Further Insight Into the Heparin-Releasable and Glycosylphosphatidylinositol-Lipid— Anchored Forms of Tissue Factor Pathway Inhibitor

Abstract

The release of tissue factor pathway inhibitor (TFPI) from human umbilical vein endothelial cells (HUVECs) was investigated using heparin and phospholipase C. The experiment included incubating HUVECs with 0, 1, or 10 U/mL heparin diluted in Dulbecco Modified Eagle's Medium plus 5% fetal calf serum for 1 or 24 hours. A statistically significant increase in TFPI activity levels was seen at 1 hour, but not at 24 hours. A 20-fold increase in the release of TFPI after phospholipase C treatment of HUVECs was demonstrated, confirming that it is glycosylphosphatidylinositol-lipid (GPI) anchored. Sequential treatment of HUVECs with phospholipase C and heparin was performed, and a trend was observed where GPI-anchored TFPI levels were increased after 1 hour of pretreatment with heparin but were decreased after 24 hours. Serum is a requirement for the heparin-dependent release of TFPI from HUVECs. Heparin pretreatment of HUVECs may affect levels of GPI anchored TFPI in a time and dose-dependent manner.

Keywords

glycosylphosphatidylinositol-lipid anchor heparin tissue factor pathway inhibitor

References

Broze GJ Jr. Tissue factor pathway inhibitor and the current concept of blood coagulation. Blood Coagul Fibrinolysis. 1995;6:S7-S13.

Broze GJ Jr. Tissue factor pathway inhibitor and the revised theory of coagulation. Annu Rev Med. 1995;46: 103-112.

Broze GJ Jr , Lange GW , Duffin KL , MacPhail L. Heterogeneity of plasma tissue factor pathway inhibitor. Blood Coagul Fibrinolysis. 1994 ;5:551-559.

Hansen JB , Huseby KR , Huseby NE , Ezban M. , Nordoy A. Tissue factor pathway inhibitor in complex with low density lipoprotein isolated from human plasma does not possess anticoagulant function in tissue factor-induced coagulation in vitro. Thromb Res. 1997;85:413-425.

Nordfang O. , Bjorn SE , Valentin S. , et al. The C-terminus of tissue factor pathway inhibitor is essential to its anticoagulant activity. Biochemistry. 1991;30:10371-10376.

Valentin S. , Nordfang O. , Bregengard C. , Wildgoose P. Evidence that the C-terminus of tissue factor pathway inhibitor (TFPI) is essential for its in vitro and in vivo interaction with lipoproteins. Blood Coagul Fibrinolysis. 1993;4:713-720.

Wesselschmidt R. , Likert K. , Girard T. , Wun TC , Broze GJ Jr. Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa. Blood. 1992;79:2004-2010.

Bajaj MS , Kuppuswamy MN , Saito H. , Spitzer SG , Bajaj SP Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that the endothelium is the primary site of its synthesis. Proc Natl Acad Sci U S A. 1990 ;87:8869-8873.

Sandset PM Tissue factor pathway inhibitor-an update. Haemostasis. 1996;26:154-165.

10.

Sandset PM , Abildgaard U. , Larsen ML Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res. 1988;50:803-813.

11.

Lindahl AK , Abildgaard U. , Stokke G. Release of extrinsic pathway inhibitor after heparin injection: increased response in cancer patients. Thromb Res. 1990 ;59:651-656.

12.

Novotny WF , Brown SG , Miletich JP , Rader DJ , Broze GJ Jr. Plasma antigen levels of the lipoprotein associated coagulation inhibitor in patient samples. Blood. 1991;78: 387-393.

13.

Giraux JL , Tapon-Bretaudiere J. , Matou S. , Fischer AM Fucoidan, as heparin, induces tissue factor pathway inhibitor release from cultured human endothelial cells. Thromb Haemost. 1998;80:692-695.

14.

Hansen JB , Svensson B. , Olsen R. , Ezban M. , Osterud B. , Paulssen RH Heparin induces synthesis and secretion of tissue factor pathway inhibitor from endothelial cells in vitro. Thromb Haemost. 2000;83:937-943.

15.

Li Y. , Rodriquez M. , Spencer FA , Becker RC Comparative effects of unfractionated heparin and low molecular weight heparin on vascular endothelial cell tissue factor pathway inhibitor release: a model for assessing intrinsic thromboresistance. J Thromb Thrombolysis . 2002;14: 123-129.

16.

Lupu C. , Poulsen E. , Roquefeuil S. , Westmuckett AD , Kakkar VV , Lupu F. Cellular effects of heparin on the Production and release of tissue factor pathway inhibitor in human endothelial cells in culture. Arterioscler Thromb Vasc Biol. 1999;19:2251-2262.

17.

Martinez-Sales V. , Vila V. , Reganon E. , Oms JG , Aznar J. Effect of unfractionated heparin and a low molecular weight heparin (enoxaparin) on coagulant activity of cultured human endothelial cells. Haematologica. 2003; 88:694-699.

18.

