This revised proposal is designed to test the hypothesis that talin and vinculin play a role in attaching GPIIb-IIIa to the platelet cytoskeleton via an interaction with the cytoplasmic domains of this integrin. GPIIb-IIIa is the platelet fibrinogen and vWF receptor. As a consequence of binding to these molecules, GPIIb-IIIa becomes associated with the platelet cytoskeleton, which may contribute to the role of GPIIb-IIIa in clot retraction, tyrosine phosphorylation, receptor clustering and redistribution. The Applicant found that talin, vinculin and actin co-purify with GPIIb-IIIa by RGD affinity chromatography and co-distribute with GPIIb-IIIa in thrombin-stimulated platelets.
The Specific Aims of this proposal are 1) To demonstrate direct binding of purified talin or vinculin to activated GPIIb-IIIa. This will be done by measuring simultaneous binding of fibrinogen and talin or vinculin to GPIIb-IIIa in double label experiments. GPIIb-IIIa will be activated by RGD peptide pretreatment of anti-LIBS6. Also, immunoprecipitation studies will be performed to detect interactions of talin and vinculin with GPIIb-IIIa. 2) To localize specific determinants within the cytoplasmic domains of GPIIb-IIIa mediating interactions with cytoskeletal proteins. This will be done with recombinant GPIIb-IIIa truncated or mutated in the cytoplasmic domain. 3) To examine the functional significance of GPIIb-IIIa association with the platelet cytoskeleton by attempting to inhibit this interaction in permeabilized cells with synthetic peptides mimicking the cytoplasmic sequences or with anti-cytoplasmic domain antibodies, and 4) To investigate the possibility that fibrinogen binding to GPIIb-IIIa changes the conformation of the receptor cytoplasmic domains. This will be accomplished with LIBS antibodies that recognize the cytoplasmic domains of GPIIb-IIIa.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL041793-09
Application #
2735146
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1989-05-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Pharmacology
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612