Enhanced Affinity for Arachidonic Acid in Platelet-Rich Plasma from Rats with Adriamycin-Induced Nephrotic Syndrome

1982 ◽  
Vol 48 (03) ◽  
pp. 260-262 ◽  
Author(s):  
F Delaini ◽  
A Poggi ◽  
M B Donati
Keyword(s):  
Arachidonic Acid ◽  
Nephrotic Syndrome ◽  
Platelet Rich Plasma ◽  
Platelet Response ◽  
Heavy Proteinuria ◽  
Washed Platelet ◽  
Cyclooxygenase Activity ◽  
Stable Product ◽  
Kinetics Of ◽  
Thrombotic Tendency

SummaryWe have measured the response to arachidonic acid (AA) in platelet-rich plasma (PRP) of rats with Adriamycin-induced nephrotic syndrome. For this purpose we measured the kinetics of generation of malondialdehyde (MDA), a stable product of cyclooxygenase activity, in response to platelet stimulation with different concentrations of the substrate. The apparent Km of platelet cyclo-oxygenase for AA was similar in PRP from control rats and rats treated 1–5 days previously, whereas it was significantly reduced, as compared to controls, in PRP of rats treated 2–5 weeks previously. Such a difference was not observed when washed platelet suspensions were tested instead of PRP. Experiments with crossed platelet/plasma systems indicated that in rats treated from 2–5 weeks, a plasmatic abnormality was indeed responsible for the increased affinity of platelets for AA.It is conceivable that in this nephrotic syndrome model characterized by heavy proteinuria, some plasmatic component would be lost with the urine which is normally modulating the platelet response to AA. The observed increase in platelet affinity for AA could at least partially contribute to the enhanced thrombotic tendency reported in the same experimental model.

Malondialdehyde Formation in Rat Platelet-Rich Plasma

1980 ◽  
Vol 44 (02) ◽  
pp. 049-051 ◽  
Author(s):  
G Rajtar ◽  
M Livio ◽  
J Merino ◽  
G de Gaetano
Keyword(s):  
Arachidonic Acid ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Membrane Phospholipids ◽  
Experimental Conditions ◽  
Malondialdehyde Formation ◽  
Km Value ◽  
Free Arachidonic Acid ◽  
Stable Product ◽  
Kinetics Of

SummaryMalondialdehyde (MDA) is a stable product of arachidonic acid metabolism, catalyzed by the enzyme cyclo-oxygenase. The experimental conditions for measuring the kinetics of MDA formation in rat citrated platelet-rich plasma were defined. Platelets were stimulated with either arachidonic acid, the substrate of MDA, or thrombin, an enzyme which induces release of free arachidonic acid from platelet membrane phospholipids. MDA formation was almost linear for a limited period of time (between 0 and 2 min with arachidonic acid and between 1 and 3 min with thrombin) and was concentration-dependent with saturation kinetics.The hyperbolic curves obtained could be recast in linear plots (according to Woolf transformation S/V versus S) when arachidonic acid was used. With thrombin, in contrast, the highest concentration at which no MDA production could be detected (3 NIH u/ml) had to be subtracted from each concentration of the enzyme used to obtain Woolf plots. The apparent Km value of arachidonic acid was 0.49 ± 0.09 mM and Vmax was 1.44 ± 0.06 nmoles MDA/1.4 × 109 platelets/min. The corresponding values in experiments with thrombin were 6.5 ± 1.5 NIH u/ml and 0.233 ± 0.012 nmoles MDA/1.4 × 109 platelets/min.

Platelet Suruival and Function in Rats with Enhanced Thrombotic Tendency

1985 ◽  
Vol 54 (04) ◽  
pp. 739-743 ◽  
Author(s):  
Federica Delaini ◽  
Elisabetta Dejana ◽  
Ine Reyers ◽  
Elisa Vicenzi ◽  
Germana De Bellis Vitti ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Nephrotic Syndrome ◽  
Platelet Function ◽  
Laboratory Tests ◽  
Thrombus Formation ◽  
The Other ◽  
Mean Survival Time ◽  
The Mean ◽  
And Function ◽  
Thrombotic Tendency

