The overall objective of this proposal is to define the molecular basis of plateletfibrin interactions, which play a major role in both the normal hemostatic process and the development of thrombosis. In response to vascular injury, platelets adhere to the exposed subendothelium, and release their granule contents which in turn activate other platelets, causing aggregate formation in a fibrinogen-dependent process. Activated platelets are critical for the catalysis of the coagulation cascade, especially the conversion of prothrombin to thrombin. Next, thrombin removes fibrinopeptides from fibrinogen, triggering rapid protofibril growth, followed by the formation of a platelet-fibrin clot. Subsequent platelet-fibrin interactions, which involve the actin-myosin system of the activated platelet, lead to a dramatic contraction of the fibrin gel. Data obtained in the first project period have raised the following questions, which are addressed in this proposal: Does removal of the fibrinopeptides from fibrinogen unmask a new binding site on the central E-domain of fibrin, which is important for platelet-fibrin binding? Do protofibrils bind to platelets through the same contacts which promote their lateral association into a network of thick fibrin fibers? Does fibrin bind to platelets through surface receptor proteins other than the glycoprotein IIb:IIIa complex involved in fibrinogen binding? What is the relationship between fibrin binding, platelet aggregation, and clot retraction? A multifaceted approach will be required to answer those questions. Measurements of the rate and equilibrium constants for binding protofibrils to stimulated platelets will be carried out by radiolabelled ligand binding and flow cytometric techniques, and the development of quantitative fluorescence microscopic techniques to study binding of platelets to the fibrin network will be undertaken. Specific inhibitory fragments derived from the D- and E-domains of fibrin(ogen) will be used to define which regions of the multinodular fibrin molecule interact with platelets. Experiments with Glanzmann's Thrombasthenia platelets and monoclonal antibodies specific for the glycoprotein IIb:IIIa complex are planned, to ascertain if fibrin binds to platelets solely through the fibrinogen receptor. In addition, experiments will determine if a platelet protein which has been isolated by its affinity for immobilized fibrin monomer is involved in fibrin binding. Characterization of the interactions of this protein with fibrinogen, protofibrils, and fibrin by light scattering and electron microscopic techniques will be carried out.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL032349-06
Application #
3343734
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1983-09-01
Project End
1990-07-31
Budget Start
1988-08-01
Budget End
1989-07-31
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
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Hantgan, R R; Braaten, J V; Rocco, M (1993) Dynamic light scattering studies of alpha IIb beta 3 solution conformation. Biochemistry 32:3935-41
Rocco, M; Spotorno, B; Hantgan, R R (1993) Modeling the alpha IIb beta 3 integrin solution conformation. Protein Sci 2:2154-66
Hantgan, R R; Endenburg, S C; Cavero, I et al. (1992) Inhibition of platelet adhesion to fibrin(ogen) in flowing whole blood by Arg-Gly-Asp and fibrinogen gamma-chain carboxy terminal peptides. Thromb Haemost 68:694-700
Hensler, M E; Frojmovic, M; Taylor, R G et al. (1992) Platelet morphologic changes and fibrinogen receptor localization. Initial responses in ADP-activated human platelets. Am J Pathol 141:707-19
Ramsamooj, P; Lively, M O; Hantgan, R R (1991) Evidence that the central region of glycoprotein IIIa participates in integrin receptor function. Biochem J 276 ( Pt 3):725-32
Ramsamooj, P; Doellgast, G J; Hantgan, R R (1990) Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa. Thromb Res 58:577-92
Hantgan, R R; Nichols, W L; Ruggeri, Z M (1990) von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets. Blood 75:889-94
Hantgan, R R; Hindriks, G; Taylor, R G et al. (1990) Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood. Blood 76:345-53
Lewis, J C; Hantgan, R R; Stevenson, S C et al. (1990) Fibrinogen and glycoprotein IIb/IIIa localization during platelet adhesion. Localization to the granulomere and at sites of platelet interaction. Am J Pathol 136:239-52

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