acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies. Cancer Res. 2008;68(24):10349-10357. 8. Grand FH, Hidalgo-Curtis CE, Ernst T, et al. Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms. Blood. 2009; 113(24):6182-6192. 9. Sanada M, Suzuki T, Shih LY, et al. Gain-of-function of mutated C-CBL tumour suppressor in myeloid neo- plasms. Nature. 2009;460(7257):904-908. 10. Kotecha N, Flores NJ, Irish JM, et al. Single-cell pro- filing identifies aberrant STAT5 activation in myeloid ma- lignancies with specific clinical and biologic correlates. Can- cer Cell. 2008;14(4):335-343. ●●● PLATELETS & THROMBOPOIESIS Comment on Ren et al, page 869 A Munc in the platelet granule works ---------------------------------------------------------------------------------------------------------------- Michael S. Marks UNIVERSITY OF PENNSYLVANIA SCHOOL OF MEDICINE The membrane fusion regulator Munc13-4 facilitates calcium-stimulated release of cytolytic and inflammatory mediators from lymphocytes and granulocytes. In this issue of Blood, Ren et al reveal a similar requirement for Munc13-4 in secretion from activated platelets and provide new insights into a human genetic disease. 1 W hen platelets are stimulated, they re- lease a variety of soluble mediators from 3 different types of intracellular storage compartments: -granules, dense granules, and lysosomes. To release their contents to the extracellular space, the membrane of each com- partment must fuse with the plasma membrane, a process that is driven by cognate interactions between soluble N-ethylmaleimide– sensitive factor attachment protein receptor (SNARE) family proteins. Formation of a 4-he- lix bundle by cytoplasmic coiled-coil domains of a tSNARE complex on the plasma membrane and a vSNARE on the granule membrane liber- ates the energy required to drive membrane fu- sion. 2 Similar SNARE-dependent fusion events mediate intracellular membrane trafficking be- tween all secretory and endosomal compart- ments, but granule release must be more tightly regulated so that it only occurs after calcium influx induced by platelet activation. The SNAREs themselves are not calcium-respon- sive; rather, regulatory proteins must ensure that granules accumulate at plasma membrane fusion sites and that appropriate SNARE complexes form at the right time. Identifying these regula- tory proteins is of paramount importance both for understanding how to manipulate platelet granule release for therapeutic means and for identifying genetic abnormalities that might underlie bleeding disorders. Ren et al identified Munc13-4 as one such SNARE regulator in platelets. 1 Munc13-4 is a member of a small family of proteins that regulate SNARE-dependent fusion at the plasma membrane. The gene en- coding Munc13-4 is mutated in familial he- mophagocytic lymphohistocytosis type 3 (FHL3), 3 a disease in which patients suffer from uncontrolled inflammation, lymphopro- liferation, and neuronal abnormalities. The hematologic symptoms of FHL3 reflect the failure of several hematopoietic cell types to secrete contents from storage compartments— referred to collectively as lysosome-related or- ganelles (LROs) 4 —in response to agonists. The affected LROs include cytolytic granules of cytotoxic T cells and natural killer cells, 3,5 azurophilic granules of basophils and mast cells, 6 and secondary and tertiary granules of neutrophils 7 ; lethality in FHL3 reflects the loss of Munc13-4 function in cytolytic granule release and consequently of cytotoxic T and natural killer cell function. Cytolytic granules in Munc13-4 – deficient T cells accumulate prop- erly at the plasma membrane but do not release their contents, 3 suggesting that Munc13-4 par- ticipates in activating calcium-induced LRO fusion with the plasma membrane. Munc13 family members bind in vitro to tSNAREs (eg, see Guan et al 8 ), suggesting that they prime fusion by either facilitating formation of 4-helix SNARE bundles or by “tethering” LROs to the plasma membrane to allow SNARE complexes to form (see figure). Ren et al built upon earlier findings by Shirakawa and colleagues 9 that platelets ex- press Munc13-4 and that addition of recombi- nant Munc13-4 to permeabilized platelets enhances calcium-induced release of a dense granule cargo, serotonin. To test whether Munc13-4 is required for platelet granule re- lease, Ren et al turned to the Unc13d Jinx mouse model of FHL3, in which the Unc13d gene encoding Munc13-4 is inactivated. The key finding is that after stimulation with thrombin, Unc13d Jinx Munc13-4 – deficient platelets se- crete dramatically less platelet factor-4 (from -granules) and beta-hexosaminidase (from lysosomes) than wild-type platelets and fail completely to secrete serotonin (from dense granules). This secretion defect correlated with 2 signs that granules failed to fuse with the plasma membrane—a loss of thrombin- stimulated surface expression of -granule and lysosomal membrane proteins, and reten- tion of intracellular granules by electron mi- croscopy— but was not a consequence of inef- fective signaling, because Unc13d Jinx platelets responded to thrombin stimulation appropri- ately by calcium influx, tyrosine phosphoryla- tion, and integrin activation. Using a beautiful in vitro reconstitution system, addition of re- combinant full-length Munc13-4 to perme- abilized Unc13d Jinx platelets fully restored secretion from all 3 granule types. A truncated Munc13-4 stimulates fusion of platelet granules with the plasma membrane to release granule contents. (Top) Presecretory phase in which secretory lyso- somes, platelet -granules or dense granules, or other LRO is “docked” at the plasma membrane. Fusion requires engagement of a vSNARE (red) on the granule membrane with a tSNARE complex (green) on the plasma membrane. Munc13-4 (orange) stimu- lates this engagement in a calcium-dependent man- ner. Upon cell activation and calcium influx (bottom), the membranes fuse, releasing the contents of the granule to the extracellular space. Highlighted are examples of LRO cargoes that are known to be regulated by Munc13-4. 864 12 AUGUST 2010 I VOLUME 116, NUMBER 6 blood For personal use only. on July 24, 2018. by guest www.bloodjournal.org From