Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent occlusive thrombosis without triggering bleeding. My work addresses this question by showing how the rates and extents of granule secretion could be regulated by various v-SNAREs. We also show that the granule cargo decondensation is an intermediate to secretion that also contributes to rates of cargo release. Platelets contain four major VAMP isoforms (-2, -3, -7, and -8), however, VAMP-8 and -7 play a primary role while VAMP-2 and -3 are ancillary in secretion. To exploit this heterogeneity in VAMP usage, platelet-specific V-2/3-/- and V-2/3/8-/- mouse models were generated and characterized to understand how secretion influences hemostasis. We found that each VAMP isoform differentially contributes by altering the rates and extents of cargo release. The loss of VAMP-2 and -3 had a minimal impact while the loss of VAMP-2, -3 and -8 significantly reduced the granule secretion. Platelet activation and aggregation were not affected though the spreading was reduced in V-2/3/8-/- platelets indicating the importance of secretion in spreading. Though coagulation pathways were unaltered, PS exposure was reduced in both V-2/3-/- and V-2/3/8-/- platelets suggesting diminished procoagulant activity. In vivo experiments showed that V-2/3/8-/- animals bled profusely upon tail transaction and failed to form occlusive thrombus upon arterial injury while V-2/3-/- animals did not display any hemostatic deficiency. These data suggest that about 40-50% reduction in secretion provides protection against thrombosis without compromising hemostasis and beyond 50% secretion deficiency, the animals fail to form functional thrombi and exhibit severe bleeding. Additionally, detailed structural analysis of activated platelets suggests that the post-stimulation cargo dissolution depends on an agonist concentration and stimulation duration. This process is VAMP-dependent and represents intermediate steps leading to a full exodus of cargo. Moreover, we also show that VAMP-8 is important for compound fusion events and regulates fusion pore size. This is a first comprehensive report that shows how manipulation of the exocytic machinery have an impact on secretion and ultimately on hemostasis. These animals will be instrumental in future investigations of platelet secretion in many other vascular processes.