This project will continue our studies on microtubule (MT) protein focusing primarily on structural investigations both within and outside MT. Specifically, we plan to study a) the exchangeable nucleotide binding site with respect to conformational changes occurring during polymerization/depolymerization, and amino acid composition at this site on the Beta subunit; b) the identification and functional characterization of microtubule-associated proteins (MAPs); c) the interaction of MT with other structural platelet proteins e.g. myosin and actin; d) the association of MT with membrane components and e) the characteristics of cold-stable MT. Items c) and d) are continuation projects of previous investigations. The hypothesis which forms the conceptual framework for many of these studies postulates an indirect linkage between membranes and MT, possibly through the interposition of certain MAPs. Binding of platelet activators to receptors that are connected to MT dissociates the latter by changing the number of phosphate groups on polyphosphoinositides. This system sensitizes platelets to the action of aggregating agents. The effect of Ca++ and Ca++ calmodulin on structural aspects of MT and their MAPs will be intensely investigated. Changes in phosphorylation of tubulin and MAPs will be identified and correlated to the state of assembly/disassembly of platelet MT. Cold-stable MT which exist in platelets will be investigated for characteristics that make them temperature-resistant. The methodology for these studies includes one and two dimensional polyacrylamide gel electrophoresis, autofluorography, nonradiative energy transfer measurements, analysis of peptides and amino acids, covalent linking of neighboring proteins by cleavable crosslinking agents and electron microscopic identification of certain proteins by ferritin-conjugated antibodies. All investigations will be performed with microtubule protein isolated from human platelets. These studies are fundamental in nature and broaden our general understanding of the molecular events early in the course of platelet activation. Their significance, however, also extends into practical aspects of platelet physiology, especially in the field of cold (4 C) storage and preservation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL019323-10
Application #
3335791
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1976-06-01
Project End
1988-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
10
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Memorial Hospital of Rhode Island
Department
Type
DUNS #
City
Pawtucket
State
RI
Country
United States
Zip Code
02860
Berry, S; Dawicki, D D; Steiner, M (1989) Time resolved analysis of tubulin phosphorylation during platelet activation. Biochem Biophys Res Commun 159:170-6
Berry, S; Dawicki, D D; Agarwal, K C et al. (1989) The role of microtubules in platelet secretory release. Biochim Biophys Acta 1012:46-56
Lineberger, B; Dawicki, D D; Agarwal, K C et al. (1989) Permeabilization of platelets: an investigation of biochemical, ultrastructural and functional aspects. Biochim Biophys Acta 1012:36-45
Berry, S; Dawicki, D; Steiner, M (1988) Agonist-induced changes of platelet tubulin phosphorylation. Biochem Biophys Res Commun 151:1250-5
Steiner, M (1987) Platelet surface glycosaminoglycans are an effective shield for distinct platelet receptors. Biochim Biophys Acta 931:286-93
Steiner, M (1985) Dynamics of platelet tubulin in response to changes in free sulfhydryl groups. Thromb Haemost 53:176-9