scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379 ◽  
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

Abstract To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

Movement of Calcium Ions and their Role in the Activation of Platelets

1978 ◽  
Vol 40 (02) ◽  
pp. 212-218 ◽  
Author(s):  
P Massini ◽  
R Käser-Glanzmann ◽  
E F Lüscher
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Calcium Ions ◽  
Binding Sites ◽  
Shape Change ◽  
Release Reaction ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Different Types ◽  
Ca Ions

SummaryThe increase of the cytoplasmic Ca-concentration plays a central role in the initiation of platelet activation. Four kinds of movements of Ca-ions are presumed to occur during this process: a) Ca-ions liberated from membranes induce the rapid shape change, b) Vesicular organelles release Ca-ions into the cytoplasm which initiate the release reaction, c) The storage organelles called dense bodies, secrete their contents including Ca-ions to the outside during the release reaction, d) At the same time a rearrangement of the plasma membrane occurs, resulting in an increase in its permeability for Ca-ions as well as in an increase in the number of Ca-binding sites.Since most processes occurring during platelet activation are reversible, the platelet must be equipped with a mechanism which removes Ca-ions from the cytoplasm. A vesicular fraction obtained from homogenized platelets indeed accumulates Ca actively. This Ca- pump is stimulated by cyclic AMP and protein kinase; it may be involved in the recovery of platelets after activation.It becomes increasingly clear that the various manifestations of platelet activation are triggered by a rise in the cytoplasmic Ca2+-concentration. The evidence for this and possible mechanisms involved are discussed in some detail in the contributions by Detwiler et al. and by Gerrard and White to this symposium. In this article we shall discuss four different types of mobilization of Ca-ions which occur in the course of the activation of platelets. In addition, at least one transport step involved in the removal of Ca2+ must occur during relaxation of activated platelets.

scholarly journals PADGEM protein in human erythroleukemia cells

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 722-728 ◽  
Author(s):  
E Yeo ◽  
BC Furie ◽  
B Furie
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Cell Content ◽  
Granule Membrane ◽  
Hel Cells

Abstract PADGEM protein, a platelet alpha granule membrane glycoprotein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion and platelet activation. PADGEM protein is expressed on the surface of activated platelets but not on the surface of resting platelets. Human erythroleukemia (HEL) cells contain platelet alpha granule-like organelles, alpha granule proteins, and express platelet membrane glycoproteins GPIIb/IIIa and GPIb. We demonstrate that HEL cells express a protein that has a molecular weight identical to that of PADGEM and binds to anti-PADGEM antibodies. The exposure of HEL cells in culture to dimethylsulfoxide (DMSO) increased the number of cells expressing PADGEM. Fluorescence activated flow cytometric analysis demonstrated an increase in mean surface expression of PADGEM in DMSO-exposed cells compared to noninduced cells. Total cell content of PADGEM was increased 5.3-fold after DMSO exposure, as determined by radioimmunoassay. Direct binding experiments with the monoclonal anti-PADGEM antibody KC4 demonstrated specific, saturable, and time-dependent interaction of KC4 with HEL cells. A Kd of 7 nM was estimated. There were 14,000 surface binding sites per cell in noninduced cells and 24,000 surface binding sites per cell in DMSO- induced HEL cells. Surface expression of PADGEM protein on HEL cells was not increased with platelet agonists, including thrombin, epinephrine, ADP, nor cytokines, including IL-1, IL-2, tissue necrosis factor. The presence of PADGEM protein in HEL cells should facilitate the elucidation of the function of PADGEM protein.

scholarly journals Role of membranes in bile formation. Comparison of the composition of bile and a liver bile-canalicular plasma-membrane subfraction

1976 ◽  
Vol 154 (3) ◽  
pp. 589-595 ◽  
Author(s):  
W H. Evans ◽  
T Kremmmer ◽  
J G. Culvenor
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Bile Acids ◽  
Secretory Process ◽  
Major Protein ◽  
Outer Face ◽  
Ph Optimum ◽  
Bile Formation ◽  
Canalicular Membrane ◽  
Bile Canalicular Membrane

