Localization of VAMP Isoforms In Platelets Reveals Separate Granule Populations with Distinct Functions

Abstract 2015

Secretion of granules from platelets is essential for normal platelet activity in hemostasis and thrombosis. Platelet granules have different morphologies and different granule subpopulations contain distinct cargos. Platelet granules not only participate in the release of granule contents, but also appear to function to increase plasma membrane surface area during platelet spreading, morphogenesis, tether formation, and membrane repair. Whether different granule subpopulations mediate these distinct functions of the granule is currently not known. To judge whether specific subpopulations of granules are involved in different granule functions, we evaluated the subcellular localization of vesicle-associated membrane proteins (VAMPs) in resting and spread platelets using immunofluorescence confocal microscopy. Imaging showed that VAMP 3- and VAMP 8-containing granules segregate from one another into distinct granule populations (Pearson (R) correlation values (R = 0.143 ± .039, n=25). Fibrinogen and von Willebrand factor (vWF) also segregated from one another (R= 0.099 ± .027, n=15), but associated with granules containing either VAMP 3 or 8 (R = 0.447 ± 0.065 and R = 0.5485 ± 0.62, n=20, respectively). To visualize the granule contribution to membrane remodeling during spreading, granule membranes were selectively labeled using the membrane dye FM 1–43 followed by washout of external dye. Following platelet spreading, FM 1–43-labeled granules coalesced at the ends of pseudopodia and appeared as punctate areas of FM-1-43 staining on the plasma membrane. Quantitation of staining within spread platelets demonstrated that 92% of granules expressing VAMP 8 translocated with their associated cargo (vWF or fibrinogen) to the central granulomere. VAMP 3 also translocated with vWF and fibrinogen to the granulomere in spread platelets. In contrast, only 31% of granules expressing VAMP 5 localized to the granulomere of spread platelets, while 69% of VAMP 5 staining localized to pseudopodia and lamellipodia at the platelet periphery. Granules expressing VAMP 5 demonstrated little colocalization with vWF and fibrinogen in spread platelets. Similarly, VAMP 7 translocated to pseudopodia and lamellipodia during spreading, displaying punctuate staining at the leading edge of plasma membrane. Studies evaluating the expression of VAMP isoforms in nonpermeabilized platelets demonstrated that a portion of VAMP 5, but not VAMPs 3, 7, or 8, resides on the extracellular face of the plasma membrane. Flow cytometry confirmed the presence of VAMP 5 on the extracellular surface of platelets. These data demonstrate that different VAMP isoforms segregate to distinct platelet granule subpopulations and subcellular locals. The colocalization of VAMPs 3 and 8 with vWF and fibrinogen is consistent with previous data demonstrating a role for these VAMP isoforms in cargo release. In contrast, granules expressing VAMP 5 or VAMP 7, which contains an actin-binding login domain, translocate to areas of cytoskeletal remodeling and may contribute membrane to growing cytoskeletal structures as the platelet spreads. These studies indicate that separate granule populations defined by expression of distinct VAMP isoforms perform different functions in platelets.