Esmon CT, D’Angelo A, Vigano-D’Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-925
    CrossrefPubMedGoogle Scholar
  • 10 Branson HE, Katz J, Marble R, Griffin JH. Inherited protein C deficiency in a coumarin-responsive chronic relapsing purpura fulminans syndrome in a neonate. Lancet 1983; 2: 1165-1167
    PubMedGoogle Scholar
  • 11 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373
    CrossrefPubMedGoogle Scholar
  • 12 Broekmans AW, Veltkamp JJ, Bertina RM. Congenital protein C deficiency and venous thromboembolism: A study of three Dutch families. N Eng J Med 1983; 309: 340-344
    CrossrefPubMedGoogle Scholar
  • 13 Griffin JH, Mosher DF, Zimmerman TS, Kleiss AJ, Protein C. an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood 1982; 60: 261-264
    PubMedGoogle Scholar
  • 14 Mannucci PM, Vigano S. Deficiencies of protein C, an inhibitor of blood coagulation. Lancet 1982; 463
    PubMedGoogle Scholar
  • 15 Gruber A, Griffin JH, Harker LA, Hanson SR. Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model. Blood 1989; 73: 639-742
    PubMedGoogle Scholar
  • 16 Orthner CL, Ralston AH, McGriff JD, Gee DM, Drohan WN. Large scale purification and preclinical studies of activated protein C (APC) as an antithrombotic agent. Blood 1990; 76: 517a (Abst.)
    PubMedGoogle Scholar
  • 17 Okajima K, Koga S, Kaji M, Inoue M, Nakagaki T, Funatsu A, Okabe H, Takatuki K, Aoki N. Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects. Thromb Haemost 1990; 63: 48-53
    Article in Thieme ConnectPubMedGoogle Scholar
  • 18 Suzuki K, Nishioka J, Kusumoto H, Hashimoto S. Mechanism of inhibition of activated protein C by protein C inhibitor. J Biochem 1984; 95: 187-195
    CrossrefPubMedGoogle Scholar
  • 19 Heeb MJ, Griffin JH. Physiologic inhibition of human activated protein C by a,-antitrypsin. J Biol Chem 1988; 263: 11613-11616
    PubMedGoogle Scholar
  • 20 Heeb MJ, Mosher D, Griffin JH. Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation. Blood 1989; 73: 455-461
    PubMedGoogle Scholar
  • 21 DeFouw NJ, van Hinsbergh VWM, de Jong YF, Haverkate F, Bertina RM. The interaction of activated protein C and thrombin with plasminogen activator inhibitor released from human endothelial cells. Thromb Haemost 1987; 57: 176-182
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Heeb JH, Gruber A, Griffin JH. Identification of divalent metal ion-dependent inhibition of activated protein C by a2-macroglobulin and a2-antiplas-min in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin. J Biol Chem 1991; 266: 17606-17612
    PubMedGoogle Scholar
  • 23 Berger Jr H, Kirstein CG, Orthner CL. Pharmacokinetics of activated protein C in guinea pigs. Blood 1991; 77: 2174-2184
    PubMedGoogle Scholar
  • 24 Hoogendoorn H, Toh CH, Nesheim ME, Giles AR. a2-Macroglobulin binds and inhibits activated protein C. Blood 1991; 78: 2283-2290
    PubMedGoogle Scholar
  • 25 Travis J, Salvesan G. Human plasma proteinase inhibitors. Annu Rev Biochem 1983; 52: 655-709
    CrossrefPubMedGoogle Scholar
  • 26 Perlmutter DH, Glover GI, Rivetna M, Schasteen CS, Fallon R. Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes. Proc Natl Acad Sci USA 1990; 87: 3753-3757
    CrossrefPubMedGoogle Scholar
  • 27 Orthner CL, Madurawe RD, Velander WH, Drohan WN, Battey FD, Strickland DK. Conformational changes in an epitope localized to the N-terminal region of protein C: Evidence for interaction of protein C domains. J Biol Chem 1989; 264: 18781-18788
    PubMedGoogle Scholar
  • 28 Tharakan J, Strickland D, Burgess W, Drohan WN, Clark DB. Development of an immunoaffinity process for factor IX purification. Vox Sang 1990; 58: 21-27
    CrossrefPubMedGoogle Scholar
  • 29 Miekka SI, Ingham KC, Menache D. Rapid method for isolation of human plasma fibronectin. Thromb Research 1982; 27: 1-14
    CrossrefPubMedGoogle Scholar
  • 30 Seegers WH, McCoy L, Kipfer RK, Murano G. Preparation and properties of thrombin. Arch Biochem Biophysics 1968; 128: 194-201
    CrossrefPubMedGoogle Scholar
  • 31 Wickerhauser M, Williams C. A single step method for the isolation of antithrombin III (Abstract). Thromb Hemost 1983; 50: 119
    PubMedGoogle Scholar
  • 32 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 33 Dahlback B, Lundwall A, Stenflo J. Localization of thrombin cleavage sites in the aminoterminal region of bovine protein S. J Biol Chem 1986; 261: 5111-5115
    PubMedGoogle Scholar
  • 34 Conway EM, Nowakowski B, Steiner-Mosonyi M. Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Blood 1992; 80: 1254-1263
    PubMedGoogle Scholar
  • 35 Esmon NL, DeBault LE, Esmon CT. Proteolytic formation and properties of T-carboxyglutamic acid-domainless protein C. J Biol Chem 1983; 256: 5548-5553
    PubMedGoogle Scholar
  • 36 Orthner CL, Bhattacharya P, Strickland DK. Characterization of a protein C activator from the venom of Agkistrodon contortrix contortrix. Biochemistry 1988; 27: 2558-2564
