Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis. To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo.

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the identity of the functional syntaxin has been controversial. Previous studies, using anti-syntaxin antibodies in permeabilized platelets, suggested roles for both syntaxin-2 and -4. These conclusions were experimentally tested using platelets from syntaxin knockout mice and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient that lacks syntaxin-11. Platelets from syntaxin-2 and syntaxin-4 single or double knockout mice had no significant secretion defect. However, platelets from the FHL4 patient had a robust defect, though their morphology, activation, and cargo levels appeared normal. Semi-quantitative western blotting showed that syntaxin-11 is the most abundant syntaxin in both human and murine platelets. Co-immunoprecipitation experiments showed that syntaxin-11 forms SNARE complexes with VAMP-8 and SNAP-23. These data conclusively demonstrate that syntaxin-11, but not syntaxin-2, or -4, is required for platelet exocytosis. We also show that a syntaxin binding protein, tomosyn-1, is important for platelet exocytosis and hemostasis. Tomosyn-1 was identified from platelet extracts using affinity chromatography, RT-PCR analysis, and western blotting analysis. Tomosyn-1 was co-immunoprecipitated with syntaxin-11/SNAP-23 from both resting and activated platelet extracts. Platelets from tomosyn-1-/- mice displayed a secretion defect, but their morphology and activation appeared normal. Tomosyn-1-/- mice showed impaired thrombus formation in two different injury models. Given the importance of platelet secretion to hemostasis, it is hoped that the insights gained from these studies in this dissertation will help to identify new and more valuable therapeutic targets to control clot formation.