Our long-term goal is to biochemically define the process of platelet formation. This goal will be achieved by analysis of molecular abnormalities associated with platelet formation disorders. The hereditary macrothrombocytopenia of the Wistar Furth (WF) rat, a platelet anomaly, which resembles gray platelet syndrome of man, will be the model for study. The autosomal recessive inheritance, and the association of both large platelet size and alpha-granule abnormalities, suggests that a defect in a single protein affects both alpha-granule formation and subdivision of megakaryocyte cytoplasm into platelets, in both these macrothrombocytopenias. Our finding of a unique epitope on WF rat talin has led us to hypothesize that talin, an actin-binding cytoskeleton protein, is the defective protein.
The specific aims and the methodology for each, are as follows: l) define the molecular basis for the unique talin antigenic determinant of the WF rat. Limited protease digestion and antibody mapping indicate that the unique antigenic determinant is within the region encoded by base-pair 2660 through 4260 of WF rat talin cDNA. This cDNA region will be amplified by PCR, and analyzed for mutations by nucleotide sequencing. 2) demonstrate that a talin defect is/is not responsible for abnormalities of the megakaryocyte-platelet system of WF rats. We will use several approaches including F1 crosses, talin antisense oligonucleotides, and microinjection of purified Wistar rat talin into WF megakaryocytes to address this aim. 3) identify any functional defect in WF rat talin. Functional defects will be identified by biochemical analysis of interactions between talin and other proteins. 4) define altered talin interactions in abnormal cytoplasmic structures of WF rat megakaryocytes and platelets. This will be done by immunogold ultrastructural analysis of WF and Wistar megakaryocytes and platelets. 5) assess alpha-granule-associated talin and its protein interactions, and characterize the alpha-granule abnormalities of WF rat megakaryocytes and platelets. We will biochemically analyze talin association with isolated alpha-granules, and use immunoelectron microscopy to examine the basis for defective alpha-granule formation and storage in the WF rat. These studies should yield basic insights into megakaryocyte differentiation and the process of platelet formation, as well as provide some understanding of platelet formation abnormalities in hereditary and acquired diseases.
| Stenberg, P E; Pestina, T I; Barrie, R J et al. (1997) The Src family kinases, Fgr, Fyn, Lck, and Lyn, colocalize with coated membranes in platelets. Blood 89:2384-93 |
| Pestina, T I; Stenberg, P E; Druker, B J et al. (1997) Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members. Arterioscler Thromb Vasc Biol 17:3278-85 |
| Pestina, T I; Jackson, C W; Stenberg, P E (1995) Abnormal subcellular distribution of myosin and talin in Wistar Furth rat platelets. Blood 85:2436-46 |
