Platelets circulate in blood as small discs which respond to stimuli by changing their forms. This shape transformation occurs by the growth of filopodia and lamellipodia from the cell surface in a process dependent on actin assembly. Greater than 98% of the barbed (fast exchanging) actin filament ends are blocked in resting platelets. Activation of platelets by thrombin leads to rapid actin assembly by causing the cytoskeletal actin filaments of the resting cell to become fragmented and then uncapped. Thrombin results in a cascade of intracellular signals that produces actin assembly and shape change in 15-75 sec. To understand the intracellular signaling system for fast actin assembly from the thrombin receptor to barbed filament ends, the Applicant developed a permeabilized system that responds to TRAP (thrombin receptor activating peptide) by uncapping barbed filament ends. Using this system, the Applicant has established the following pathway: TRAP-->racGTPase-->PI-kinases -- >phosphatidylinositol 4,5-bisphosphate (PI4,5P2)-->filament uncapping. The first goal of this proposal is to define if PI-4 and/or PI-5 kinase are activated by TRAP to produce PI4,5P2 in the permeabilized platelets. The functional kinase and its product, PI4,5P2, will be located in the platelet, and upstream intermediates of this pathway lead to activation of this PI- kinase and actin capping proteins released by ppis and upstream signaling intermediates will be identified. The second goal of this proposal will focus on actin assembly in a signal transduction pathway that begins by activating protein kinase C (PKC). Phorbol myristate acetate (PMA) activates PKC to generate a slow morphological transformation and actin assembly. This pathway differs dramatically from that activated by thrombin as it requires minutes to initiate, is calcium independent, and has PI-3 kinase and its product, PI3,4P2, as essential components. Thrombin-induced actin assembly does not require PI3,4P2. The PMA- induced pathway, however, also initiates actin assembly by uncapping barbed actin filament ends, although the extent of uncapping is small compared with thrombin. The Applicant will define if PI3,4P2 is essential for filament uncapping and if the GPIIb/IIIa receptor, which in its ligated and active form causes a shape change, is involved in this pathway. The location of the signal transduction proteins, PI-3 kinase, GPIIb/IIIa, and tensin in resting and active platelet cytoskeleton will be determined to see if complex formation occurs during activation.
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