This revised proposal seeks to understand the mechanisms of platelet adhesion and activation by collagen. Collagen is one of the most thrombogenic components of the blood vessel wall. Therefore, the mechanisms by which platelets adhere to and are activated by collagen is important to our understanding of thrombosis and hemostasis. Dr. Santoro has previously established the role of the alpha2beta1 integrin as a receptor for collagen on platelets and other cell types. The DGEA sequence within the alpha1 chain of collagen has been tentatively identified as a recognition sequence for alpha2beta1. The DGEA sequence occurs twice within the alpha1 chain of collagen. As the first Specific Aim of this proposal, Dr. Santoro will utilize antipeptide antibodies directed at the DGEA and adjacent flanking sequences and site-directed mutagenesis of recombinant alpha1 collagen chains as independent and complimentary approaches to dissect the role of the DGEA sequences in alpha2beta1 recognition. The second Specific Aim relates to the identification of a non-DGEA sequence in recognition of the alpha2 (I) chain of collagen in alpha2beta1 integrin recognition. This chain of collagen lacks a DGEA sequence but still interacts with the receptor. Protein fragmentation, synthetic peptide, inhibitory antibodies and mutant recombinant protein approaches will be utilized to identify the recognition sequence.
The third Aim of this proposal relates to identification of a co-receptor that participates in concert with alpha2beta1, to produce signalling when platelets interact with collagen. There is substantial evidence that secretory pathway is not initiated by interaction of platelets with collagen via alpha2beta1 alone. Monoclonal antibody approaches will be implemented as a means of identifying this co-receptor. Taken together, these studies will potentially lead to an understanding of the molecular mechanisms by which platelets and other cells adhere and respond to collagen.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL040506-08
Application #
2459950
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-04-01
Project End
1999-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
8
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Washington University
Department
Pathology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Dickeson, S K; Mathis, N L; Rahman, M et al. (1999) Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins. J Biol Chem 274:32182-91
Zutter, M M; Santoro, S A; Wu, J E et al. (1999) Collagen receptor control of epithelial morphogenesis and cell cycle progression. Am J Pathol 155:927-40
Bhattacharyya-Pakrasi, M; Dickeson, S K; Santoro, S A (1998) Alpha2beta1 integrin recognition of the carboxyl-terminal propeptide of type I procollagen: integrin recognition and feed-back regulation of matrix biosynthesis are mediated by distinct sequences. Matrix Biol 17:223-32
Dickeson, S K; Bhattacharyya-Pakrasi, M; Mathis, N L et al. (1998) Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy. Biochemistry 37:11280-8
Staatz, W D; Walsh, J J; Santoro, S A (1997) The alpha 2 beta 1 integrin binds to the CB4 peptide of the alpha 2(I) collagen chain. Biochem Mol Biol Int 42:577-82
Dickeson, S K; Walsh, J J; Santoro, S A (1997) Contributions of the I and EF hand domains to the divalent cation-dependent collagen binding activity of the alpha2beta1 integrin. J Biol Chem 272:7661-8
Wu, J E; Santoro, S A (1996) Differential expression of integrin alpha subunits supports distinct roles during lung branching morphogenesis. Dev Dyn 206:169-81
Zutter, M M; Fong, A M; Krigman, H R et al. (1992) Differential regulation of the alpha 2 beta 1 and alpha IIb beta 3 integrin genes during megakaryocytic differentiation of pluripotential K562 cells. J Biol Chem 267:20233-8
Burger, S R; Zutter, M M; Sturgill-Koszycki, S et al. (1992) Induced cell surface expression of functional alpha 2 beta 1 integrin during megakaryocytic differentiation of K562 leukemic cells. Exp Cell Res 202:28-35
Staatz, W D; Fok, K F; Zutter, M M et al. (1991) Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen. J Biol Chem 266:7363-7

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