SummaryWe have investigated the relevance of some laboratory tests of platelet function in predicting conditions of thrombotic tendency. For this purpose, we studied platelet survival, platelet aggregation in response to different stimuli, TxB2 and 6-keto-PGFlα production in serum of rats bearing a nephrotic syndrome induced by adriamycin. These animals show a heavy predisposition to the development of both arterial and venous thrombosis. The mean survival time was normal in nephrotic rats in comparison to controls. As to aggregation tests, a lower aggregating response was found in ADR-treated rats using ADP or collagen as stimulating agents. With arachidonic acid (AA) we observed similar aggregating responses at lower A A concentrations, whereas at higher AA concentrations a significantly lower response was found in nephrotic rats, despite their higher TxB2 production. Also TxB2 and 6-keto-PGFlα levels in serum of nephrotic rats were significantly higher than in controls. No consistent differences were found in PGI2-activity generated by vessels of control or nephrotic rats.These data show that platelet function may appear normal or even impaired in rats with a markedly increased thrombotic tendency. On the other hand, the significance of high TxB2 levels in connection with mechanisms leading to thrombus formation remains a controversial issue.

Disturbed Plasmatic Modulation Of Arachidonic Acid (AA) Metabolism In Platelets From Rats With Nephrotic Syndrome: An Early Parameter Of Thrombotic Risk?

1981 ◽  
Author(s):  
F Delaini ◽  
L Reyers ◽  
G de Beilis Vitti ◽  
A Poggi ◽  
M B Donati
Keyword(s):  
Arachidonic Acid ◽  
Nephrotic Syndrome ◽  
Abdominal Aorta ◽  
Fibrinolytic Activity ◽  
Platelet Rich Plasma ◽  
Spectrophotometric Assay ◽  
Thrombotic Risk ◽  
Occlusion Time ◽  
Increased Risk ◽  
Increased Susceptibility

The nephrotic syndrome in humans is associated with an increased risk for arterial and venous thrombosis, the pathogenesis of which appears to be very complex. We have developed an experimental model of nephrotic syndrome in rats which follows a single i.v. injection of adriamycin (AIM, 5-10 mg/kg b.w.). Starting from 2 weeks after ADM-injection, rats showed a remarkably increased susceptibility to thrombotic stimuli; indeed, the occlusion time of a polyethylene cannula inserted in the abdominal aorta was 22±15 hours in treated animals versus 62±5 in control animals (n=16, p< 0.001). Concomitantly the rats showed: increased plasma fibrinogen and factor VIII: C activity, shortened APTT, low antithrombin and markedly depressed fibrinolytic activity. Moreover, platelet-rich plasma (PRP) of treated rats produced significantly higher amounts of both malondialdehyde (MDA) (spectrophotometric assay) and thromboxane B2 (RIA) in response to AA. When MDA generation was studied with a kinetic approach, the PRP of nephrotic animals had significantly lower apparent Km for AA in respect to the controls (0.15 ± 0.07 mM AA versus 0.90 ± 0.25 mM AA, p<0.01). Washed platelets from both groups, in contrast, showed similar Km values for AA (0.10 mM ± 0.04 versus 0.12 ± 0.03).MDA generation by washed normal platelets resuspended in plasma was normal if plasma had been collected 1 or 3 days after ADM administration but was already significantly higher with plasma collected 5, 7 or 14 days after treatment. The concomitant mild hypoalbuminemia does not completely account for this plasma abnormality. Thus, in the model, an abnormal plasmatic modulation of platelet AA metabolism could be detected much earlier than the coagulation/fibrinolysis changes and the hypersensitivity to thrombotic stimuli.

Hyperaggregability Of Serotonin-Depleted Platelets To Arachidonic Acid (AA) In SLE And Transplant Recipients

1981 ◽  
Author(s):  
D Marchesi ◽  
A Parbtani ◽  
G Frampton ◽  
M Livio ◽  
G Remuzzi ◽  
...  
Keyword(s):  
Renal Function ◽  
Arachidonic Acid ◽  
Nephrotic Syndrome ◽  
Platelet Activation ◽  
Great Majority ◽  
Transplant Recipients ◽  
Renal Microvasculature ◽  
Thrombotic Tendency ◽  
Reduced Renal Function

