Page 163 - Haematologica July

Munc13-4 in hemostasis and airway inflammation
AB
Figure 5. Deletion of Munc13-4 impedes platelet aggregation and interferes with thrombus formation in vitro. Samples from Munc13-4 mutant mice were stimulated with
unless otherwise specified.
CD collagen. Representative tracings of platelet aggregation (A) and average maximum aggregation measured by light transmittance (B) on platelet- rich plasma. N=3-6. Whole blood was fluorescently-labeled and perfused over collagen-coated plates at low (C) or high (D) sheer stress. Thrombus buildup was monitored by fluores- cence intensity and compared at 200 seconds (s). N=5-10. Color legend in (A) applies to all panels. Bar or circle: mean; error bar: Standard Error of Mean. #P<0.05; †P<0.01; *P<0.001; comparisons are with Munc13-4+/+
ABC
Figure 6. Deletion of Munc13-4 disrupts hemostasis and thrombus formation in vivo. Arterial (A) or venous (B) bleeding was measured in tails from Munc13-4 mutant mice. N=8-10. (C) Time to carotid flow occlusion after applying FeCl3 for 3 minutes (min). N=6-9. Circle: individual mouse; horizontal line or bar: mean; error bar: Standard Error of Mean. †P<0.01; *P<0.001; comparisons are with Munc13-4+/+ unless otherwise specified.
While in mast cells the residual exocytosis observed after removing Munc13-4 was mediated by Munc13-2,32 the priming component that mediates alpha granule exocyto- sis in platelets remains unknown. Though generally thought of as a neuronal protein, Munc13-1 has been shown to regulate exocytosis in cytotoxic T lympho- cytes,36 and it could be involved in platelet alpha granule release. In contrast to Munc13-4, Munc13-1 contains a regulatory diacylglycerol (DAG)-binding C1 domain.37 Collagen stimulation of platelets causes more sustained DAG formation than thrombin.38 It is possible that Munc13-1 plays a compensatory role in lysosomal granule
release, which would explain why we only observed a thrombin-dependent defect in the absence of Munc13-4.
Hemostasis and thrombosis
Munc13-4 global KO mice have a severe hemostatic defect, and the hypothesis is that this is due to impaired ADP release from platelets.11,13 This cannot be tested with a regular global KO mouse given that Munc13-4 is expressed in other tissues relevant to hemostasis (e.g. endothelium).14 Our finding that the bleeding diathesis is identical in global and conditional KO mice (Figure 6) sup- ports the notion that this Munc13-4-dependent phenome-
haematologica | 2018; 103(7)
1241