Thrombospondin (Tsp) is a 450 kDa protein with 3 disulfide-linked chains. It is secreted by platelets and certain cultured cells. It binds to cells and many different proteins; it may function as an adhesive or sub-cellular matrix protein. We have observed that Tsp i) has a conformation-sensitive thiol, ii) has an intrachain disulfide bond with a conformation-dependent stability, and iii) readily forms disulfide-linked complexes with other proteins and with other molecules of Tsp. I hypothesize that this reflects a dynamic function of Tsp as a covalent crosslinking protein. As a test of this hypothesis and for further characterization of Tsp, the number and locations of thiols and conformation-sensitive disulfide bonds will be determined, and the specificity for protein and for location of disulfide bonds for the thiol-disulfide exchange will be examined. The thiol-disulfide exchange was shown to have a pH optimum below 7 (8-10 would be predicted); this will be studied to determine if the pH dependence reflects a pH- dependent conformational change or the influence of another ionizable group on the dissociation of the thiol. Finally, attempts will be made to determine whether the thiol is necessary for Tsp binding to cells (including platelets), for its lectin activity or for platelet aggregation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
1R37HL037250-01
Application #
3486143
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1986-12-01
Project End
1991-11-30
Budget Start
1986-12-01
Budget End
1987-11-30
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Suny Downstate Medical Center
Department
Type
Schools of Medicine
DUNS #
068552207
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
Essex, D W; Miller, A; Swiatkowska, M et al. (1999) Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin. Biochemistry 38:10398-405
Milev, Y; Essex, D W (1999) Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III. Arch Biochem Biophys 361:120-6
Huang, E M; Detwiler, T C; Milev, Y et al. (1997) Thiol-disulfide isomerization in thrombospondin: effects of conformation and protein disulfide isomerase. Blood 89:3205-12
Chen, K; Detwiler, T C; Essex, D W (1995) Characterization of protein disulphide isomerase released from activated platelets. Br J Haematol 90:425-31
Essex, D W; Chen, K; Swiatkowska, M (1995) Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane. Blood 86:2168-73
Chen, K; Essex, D W (1995) Purification of secreted platelet protease nexin I. Thromb Res 79:527-9
Chang, A C; Detwiler, T C (1992) Reactions of thrombin-serpin complexes with thrombospondin. Arch Biochem Biophys 299:100-4
Chen, K; Lin, Y; Detwiler, T C (1992) Protein disulfide isomerase activity is released by activated platelets. Blood 79:2226-8
Chang, A C; Detwiler, T C (1991) The reaction of thrombin with platelet-derived nexin requires a secondary recognition site in addition to the catalytic site. Biochem Biophys Res Commun 177:1198-204
Speziale, M V; Detwiler, T C (1991) Reduction of a disulfide bond of thrombospondin in the supernatant solution of activated platelets. Arch Biochem Biophys 286:546-50

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