scholarly journals Lipoxin synthase activity of human platelet 12-lipoxygenase

1993 ◽  
Vol 296 (1) ◽  
pp. 127-133 ◽  
Author(s):  
M Romano ◽  
X S Chen ◽  
Y Takahashi ◽  
S Yamamoto ◽  
C D Funk ◽  
...  
Keyword(s):  
Human Platelet ◽  
Fold Increase ◽  
Eicosatrienoic Acid ◽  
Human Platelets ◽  
Dual Function ◽  
Lipoxin A4 ◽  
Suicide Inactivation ◽  
Leukotriene A4 ◽  
12 Lipoxygenase

Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity.

Human platelet chemotaxis: requirement for plasma factor(s) and the role of collagen

1978 ◽  
Vol 235 (1) ◽  
pp. H23-H28 ◽  
Author(s):  
R. W. Lowenhaupt
Keyword(s):  
Structural Integrity ◽  
Human Platelet ◽  
Human Platelets ◽  
Factor Xi ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Plasma Factors ◽  
Short Time

Platelets are actively mobile in plasma in vitro and, in addition, they migrate specifically and directionally toward added intact collagen (chemotaxis). Native human, bovine, and equine collagen, suspended in plasma, induce a chemotactic response in human platelets. However, heat-denatured and dinitrofluorobenzene-treated collagen fail to attract platelets. Platelets migrate directionally and specifically to intact native collagen incubated in plasma over a large distance (6 mm) in a very short time (total 15 min), as observed in a newly designed micromaze apparatus. Platelets obtained from donors deficient in plasma factors XII, IX, and VIII showed normal migration and chemotaxis in normal plasma and in their respective factor-deficient plasmas. Although nondirectional movement (mobility) was normal, platelets from a donor deficient in factor XI did not exhibit chemotaxis toward collagen in either factor XI-deficient plasma or in normal plasma. The results indicate that 1) collagen is a physiological substrate for the chemotactic phenomenon, 2) intact chemical and/or structural integrity of collagen is required for the induction of platelet chemotaxis, 3) at least one plasma constituent, factor XI, plays an essential role in the chemotactic phenomenon, and 4) contact between collagen and a plasma factor is essential for normal chemotaxis.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

scholarly journals 14,15-Dehydroleukotriene A4: a specific substrate for leukotriene C4 synthase

1997 ◽  
Vol 328 (1) ◽  
pp. 225-229 ◽  
Author(s):  
Angelo SALA ◽  
Mylene GARCIA ◽  
Simona ZARINI ◽  
Jean Claude ROSSI ◽  
Giancarlo FOLCO ◽  
...  
Keyword(s):  
Human Platelet ◽  
Leukotriene B4 ◽  
Reversed Phase ◽  
Human Platelets ◽  
Polymorphonuclear Leucocyte ◽  
Specific Substrate ◽  
Leukotriene C4 ◽  
Synthase Inhibitor ◽  
Leukotriene A4 ◽  
Leukotriene C4 Synthase

We studied the metabolism of 14,15-dehydro-leukotriene A4 (14,15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14,15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14,15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 μM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme.

The Role of Ca2+I in Procoagulant Surface Expression and Apoptosis in Human Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3569-3569
Author(s):  
Adam M. Gwozdz ◽  
Hong Wang ◽  
K.W. Annie Bang ◽  
Marian A. Packham ◽  
John Freedman ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Fold Increase ◽  
Surface Expression ◽  
Human Platelets ◽  
Apoptotic Pathway ◽  
Outer Leaflet ◽  
Activated Platelets

