Recent studies have indicated that stimulation of endothelium by physiologic mediators results in suppression of anticoagulant and enhancement of procoagulant properties. One endothelial cell procoagulant cofactor whose expression is enhanced by tumor necrosis factor (TNF) is a cell surface binding site for Factors IX and IXa, which in the presence of Factors VIII and X is relatively selective for the enzyme (Factors IXa) and promotes it procoagulant activity. Previous work has indicated that this binding site is comprised, at least in part, of a trypsin- sensitive, cell surface protein Mr 140,000 and has established a protocol for solubilization and partial purification in a form which retains activity for several weeks. This proposal concerns completion of the isolation of this protein and characterization of its role in regulating the coagulant activity of Factor IX/IXa on quiescent and tumor necrosis factor (TNF)-stimulated endothelium. Isolation of the binding site will be followed by production of antisera, both monoclonal and polyclonal, to assess its role in kinetic studies of Factor IX and Factor X activation, and the binding of radiolabelled Factor IX/IXa to endothelium. Factor X activation on endothelium, using confluent and pre- confluent cultures, will be compared with that observed on other vascular and vessel wall-derived cells, phospholipid vesicles of varying composition, and in the presence of purified binding site. Mechanisms through which TNF up-regulates and excess Factor IX suppresses binding site expression will be examined. Regulation of endocytosis and degradation of Factor IX/IXa on quiescent and stimulated endothelium will be compared and the role of the Factor IX/IXa binding protein and of Factors VIII and X assessed. Complementary studies will examine domains of the Factor IX/IXa molecule involved in interaction with cellular binding site. Preliminary studies using a modified form of Factor IX with the delta-carboxyglutamic acid residues removed, a mutant Factor IX molecule with a defect at the start of the growth factor region, and purified activation peptide have implicated each of these domains in recognition of the cellular binding site. These studies will be extended using recombinant Factor IX molecules, naturally occurring mutants and peptides prepared from the growth factor and activation peptide domains. Using the approach one peptide, comprising residues 41-50 from Factor IX, which blocks Factor IX/IXa-binding site interaction has already been identified. Although Factor IX binding to the vessel wall has been demonstrated in vivo, a role of the Factor IX/IXa binding site in thrombosis has not been demonstrated. To address this issue, two experimental thrombosis models in which Factor IXa makes a major contribution, Wessler, stasis and TNF-induced thrombosis, will be studied. Protection from thrombosis by agents which antagonize Factor IX/IXa-cell surface interaction in vitro, such as the peptide comprising residues 41-50 of Factor IX, blocking antibodies to the binding site and active site-blocked Factor IXa, will be examined.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL034625-06
Application #
3347712
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1987-08-01
Project End
1993-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Kuwabara, K; Pinsky, D J; Schmidt, A M et al. (1995) Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries. J Biol Chem 270:8179-87
Kuwabara, K; Ogawa, S; Matsumoto, M et al. (1995) Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells. Proc Natl Acad Sci U S A 92:4606-10
Benedict, C R; Pakala, R; Willerson, J T (1994) Endothelial-dependent procoagulant and anticoagulant mechanisms. Recent advances in understanding. Tex Heart Inst J 21:86-90
Karakurum, M; Shreeniwas, R; Chen, J et al. (1994) Hypoxic induction of interleukin-8 gene expression in human endothelial cells. J Clin Invest 93:1564-70
Pinsky, D; Oz, M; Liao, H et al. (1993) Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model. J Clin Invest 92:2994-3002
Kao, J; Fan, Y G; Haehnel, I et al. (1993) Endothelial-monocyte activating polypeptides (EMAPs): tumor derived mediators which activate the host inflammatory response. Behring Inst Mitt :92-106
Yuzawa, Y; Brentjens, J R; Brett, J et al. (1993) Antibody-mediated redistribution and shedding of endothelial antigens in the rabbit. J Immunol 150:5633-46
Cozzolino, F; Torcia, M; Lucibello, M et al. (1993) Interferon-alpha and interleukin 2 synergistically enhance basic fibroblast growth factor synthesis and induce release, promoting endothelial cell growth. J Clin Invest 91:2504-12
Oz, M C; Pinsky, D J; Koga, S et al. (1993) Novel preservation solution permits 24-hour preservation in rat and baboon cardiac transplant models. Circulation 88:II291-7
Schmidt, A M; Yan, S D; Brett, J et al. (1993) Regulation of human mononuclear phagocyte migration by cell surface-binding proteins for advanced glycation end products. J Clin Invest 91:2155-68

Showing the most recent 10 out of 49 publications