Platelets, when activated by a variety of extracellular agents, undergo a complex series of physiological reactions including shape change, aggregation and secretion. The extracellular agents interact with specific receptors resulting in the elevation of intraplatelet free Ca2+. The Ca2+ signal then triggers numerous biochemical reactions which culminate in the physiological response. The regulation by Ca2+ of many of these biochemical reactions is mediated by the Ca2+-binding protein calmodulin. Understanding the mechanisms by which Ca2+ and calmodulin regulate the biochemical reactions is the primary objective of this research project. The specific research objectives are as follows: (1) purify to homogeneity and characterize the 60K platelet calmodulin-binding protein which we have identified as a calmodulin-dependent protein phosphatase, (2) identify endogenous substrates for the phosphatase and correlate their Ca2+- and calmodulin-dependent dephosphorylation with specific platelet functions, (3) investigate further the stimulatory effect of calmodulin antagonists on the level of 32P-labeled polyphosphatidylinositols in the platelet and determine if they are substrates for the calmodulin-dependent phosphatase, (4) isolate, characterize and investigate the mechanism of action of the factor which imparts calmodulin-sensitivity to both the kinetics of actin polymerization and the structural characteristics of actin-filaments, (5) further characterize the 150K, 90K, 82K and 41K calmodulin-binding proteins in the platelet and attempt to correlate them with specific calmodulin-dependent enzymes and structural proteins, and (6) identify and characterize platelet Ca2+-binding proteins which, although distinct from calmodulin, may also be important in the regulation of platelet function. Investigating the basic components of the Ca2+-dependent reactions in the platelet will provide new insights into Ca2+-regulatory mechanisms in general as well as their role in regulating platelet functions. Both objectives are important; Ca2+ is an important regulator in all eukaryotic cells, and platelets play a central role in the maintenance of hemostasis as well as in a variety of pathological conditions such as thrombosis and atherosclerosis. Understanding the basic Ca2+ regulatory mechanisms may provide a rationale for the development of new pharmacological reagents to manipulate this important group of reactions.
