Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Abstract Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha- granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha- granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb- IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.

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Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Blood ◽

10.1182/blood.v75.6.1220.bloodjournal7561220 ◽

1990 ◽

Vol 75 (6) ◽

pp. 1220-1227 ◽

Cited By ~ 1

Author(s):

EM Cramer ◽

GF Savidge ◽

W Vainchenker ◽

MC Berndt ◽

D Pidard ◽

...

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Polyclonal Antibodies ◽

Staining Technique ◽

Membrane System ◽

Type I ◽

Immunogold Staining ◽

The Third ◽

Granule Membrane ◽

Gray Platelet Syndrome

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha- granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha- granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb- IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.

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Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and megakaryocytes

Blood ◽

10.1182/blood.v82.10.3034.3034 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3034-3044 ◽

Cited By ~ 41

Author(s):

G Berger ◽

JP Caen ◽

MC Berndt ◽

EM Cramer

Keyword(s):

Plasma Membrane ◽

Subcellular Distribution ◽

Critical Role ◽

Staining Technique ◽

Ultrastructural Observation ◽

Type I ◽

Cell Content ◽

Major Band ◽

Immunogold Staining ◽

Granule Membrane

Abstract CD36 (glycoprotein [GP] IV) is a membrane GP of 88 kD found on monocytes, endothelial cells, and platelets. It may serve as a receptor for collagen and is also able to bind thrombospondin (TSP), because a monoclonal antibody to CD36 inhibits TSP binding to thrombin-stimulated platelets. In the following study, we investigated the subcellular distribution of CD36 within normal resting platelets, thrombin- stimulated platelets, and in cultured megakaryocytes (MK) by an immunogold staining technique and electron microscopy. We used an affinity-purified monospecific polyclonal antibody showing a single major band of precipitation at 88 kD via immunoblot analysis. In normal platelets, ultrastructural observation detected immunolabeling for CD36, homogeneously distributed along the platelet plasma membrane and in the luminal side of the open canalicular system (OCS). Moreover, some labeling was found around the alpha-granules along the inner face of their limiting membrane. An average of 70% of granules were labeled. The granule-associated pool of CD36 was estimated at approximately 25% of the total cell content. To exclude the possibility of a cross- reaction with GPIIb-IIIa, platelets from a patient with type I Glanzmann's thrombasthenia (which completely lack GPIIb-IIIa) were studied and showed a similar subcellular distribution of CD36, including alpha-granule membrane labeling. In activated platelets, CD36 was shown to be redistributed to the OCS and pseudopods of the plasma membrane. Platelets from a patient with the Gray platelet syndrome expressed CD36 on their plasma membrane, and some immunolabeling was also found within small abnormal alpha-granules. In cultured MK, CD36 immunolabeling was detected in the Golgi saccules, associated vesicles, immature alpha-granules, and demarcation membranes. In conclusion, this study shows the existence of a significant intragranular pool of CD36 in platelets that may play a critical role in the surface expression of alpha-granule TSP during platelet activation.

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Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and megakaryocytes

Blood ◽

10.1182/blood.v82.10.3034.bloodjournal82103034 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3034-3044 ◽

Cited By ~ 1

Author(s):

G Berger ◽

JP Caen ◽

MC Berndt ◽

EM Cramer

Keyword(s):

Plasma Membrane ◽

Subcellular Distribution ◽

Critical Role ◽

Staining Technique ◽

Ultrastructural Observation ◽

Type I ◽

Cell Content ◽

Major Band ◽

Immunogold Staining ◽

Granule Membrane

CD36 (glycoprotein [GP] IV) is a membrane GP of 88 kD found on monocytes, endothelial cells, and platelets. It may serve as a receptor for collagen and is also able to bind thrombospondin (TSP), because a monoclonal antibody to CD36 inhibits TSP binding to thrombin-stimulated platelets. In the following study, we investigated the subcellular distribution of CD36 within normal resting platelets, thrombin- stimulated platelets, and in cultured megakaryocytes (MK) by an immunogold staining technique and electron microscopy. We used an affinity-purified monospecific polyclonal antibody showing a single major band of precipitation at 88 kD via immunoblot analysis. In normal platelets, ultrastructural observation detected immunolabeling for CD36, homogeneously distributed along the platelet plasma membrane and in the luminal side of the open canalicular system (OCS). Moreover, some labeling was found around the alpha-granules along the inner face of their limiting membrane. An average of 70% of granules were labeled. The granule-associated pool of CD36 was estimated at approximately 25% of the total cell content. To exclude the possibility of a cross- reaction with GPIIb-IIIa, platelets from a patient with type I Glanzmann's thrombasthenia (which completely lack GPIIb-IIIa) were studied and showed a similar subcellular distribution of CD36, including alpha-granule membrane labeling. In activated platelets, CD36 was shown to be redistributed to the OCS and pseudopods of the plasma membrane. Platelets from a patient with the Gray platelet syndrome expressed CD36 on their plasma membrane, and some immunolabeling was also found within small abnormal alpha-granules. In cultured MK, CD36 immunolabeling was detected in the Golgi saccules, associated vesicles, immature alpha-granules, and demarcation membranes. In conclusion, this study shows the existence of a significant intragranular pool of CD36 in platelets that may play a critical role in the surface expression of alpha-granule TSP during platelet activation.

