Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Sander, H.J.; Slot, Jan W.; Bouma, Bonno N.; Bolhuis, P. A.; Pepper, Duncan S.; Sixma, Jan J.

doi:10.1172/jci111084

Cited by 72 publications

(27 citation statements)

References 43 publications

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“…37 Four distinct regions can be distinguished within ␣-granules based on ultrastructural and immunolabeling studies. 37,[46][47][48] These regions include the peripheral membrane (containing P-selectin), tubular structures (containing multimeric VWF), an electron dense nucleoid (containing ␤-TG and PF4), and an electron lucent region (containing fibrinogen and TSP-1). Proteomic analysis of proteins released from activated human platelets has identified more than 300 proteins, many of which reside within ␣-granules.…”

Section: Discussionmentioning

confidence: 99%

Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet -granule biogenesis

2005

Blood

139

162

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Bleeding problems are associated with defects in platelet ␣-granules, yet little is known about how these granules are formed and released. Mutations affecting VPS33B, a novel Sec1/Munc18 protein, have recently been linked to arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. We have characterized platelets from patients with ARC syndrome and observed reduced aggregation with arachidonate and adenosine diphosphate (ADP). Structural abnormalities seen in ARC platelets included increased platelet size, a pale appearance in blood films, elevated numbers of ␦-granules, and completely absent ␣-granules. Soluble and membrane-bound ␣-granule proteins were significantly decreased or undetectable in ARC platelets, suggesting that both the releasable protein pools and membrane components of ␣-granules were absent. The role of VPS33B in platelet granule biogenesis was evaluated by immunofluorescence microscopy in normal human megakaryocytes. VPS33B colocalized appreciably with markers of ␣-granules, moderately with late endosomes/lysosomes, minimally with ␦-granules/lysosomes, and not with cis-Golgi complexes. VPS33B protein expression determined by immunoblotting confirmed the presence of VPS33B in control fibroblasts but not in ARC fibroblasts, and in normal megakaryocytes but not in platelets. We conclude that like other Sec1/Munc18 proteins, VPS33B is involved in intracellular vesicle trafficking, being essential for the development of platelet ␣-granules but not for granule secretion. IntroductionCongenital platelet disorders involving abnormalities in ␣ and/or dense (␦) granules are an important cause of inherited bleeding disorders. A number of genes have been linked to the ␦-granule defects associated with the Hermansky-Pudlak and ChediakHigashi syndromes, 1-6 and 16 genes have been identified in mice that regulate vesicle trafficking to platelet ␦-granules and melanosomes; some of these genes encode known vesicle trafficking proteins whereas others encode components of BLOC (biogenesis of lysosome-related organelles complexes) protein complexes. 3,7 Much less is known about the genes or proteins involved in ␣-granule defects, although some transcription factors and cellsurface glycoproteins have been implicated. For example, a family with X-linked macrothrombocytopenia and reduced ␣-granules was found to have an aspartate-to-glycine mutation in the transcription factor GATA1, 8 and hemizygous loss of the FLI1 transcription factor causes the Paris-Trousseau syndrome, characterized by abnormal megakaryocytes and giant ␣-granules. 9,10 In mice, loss of transcription factor NF-E2 expression produces abnormal megakaryocytes with absent ␣-granules, 11 and hematopoietic zinc finger (Hzf)-deficient mice have megakaryocytes and platelets with reduced ␣-granules and ␣-granule proteins. 12 Glycoprotein (GP) Ib␤-deficient mouse platelets contain abnormally large ␣-granules, 13 and giant ␣-granules have also been observed in platelets from a patient with Bernard-Soulier syndrome. 14 A deficiency of both platel...

