Dynamin-Related Protein-1 Controls Fusion Pore Dynamics During Platelet Granule Secretion and Thrombus Formation In Vivo

Abstract 361

Platelet granule secretion serves a central role in hemostasis and thrombosis. During platelet secretion, fusion of granule membranes with those of the plasma membrane results in the release of granule contents. Recently electrochemical techniques using single-cell amperometry have shown that platelet membrane fusion results in the formation of a fusion pore. The fusion pore subsequently expands to enable the complete extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore formation and expansion are not known. To discover novel components of the platelet secretory machinery, we tested >300,000 compounds in a forward chemical genetic screen designed to identify inhibitors of dense granule secretion. A compound, ML160, was found that blocked dense granule release with an IC₅₀ of approximately 0.5 μM. ML160 was also identified in an unrelated high throughput screen designed to detect inhibitors of dynamin-related protein-1 (Drp-1). Although best known as mediators of membrane fission, dynamins also contribute to granule exocytosis by controlling fusion pore expansion. Immunoblot analysis of platelet pellets and supernatants confirmed the presence of Drp-1 in platelets and demonstrated nearly equal distribution between platelet membranes and cytosol. mDivi-1, a well-characterized small molecule inhibitor of Drp-1 that acts outside of the GTP binding site, blocked PAR1-mediated platelet dense granule and α-granule release with an IC₅₀ of approximately 20 μM. mDivi-1 also inhibited granule release induced by the thromboxane receptor agonist U46619, PMA, or Ca²⁺ ionophore, indicating that Drp-1 acts distally in the secretory pathway. To assess whether Drp-1 functions in platelet fusion pore dynamics, we tested the effect of mDivi-1 on the release of dense granules from rabbit platelets using single-cell amperometry. This technique monitors the release of serotonin from single granules in real-time with sub-millisecond temporal resolution. mDivi-1 exposure (10 μM) retarded each release event, resulting in a prolonged spike width of 23.00 ± 1.702 msec compared to the control value of 14.71 ± 1.194 msec. Although this concentration of mDivi-1 did not change the overall percentage of the fusion pore events or the amount of serotonin released through the fusion pore, it showed a distinct effect on the transition from stable fusion pore to maximal fusion pore dilation (% foot= 17.46 ± 1.809%, 9.464 ± 2.014% for control and mDivi-1 conditions, respectively). Evaluation of fluorescein-dextran incorporation into activated platelets by fluorescence microscopy enabled visualization of fusion pore dynamics and confirmed the effect of mDivi-1 on fusion pore expansion. To assess whether Drp-1 participates in platelet function in vivo, we determined the effect of mDivi-1 on thrombus formation following laser-induced injury of mouse cremaster arterioles. mDivi-1 inhibited platelet accumulation at the site of vascular injury by 74%. In contrast, mDivi-1 had no significant effect on fibrin formation under the same conditions. These results identify Drp-1 in platelets, demonstrate a role for Drp-1 in fusion pore dynamics, and indicate that pharmacological regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo.