Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo

Abstract

Individuals whose platelets lack dense or alpha-granules suffer various degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. In platelets, SNARE proteins mediate the membrane fusion events required for granule cargo release. Endobrevin/VAMP-8 is the primary vesicle-SNARE (v-SNARE) responsible for efficient release of dense and alpha-granule contents; thus, VAMP-8(-/-) mice are a useful model to evaluate the importance of platelet granule secretion in thrombus formation. Thrombus formation, after laser-induced vascular injury, in these mice is delayed and decreased, but not absent. In contrast, thrombus formation is almost completely abolished in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lacks dense granules. Evaluation of aggregation of VAMP-8(-/-) and ruby-eye platelets indicates that defective ADP release is the primary abnormality leading to impaired aggregation. These results demonstrate the importance of dense granule release even in the earliest phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for antiplatelet therapies.

Figure 1

Platelet accumulation in VAMP-8^−/− mice after laser-induced injury of cremaster arterioles. (A) Platelet accumulation was imaged at the indicated time intervals after injury in both wild-type (WT) and VAMP-8–deficient (VAMP-8^−/−) mice. (B) Median integrated platelet fluorescence intensity was plotted at each second for 180 seconds after laser injury in VAMP-8^−/− mice. (C) At 30 seconds, median platelet accumulation in VAMP-8^−/− mice was 4% of WT (P = .01). There was a trend toward a decrease in maximal thrombus size in VAMP-8^−/− mice compared with WT mice, but the difference was not statistically significant (74% of WT; P = .061). By 180 seconds, median platelet accumulation in VAMP-8^−/− mice was 28% of WT mice (P = .01).

Figure 2

Role of ADP in aggregation of VAMP-8^−/− platelets. Wild-type (A) and VAMP-8^−/− (B) platelets were incubated with the indicated concentrations of thrombin and percentage of light transmission was recorded. Panel C represents the mean percentage of aggregation versus thrombin concentration with SD indicated (3 aggregations/condition, n = 8 mice). Thrombin (D: 15 mU/mL; E: 100 mU/mL) was used to stimulate WT (black line) or VAMP-8^−/− platelets (gray line). Aggregation (upper trace) and release of ATP (lower trace) were monitored. (F) Aggregations were measured for VAMP-8^−/− platelets stimulated with 15 mU/mL thrombin and wild-type platelets stimulated with 15 mU/mL thrombin plus 1 U/mL apyrase. (G) Aggregations were measured for VAMP-8^−/− platelets stimulated with 15 mU/mL thrombin alone, 15 mU/mL thrombin plus 2 μM ADP, or 2 μM ADP alone. All aggregation measurements were performed with constant stirring.

Figure 3

Platelet accumulation in ruby-eye mice after laser-induced injury of cremaster arterioles. (A) Platelet accumulation was imaged at the indicated time intervals, after injury, in both wild-type (WT) and ruby-eye mice. (B) Median integrated platelet fluorescence intensity was recorded at each second for 180 seconds after laser injury in ruby-eye mice. (C) At 30 seconds, median platelet accumulation in ruby-eye mice was 22% of WT (P = .001). Maximal thrombus size in ruby-eye mice was decreased compared with WT mice (12% of WT; P = .001). By 180 seconds, median platelet accumulation in ruby-eye mice was 31% of WT mice (P = .007).

Figure 4

Thrombin-mediated aggregation of ruby-eye platelets. Wild-type (A) and ruby-eye (B) platelets were incubated with the indicated concentrations of thrombin and percentage of light transmission was recorded. (C) Mean percentage aggregation in wild-type and ruby-eye platelets was plotted as a function of thrombin concentration with standard deviation indicated (n = 3-8 aggregations/condition, n = 5 mice). (D) Ruby-eye platelets were incubated with 2 μM ADP alone, 25 mU/mL thrombin alone, or 25 mU/mL thrombin plus 2 μM ADP as indicated. Platelet aggregation was measured with constant stirring.