The purpose of this research proposal is to acquire a better understanding of the molecular events occurring in the human platelet membrane during the transition of platelets from the resting to the activated, hemostatically-effective state. Membrane glycoproteins (GP), especially GP Ib, GP IIb and GP IIIa, are thought to act as receptors for platelet binding proteins, such as factor VIII/von Willebrand factor and fibrinogen, which are requisite cofactors of platelet-vessel wall interaction and platelet-platelet cohesion, respectively. Recent evidence suggests that the induction of fibrinogen receptor activity involves calcium-mediated conformational changes and/or reassociations of GP IIb and GP IIIa within the membrane. In order to ascertain the extent of complex formation in resting as opposed to activated platelets, and thus the role of complex formation in fibrinogen receptor induction, I propose to study the calcium-mediated interaction of GP IIb and IIIa in greater detail using site-specific murine monoclonal antibodies and immunoelectron microscopy, immunofluorescence microscopy and electron spin resonance techniques. Since recent evidence indicates that GP Ib exists in the membrane of intact platelets in a form that may be structurally and functionally different from that hitherto analyzed in vitro, similar methods will be applied to an analysis of GP Ib and its """"""""structural analogues,"""""""" including glycocalicin, with the membrane of intact platelets. The endogenous calcium-activated protease (CAP) of platelets is thought to modulate platelet activation by cleavage of certain cytoskeletal elements (actin-binding protein and P235) or glycoprotein Ib. Having recently obtained evidence suggesting that endogenous platelet fibrinogen and factor VIII/von Willebrand factor are also substrates of this protease, I propose to investigate the potential role of CAP in the modulation of platelet activation via cleavage of these proteins. These investigations should lead to a better understanding of the phenomena of platelet adhesion and platelet aggregation at a molecular level and thus provide new insight into the etiology of thrombotic disorders and other pathologic conditions in which primary hemostasis and platelet function are compromised.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL032279-03
Application #
3343639
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-04-01
Project End
1987-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Bloodcenter of Wisconsin, Inc.
Department
Type
DUNS #
City
Milwaukee
State
WI
Country
United States
Zip Code
53233
Kouns, W C; Steiner, B; Kunicki, T J et al. (1994) Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia. Blood 84:1108-15
Kunicki, T J; Orchekowski, R; Annis, D et al. (1993) Variability of integrin alpha 2 beta 1 activity on human platelets. Blood 82:2693-703
Tomiyama, Y; Tsubakio, T; Piotrowicz, R S et al. (1992) The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules. Blood 79:2303-12
Tomiyama, Y; Brojer, E; Ruggeri, Z M et al. (1992) A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3. J Biol Chem 267:18085-92
Wencel-Drake, J D; Okita, J R; Annis, D S et al. (1991) Activation of calpain I and hydrolysis of calpain substrates (actin-binding protein, glycoprotein Ib, and talin) are not a function of thrombin-induced platelet aggregation. Arterioscler Thromb 11:882-91
Smith, J W; Ruggeri, Z M; Kunicki, T J et al. (1990) Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites. J Biol Chem 265:12267-71
Okita, J R; Frojmovic, M M; Kristopeit, S et al. (1989) Montreal platelet syndrome: a defect in calcium-activated neutral proteinase (calpain). Blood 74:715-21
Kunicki, T J; Beardsley, D S (1989) The alloimmune thrombocytopenias: neonatal alloimmune thrombocytopenic purpura and post-transfusion purpura. Prog Hemost Thromb 9:203-32
Furihata, K; Hunter, J; Aster, R H et al. (1988) Human anti-PlE1 antibody recognizes epitopes associated with the alpha subunit of platelet glycoprotein Ib. Br J Haematol 68:103-10
Kunicki, T J; Nugent, D J; Staats, S J et al. (1988) The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex. J Biol Chem 263:4516-9

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