COMManding platelet α-granule cargo.
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ABSTRACT:
SUBMITTER: Lykins JT
PROVIDER: S-EPMC8832477 | biostudies-literature | 2022 Feb
REPOSITORIES: biostudies-literature
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Project description:Platelet α-granules play important roles in platelet function. They contain hundreds of proteins that are synthesized by the megakaryocyte or taken up by endocytosis. The trafficking pathways that mediate platelet α-granule biogenesis are incompletely understood, especially with regard to cargo synthesized by the megakaryocyte. Vacuolar-protein sorting 33B (VPS33B) and VPS16B are essential proteins for α-granule biogenesis, but they are largely uncharacterized. Here, we adapted a powerful method to directly map the pathway followed by newly synthesized cargo proteins to reach α-granules. Using this method, we revealed the recycling endosome as a key intermediate compartment in α-granule biogenesis. We then used CRISPR/Cas9 gene editing to knock out VPS33B in pluripotent stem cell-derived immortalized megakaryocyte cells (imMKCLs). Consistent with the observations in platelets from patients with VPS33B mutation, VPS33B-knockout (KO) imMKCLs have drastically reduced levels of α-granule proteins platelet factor 4, von Willebrand factor, and P-selectin. VPS33B and VPS16B form a distinct and small complex in imMKCLs with the same hydrodynamic radius as the recombinant VPS33B-VPS16B heterodimer purified from bacteria. Mechanistically, the VPS33B-VPS16B complex ensures the correct trafficking of α-granule proteins. VPS33B deficiency results in α-granule cargo degradation in lysosomes. VPS16B steady-state levels are significantly lower in VPS33B-KO imMKCLs, suggesting that VPS16B is destabilized in the absence of its partner. Exogenous expression of green fluorescent protein-VPS33B in VPS33B-KO imMKCLs reconstitutes the complex, which localizes to the recycling endosome, further defining this compartment as a key intermediate in α-granule biogenesis. These results advance our understanding of platelet α-granule biogenesis and open new avenues for the study of these organelles.
| S-EPMC6737417 | biostudies-literature
Project description:Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.
| S-EPMC4513253 | biostudies-literature
Project description:Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures.To explore the effect of higher temperatures on platelet physiology.Platelet responses including adhesion, spreading (fluorescence microscopy), ?(IIb)?(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied.Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion.Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.
| S-EPMC3155010 | biostudies-literature
Project description:Dense granules are important in platelet aggregation to form a hemostatic plug as evidenced by the increased bleeding time in mice and humans with dense granule deficiency. Dense granules also are targeted by antiplatelet agents because of their role in thrombus formation. Therefore, the molecular understanding of the dense granule and its biogenesis is of vital importance. In this work, we establish a human megakaryocytic cell line (MEG-01) as a model system for the study of dense granule biogenesis using a variety of cell biology and biochemical approaches. Using this model system, we determine the late endocytic origin of these organelles by colocalization of the internalized fluid phase marker dextran with both mepacrine and transmembrane dense granule proteins. By mistargeting of mutant dense granule proteins, we demonstrate that sorting signals recognized by adaptor protein-3 are necessary for normal transport to dense granules. Furthermore, we show that tissue-specific Rab32 and Rab38 are crucial for the fusion of vesicles containing dense granule cargo with the maturing organelle. This work sheds light on the biogenesis of dense granules at the molecular level and opens the possibility of using this powerful model system for the investigation of new components of the biogenesis machinery.
| S-EPMC3496959 | biostudies-literature
Project description:There has been recent controversy as to whether platelet α-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 α-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, α-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that α-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked α-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 α-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that α-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading.
| S-EPMC3390958 | biostudies-literature
Project description:Cancer is associated with a prothrombogenic state capable of platelet activation. Platelets, on the other hand, can support angiogenesis, a process involved in the progression of tumor growth and metastasis. However, it is unclear whether platelet/tumor interactions substantially contribute to tumor physiology. We investigated whether platelets stabilize tumor vessels and studied the underlying mechanisms. We induced severe acute thrombocytopenia in mice bearing s.c. Lewis lung carcinoma or B16F10 melanoma. Intravital microscopy revealed that platelet depletion led to a rapid destabilization of tumor vessels with intratumor hemorrhage starting as soon as 30 min after induction of thrombocytopenia. Using an inhibitor of glycoprotein Ibalpha (GPIbalpha) and genetically engineered mice with platelet adhesion defects, we investigated the role of platelet adhesion receptors in stabilizing tumor vessels. We found that a single defect in either GPIbalpha, von Willebrand factor, P-selectin, or platelet integrin activation did not lead to intratumor hemorrhage. We then compared the ability of transfused resting and degranulated platelets to prevent intratumor hemorrhage. Whereas resting platelets prevented thrombocytopenia-induced tumor bleeding, circulating degranulated platelets did not. This suggests that the prevention of intratumor hemorrhage by platelets relies on the secretion of the content of platelet granules. Supporting this hypothesis, we further found that thrombocytopenia dramatically impairs the balance between propermeability and antipermeability factors in tumor-bearing animals, in particular depleting blood of angiopoietin-1 and serotonin. Our results show a crucial contribution of platelets to tumor homeostasis through continuous prevention of severe intratumor hemorrhage and consequent cell death. The study also suggests platelet function as a reasonable target for specific destabilization of tumor vessels.
| S-EPMC2547489 | biostudies-literature
Project description:Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.
| S-EPMC6397092 | biostudies-literature
Project description:The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.
| S-EPMC10144907 | biostudies-literature
Project description:Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.
| S-EPMC7839360 | biostudies-literature
Project description:IntroductionPlatelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy - the so-called 'super-resolution' microscopies - as a possible solution. Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application. Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.
| S-EPMC5942152 | biostudies-literature