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Section: Discussionmentioning

confidence: 99%

Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet -granule biogenesis

2005

Blood

139

162

View full text Add to dashboard Cite

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“…Particularly relevant to the present study is the demonstration of an actively cycling pool of ajIb33 in platelets (63, 64) and its presence on the a-granule membrane (65,66). However, the intragranular storage pattern determined by immunoelectron microscopy shows fibrinogen to be randomly distributed in the matrix of a-granules except the nucleoid (67,68). This suggests that at least some ofthe fibrinogen molecules within a-granules may dissociate from their membrane-bound receptor.…”

Section: Discussionmentioning

confidence: 99%

Kistrin, an integrin antagonist, blocks endocytosis of fibrinogen into guinea pig megakaryocyte and platelet alpha-granules.

Handagama¹,

Bainton²,

Jacques³

et al. 1993

J. Clin. Invest.

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Recent data indicate that megakaryocyte/platelet a-granule fibrinogen is endocytosed from plasma. Because fibrinogen is the major platelet protein present in high concentrations in a-granules, fibrinogen uptake into a-granules may occur via specific receptors. In that cells of the megakaryocyte/platelet lineage contain two integrins-am63 (GP IIb-Illa) and the vitronectin receptor (a'83)-that can bind fibrinogen, one or both of these receptors may mediate the endocytic uptake of fibrinogen. To test this hypothesis, we examined the effect of Kistrin, an RGD-containing protein purified from the venom of Agkistrodon rhodostoma that inhibits fibrinogen binding to human platelet receptors, on endocytosis of fibrinogen by megakaryocytes and platelets. Continuous intravenous infusion of kistrin into guinea pigs (200 ag/h) over a 24-h period inhibited collagen-induced platelet aggregation. When biotinylated fibrinogen was injected intravenously into animals receiving Kistrin, megakaryocytes failed to endocytose the labeled fibrinogen. Endocytosis of fibrinogen into platelets was also inhibited in these animals. In contrast, platelets and megakaryocytes obtained from shaminfused control animals contained the injected biotinylated fibrinogen. We conclude that, in addition to the well-known extracellular function of cell adhesion, integrins can also act as receptors that mediate endocytosis of exogenous proteins and incorporate them into regulated secretory granules. (J. Clin. Invest. 1993. 91:193-200.)

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“…35 In Figure 3A PF4 appear as clusters rather than granules. These clusters are randomly distributed in the platelets.…”

Section: Pf4mentioning

confidence: 96%

Microparticles are the basic storage units for different proteins in platelet granules

Zhang

Yang²

2012

Blood

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Footnote:To avoid confusion, the term of microparticles in this study refers to the miscellaneous vesicles derived or released from platelets, including exosomes and shedding vesicles from plasma membranes. Blood First Edition AbstractPlatelets are circulating carriers of numerous proteins including coagulation and inflammation factors, cytokines, and angiogenesis regulatory factors. Understanding how platelets store and organize the proteins is crucial to understanding their role and function in the vast involved pathophysiological events. Therefore, in this study we hypothesized that microparticles are the basic storage units for different proteins in platelet granules. We utilized stimulated emission depletion microscopy to study the ultrastructure of platelets and the protein storage in granules. Our results demonstrated that P-selectin, fibrinogen, platelet factor 4 and vascular endothelial growth factor are all packaged and stored in individual sub-granule vesicles. The four proteins all exhibited ring-like distribution in the vesicles with a diameter of approximately 90 nm. We further discovered that one microparticle stores only one type of proteins. These results supported our hypothesis and have provided novel insights of how platelets systematically store and organize proteins in the granules. These insights have not only reevaluated the ultrastructure of platelets, but also provided a link connecting microparticles and platelets from structure to function and pathologies. The link is believed to benefit the understanding of how platelets regulate their pathophysiological participations, such as inflammation, angiogenesis, and tumor growth.

show abstract

Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Cited by 72 publications

References 43 publications

Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet -granule biogenesis

Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet -granule biogenesis

Kistrin, an integrin antagonist, blocks endocytosis of fibrinogen into guinea pig megakaryocyte and platelet alpha-granules.

Microparticles are the basic storage units for different proteins in platelet granules

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