1. Enzymes, proteins, glycoproteins and lipids of rodent bile were compared with those of a plasma-membrane subfraction originating from the hepatocyte bile-canalicular membrane. 2. Three bile-canalicular glycoprotein enzyme activities were detected in bile. Comparison of the pH optimum and immunoinhibition properties of membrane and bile 5′-nucleotidase activity indicated that they were the same enzyme. Correspondence between membrane and bile alkaline phosphodiesterases also suggested that they were the same enzymes. Activities of Mg2+-stimulated adenosine triphosphatase, a lipid-dependent intrinsic membrane protein, and galactosyltransferase, a Golgi membrane marker, were not detected in bile. 3. Rodent bile contained 15 polypeptide bands that differed radically from those of bile-canalicular membranes. Bands that may correspond in molecular weight to liver plasma-membrane glycoproteins were present at low staining intensities in bile. A major protein of apparent molecular weight 49 500 was present, and albumin was detected by immunodiffusion. 4. The lipid composition of bile and bile-canalicular membrane also differed. Phosphatidylcholine accounted for 82% of rat bile phospholipids, and only trace amounts of phosphatidylinositol, phosphatidylserine and sphingomyelin were present. 5. The results indicate that in healthy animals, the bile-canalicular membrane is refractory to the action of bile acids during the secretory process. The presence of only small amounts of bile-canalicular membrane components, especially glycoprotein enzymes located at the outer face of the membrane, suggests that these are released from the membrane by bile acids after secretion of bile into the canalicular spaces.

scholarly journals A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

1990 ◽  
Vol 1 (11) ◽  
pp. 843-852 ◽  
Author(s):  
H McNeill ◽  
P J Jensen
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Human Keratinocytes ◽  
Urokinase Plasminogen Activator ◽  
Receptor Expression ◽  
Epithelial Migration ◽  
High Affinity ◽  
Upa Receptor

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.

scholarly journals Zinc binding in cow's milk and human milk

1983 ◽  
Vol 209 (2) ◽  
pp. 505-512 ◽  
Author(s):  
P Blakeborough ◽  
D N Salter ◽  
M I Gurr
Keyword(s):  
Molecular Weight ◽  
Human Milk ◽  
Average Molecular Weight ◽  
Human Infant ◽  
Zinc Binding ◽  
Cow’S Milk ◽  
Major Protein ◽  
Cow's Milk ◽  
Beta Casein ◽  
A Minor

In both cow's milk and human milk, zinc was associated with proteins of high molecular weight (greater than 100 000), as judged by analysis with Sephadex G-75. Precipitation of the casein at pH 4.6 and filtration of the resultant acid whey on Sephadex G-25 led, however, to the recovery of about 90% of the zinc as a compound of low molecular weight, which was tentatively identified as zinc citrate. Over 95% of the zinc of cow's milk was sedimented with the casein micelles on ultracentrifugation. Filtration of these micellar caseins on Sephadex G-150 gave two peaks containing zinc, which corresponded to aggregates of alpha-casein-kappa-casein and of alpha-casein-beta-casein. Ultracentrifugation of human milk sedimented only approx. 40% of total zinc. Analysis of sediment and supernatant on Sephadex G-150, however, indicated that about 85% of the zinc was associated with a protein complex of molecular weight greater than 150 000. The major protein of this complex was identified as lactoferrin. A minor zinc-binding component of average molecular weight 30 000 was also observed in the supernatant. The results indicated that zinc is bound to different macromolecules in cow's and human milk. This may be a factor affecting the bioavailability to the human infant of zinc from the two milks, and it is suggested that in human milk lactoferrin may be involved in the uptake of zinc.

