Date Available

4-30-2012

Year of Publication

2012

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Biochemistry

First Advisor

Dr. Sidney W. Whiteheart

Abstract

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.

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