In a variety of renal disorders, including SLE and transplantation, in vivo platelet activation is present. This is in part a result of the nephrotic syndrome, but is also observed in patients with g.n. who are not nephrotic. As a result, intraplatelet concentrations of serotonin and other amines are depleted in patients with active nephritis. The functional capacity of such amine-depleted platelets has been little studied; Pareti et al (1980) reported defective aggregation to ADP, adrenaline and collagen in such “acquired storage pool deficiencies”. We studied aggregation thresholds to ADP, collagen and AA in 25 patients with SLE nephritis, none of whom had a nephrotic syndrome or reduced renal function. In these patients platelet activation is due principally to circulating factors. Sixteen transplant recipients were also studied. In them, activation is principally within the renal microvasculature of the allograft. We also measured intraplatelet serotonin concentrations, which were low or zero in the great majority of patients in both groups. Bleeding times were normal in all, as were platelet aggregation thresholds to ADP and collagen. AA thresholds, however, were markedly depressed (<0.32 mmol) in all transplant recipients and most patients with SLE. This specific hyperaggregability may be responsible for both persistence of disease and the thrombotic tendency observed in some patients, including those with circulating anticoagulant.

A Kinetic Study of Platelet Malondialdehyde (MDA) Formation and its Pharmacological Inhibition in rat Platelet-Rich Plasma (PRP)

1979 ◽  
Author(s):  
G. Rajtar ◽  
M. Livio ◽  
J. Merino ◽  
de G. Gaetano
Keyword(s):  
Arachidonic Acid ◽  
Acetylsalicylic Acid ◽  
Kinetic Study ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Suitable Model ◽  
Experimental Conditions ◽  
Intracellular Mechanism ◽  
Kinetics Of ◽  
Rat Platelets

Experimental conditions were defined for kinetic analysis of MDA formation and inhibition in PRP prepared from citrated blood of male CD rats. Portions of PRP were stirred at 37°C with different concentrations of thrombin or arachidonic acid (AA) and the reaction was stopped by TCA. The rate of MDA generation (measured by a thiobarbiturate reaction) increased linearly during the first few min, reaching a plateau within 5 min. In all subsequent experiments PRP was incubated with thrombin for 2 ½ min or AA for 2 min. Km for thrombin was about 10 NIH U/ml and for AA 0.35 mM. Vmax were 0.27 and 0.40 nmol. MDA/1.4 × 109 platelets/min. Acetylsalicylic acid (ASA) or indomethacin (I) were incubated with PRP at 37°C for either 1 or 10 min before addition of thrombin or AA. Both drugs inhibited MDA formation in an apparently competitive way. Ki values were 10-30 times lower for I than for ASA. Ki values Of both drugs were about 20 times lower after 10 than after 1 min incubation. The inhibitory effect of either drug could be decreased by diluting the PRP samples, and appeared to be additive. These data suggest that: 1) rat platelets kept in their own plasma are a suitable model for studying the kinetics of MDA generation; 2) in rat platelets ASA and I act on the same enzymatic intracellular mechanism; 3) irreversible acetylation of platelet cyclo-oxygenase does not account for ASA’s initial inhibitory effect on platelet MDA generation.

Inhibitory Effect of Staphylokinase on Platelet Aggregation

1993 ◽  
Vol 70 (05) ◽  
pp. 834-837 ◽  
Author(s):  
Akira Suehiro ◽  
Yoshio Oura ◽  
Motoo Ueda ◽  
Eizo Kakishita
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Degradation Products ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Effective Concentration ◽  
Inhibit Platelet Aggregation ◽  
Platelet Suspension ◽  
Washed Platelet ◽  
Human Platelet Aggregation

SummaryWe investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37° C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10-4 g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10-6 g/ml. The effect could be inhibited by adding aprotinin or α2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10-6 g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of α2-antiplasmin.

Stimulated Platelet Aggregation, Thromboxane B2 Formation and Platelet Sensitivity to Prostacyclin - A Critical Evaluation

1981 ◽  
Vol 45 (03) ◽  
pp. 204-207 ◽  
Author(s):  
Wolfgang Siess ◽  
Peter Roth ◽  
Peter C Weber
Keyword(s):  
Arachidonic Acid ◽  
Platelet Count ◽  
Platelet Aggregation ◽  
Reliable Method ◽  
Platelet Rich Plasma ◽  
Critical Evaluation ◽  
Vascular Diseases ◽  
Thromboxane B2 ◽  
Test Time ◽  
Stimulation Of

SummaryPlatelets have been implicated in the development of atherosclerotic and thrombotic vascular diseases. Evaluation of platelet aggregation in relation to endogenously formed compounds which affect platelet function may provide information of clinical and pharmacological relevance. We describe a method in which thromboxane B2 (TXB2) formation was analyzed following stimulation of platelet-rich plasma (PRP) with ADP, 1-epinephrine, collagen, and arachidonic acid. In addition, we determined platelet sensitivity to prostacyclin following ADP- and collagen-induced platelet aggregation. The parameters under study were found to depend on the platelet count in PRP, on the type and dose of the aggregating agent used, and on the test time after blood sampling. By standardization of these variables, a reliable method was established which can be used in clinical and pharmacological trials.

