Fellowship/Schraw: Establishing the Ground Rules for Regulation of Platelet Membrane Fusion

Platelets are small cell fragments that recognize sites of damage in blood vessels and are a critical component in maintaining haemostatsis. Upon sensing vascular damage platelets undergo a series of reactions that involves release of content from the granule stores (alpha, dense core & Iysosoma/), sequestering additional platelets and inevitably clot formation. A central component of granule release is the action of Soluble NSF Attachment Protein Receptors (SNAREs) that are required for membrane fusion. The SNARE molecules are integrated into the membranes with the t-SNAREs located on the target membrane and the v-SNAREs on the granules. These proteins bind to each other forming the trans-membrane core complex that is a requirement of granule fusion. The hypothesis of this proposal is that syntaxin, a t-SNARE, is regulated by several molecules that control its ability to bind to other SNARE molecules. To understand how syntaxin is regulated three specific aims are proposed. Aim1 :Identify potential syntaxin interacting proteins in platelets and determine their specificity. Syntaxin2,4,& 7 have been identified in platelets. The effector molecules identified to date are Munc18(a,b,c), Doc2 and Munc13. This aim will attempt to find the remaining syntaxin interacting proteins present in platelets. Aim2:Determine if the interactions between syntaxin and syntaxin-binding proteins are modulated during the platelet's response to stimuli. Upon activation of platelets the set of molecules that associates with syntaxin shifts to facilitate trans-membrane core complex formation. This aim will establish which effector molecules are involved in maintaining syntaxin in the resting state versus the activated state. Aim3:Determine the physiological relevance of the syntaxin interactions by introducing perturbents into a permeabilized platelet release assay. How the potential SNARE regulatory proteins affect the t-SNARE syntaxin in platelets and how these regulators may be controlled by upstream signaling events will be explored by using recombinant proteins, peptides and specific antibodies in the exocytosis assay. This aim attempts to place the effector molecules in an ordered arrangement of steps that leads to regulated granule fusion. Understanding the process of platelet granule release will aid in identification of therapeutic targets for controlling clot formation and in diagnosis of patients with bleeding time disorders due to defective platelet secretion machinery.