Identification of cytochrome P450s involved in the metabolism of arachidonic acid in human platelets

SummaryImmunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 109 platelets with a mean value of 70.3 ± 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

Download Full-text

Pretreatment of Human Platelets with Plasmin Inhibits Responses toThrombin, but Potentiates Responses to Low Concentrations of Aggregating Agents, Including the Thrombin Receptor Activating Peptide, SFLLRN

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656044 ◽

1997 ◽

Vol 77 (04) ◽

pp. 741-747 ◽

Cited By ~ 12

Author(s):

R L Kinlough-Rathbone ◽

D W Perry ◽

M L Rand ◽

M A Packham

Keyword(s):

Arachidonic Acid ◽

Platelet Activating Factor ◽

Human Platelets ◽

Thrombin Receptor ◽

Fibrinolytic Agents ◽

Albumin Solution ◽

Fibrinogen Receptor ◽

Low Concentrations ◽

Induced Aggregation

SummaryEffects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37° C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2C1 (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.

Download Full-text

Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657338 ◽

1983 ◽

Vol 49 (02) ◽

pp. 132-137 ◽

Cited By ~ 16

Author(s):

A Eldor ◽

G Polliack ◽

I Vlodavsky ◽

M Levy

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Competitive Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

Ionophore A23187 ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

Download Full-text

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661603 ◽

1986 ◽

Vol 56 (01) ◽

pp. 057-062 ◽

Cited By ~ 56

Author(s):

Martine Croset ◽

M Lagarde

Keyword(s):

Arachidonic Acid ◽

Fatty Acid ◽

Platelet Aggregation ◽

Fatty Acid Distribution ◽

Thromboxane A2 ◽

Human Platelets ◽

Aggregation Response ◽

Docosahexaenoic Acids ◽

Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

Download Full-text

Arachidonic acid stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release, and aggregation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44408-5 ◽

1983 ◽

Vol 258 (18) ◽

pp. 11236-11242 ◽

Cited By ~ 21

Author(s):

W Siess ◽

F L Siegel ◽

E G Lapetina

Keyword(s):

Arachidonic Acid ◽

Phosphatidic Acid ◽

Protein Phosphorylation ◽

Phospholipase C ◽

Shape Change ◽

Human Platelets ◽

Serotonin Release ◽

Platelet Shape

Download Full-text

Transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine during storage of human platelets for 5 days

Biochemistry and Cell Biology ◽

10.1139/o90-016 ◽

1990 ◽

Vol 68 (1) ◽

pp. 117-122 ◽

Cited By ~ 1

Author(s):

Julie Lacasse ◽

Rosalind S. Labow ◽

Morris Kates ◽

George A. Adams

Keyword(s):

Arachidonic Acid ◽

Storage Conditions ◽

Phospholipid Metabolism ◽

Human Platelets ◽

Acyl Transfer ◽

Acid Uptake ◽

Molar Composition ◽

Platelet Storage ◽

Head Groups ◽

Glycerol Uptake

Human platelets are routinely stored for 5 days prior to transfusion, but they deteriorate during storage. Since very little information is available concerning the effect of storage on platelet phospholipid metabolism, the biosynthesis and remodelling of platelet phospholipids were studied. Platelets were incubated separately with [14C]glycerol, [14C]arachidonic acid, or a mixture of [14C]glycerol and [3H]arachidonic acid, and stored in a platelet storage medium at 22 °C. Maximum glycerol uptake (20%) was attained after 6 h. [14C]Glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, and to a much lesser extent phosphatidylserine, under storage conditions for 5 days. The distribution of the initial arachidonic acid uptake was not as would be expected based on the molar composition of endogenous phospholipids. The arachidonic acid (75%) which was taken up within 10 min of incubation distributed 55% into the phosphatidylcholine and only 14% into the phosphatidylethanolamine; the molar composition is actually 18% phosphatidylcholine and 47% phosphatidylethanolamine. During storage, there was a continuous transfer of the radiolabeled arachidonic acid from phosphatidylcholine to phosphatidylethanolamine until, after 5 days, the distribution of arachidonic acid was identical to the endogenous distribution. In contrast, no change in the glycerol incorporation pattern was detected during storage. This suggested that the mechanism for arachidonic acid redistribution was not through exchange of polar head groups, but through acyl transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine.Key words: human, platelet, storage, arachidonate, phospholipids.

Download Full-text

THE REDUCED CAPACITY OF THROMBOXANE FORMATION IN THROMBIN-PREACTIVATED PLATELETS IS NOT CAUSED BY DEPLETION OF THEIR ARACHIDONIC ACID POOL

10.1055/s-0038-1643393 ◽

1987 ◽

Author(s):

R E Scharf ◽

M Stockschläder ◽

H J Reimers ◽

W Schneider

Keyword(s):

Arachidonic Acid ◽

Human Platelets ◽

Thrombin Receptor ◽

Precursor Pool ◽

Acid Pool ◽

Exogenous Arachidonic Acid ◽

High Concentrations ◽

Short Time ◽

Thromboxane Formation

Thromboxane (TX) synthesis of washed human platelets pretreated with high concentrations of thrombin (0.5-2.0 U/ml) for 20 sec is significantly reduced upon further thrombin stimulation. Compared to controls (tyrode-pretreated platelets), thrombin-preactivated platelets recover normal TX synthesis following exposure to exogenous arachidonic acid (AA) indicating that short-time thrombin treatment does not inactivate platelet cyclooxygenase or TX synthetase (Blood 63: 858, 1984). To evaluate whether the reduced TX synthesis upon -the second thrombin exposure is due to depletion of their AA precursor pool, thrombin-pretreated platelets and tyrode-pretreated platelets (5×108/ml) were resuspended in autologous ACD plasma and incubated at 37°C with 0.2 μCi 14C-AA (20 μM) for 60 to 90 min in the presence of PGE1 (10 μM). Mean platelet uptake of 14C-AA (disappearance of radioactivity from the supernatant) was 12+3 nmoles AA/109 platelets and did not differ significantly between thrombin-pretreated platelets and controls. Thrombin-pretreated platelets released 10% or 4.5% of their 14c-activity upon further exposure to thrombin (2 U/ml) or collagen (8 μg/ml), respectively. The release from control platelets (15% with thrombin, 6.5% with collagen) did not differ from that of thrombin-pretreated platelets. However, even after incubation in ACD plasma, thrombin-pretreated platelets continued to form significantly less TXB2 (5.0±1.6 nmoles/109 platelets) than controls (9.7±2.2 nmoles/109 platelets, p< 0.05). These data indicate that the reduced capacity of thrombin-pretreated platelets is due neither to a depletion of the endogenous AA pool nor to an inactivation of cyclooxygenase or TX synthetase. The reduced TX synthesis capacity may be caused by a modification, destruction or desensitization of the platelet thrombin receptor as a consequence of the preceding thrombin stimulation.

Download Full-text