scholarly journals A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase

1988 ◽  
Vol 250 (2) ◽  
pp. 355-361 ◽  
Author(s):  
N Hack ◽  
J M Wilkinson ◽  
N Crawford
Keyword(s):  
Human Platelet ◽  
Blood Platelets ◽  
Membrane Vesicles ◽  
Intracellular Membrane ◽  
Dose Dependency ◽  
Intracellular Membranes ◽  
Maximum Inhibition ◽  
Igg1 Subclass ◽  
Energy Dependent ◽  
Human Blood Platelets

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

scholarly journals Localization of cyclo-oxygenase and thromboxane synthetase in human platelet intracellular membranes

1982 ◽  
Vol 204 (3) ◽  
pp. 847-851 ◽  
Author(s):  
F Carey ◽  
S Menashi ◽  
N Crawford
Keyword(s):  
Arachidonic Acid ◽  
Human Platelet ◽  
Surface Membrane ◽  
Membrane Vesicles ◽  
Intracellular Membrane ◽  
Intracellular Membranes ◽  
Selective Enrichment ◽  
Thromboxane Synthetase ◽  
Tubular Membranes

Platelet mixed membrane fractions can be separated into discrete vesicle subpopulations of surface and intracellular origin. Intracellular membrane vesicles are the predominant site of phospholipid-modifying enzymes that liberate arachidonic acid. We report the selective enrichment in intracellular membranes of cyclo-oxygenase and thromboxane synthetase activities. Surface membrane fractions show no such enrichment. These results suggest that a sequence of activities leading to the biosynthesis of thromboxane from arachidonate is associated with the intracellular membrane elements known as dense tubular membranes.

scholarly journals Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes

1986 ◽  
Vol 233 (3) ◽  
pp. 661-668 ◽  
Author(s):  
N Hack ◽  
M Croset ◽  
N Crawford
Keyword(s):  
Atpase Activity ◽  
Blood Platelets ◽  
Present Report ◽  
Surface Membrane ◽  
Storage Site ◽  
Intracellular Membrane ◽  
Bivalent Cation ◽  
Platelet Surface ◽  
Intracellular Membranes ◽  
Dependent Transport

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

scholarly journals Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Y Miyakawa ◽  
A Oda ◽  
BJ Druker ◽  
H Miyazaki ◽  
M Handa ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Human Platelet ◽  
Blood Platelets ◽  
Human Platelets ◽  
Genetically Engineered ◽  
Hematopoietic Growth Factors ◽  
Signal Transducers ◽  
Stat Proteins ◽  
Human Blood Platelets

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c- Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.

The Uptake and Metabolism of Adenosine in Human Blood Platelets

1975 ◽  
Author(s):  
J. P. M. Lips ◽  
J. J. Sixma ◽  
A. M. C. Trieschnigg ◽  
H. Holmsen
Keyword(s):  
Human Blood ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Uptake Mechanism ◽  
Low Concentrations ◽  
Adenosine Transport ◽  
Energy Dependent ◽  
Unchanged Form ◽  
Uptake And Metabolism ◽  
Human Blood Platelets

Adenosine is taken up tino intact washed human blood platelets by two independent carrier-mediated processes. The ‘low Km system’ has a Km of 9 uM, a V of 792 pMol/min/109, is energy dependent and has a Q10 of 1.71. This system is competitively inhibited by papaverine and dipyridamol. The high Km system has a Km of 9.4 mM, as V of 106 nMol/min/109 is also energy dependent and has a Q10 of 1.31. This system is competitively inhibited by adenine, that has a very high affinity (K1 = 5,8 uM).Evidence is presented that the low Km system transports adenosine in such a way, that it is directly incorporated into nucleotides. Adenosine transported by way of the high Km system arrives unchanged inside the platelets. At low concentrations all this adenosine is deaminated. At higher concentrations (5 mM) part of it is converted into adenine nucleotides and part of it is present in an unchanged form. Only a relatively small part is deaminated at high adenosine concentrations.The high Km system is inhibited by purines. The low Km system is only inhibited by purine ribosides. The presence of a double uptake mechanism and metabolic pathway has direct bearing on experiments that have been effected about the relation between adenosine transport and inhibition of ADP aggregation.

