Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

Platelet exocytosis, mediated by SNAREs and Ca2+-dependent regulators, is critical for haemostasis. Munc13-4 binds membranes in a Ca2+- and phosphatidylserine (PS)-dependent manner and acts as a tethering factor for pre-docked platelet dense granule secretion to mediate rapid response to vascular damage.

Platelet secretion is kinetically heterogeneous in an agonist-responsive manner

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? Here we sought to describe the platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (ie, 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, whereas thrombin rapidly induced the greatest release. Cargo with opposing actions (eg, proangiogenic and antiangiogenic) 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 3 classes of release events, differing in their rates. The distribution of cargo into these 3 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.

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. Disclosures: No relevant conflicts of interest to declare.

Munc18b/STXBP2 is required for platelet secretion

Platelets are vital for hemostasis because they release their granule contents in response to vascular damage. Platelet exocytosis is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), whose interactions are governed by regulators, eg, Sec/Munc18 proteins. These proteins chaperone syntaxin t-SNAREs and are required for exocytosis. Platelets contain 3 Munc18 isoforms: Munc18a, Munc18b, and Munc18c. We report that Munc18b is the major isoform and is required for platelet secretion. Familial hemophagocytic lymphohistiocytosis type 5 (FHL5) is caused by defects in the Munc18b/STXBP2 gene. We confirm a previous report showing that platelets from FHL5 patients have defective secretion. Serotonin, ADP/ATP, and platelet factor 4 release was profoundly affected in the 2 biallelic patients and partially in a heterozygous patient. Release of lysosomal contents was only affected in the biallelic platelets. Platelets from the FHL5 biallelic patients showed decreased Munc18b and syntaxin-11 levels were significantly reduced; other syntaxins were unaffected. Munc18b formed complexes with syntaxin-11, SNAP-23, and vesicle-associated membrane protein-8 in human platelets. Other potential secretion regulators, Munc13-4 and Rab27, were also found associated. These data demonstrate a key role for Munc18b, perhaps as a limiting factor, in platelet exocytosis and suggest that it regulates syntaxin-11.

Syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet secretion

The platelet release reaction plays a critical role in thrombosis and contributes to the events that follow hemostasis. Previous studies have shown that platelet secretion is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins from granule and plasma membranes. The SNAREs form transmembrane complexes that mediate membrane fusion and granule cargo release. Although VAMP-8 (v-SNARE) and SNAP-23 (a t-SNARE class) are important for platelet secretion, the identity of the functional syntaxin (another t-SNARE class) has been controversial. Previous studies using anti-syntaxin Abs in permeabilized platelets have suggested roles for both syntaxin-2 and syntaxin-4. In the present study, we tested these conclusions using platelets from syntaxin-knockout mouse strains and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient. Platelets from syntaxin-2 and syntaxin-4 single- or double-knockout mice had no secretion defect. Platelets from a FHL4 patient deficient in syntaxin-11 had a robust defect in agonist-induced secretion although their morphology, activation, and cargo levels appeared normal. Semiquantitative Western blotting showed that syntaxin-11 is the more abundant syntaxin in both human and murine platelets. Coimmunoprecipitation experiments showed that syntaxin-11 can form SNARE complexes with both VAMP-8 and SNAP-23. The results of the present study indicate that syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet exocytosis.