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E.I. Cardenas et al.
Munc13-4-regulates platelet aggregation and thrombus stability
To determine how the observed exocytic defects affect platelet function, we first studied platelet aggregation. When using a low concentration of collagen, platelets not expressing Munc13-4 did not undergo shape change (as evidenced by the flat aggregometry recordings) and were unable to form aggregates (Figure 5A and B). While hypo- morphic Munc13-4F/F platelets underwent some shape change, they were still unable to form aggregates. Platelets from all genotypes underwent shape change and formed aggregates at similar levels when using a high dose of col- lagen (Figure 5A and B).
We then assessed platelet thrombus formation under shear stress in a collagen-coated flow chamber and docu- mented a severe defect in Munc13-4-deficient platelets (Figure 5C). The severity of this defect increased at a high- er shear stress (Figure 5D), which suggests that platelets lacking Munc13-4 are unable to form stable thrombi. At high shear, platelets with approximately 50% expression of Munc13-4 (Munc13-4+/−) behaved normally, but those with approximately 20% expression (Munc13-4F/F) showed an intermediate defect.
In all our in vitro studies (Figures 2-5) platelets from Munc13-4Δ/Δ mice behaved almost identically to those from Munc13-4−/− mice, eliminating inefficient Cre recom-
bination as an explanation for the findings in the following in vivo studies.
Lack of Munc13-4 disrupts hemostasis and thrombosis
We used two tail-bleeding tests to study hemostasis. In the classical transection model, Munc13-4 deletant mice were unable to stop bleeding and were euthanized after 20 min (Figure 6A). Furthermore, the hypomorphic Munc13-4F/F mice displayed an identical bleeding diathesis. Because we did not observe a defect in thrombus forma- tion ex vivo at low shear stress, we hypothesized that the Munc13-4F/F mice would stop bleeding if we avoided sec- tioning the tail ventral artery and induced only venous bleeding. With this in mind, we developed a device to make a reproducible incision on the tail dorsal venous plexus only (Online Supplementary Figure S1). Munc13-4−/− and Munc13-4Δ/Δ mice still showed prolonged bleeding, but Munc13-4F/F mice behaved similarly to their WT coun- terparts (Figure 6B). This phenotype was not due to an abnormal number of platelets in the mutant mice (Table 1).
Finally, we assessed thrombosis in vivo with the FeCl3 model of carotid thrombosis. In accordance with our in vitro results, we did not observe vessel occlusion in Munc13-4Δ/Δ mice, while WT arteries occluded in less than 5 min, and hypomorphic Munc13-4F/F mice presented an
AB
C
E
D
Figure 2. Deletion of Munc13-4 impairs dense granule release in collagen-stimulated platelets. Samples from wild-type mice for Munc13-2 and -4 (WT), Munc13-4 +/−, −/−, F/F, Δ/Δ, Munc13-2 −/−, and Munc13-2 and 4 double KO (DKO) mice were stimulated with collagen. Representative tracings (A) and mean release of ATP (dense granules) (B) meas-
F ured by luminometry in whole blood. N=5-14. Representative tracings (C and E) and mean fluo- rescence intensity over baseline (ΔMFI) (D and F) of P-selectin (alpha granules) (C and D) and LAMP-1 (lysosomal granules) (E and F) translocated to the surface of washed platelets measured by flow cytometry. N=8-16. Color leg- end in (A) applies to all panels. Bar: mean; error bar: Standard Error of Mean. #P<0.05; †P<0.01; *P<0.001; comparisons are with
WT unless otherwise specified.
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