The aims of this resubmitted competitive renewal follow on results demonstrating that the ability of adherent platelets to assemble plasma fibronectin is determined by the ligands that mediate platelet adhesion. Thus, platelets adherent of fibrinogen, vitronectin, or von Willebrand factor are suppressed in their ability to assemble fibronectin whereas platelets adherent to fibrin, laminin, collagen, or fibronectin itself assemble fibronectin robustly. Further, assembly of fibronectin by platelets in an ex vivo flow system is a strong determinant of the extent of platelet thrombus formation on matrices of fibrin or collagen. The hypotheses are (i) platelet integrins and other cell surface proteins recognize critical features of adhesive ligands, initiating subtly different signaling pathways that support or suppress subsequent assembly of fibronectin by adherent platelets;(ii) as yet unappreciated differences in the microscopic and biochemical consequences of platelet adhesion correlate with the ability of adherent platelets to assemble fibronectin;and (iii) the N- terminal portion of fibronectin interacts specifically with unstructured stretches of certain proteins, among which are the cell surface molecules on adherent platelets and fibroblasts that drive fibronectin assembly.
Specific aims are to: 1. Identify features of fibronectin, fibrinogen/fibrin, vitronectin, and von Willebrand factor that account for their supportive or suppressive activity. 2. Characterize differences other than ability to assemble fibronectin that distinguish platelets adherent to supportive and suppressive ligands. 3. Discover the molecular features of fibronectin assembly sites present on adherent assembly- competent platelets and absent on adherent assembly-incompetent platelets. Methods to accomplish these aims include in vitro mutagenesis to dissect the structure/function of the supportive and suppressive adhesive ligands, microscopic and proteomic characterization of assembly- competent and assembly-incompetent adherent platelets, utilization of a reversible cross-linking strategy to identify the platelet surface proteins that interact with supportive and suppressive adhesive ligands, and development of activated blood coagulation Factor XIII as a tool to identify the platelet surface molecules that initiate fibronectin assembly.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL021644-31
Application #
7759177
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
1977-12-01
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2011-12-31
Support Year
31
Fiscal Year
2010
Total Cost
$356,220
Indirect Cost
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Ma, Wenjiang; Ma, Hanqing; Mosher, Deane F (2015) On-Off Kinetics of Engagement of FNI Modules of Soluble Fibronectin by ?-Strand Addition. PLoS One 10:e0124941
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Shen, Bo; Estevez, Brian; Xu, Zheng et al. (2015) The interaction of G?13 with integrin ?1 mediates cell migration by dynamic regulation of RhoA. Mol Biol Cell 26:3658-70
Ma, Wenjiang; Ma, Hanqing; Fogerty, Frances J et al. (2015) Bivalent ligation of the collagen-binding modules of fibronectin by SFS, a non-anchored bacterial protein of Streptococcus equi. J Biol Chem 290:4866-76
Harris, Gemma; Ma, Wenjiang; Maurer, Lisa M et al. (2014) Borrelia burgdorferi protein BBK32 binds to soluble fibronectin via the N-terminal 70-kDa region, causing fibronectin to undergo conformational extension. J Biol Chem 289:22490-9
Sabatier, Laetitia; Djokic, Jelena; Fagotto-Kaufmann, Christine et al. (2013) Complex contributions of fibronectin to initiation and maturation of microfibrils. Biochem J 456:283-95
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Maurer, Lisa M; Annis, Douglas S; Mosher, Deane F (2012) IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly. PLoS One 7:e30615
Tomasini-Johansson, Bianca R; Johnson, Ian A; Hoffmann, F Michael et al. (2012) Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds. Matrix Biol 31:360-7
Maurer, Lisa M; Ma, Wenjiang; Eickstaedt, Nathan L et al. (2012) Ligation of the fibrin-binding domain by ?-strand addition is sufficient for expansion of soluble fibronectin. J Biol Chem 287:13303-12

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