Continued support is requested for studies of the interaction of Fibronectin (Fn) with platelet GPIIb-IIIa and other integrins of significance in hemostasis, thrombosis, and vascular biology. Fn, a mosaic protein, is a prototype adhesive glycoprotein, and the cell binding region of Fn is well suited to structure-function analysis by expression in prokaryotic systems. It is proposed that the recognition of Fn, and possibly other macromolecules by integrins, involves extended complementary surfaces of the 2 proteins. In the case of GPIIb-IIIa, these complementary surfaces in Fn include the RGD sequence as well as additional sequences and may involve multiple type III homology units. This hypothesis will be tested by identifying the recognition sequences in Fn utilized by platelet GPIIb-IIIa, alphavbeta3, alpha5beta1, and alpha4beta1 by assaying the binding of Fn to purified integrins in vitro. Initial identification of sites in Fn will be achieved by mapping the epitopes of monoclonal anti-Fn antibodies which inhibit the Fn integrin interaction by use of recombinant Fn fragments containing type III homology units and nested deletions thereof and by use of synthetic peptides. The capacity of identified recombinant Fn fragments and peptides to bind to the integrins and to inhibit Fn binding will be assessed to confirm their identification as recognition sequences. Cross competition experiments will provide initial insight into whether these Fn fragments or peptides utilize shared recognition sites in the integrin and whether they compete for other ligands, e.g., Fg, vWF. The effect of these Fn fragments and peptides on the aggregation of platelets and the adhesion of cells overexpressing recombinant alphavbeta3, alpha5beta1, and alpha4beta1 will be assessed. The sites in integrin which recognize these regions of Fn will be identified by analyzing the ability of the Fn fragments to bind to synthetic peptides containing discrete ligand recognition regions of the integrins. Chemical crosslinking of the Fn fragments or peptides to the integrins followed by fragmentation of the crosslinked complexes and mapping of the crosslinking sites will provide initial identification of novel fn binding regions in the integrins. The capacity of the identified Fn fragments and peptides to bind to recombinant integrins with altered ligand binding function will provide an additional means of evaluating the integrin recognition mechanism for Fn. The studies will provide fundamental information about protein/protein interactions which govern key cellular events in hemostasis, thrombosis, and vascular biology. As with RGD peptides, the functional properties of the Fn sequences to be identified may permit development of novel means of inhibiting or enhancing adhesive functions of platelets, leukocytes, and endothelial cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL028235-12
Application #
3339664
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1982-01-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
12
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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