Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7

Abstract

There has been recent controversy as to whether platelet ␣-granules represent a single granule population or are composed of different subpopulations that serve discrete functions. To address this question, we evaluated the localization of vesicle-associated membrane proteins (VAMPs) in spread platelets to determine whether platelets actively sort a specific subpopulation of ␣-granules to the periphery during spreading. Immunofluorescence microscopy demonstrated that granules expressing VAMP-3 and VAMP-8 localized to the central granulomere of spread platelets along with the granule cargos von Willebrand factor and serotonin. In contrast, ␣-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that ␣-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked ␣-granules and demonstrated only minimal spreading. Similarly, spreading was impaired in platelets obtained from Unc13d Jinx mice, which are deficient in Munc13-4 and have an exocytosis defect. These studies identify a new ␣-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that ␣-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading. IntroductionPlatelets are replete with granules containing cargo that is required for platelet function in hemostasis, thrombosis, inflammation, angiogenesis, and malignancy. [1][2][3][4] Platelet granule types include ␣-granules, dense granules, and lysosomes. Of these granule types, the ␣-granule is by far the most abundant with 50 to 80 granules/ platelet, compared with 3 to 6 dense granules/platelet and 0 to 3 lysosomes/platelet. Recent studies indicate that ␣-granules may not constitute a homogenous population. There is evidence that ␣-granule subpopulations can be distinguished on the basis of morphology, 5 cargo type, 6-8 and response to agonists. 7-10 However, experiments using high resolution immunofluorescence microscopy have raised the possibility that the distribution of cargo among ␣-granules is largely stochastic and that the apparent segregation observed by standard immunofluorescence microscopy could result from segregation within granules. 11,12 Although the study of ␣-granule heterogeneity has focused primarily on the localization and release of granule cargo, granules also serve an essential role in membrane remodeling. In nucleated cells, granules provide an internal reservoir of membrane to expand and reshape the plasma membrane during cell movement, 13 membrane resealing, 14,15 neurite outgrowth, [16][17][18] and development of the phagocytotic cup in macrophages. 19,20 Different subpopulations of granules demonstrate different behaviors during membrane remodeling. Recent studies demonstrate that these different granule types can be distinguished by the vesicle-associated membrane proteins (VAMPs) that th...

Should any genetic defect affecting α‐granules in platelets be classified as gray platelet syndrome?

“…36]. One example of proven heterogeneity concerns a specific subpopulation of granules (termed T-granules by some) that specifically label for vesicle-associated membrane protein-7 (VAMP-7) and which contain tissue inhibitor of matrix metalloprotease-2 (TIMP-2) and vascular endothelial growth factor (VEGF) among stored proteins [36,37]. In view of this evolution, ands the fact that classic GPS remains apart as a specific disease of a-granule biogenesis; it seems premature at the present time to group patients with ARC syndrome (and defects of VPS33B or VIPAS39), GFI1B or GATA1 deficiencies within a single named disease even if some platelet defects overlap.…”

WITHDRAWN: "Exogenous carbon monoxide suppresses LPS-induced platelet SNAREs complex assembly and α-granule exocytosis via integrin αIIbβ3-mediated PKCθ/Munc18a pathway"

“…There are three granule types (α-granules, dense granules, and lysosomes) in platelets. Of these granule types, the α-granules are the most abundant granules with 50 to 80 granules/platelet, compared with 3 to 6 dense granules/platelet and 0 to 3 lysosomes/platelet [8]. During sepsis, lipopolysaccharides (LPS) and inflammatory cytokines (e.g.,TNF-α) accelerate the activation of platelets, which further leads to the formation of micro-thrombi in the capillaries [4,9].…”

VAMP-7 links granule exocytosis to actin reorganization during platelet activation

“…39 In platelets, a granules expressing VAMP-7 move to the periphery of the platelets during spreading. 40 However, the role of VAMP-7 in platelet function is unknown.…”

“…43 Conversely, granules contribute to actin-mediated platelet spreading, as evidenced by the fact that spreading is impaired in platelets from patients with gray platelet syndrome or in mouse platelets that lack Munc13-4, a chaperone protein required for granule exocytosis. 40 Yet the molecular underpinnings of the interactions between granule exocytosis and plateletshape change remain poorly understood.…”