I. Signal transduction in Platelet Activation. Elevation of cytosolic calcium plays a major role in platelet activation by physiologic agonists or by extended storage. The rise in cytosolic calcium induces a variety of biochemical responses including tyrosine phosphorylation and dephosphorylation of specific proteins. Activation of platelets leads to an expression of a fibrinogen-binding integrin on the surface of platelets, followed by binding of a bivalent fibrinogen molecule and crosslinking of adjacent platelets by fibrinogen to produce a platelet aggregate. Additional tyrosine phosphorylation of internal platelet proteins takes place after aggregation and is thought to stabilize the forming aggregate. A tyrosine kinase inhibitor, erbstatin, was found to selectively potentiate fibrinogen binding-mediated tyrosine phosphorylation of a single 95 kDa protein following platelet activation. The mechanism of selective tyrosine phosphorylation of this protein appears to be a result of the disruption of the translocation of phosphatases to the cytoskeleton during platelet activation. Isolation and sequencing of the 95 kDa protein is being pursued. II. Nitric Oxide and sickle cell disease platelets. Nitric oxide (NO) mediates relaxation of arterioles and inhibits platelet activation. Excessive thrombosis found in certain clinical conditions may be due to an insufficient availability of nitric oxide. Sickle cell disease is associated with frequent pain crisis of which the etiology is associated with frequent vasoocclusive phenomenon. Recently the focus of clinical investigations has turned to the role of nitric oxide in the pathophysiology of this disease. We have compared the responsiveness of platelets isolated from sickle cell patients and normal volunteers to platelet agonists and to chemical NO donors. Sickle cell platelets appear to be more responsive to NO donors which suggests that physiologic production of NO in sickle cell patients may be decreased. III. Prion Proteins and Platelets. The prion protein (PrPC) is expressed on many cell types and tissues and is thought to be the infectious agent in the fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE). We have investigated the role of PrPc in platelet physiology and found that platelet PrPc appears to be associated with an intracellular membrane vesicle in resting platelets and that platelet activation increases PrPc expression on the platelet surface. The surface expression of PrPc, as detected by flow cytometry with a PrPc-specific monoclonal antibody, increased more than twofold after platelet activation. When compared to the concentration of agonist dependent expression of a lysosomal protein Gp53 and an alpha-granular protein P-selectin, and the normal expression of prion in Hermansky-Pudlak patients which do not have dense granules, the platelet PrPc appear to be localized in alpha-granules of resting platelets. The function of PrPc may be in mediating heterozygous platelet-cell interaction and/or adhesion. We are also investigating the ability of platelets to carry infectivity from scrapie infected hamsters to healthy animals in an attempt to model the risk of transmitting TSE diseases in humans by platelet transfusion. Hamster platelets and leukocytes express none or very low quantities of monomeric PrPc either on their surface or inside the cells, as detected by a monoclonal antibody against PrPc (3F4). This is in contrast to human platelets and leukocytes, both of which carry the prion protein on their surface. To determine whether TSE infectivity is associated with platelets or leukocytes, we isolated them from symptomatic scrapie-infected hamsters and inoculated them into the brains of healthy hamsters. Preliminary results suggest that platelets of scrapie infected hamsters contain little, if any, infectivity while infectivity in leukocytes is enriched. The correlation of cellular PrPc distribution and the infectivity of different cell types will confirm whether PrPc is required for transfer of infectivity.