scholarly journals Deletion of Mfsd2b impairs thrombotic functions of platelets

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Madhuvanthi Chandrakanthan ◽  
Toan Quoc Nguyen ◽  
Zafrul Hasan ◽  
Sneha Muralidharan ◽  
Thiet Minh Vu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Knockout Mice ◽  
Blood Cells ◽  
Thrombus Formation ◽  
Whole Body ◽  
Minor Amount ◽  
Platelet Morphology ◽  
Activated Platelets ◽  
A Minor ◽  
Platelet Functions

AbstractWe recently discovered that Mfsd2b, which is the S1P exporter found in blood cells. Here, we report that Mfsd2b is critical for the release of all S1P species in both resting and activated platelets. We show that resting platelets store S1P in the cytoplasm. After activation, this S1P pool is delivered to the plasma membrane, where Mfsd2b is predominantly localized for export. Employing knockout mice of Mfsd2b, we reveal that platelets contribute a minor amount of plasma S1P. Nevertheless, Mfsd2b deletion in whole body or platelets impairs platelet morphology and functions. In particular, Mfsd2b knockout mice show significantly reduced thrombus formation. We show that loss of Mfsd2b affects intrinsic platelet functions as part of remarkable sphingolipid accumulation. These findings indicate that accumulation of sphingolipids including S1P by deletion of Mfsd2b strongly impairs platelet functions, which suggests that the transporter may be a target for the prevention of thrombotic disorders.

ATP is required in platelet serotonin exocytosis for protein phosphorylation and priming of secretory vesicles docked on the plasma membrane

1996 ◽  
Vol 109 (1) ◽  
pp. 113-118
Author(s):  
T. Morimoto ◽  
S. Ogihara
Keyword(s):  
Plasma Membrane ◽  
Protein Phosphatase ◽  
Secretory Activity ◽  
Electron Microscopic ◽  
Dense Granules ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Amorphous Structures ◽  
Gamma S

Calcium-evoked secretion generally requires the presence of millimolar concentrations of Mg-ATP. We investigated the role of Mg-ATP in the secretion of serotonin from electropermeabilized bovine platelets. The secretion of serotonin was lost within 5 minutes when the Mg-ATP concentration was diluted to less than 0.1 mM, but was maintained when ATP-gamma S (adenosine 5′-O-3-thiotriphosphate) was used instead of ATP. Okadaic acid, a potent inhibitor of protein phosphatase, could also maintain the exocytotic activity even when ATP was diluted. Decrease in the secretory activity was paralleled by a decrease in phosphorylation level of four proteins after dilution of ATP, but the activity was maintained when the thiophosphorylation level of these proteins was maintained. Two of these proteins were digested by a protease, calpain, which has been shown to lead to a loss in the exocytotic activity. Electron microscopic studies showed that calcium did not induce the formation of distinct bridge-like structures between the granule membrane and the plasma membrane in Mg-ATP-diluted cells, previously shown as the structure transiently formed prior to fusion of the two membranes. Anchorage of the secretory dense granules to the plasma membrane and the presence of the amorphous structures between the granules and the plasma membrane were unchanged by dilution of ATP. These results indicate that ATP is not required for the anchorage itself, but is required to prime anchored granules for calcium-triggered secretion. Maintenance of the phosphorylated state of proteins by ATP enables the calcium trigger to form the bridge-like structures preceding membrane fusion events.

The 33-kDa Platelet α-granule Membrane Protein (GMP-33) Is an N-terminal Proteolytic Fragment of Thrombospondin

2001 ◽  
Vol 86 (09) ◽  
pp. 887-893 ◽  
Author(s):  
Conchi Damas ◽  
Tom Vink ◽  
Karel Nieuwenhuis ◽  
Jan Sixma
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Membrane Protein ◽  
Immunoaffinity Chromatography ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Proteolytic Fragment ◽  
Activated Platelets ◽  
N Terminus ◽  
Granule Membrane

SummaryGMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet α-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.

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