Perez-Ruiz A. , Montes R. , Carrasco P. , Rocha E. Effects of a low molecular weight heparin, bemiparin, and unfractionated heparin on hemostatic properties of endothelium . Clin Appl Thromb Hemost. 2002;8:65-71.

19.

Mast AE , Acharya N. , Malecha MJ , Hall CL , Dietzen DJ Characterization of the association of tissue factor pathway inhibitor with human placenta. Arterioscler Thromb Vasc Biol. 2002;22:2099-2104.

20.

Zhang J. , Piro O. , Lu L. , Broze GJ Jr. Glycosyl phosphatidylinositol anchorage of tissue factor pathway inhibitor . Circulation. 2003;108:623-627.

21.

Novotny WF , Palmier M. , Wun TC , Broze GJ Jr , Miletich JP Purification and properties of heparin-releasable lipoprotein-associated coagulation inhibitor. Blood. 1991; 78:394-400.

22.

Jaffe EA , Nachman RL , Becker CG , Minick CR Culture of human endothelial cells derived from umbilical veins. J Clin Invest. 1973;52:2745-2756.

23.

Sandset PM , Larsen ML , Abildgaard U. , Lindahl AK , Odegaard OR Chromogenic substrate assay of extrinsic pathway inhibitor (EPI): levels in the normal population and relation to cholesterol. Blood Coagul Fibrinolysis. 1991;2:425-433.

24.

Adams MJ , Oostryck R. A comparative study of functional assays for tissue factor pathway inhibitor using normal plasma and clinical samples. Blood Coagul Fibrinolysis . 2000;11:327-333.

25.

Adams MJ , Donohoe S. , Mackie IJ , Machin SJ Anti-tissue factor pathway inhibitor activity in patients with primary antiphospholipid syndrome. Br J Haematol. 2001;114:375-379.

26.

Adams MJ , Cardigan RA , Marchant WA , et al. Tissue factor pathway inhibitor antigen and activity in 96 patients receiving heparin for cardiopulmonary bypass. J Cardiothorac Vasc Anesth. 2002;16:59-63.

27.

Adams M. , Breckler L. , Stevens P. , Thom J. , Baker R. , Oostryck R. Anti-tissue factor pathway inhibitor activity in subjects with antiphospholipid syndrome is associated with increased thrombin generation. Haematologica. 2004; 89:985-990.

28.

Sevinsky JR , Rao L. , Ruf M. Ligand-induced protease receptor translocation into caveolae: A mechanism for regulating cell surface proteolysis of the tissue-factor dependent coagulation pathway. J Cell Biol. 1996;133: 293-304.

29.

Lupu C. , Goodwin CA , Westmuckett AD , et al. Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium. Arterioscler Thromb Vasc Biol . 1997;17: 2964-2974.

30.

Chang JY , Monroe DM , Oliver JA , Roberts HR TFPIbeta a second product from the mouse tissue factor pathway inhibitor (TFPI) gene. Thromb Haemost. 1999;81:45-49.

31.

Ott I. , Miyagi Y. , Miyazaki K. , et al. Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor. Arterioscler Thromb Vasc Biol. 2000;20:874-882.

32.

Ott I. , Vukovich R. , Schomig A. , Neumann FJ Overexpression of glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor-1 inhibits tissue factor activity. Blood Coagul Fibrinolysis. 2003;14:539-544.

33.

Unger RE , Krump-Konvalinkova V. , Peters K. , Kirkpatrick CJ In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMEC-ST1.6R . Microvasc Res. 2002;64:384-397.

34.

Dirks WG , MacLeod RA , Drexler HG ECV304 (endothelial) is really T24 (bladder carcinoma): cell line cross- contamination at source. In Vitro Cell Dev Biol Anim. 1999 ;35:558-559.

35.

Brown J. , Reading SJ , Jones S. , et al. Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: a comparison with the human bladder cancer derived epithelial cell line T24/83. Lab Invest. 2000;80:37-45.

36.

Hansen JB , Naalsund T. , Sandset PM , Svensson B. Rebound activation of coagulation after treatment with unfractionated heparin and not with low molecular weight heparin is associated with partial depletion of tissue factor pathway inhibitor and antithrombin. Thromb Res. 2000;100:413-417.

37.

Bendz B. , Hansen JB , Andersen TO , Ostergaard P. , Sandset PM Partial depletion of tissue factor pathway inhibitor during subcutaneous administration of unfractionated heparin, but not with two low molecular weight heparins . Br J Haematol. 1999;107:756-762.

38.

Pendurthi UR , Rao LV , Williams JT , Idell S. Regulation of tissue factor pathway inhibitor expression in smooth muscle cells. Blood. 1999;94:579-586.

39.

Edstrom CS , Calhoun DA , Christensen RD Expression of tissue factor pathway inhibitor in human fetal and placental tissues. Early Hum Dev. 2000;59:77-84.

40.

Mast AE , Higuchi DA , Huang ZF , Warshawsky I. , Schwartz AL , Broze GJ Jr. Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor. Biochem J. 1997;327:577-583.