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A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation.

The Journal of Cell Biology ◽

10.1083/jcb.101.3.880 ◽

1985 ◽

Vol 101 (3) ◽

pp. 880-886 ◽

Cited By ~ 565

Author(s):

P E Stenberg ◽

R P McEver ◽

M A Shuman ◽

Y V Jacques ◽

D F Bainton

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Plasma Membranes ◽

Polyclonal Antibodies ◽

Fold Increase ◽

Immunogold Label ◽

Thin Sections ◽

Granule Membrane ◽

Immunocytochemical Techniques ◽

Intracellular Organelles

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.

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Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1

Blood ◽

10.1182/blood.v84.6.1722.bloodjournal8461722 ◽

1994 ◽

Vol 84 (6) ◽

pp. 1722-1730 ◽

Cited By ~ 1

Author(s):

EM Cramer ◽

G Berger ◽

MC Berndt

Keyword(s):

Plasma Membrane ◽

Polyclonal Antibodies ◽

Present Report ◽

Cross Reactivity ◽

Membrane Receptors ◽

Type I ◽

Double Labeling ◽

Platelet Surface ◽

Platelet Receptor ◽

Granule Membrane

CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha- granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha- granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule- specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).

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Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1

Blood ◽

10.1182/blood.v84.6.1722.1722 ◽

1994 ◽

Vol 84 (6) ◽

pp. 1722-1730 ◽

Cited By ~ 43

Author(s):

EM Cramer ◽

G Berger ◽

MC Berndt

Keyword(s):

Plasma Membrane ◽

Polyclonal Antibodies ◽

Present Report ◽

Cross Reactivity ◽

Membrane Receptors ◽

Type I ◽

Double Labeling ◽

Platelet Surface ◽

Platelet Receptor ◽

Granule Membrane

Abstract CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha- granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha- granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule- specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).

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Alternaria toxins and their effects on host plants

Canadian Journal of Botany ◽

10.1139/b95-282 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 453-458 ◽

Cited By ~ 91

Author(s):

Hiroshi Otani ◽

Keisuke Kohmoto ◽

Motoichiro Kodama

Keyword(s):

Plasma Membrane ◽

Host Plants ◽

Plasma Membranes ◽

Early Effect ◽

The Third ◽

The Matrix ◽

Electrolyte Loss ◽

Host Specific Toxins ◽

Acid Structure ◽

Malate Oxidation

There are now nine or more Alternaria pathogens that produce host-specific toxins, and the structures of most of the toxins have been elucidated. Alternaria host-specific toxins are classified in three groups in terms of the primary site action. ACT-, AF-, and AK-toxins have in common an epoxy-decatrienoic acid structure and exert their primary effect on the plasma membrane of susceptible cells. A rapid increase in electrolyte loss from tissues and invaginations in the plasma membranes are common effects of these toxins. The second group is represented by ACR(L)-toxin, which induces changes in mitochondria, including swelling, vesiculation of cristae, decrease in the electron density of the matrix, increase in the rate of NADH oxidation, and inhibition of malate oxidation. The third group consists of AM-toxin, which appears to exert an early effect on both chloroplasts and plasma membranes. AM-toxin induces vesiculation of grana lamellae, inhibition of CO2 fixation, invagination of plasma membranes, and electrolyte loss. The roles of host-specific toxins in pathogenesis are discussed. Key words: Alternaria, host-specific toxin, plasma membrane, mitochondrion, chloroplast.