Abstract Asymmetry of phospholipids across the plasma membrane bilayer is a feature of all eukaryotic cells. When platelets are stimulated with certain agonsists, phospholipids are randomized by the action of a Ca2+-dependent scramblase enzyme, resulting in exposure of the anionic aminophospholipid phosphatidylserine (PS) on the outer leaflet that provides a procoagulant surface, catalyzing thrombin formation. We have previously demonstrated that the procoagulant surface of activated platelets persists in vitro for at least 4 hrs (Blood100:63b, 2002). Such persistence may propagate thrombosis in vivo when activated procoagulant platelets re-enter the circulation after fibrinolysis. There is currently little information concerning the mechanisms by which the procoagulant surface persists on activated platelets. In this in vitro study, the Ca2+-chelator BAPTA (0.1 μmol/109 platelets) was used to investigate the role of intracellular Ca2+ (Ca2+i) in procoagulant surface expression and persistence; PS expression was determined flow cytometrically by the binding of annexin A5-FITC. Unexpectedly, chelation of Ca2+i resulted in a 2–2.5x-fold increase in PS expression on the surface of platelets 5 min after activation with thrombin or thrombin+collagen (T+C), and this persisted for up to 4 hrs (last time point tested). Since PS expression is a hallmark of apoptosis in nucleated cells, we also examined another platelet apoptosis marker, the collapse of the mitochondrial inner membrane potential (ΔΨm), by flow cytometry using the potential-sensitive dye TMRM; PS expression was measured concurrently. This allowed us to distinguish between activated platelets expressing PS with an intact ΔΨm and apoptotic platelets expressing PS with a dissipated ΔΨm. 70–85% of the thrombin- or T+C-activated platelets expressing PS had an intact ΔΨm, which persisted for up to 4 hrs after activation. Thus, PS expression can occur independently of ΔΨm loss. However, chelation of Ca2+i with BAPTA resulted in 60–70% of the thrombin- or T+C-activated platelets persistently expressing PS to also have a collapsed ΔΨm, indicating that apoptotic pathways similar to those found in nucleated cells may modulate PS expression in platelets and may depend on Ca2+i concentrations. Caspases and calpain are centrally involved in apoptotic signaling and execution in nucleated cells. Caspases-9 and -3 have been identified in human platelets and may be responsible for downstream activation of calpain. We examined the effects of Ca2+i chelation in thrombin- and T+C- activated platelets on the activation of procaspases and calpain by Western blotting. In keeping with our observations of increased PS expression with concurrent ΔΨm loss in activated platelets with Ca2+i chelation, we observed cleavage of both procaspase-9, procaspase-3 and calpain, which did not occur in activated platelets without Ca2+i chelation. Taken together, our results indicate that Ca2+i levels in activated platelets may serve as a decisional checkpoint for the apoptotic pathway in human platelets, where procaspase-9 and procaspase-3 along with downstream calpain may function in a Ca2+-sensitive manner to protect platelets against PS exposure and ΔΨm collapse.

Enzymatic Characteristics of In-Vitro Incorporation of P32 Orthophosphate into Human Platelet Phosphatide

1965 ◽  
Vol 13 (01) ◽  
pp. 126-135 ◽  
Author(s):  
C. M Grossman ◽  
R Kohn
Keyword(s):  
Human Platelet ◽  
Human Platelets ◽  
Artificial Medium ◽  
Enzymatic Characteristics

SummaryThe incorporation of P32 orthophosphate into phosphatide by isolated human platelets in an artificial medium was studied. The addition of homologous plasma increased the incorporation and the formation of labelled phosphatide was significantly reduced in the absence of oxygen or in the presence of cyanide, and by varying the pH from 7.4 to 5.6 and 8.6. The enzymatic character of the incorporation and the role of platelet respiration are briefly discussed.

Further Observations On The Possible Association Of Human Platelet Membrane Glycoproteins IIb And IIIa

1981 ◽  
Author(s):  
J-P Rosa ◽  
D Pidard ◽  
T Kunicki ◽  
A T Nurden
Keyword(s):  
Platelet Aggregation ◽  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Normal Human ◽  
Triton X

Studies are described which represent a continuation of our investigation into the role of membrane glycoproteins (GP’s) IIb and IIIa during human platelet aggregation. The surface proteins of washed platelets were labelled with 125I by the lactoperoxidase-catalysed method prior to membrane isolation by the glycerol lysis procedure. Solubilisation of the membrane proteins by triton X-100 was followed by their analysis by crossed immunoelectrophoresis (CIE) using a rabbit antibody prepared against normal human platelets. In the absence of divalent cation chelation GP IIb and Ilia were contained within a single 125I-labelled immunoprecipitate. When the isolated membranes were solubilised by triton X-100 in the presence of 5mM EDTA, GP IIb and IIIa formed distinctand separate immunoprecipitates during CIE. In order to further investigate this finding 125I-labelled membrane proteins solubilised by triton X-100 in the presence or absence of EDTA were subjected to centrifugation for 18 h at 100,000 g over a 10-40% sucrose gradient containing the nonionic detergent. The results confirmed that in the presence of divalent cations lib and Ilia were associated in a complex, and that this complex is dissociated by EDTA. The IgG..L is an alloantibody isolated from a polytransfused thrombasthenic patient that has been shown in previous studies to inhibit ADP-induced platelet aggregation and the binding of 125I-fibrinogen to normal human platelets in the presence of ADP. When the IgG..L was incorporated in an intermediate gel during CIE it was shown to precipitate the complex containing IIb/IIIa but under identical conditions it did not precipitate the individual glycoproteins dissociated by EDTA. Divalent cation-mediated changes in the orientation of lib and Ilia in the platelet membrane should be considered in assessing the role of these GP’s in platelet function.

