Inverse immunostaining pattern for synthesized versus endocytosed alpha‐granule proteins in human bone marrow megakaryocytes

Larouzière, Vanina De; Brouland, Jean‐Philippe; Souni, Fadila; Drouet, Ludovic; Cramer, Elisabeth M.

doi:10.1046/j.1365-2141.1998.00755.x

Cited by 10 publications

(5 citation statements)

References 43 publications

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“…Platelet secretory granule protein cargo is synthesized in the MK ER and transported via vesicles derived from the trans-Golgi network 31 to early MVBs, which according to current models also receive cargo in endocytic vesicles as they mature and give rise to platelet lysosomes and α and dense 32 granules. 31, 33, 34 Transferrin receptor (TFR; CD71) undergoes clathrin-mediated endocytosis and is frequently used as marker of the MK recycling endocytic compartment, 35 while TGN46 is a marker of the trans-Golgi network.…”

Section: Resultsmentioning

confidence: 99%

“…31, 33, 34 Transferrin receptor (TFR; CD71) undergoes clathrin-mediated endocytosis and is frequently used as marker of the MK recycling endocytic compartment, 35 while TGN46 is a marker of the trans-Golgi network. IFM examination of thiol isomerase expression in human MKs in serum-free culture revealed little overlap between ERp57, TFR and TGN46 (Figure 2 A; Video 5) or between PDI and the α-granule cargo proteins VWF (Figure S1A; Video 6) and thrombospondin-1 (TSP-1; not shown).…”

Section: Resultsmentioning

confidence: 99%

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Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases

Crescente

Pluthero

et al. 2016

ATVB

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Objective Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including PDI and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation and thrombus formation. In this study we examined for the first time the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets and identified the cellular events responsible for their movement to the platelet surface upon activation. Approach and Results Immunofluorescence microscopy (IFM) imaging was used to localize PDI and ERp57 in murine and human megakaryocytes at various developmental stages. IFM and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and SERCA3. IFM and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin), and in the presence or absence of membrane fusion mediated by Munc 13-4 (absent in platelets from Unc13dJinx mice). Conclusions Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes, and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not SNARE/Munc 13-4-dependent vesicular-plasma membrane fusion.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases

Crescente

Pluthero

et al. 2016

ATVB

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“…22 In vivo studies have recently confirmed these data. 25 vWF appears early also during MK maturation and is stored in a specific compartment at one pole of the ␣-granules. 3 In gray platelets, this polar distribution of vWF within ␣-granules is not observed.…”

Section: Discussionmentioning

confidence: 99%

Newly recognized cellular abnormalities in the gray platelet syndrome

Drouin

Favier

Massé

et al. 2001

Blood

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The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased ␣-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34 ؉ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in ␣-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of Pselectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal ␣-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS. IntroductionThe gray platelet syndrome (GPS) is an inherited disorder of primary hemostasis with associated bleeding tendency, thrombocytopenia, and classical abnormal platelet morphology. 1 The platelets appear characteristically gray after Romanovsky staining as a result of an abnormally low ␣-granule content. Immunoelectron microscopic analysis of gray platelets confirms that the ␣-granule contents appear absent. 2 In contrast, the granule-limiting membranes appear present with normally expressed protein components. 3 This syndrome is distinct from other inherited disorders that also affect platelet ␣-granules, such as ␣-␦ storage pool deficiency and the Quebec platelet disorder. The former is combined with dense granule deficiency 4 and the latter with an abnormal protease activity responsible for ␣-granule protein degradation. 5 The ␣-granules are the principal storage site for hemostatic proteins such as fibrinogen, von Willebrand factor (vWF), thrombospondin, and factor V and for growth factors such as platelet-derived growth factor and transforming growth factor-␤. 6 In addition, plasma proteins such as albumin and immunoglobulins are endocytosed by platelets and stored in ␣-granules. 7 The ␣-granule proteins are released into the open canalicular system on platelet activation and play a key role both in hemostasis and wound healing. In this report, we ...

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“…Spleen and lung biopsies were withdrawn and embedded in paraffin. Four-m sections were cut and left for 24 hours at 37 Њ C. After deparaffinization and rehydration, immunochemistry was performed with the APAAP method and enzyme activity revealed by fast red staining (Dako A/S, Glostrup, Denmark) [26]. Measurements were performed on APAAP stained bone marrow.…”

Section: Alkaline Phosphatase Anti-alkaline Phosphatase (Apaap)mentioning

confidence: 99%