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Megakaryocytes package contents into separate α-granules that are differentially distributed in platelets.

ABSTRACT: In addition to their primary roles in hemostasis and thrombosis, platelets participate in many other physiological and pathological processes, including, but not limited to inflammation, wound healing, tumor metastasis, and angiogenesis. Among their most interesting properties is the large number of bioactive proteins stored in their α-granules, the major storage granule of platelets. We previously showed that platelets differentially package pro- and antiangiogenic proteins in distinct α-granules that undergo differential release upon platelet activation. Nevertheless, how megakaryocytes achieve differential packaging is not fully understood. In this study, we use a mouse megakaryocyte culture system and endocytosis assay to establish when and where differential packaging occurs during platelet production. Live cell microscopy of primary mouse megakaryocytes incubated with fluorescently conjugated fibrinogen and endostatin showed differential endocytosis and packaging of the labeled proteins into distinct α-granule subpopulations. Super-resolution microscopy of mouse proplatelets and human whole-blood platelet α-granules simultaneously probed for 2 different membrane proteins (VAMP-3 and VAMP-8), and multiple granular content proteins (bFGF, ENDO, TSP, VEGF) confirmed differential packaging of protein contents into α-granules. These data suggest that megakaryocytes differentially sort and package α-granule contents, which are preserved as α-granule subpopulations during proplatelet extension and platelet production.

SUBMITTER: Battinelli EM

PROVIDER: S-EPMC6849942 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledESSENTIALS: How platelets organize their α-granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis.SummaryBackgroundPlatelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their α-granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences.ObjectivesOur aim was to address how the structural organization of α-granules supports their dynamics.MethodsTo preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image nearly entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM).Results and conclusionsIn resting platelets, we observed a morphologically homogeneous α-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, ~4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing α-granules and the canalicular system remained as separate membrane domains. Decondensing α-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by about three-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet α-granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of α-granule proteins.

Project description:AbstractFibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

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Megakaryocytes package contents into separate α-granules that are differentially distributed in platelets.

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