Human Platelet Aggregation Without Thromboxane (Tx) Formation

1981 ◽  
Author(s):  
J Westwick ◽  
J B Smith ◽  
V V Kakkar
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Human Platelets ◽  
Synthesis Inhibitor ◽  
Thromboxane Receptor ◽  
Human Platelet Aggregation ◽  
Thromboxane Receptor Antagonist ◽  
Induced Aggregation ◽  
Carbocyclic Thromboxane A2

It is not known whether PG endoperoxides have to be converted to TxA2 in order to induce aggregation and secretion We have examined this crucial question by measuring human platelet aggregation, 14C-5HT release and TxB2 formation induced by collagen, arachidonic acid (AA), adrenaline, U46619 and PGH2 in presence of either 1) 1-2-(4-carboxy-phenoxy) ethyl imidazole hydrochloride, a potent and selective thromboxane synthesis inhibitor (TSI) or a carbocyclic thromboxane A2 (CTA) - a so called thromboxane receptor antagonist. TSI, 1.5 to 75μM produced a dose related inhibition (IC50 5μM n=5) of Tx and elevation of PGE2 synthesis in adrenaline (8μM) and collagen (1.5μg/ml) stimulated platelets. When 14C-SHT labelled platelets were stimulated with 4.5μM adrenaline in the presence of 0, 2.5, 15 and 300μM TSI a 35±2.8, 27±0.5, 24.5±5.0% release of 14C-5HT release resulted and a 10014, 9612, 9711 and 9114% of control aggregation occurred. Similarly when human platelets were stimulated with 0.8mMAA a 51±1.4% (mean ±SE) release of 14C-5HT occurred, while in the presence of 15, 75 and 300μM of UK, a 42±0.4, 32±1, 33±3% of 14C-5HT release resulted. Aggregation induced by the PG endoperoxide analogue U46619 (lμM) was not inhibited by 300μM TSI or l00μM indomethacin, although 99.5% inhibition of Tx formation resulted, and 50 to 60% inhibition of 14C-5HT release was produced both by TSI and indomethacin. However CTA (l-3±M) produced a dose related inhibition of both aggregation and release induced by U46619. CTA (l-10μM) was found to produce superimposable dose related inhibition of collagen induced aggregation and secretion of platelets, whether the platelets were pretreated with 300μM TSI, or not.These results suggest that Tx formation is not necessary for human platelet aggregation, although is contributory to 14C-5HT release induced by collagen, adrenaline, AA and U46619.Also caution must be employed when CTA is used to elucidate the role of TxA2, as it appears to be an effective PG endoperoxide antagonist.

scholarly journals Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

2012 ◽  
Vol 53 (12) ◽  
pp. 2546-2559 ◽  
Author(s):  
Kenneth N. Ikei ◽  
Jennifer Yeung ◽  
Patrick L. Apopa ◽  
Jesús Ceja ◽  
Joanne Vesci ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Human Platelet ◽  
Lipoxygenase Products ◽  
12 Lipoxygenase

scholarly journals Isolation and characterization of the major oligosaccharide of human platelet membrane glycoprotein GPIb

1982 ◽  
Vol 205 (1) ◽  
pp. 81-90 ◽  
Author(s):  
P A Judson ◽  
D J Anstee ◽  
J R Clamp
Keyword(s):  
Human Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Molar Ratios ◽  
Isolation And Characterization ◽  
Molar Basis ◽  
Membrane Bound ◽  
Almost All

Treatment of intact human platelets with chymotrypsin released a glycopolypeptide that was shown to be derived from the major membrane glycoprotein, GPIb. The glycopolypeptide contained 59% carbohydrate on a molar basis and was rich in serine, threonine and proline. Almost all the carbohydrate could be released from the glycopolypeptide by treatment with alkali in the presence of NaBH4. The major component (comprising 80% of the released sugar) was purified and shown to be a hexasaccharide containing sialic acid, galactose, N-acetylglucosamine and N-acetylgalactosaminitol in the molar ratios 2:2:1:1. Two possible structures for this hexasaccharide are proposed on the basis of the known biosynthetic pathways of mucus-type glycoproteins. Our data is consistent with the occurrence of an O-glycosidically linked oligosaccharide on one amino acid in four of the glycopolypeptide. These results suggest that glycoprotein Ib can best be described as a membrane-bound mucus-type glycoproteins. Our data are consistent with the occurrence of an O- in the process by which platelets adhere to the exposed subendothelium of damaged blood-vessel walls. The possible role of the glycopolypeptide portion of GPIb in this process was investigated. Neither the major oligosaccharide nor the glycopolypeptide itself inhibited ristocetin-induced platelet agglutination at the concentrations tested. It is suggested that the carbohydrate moieties of GPIb molecules at the cell surface interact to form a barrier to macromolecules. Such a barrier could play a major role in modulating platelet function.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

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