A MONOCLONAL ANTIBODY (PL/IM 430) THAT BLOCKS THE ACTIVE TRANSL0CATI0N OF Ca2+ INTO HUMAN PLATELET INTRACELLULAR MEMBRANE (ER) VESICLES

1987 ◽  
Author(s):  
N Hack ◽  
J M Wilkinson ◽  
N Crawford
Keyword(s):  
Human Platelet ◽  
Human Monocyte ◽  
Biological Properties ◽  
Intracellular Membrane ◽  
Maximum Inhibition ◽  
Function Relationship ◽  
A Site ◽  
Relationship Of ◽  
Heart Foundation ◽  
Er Membrane

In earlier studies [1] we identified a number of important biological properties associated with highly purified human platelet intracellular membrane (ER), isolated by continuous flow electrophoresis. These included a high affinity Ca2+Mg2+ ATPase and protein phosghorylation both of which are involved inthe active uptake of Ca into ER vesicles. The stored Ca2+ could be released with inositol(1,4,5)trisphosphate, (IP ), (approx. 50% release in 30 s 1/2 max. for release - 0.253 μM IP3,) [2]. To probe the structure-function relationship of proteins in these ER vesicles, a panel of monoclonal antibodies (Mabs) has been raised, using the ER membrane preparation as immunogen. Four of these Mabs recognise a single 100 kDa polypeptide by immunoblotting. This protein is present in platelet membranes and can also be identified in cultured human monocyte, macrophage and endothelial cell lines. None of the M^bs showed any significant effect upon the ER membrane Ca2+ Mg2+ ATPase activity but one, PL/IM 430 (of IgGl subclass), inhibited the Ca2+sequestration by the vesicles significantly (approx. 70% inhibition at 10 μM IgG). This inhibition was independent of the ATP concentration over a range2of 0-2 mM ATP, but was2dose-dependent for external free Ca 2between 30-300 nM Ca2+, giving maximum inhibition at 300 nM Ca with 10 pM IgG2+ Binding of the antibody substantially lowers the Vmax for Ca2+for Ca2+ uptake but is without effect upon the Km. PL/IM 430 therefore appears to recognise a 100 kDa polypeptide closely involved with Ca2+ trnslocation but at a site which i, s without effect upon the Ca2+Mg− ATPase associated with the Ca pump.We are grateful to the Wellcome Trust and the British Heart Foundation for financial support for these studies.[1] Hack, N., Croset, M. and Crawford, N. (1986) Biochem. J. 233, 661-668.[2] Authi, K. S. and Crawford, N. (1985) Biochem. J. Z3O, 247-253

VASOCONSTRICTION IN RESPONSE TO HUMAN PLATELET-VESSEL WALL INTERACTIONS

1987 ◽  
Author(s):  
J M Van Nueten ◽  
W J Janssens ◽  
F De Clerck
Keyword(s):  
Calcium Ions ◽  
Vessel Wall ◽  
Human Platelet ◽  
Blood Platelets ◽  
Thromboxane A2 ◽  
Period 2 ◽  
Acting In Concert ◽  
Human Blood Platelets ◽  
Serotonergic Antagonist ◽  
Wall Interactions

Human blood platelets, stimulated with thrombin, induced contractions of isolated basilar artery segments of the dog. These platelet-mediated vascular contractions were inhibited in a concentration-dependent way by flunarizine, a Ca2+-entry blocker, selective for vascular tissues. This inhibition increased gradually as a function of time after contact with flunarizine to reach its maximum after 60-90 min. Biochemical and pharmacological analyses, using the 5-HT2-serotonergic antagonist ritanserin, the thromboxane A2/prostaglandin endo-peroxide antagonist BM 13.177 and the fatty acid cyclo-oxygenase inhibitor suprofen, showed that 5-hydroxytryptamine and prostanoids (thromboxane A2, prostaglandine endoperoxides) were the main mediators involved. They further suggested amplification between 5-hydroxytryptamine and prostanoids at the vascular level.(1) Incubation period; (2) Inhibition of platelet-mediated vascular contractions.This study demonstrates that 5-hydroxytryptamine, acting in concert with thromboxane A2 and/or prostaglandine-endoper-oxides, is responsible for the vasoconstrictor effects of aggregating platelets. It further indicates that influx of calcium ions is involved in these vasoconstrictor responses.

Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+

1988 ◽  
Vol 8 (4) ◽  
pp. 379-388 ◽  
Author(s):  
Nashrudeen Hack ◽  
Kalwant Singh Authi ◽  
Neville Crawford
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Vesicles ◽  
Intracellular Membrane ◽  
Channel Protein ◽  
Maximum Inhibition ◽  
Ldh Release ◽  
Intracellular Binding ◽  
Specific Control ◽  
Ca2 Channels ◽  
Intracellular Sequestration

A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca2+ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J. M. and Crawford, N. 1988, Biochem. J.250, 355–361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20–25 μg/ml) commensurate with total LDH release, sequestration of Ca2+ into intracellular non-mitochondrial stores is inhibited by the antibody (~50% inhibition at 20 μg/ml IgG). At higher saponin concentrations when intracellular binding of125I-labelled mAb is maximum, inhibition of Ca2+ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca2+ sequestration. No effect of the antibody were observed against IP3-induced release of prestored Ca2+, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca2+ translocating channel protein associated with the intracellular membrane (Ca2++Mg2+) ATPase and not to Ca2+ channels responsive to IP3.

Factors Influencing Thrombin Digestion Of Human Platelet Thrombospondin

1981 ◽  
Author(s):  
J W Lawler ◽  
F C Chao ◽  
J Palek
Keyword(s):  
Molecular Weight ◽  
Disulfide Bonds ◽  
Human Platelet ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Platelet Protein ◽  
Flow Through ◽  
Proteolytic Cleavages ◽  
Human Blood Platelets

Exposure of human blood platelets to thrombin results in the rapid release of a 420,000 dalton glycoprotein, designated thrombospondin, which is comprised of three polypeptides of equivalent molecular weight which are cross- linked by disulfide bonds. When purified human platelet thrombospondin is exposed to thrombin (4 units/ml) or plasmin (2 units/ml) for prolonged periods of time, proteolytic cleavages occur which result in the removal of 10,000 dalton and 30,000 dalton polypeptides with a concomitant decrease in the apparent molecular weight of the thrombospondin chains (Lawler, J.W. and Slayter, H.S., submitted for publication). In contrast, when the supernatant from thrombin- treated platelets is exposed to additional thrombin (4 units/ml) the 30,000 dalton, but not the 10,000 dalton fragment is released from thrombospondin. Proteolytic release of the 10,000 dalton polypeptide was inhibited by calcium and a nondialyzable component of the supernatant preparations. To further characterize the nondialyzable component, the supernatant was subjected to heparin-Sepharose affinity chromatography. Stepwise elution with varying NaCl concentrations yields a non-affinity flow through peak, a low affinity peak (eluted with 0.45 M NaCl) and a high affinity peak (eluted with 2.0 M NaCl). In addition to thrombospondin, the low affinity peak contains a β-thromboglobulin like protein as judged by RIA, apparent molecular weight on SDS-polyacrylamide gel electrophoresis (7,500-9,000 dalton) and heparin affinity. After dialysis of the low affinity peak against 0.14 M NaCl, 15 mM Tris-HCl (pH 7.6), 2 mM CaCl2 containing 0.02% NaN3, thrombin treatment resulted in the proteolytic release of the 30,000 dalton polypeptide, but not the 10,000 dalton polypeptide, from thrombospondin. These results suggest that calcium and a low molecular weight platelet protein can affect the pattern of thrombin digestion of thrombospondin.

The Isolation Procedure and Some Characteristics of c-AMP Dependent Protein Kinases of Human Blood Platelets

1975 ◽  
Author(s):  
L. H. Kahlé ◽  
B. W. König ◽  
J. W. ten Cate
Keyword(s):  
Protein Kinases ◽  
Kinase Activity ◽  
Human Platelet ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Terminal Position ◽  
Linear Gradient ◽  
Acceptor Activity ◽  
Dependent Protein ◽  
Human Blood Platelets

Several protein kinases have been isolated from human platelet which bind cyclic AMP and transfer a phosphate group from the terminal position of ATP to a protein acceptor molecule.The purification was performed by homogenization in a 50 mM Tris MCI buffer at pH 7.0 which contained 2 M.KC1, subsequent centrifugation at 100,000 g for 1 hr., ammonium sulfate precipitation of the supernate, gel filtration over Sephadex G 25 and DEAE Sephadex chromatography with a linear gradient of 0-1 M.NaCl in Tris HCl buffer at pH 7.0. DEAE Sephadex chromatography resulted in three peaks, which contained kinase activity. Only peak II and III contained c-AMP acceptor activity, which reached an optimum at pH 5.5 in phosphate buffer.The kinase activity was stimulated by c-AMP in all three peaks while the first peak also had some c-AMP independent kinase activity. The kinase activity was dependent on Mg ions. Difference in activity in the three peaks was observed depending on the acceptor protein that were used. Acceptor proteins studied so far were hist on and protamine. Isolation of an acceptor protein from the platelets is in progress.

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