Platelet Munc13-4 regulates hemostasis, thrombosis and airway inflammation

Abstract

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4. We assessed platelet granule exocytosis in Munc13-2 and -4 global and conditional knockout (KO) mice, and observed that deletion of Munc13-4 ablates dense granule release and indirectly impairs alpha granule exocytosis. We found no exocytic role for Munc13-2 in platelets, not even in the absence of Munc13-4. In vitro, Munc13-4-deficient platelets exhibited defective aggregation at low doses of collagen. In a flow chamber assay, we observed that Munc13-4 acted as a rate-limiting factor in the formation of thrombi. In vivo, we observed a dose-dependency between Munc13-4 expression in platelets and both venous bleeding time and time to arterial thrombosis. Finally, in a model of allergic airway inflammation, we found that platelet-specific Munc13-4 KO mice had a reduction in airway hyper-responsiveness and eosinophilic inflammation. Taken together, our results indicate that Munc13-4-dependent platelet dense granule release plays essential roles in hemostasis, thrombosis and allergic inflammation.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

“…We measured the release of ATP (dense granules) and the translocation of P-selectin (alpha granules) and LAMP-1 (lysosomal granules) to the plasma membrane upon platelet activation. We stimulated platelets with a low and high dose of thrombin as described previously (25). We observed that Munc18-2 deletion severely impaired exocytosis of dense, alpha, and lysosomal granules (Fig.…”

“…Finally, we assessed thrombosis in vivo with the FeCl 3 model of carotid artery thrombosis (25,28). Although most Munc18-2 ϩ/ϩ and Munc18-2 F/F mice showed arterial occlusion in less than 5 min, we did not observe any vessel occlusion in Munc18-2 ⌬/⌬ mice (Fig.…”

“…The impaired secretion of ADP from dense granules in Munc13-4 -deficient platelets indirectly affects the exocytosis of their alpha granules. This was proven when we were able to rescue alpha granule release with exogenous ADP in platelets lacking Munc13-4 (25). However, the severe exocytic defects observed in Munc18-2 ⌬/⌬ platelets could not be rescued, even in the presence of high doses of collagen or thrombin, or by addition of ADP (Fig.…”

“…Alterations in ADP release from dense granules can indirectly affect the exocytosis of other platelet granules (25). To test whether the defects observed in alpha and lysosomal gran-ule exocytosis from Munc18-2 ⌬/⌬ platelets was indirect, we added exogenous ADP to the stimulated platelets and found that it could rescue neither the secretion of PF4 (Fig.…”

“…6A). We then used a device we had described previously that reproducibly makes a 0.8-mm-deep dorsal cut at the level where the mouse tail is 3.8 mm in diameter (25), sectioning only the dorsal venous plexus to induce venous bleeding, and observed that Munc18-2 ⌬/⌬ mice bled significantly longer than the controls (Fig. 6B).…”