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Exercise increases the plasma membrane content of the Na+ -K+ pump and its mRNA in rat skeletal muscles

Journal of Applied Physiology ◽

10.1152/jappl.1996.80.2.699 ◽

1996 ◽

Vol 80 (2) ◽

pp. 699-705 ◽

Cited By ~ 47

Author(s):

T. Tsakiridis ◽

P. P. Wong ◽

Z. Liu ◽

C. D. Rodgers ◽

M. Vranic ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Skeletal Muscles ◽

Plasma Membranes ◽

Acute Exercise ◽

Treadmill Running ◽

Type I ◽

Mrna Levels ◽

Subunit Mrna ◽

White Type

Muscle fibers adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exercise training has been shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercise has not been determined, and it is not known whether it involves alterations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of treadmill running (20 m/min, 10% grade) on the subcellular distribution and expression of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and IIb (white-IIa/IIb) hindlimb muscles from resting and exercised female Sprague-Dawley rats were removed for subcellular fractionation. By homogenization and gradient centrifugation, crude membranes and purified plasma membranes were isolated and subjected to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthermore, mRNA was isolated from specific red type I (red-I) and white type IIb (white-IIb) muscles and subjected to Northern blotting by using subunit-specific probes. In both red-I/IIa and white-IIa/IIb muscles, exercise significantly raised the plasma membrane content of the alpha1-subunit of the pump by 64 +/- 24 and 55 +/- 22%, respectively (P < 0.05), and elevated the alpha2-polypeptide by 43 +/- 22 and 94 +/- 39%, respectively (P < 0.05). No significant effect of exercise could be detected on the amount of these subunits in an internal membrane fraction or in total membranes. In addition, exercise significantly increased the alpha1-subunit mRNA in red-I muscle (by 50 +/- 7%; P < 0.05) and the beta2-subunit mRNA in white-IIb muscles (by 64 +/- 19%; P < 0.01), but the alpha2- and beta1-mRNA levels were unaffected in this time period. We conclude that increased presence of alpha1- and alpha2-polypeptides at the plasma membrane and subsequent elevation of the alpha1- and beta2-subunit mRNAs may be mechanisms by which acute exercise regulates the Na+-K+ pump of skeletal muscle.

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The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

Biochemical Journal ◽

10.1042/bj2730429 ◽

1991 ◽

Vol 273 (2) ◽

pp. 429-434 ◽

Cited By ~ 10

Author(s):

A Darnanville ◽

R Bredoux ◽

K J Clemetson ◽

N Kieffer ◽

N Bourdeau ◽

...

Keyword(s):

Plasma Membrane ◽

Time Course ◽

Plasma Membranes ◽

Polyclonal Antibodies ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Glycoprotein Ib ◽

Dependent Protein ◽

Dose Dependent ◽

Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

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Ascospore development in the fission yeasts Schizosaccharomyces pombe and S. japonicus

Journal of Cell Science ◽

10.1242/jcs.56.1.263 ◽

1982 ◽

Vol 56 (1) ◽

pp. 263-279 ◽

Cited By ~ 2

Author(s):

K. Tanaka ◽

A. Hirata

Keyword(s):

Plasma Membrane ◽

Schizosaccharomyces Pombe ◽

Periodic Acid ◽

Staining Technique ◽

Membrane System ◽

Spore Wall ◽

Wall Material ◽

Dense Granules ◽

Thin Sectioning ◽

Ascospore Development

The fine structure of ascospore formation in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus var. japonicus was studied by serial thin-sectioning and electron microscopy. The morphogenetic events were almost the same in both species. Ascospore development was initiated by the formation of the forespore membrane on the cytoplasmic side of the differentiated nucleus-associated organelle (NAO) in the interval between meiosis I and II in S. pombe, or during the post-meiotic nuclear division in S. japonicus, and the process proceeded almost synchronously through the two or four nuclei in the ascus. The forespore membrane developed by fusion of the cytoplasmic vesicles and this was clearly demonstrated in S. japonicus where the behaviour of vesicles involved in the forespore membrane development could be traced as they were marked by the presence of electron-dense granules. The staining technique, by phosphotungustic acid—chromic acid (PTA-CA) after treatment with periodic acid, was used to attempt to elucidate the origin and the nature of the forespore membrane. The method specific to plasmalemma-type membranes stained both ascus and ascospore plasmalemmas; the forespore membrane was not stained at first but developed the same affinity for stain as the plasma membrane in the course of ascospore development. The results suggest that the forespore membrane did not come directly from the ascus plasma membrane, but from another membrane system such as the endoplasmic reticulum. Spore wall material was deposited in the space between the inner and outer leaflets of the forespore membrane.

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