The overall objectives are two-fold: 1) to continue to explore the biochemical, rheologic, and physiologic factors responsible for congenital disorders of hemostasis due to platelet dysfunction and 2) to utilize patients with well-characterized platelet dysfunction as a means of further defining the mechanisms which regulate normal platelet function. The specific studies toward these objectives are: 1) to examine activation mechanisms which could be abnormal in 7 patients with a new type of platelet dysfunction characterized by impaired platelet responses to weak platelet agonists, including studies on fibrinogen receptor exposure, calcium mobilization and responses, phospholipase C-mediated pathways, Na+/H+ exchange, and G-protein interactions. 2) to study, in patients with dense granule storage pool deficiency (delta-SPD), the basis for the storage defect and to examine no further detail (by measuring alpha-granule release and activation pathways linked to receptor occupancy) the secretion defect in these patients. 3) in SPD patients with combined defects of dense and alpha-granules (alpha/delta-SPD), to study whether the alpha- granule is absent (as distinct form the functionally defective granule in the gray platelet syndrome, alpha-SPD) by utilizing monoclonal antibodies to an alpha-granule specific protein, GMP-140. 4) to study further mechanisms involved in platelet adhesion and thrombus formation on subendothelium under conditions of controlled flow by examining a) the role of von Willebrand factor (vWf) and other adhesion molecules binding to GPIIb-IIIa in platelet adhesion and thrombus formation on subendothelium, utilizing antibodies to the 1737-1750 RGD-containing sequence in vWf, and homologous sequences in other adhesion molecules, which bind uniquely to each adhesive protein; b) the role and mechanism of platelet vWf utilizing platelets from patients with various types of von Willebrand's disease (vWd); c) the structure-function relationships of vWf that may be important for adhesion and thrombus formation utilizing plasmas from vWd patients (Type I, II variants, IIB, I-NY, and pseudo-vWd) that have been carefully characterized for vWf content, multimer characteristics, and platelet- binding properties; d) the hematocrit-dependent platelet adhesion defect in delta-SPD in order to assess further the role of ADP and red cells in promoting platelet adhesion. 4) to investigate further the basis for impaired platelet procoagulant activity (PPA) in Scott Syndrome (reduced Factor Xa binding and impaired phosphatidylserine expression) and to utilize platelets from Scott Syndrome and alpha/delta-SPD to test the hypothesis that the expression of PPA in response to agonists may be related to the fusion of alpha-granules with the plasma membrane and the formation of microvesicles with procoagulant activity. 5) to utilize a newly developed technique for assessing primary hemostasis, by obtaining, in a wide variety of patients with prolonged bleeding times and/or known platelet dysfunction, time-course studies on blood volume, thromboxane B2, platelet factor 4 and fibrinopeptide A in bleeding time blood.
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