Temporal Secretion of α-Granular Products: Insights into the Mechanisms of Release Reaction

Abstract SCI-36

Upon activation, platelets secrete numerous molecules that affect the local vascular microenvironment. Many of these components are important for hemostasis; however, others play key roles in the sequelae of thrombosis, such as angiogenesis, inflammation, and wound repair. These molecules are stored in three general types of platelet granules: dense, α, and lysosome. Analysis of platelet exocytosis shows that there is a direct correlation between agonist potency (thrombin > convulxin > PAR1 agonist > PAR4 agonist) and the extent and rate of content release. Release from dense granules is most rapid, while lysosome content release is the slowest; release of α-granule contents is heterogeneous. Detailed analysis of α-granule cargo release identified three kinetic classes of release events that differ in their rate. The content released in each of these three classes appeared to be random. Mechanistically, secretion from each granule requires integral membrane proteins called soluble NSF attachment protein receptors (SNAREs). v-SNAREs (vesicle-associated membrane proteins [VAMPs] from the granules) and heterodimeric t-SNAREs (syntaxins and SNAP-23 from the plasma membrane) form a trans-membrane, trimeric complex that mediates fusion and content release. How and where the SNAREs interact is controlled by regulatory proteins that in turn are affected during platelet activation. By analyzing platelet secretion from knockout mice and patients with familial hemophagocytic lymphohistiocytosis (FHL), it has been possible to identify the machinery required for each granule secretion event. Platelets contain four v-SNAREs (VAMP-2, −3, −7, and −8), but only VAMP-8 is required for content release. Platelets contain six t-SNAREs (syntaxin 2, 4, 7, 11, 13, and SNAP-23). Deletion of only syntaxin 11 in patients with FHL4 is sufficient to diminish platelet secretion. SNAP-23 is regulated by phosphorylation on Ser 95 by IκB kinase (IKK). This is important for SNARE complex formation, membrane fusion, and secretion. Intriguingly, mice treated with IKK inhibitors or genetically engineered with a platelet-specific deletion of Ikkb show increased tail-bleeding times. A number of syntaxin regulators have been identified in platelets. MUNC18b is a syntaxin 11 chaperone that is essential for platelet secretion in FHL5 patients. MUNC13 proteins, which facilitate t-SNARE/v-SNARE binding, are also important for platelet granule release. Deletion of Munc13-4 results in a significant platelet secretion defect and a robust bleeding diathesis in mice. Biochemical analysis shows that MUNC13-4 is a docking/tethering factor required to increase the efficacy of membrane fusion. Both MUNC18b and MUNC13-4 appear to be limiting in platelets, suggesting that they might be useful therapeutic targets. A greater understanding of platelet exocytosis is unfolding. This knowledge will undoubtedly lead to the development of better antithrombotic drugs. This work is supported by HL56652 and HL091893 from the National Institutes of Health.