    CrossrefPubMedGoogle Scholar
  • 37 Markwell MAK. A new solid state reagent to iodinate proteins: Conditions for efficient labeling of antiserum. Anal Biochem 1982; 125: 427-432
    CrossrefPubMedGoogle Scholar
  • 38 Orthner CL, Highsmith FA, Tharakan JP, Madurawe RD, Morcol T, Velander WH. Comparison of the performance of immunosorbents prepared by site-directed or random coupling of monoclonal antibodies. J Chromatogr 1991; 558: 55-70
    CrossrefPubMedGoogle Scholar
  • 39 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-2756
    CrossrefPubMedGoogle Scholar
  • 40 Schwartz AL, Fridovich SE, Knowles BB, Lodish HF. Characterization of the asialoglycoprotein receptor in a continuous hepatoma cell line. J Biol Chem 1981; 256: 8878-8881
    PubMedGoogle Scholar
  • 41 Rubin H, Xu K. Evidence for the progressive and adaptive nature of spontaneous transformation in the NIH/3T3 cell line. Proc Natl Acad Sci USA 1989; 86: 1860-1864
    CrossrefPubMedGoogle Scholar
  • 42 Rijken DC, Wijngaards C, Zaal-de-Jong M, Welbergen J. Purification and partial characterization of plasminogen activator from human uterine tissue. Biochim Biophys Acta 1979; 580: 140-153
    CrossrefPubMedGoogle Scholar
  • 43 Okuda K, Druker B, Kanakura Y, Koenigsman M, Griffin JD. Internalization of the granulocyte-macrophage colony-stimulating factor receptor is not required for the induction of protein tyrosine phosphorylation in the human myeloma cells. Blood 1991; 78: 1928-1935
    PubMedGoogle Scholar
  • 44 Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. Endotoxin enhances tissue factor and suppresses thrombomodulin expression on human vascular endothelial cells in vitro. J Clin Invest 1987; 79: 124-130
    CrossrefPubMedGoogle Scholar
  • 45 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
  • 46 Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1959; 51: 660-672
    PubMedGoogle Scholar
  • 47 Nawroth PP, Stem DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 1986; 163: 740-745
    CrossrefPubMedGoogle Scholar
  • 48 Dixon M, Webb EC. Enzymes. Academic Press 1979; 60-61
    PubMedGoogle Scholar
  • 49 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16
    PubMedGoogle Scholar
  • 50 Nelsestuen GL, Kisiel W, DiScipio RG. Interaction of vitamin K dependent proteins with membranes. Biochemistry 1978; 17: 2134-2138
    CrossrefPubMedGoogle Scholar
  • 51 Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-4677
    CrossrefPubMedGoogle Scholar
  • 52 Miletich JP, Broze Jr GJ. (3 protein C is not glycosylated at asparagine 329. J Biol Chem 1990; 265: 11397-11404
    PubMedGoogle Scholar
  • 53 Yan SCB, Razzano P, Chao YB, Walls JD, Berg DT, McClure DB, Grinnell BW. Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Bio/Technology 1990; 8: 655-661
    CrossrefPubMedGoogle Scholar
  • 54 Stubbs JD, Lekutis C, Singer KL, Bui A, Yuzuki D, Srinivasan U, Parry G. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences. Proc Natl Acad Sci USA 1990; 87: 8417-8421
    CrossrefPubMedGoogle Scholar
  • 55 Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. J Biol Chem 1985; 260: 15432-15438
    PubMedGoogle Scholar
  • 56 Hogg PJ, Ohlin AK, Stenflo J. Identification of structural domains in protein C involved in its interaction with thrombin-thrombomodulin on the surface of endothelial cells. J Biol Chem 1991; 267: 703-706
    PubMedGoogle Scholar
  • 57 Fair DS, Marlar RA, Levin EG. Human endothelial cells synthesize protein. S. Blood 1986; 67: 1168-1171
    PubMedGoogle Scholar
  • 58 Tanabe S, Sugo T, Matsuda M. Synthesis of protein C in human umbilical vein endothelial cells. J Biochem 1991; 109: 924-928
    CrossrefPubMedGoogle Scholar
  • 59 Harris KW, Esmon CT. Protein S is required for bovine platelets to support activated protein C binding and activity. J Biol Chem 1985; 260: 2007-2010
    PubMedGoogle Scholar
  • 60 Stem DM, Nawroth PP, Harris K, Esmon CT. Cultured bovine aortic endothelial cells promote activated protein C-protein S-mediated inactivation of factor Va. J Biol Chem 1986; 261: 713-718
    PubMedGoogle Scholar
  • 61 Solymoss S, Tucker MM, Tracy PB. Kinetics of inactivation of membrane-bound factor Va by activated protein C: Protein S modulates factor Xa protection. J Biol Chem 1988; 263: 14884-14890
    PubMedGoogle Scholar
  • 62 Hackeng TM, Hessing M, van’t Veer C, Meijer-Huizinga F, Meijers JCM, deGroot PG, van Mourik JA, Bouma BN. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C. J Biol Chem 1993; 268: 3993-4000
    PubMedGoogle Scholar
  • 63 Helenius A, Mellman I, Wall D, Hubbard A. Endosomes. Trends Biochem Sci 1983; 8: 245-250
    CrossrefPubMedGoogle Scholar
  • 64 Owensby DA, Morton PA, Schwartz AL. Quantitative evaluation of receptor-mediated endocytosis. Methods in Cell Biology 1989; 32: 305-328
    PubMedGoogle Scholar
  • 65 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340-2348
    PubMedGoogle Scholar