Prostanoid Release from Ex Vivo Perfused Canine Arteries and Veins: Effects of Prolonged Perfusion, Intermittent Perfusion, as well as Exposure to Exogenous Arachidonic Acid, Thrombin and Bradykinin

1989 ◽  
Vol 62 (03) ◽  
pp. 1034-1039 ◽  
Author(s):  
Jan S Brunkwall ◽  
James C Stanley ◽  
Timothy F Kresowik ◽  
Linda M Graham ◽  
William E Burkel ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Ex Vivo ◽  
Fold Increase ◽  
Ex Vivo Perfusion ◽  
Cyclooxygenase Activity ◽  
Perfusion Model ◽  
Prostanoid Production ◽  
Slow Decline ◽  
Arteries And Veins ◽  
Nonpulsatile Flow

SummaryRegulation of prostanoid release from ex vivo perfused vessel segments is not fully understood. A series of perfusion experiments were performed with canine arteries and veins to define certain regulatory phenomena. Arteries were perfused with pulsatile flow of 90 ml/min at a pressure of 100 mmHg, and veins with nonpulsatile flow of 90 ml/min at a pressure of 7 mmHg. Segments were perfused with Hanks' balanced salt solution for five 15-min periods with the perfusate exchanged after each study period. With onset of perfusion, there was an initial burst of prostacyclin release to 127 ± 40 pg/mm2, declining to 32 ± 10 pg/mm2 after 60 minutes (p <0.005). If perfusion continued for 5.5 hours, there was a stable release period between 1 and 3 hours, followed by a very slow decline. At that time addition of arachidonic acid (AA) increased prostacyclin release six-fold (p <0.01). Vessels perfused for 1 hour and then rested for another hour, responded to reperfusion at the second onset of flow with a two-fold increase in prostacyclin release (p <0.01). Vessels perfused with thrombin, bradykinin or A A (either added to each perfusate or only to the last perfusate) exhibited greater prostacyclin release than did control segments. Release of thromboxane steadily declined with time in all parts of the study, and only increased with the addition of A A to the perfusate. These data indicate that vessel segments subjected to ex vivo perfusion do not maximally utilize enzyme systems responsible for prostanoid production, and after 1 hour perfusion have not depleted their phospholipids, and maintain functioning levels of phospholipase and cyclooxygenase activity. This perfusion model allows for the study of prostacyclin and thromboxane release from arteries and veins and their response to various drugs and other stimuli.

Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

1981 ◽  
Vol 46 (02) ◽  
pp. 538-542 ◽  
Author(s):  
R Pilo ◽  
D Aharony ◽  
A Raz
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Unsaturated Fatty Acids ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

A Thiazole Compound with Potential Antithrombotic Activity

1982 ◽  
Vol 47 (02) ◽  
pp. 173-176 ◽  
Author(s):  
E E Nishizawa ◽  
A R Mendoza ◽  
T Honohan ◽  
K A Annis
Keyword(s):  
Platelet Aggregation ◽  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Development Program ◽  
Antithrombotic Agent ◽  
Antithrombotic Activity ◽  
Thiazole Derivative ◽  
Cyclooxygenase Activity ◽  
Drug Development Program

SummaryA thiazole derivative, 4,5-bis(p-methoxyphenyl)-2-(trifluoromethyl)-thiazole was found to be a potent inhibitor of collagen-induced platelet aggregation, in vitro, using platelets from at least six species, including man. It was active in human platelet-rich plasma at a concentration of 1 ng/ml. While its antiplatelet activity was greater than that of flurbiprofen, its cyclooxygenase activity was equivalent to that of flurbiprofen. Also, compared to flurbiprofen, the thiazole had less anti-inflammatory activity in the hind-paw edema test. The thiazole derivative inhibited platelet aggregation following oral administration in five laboratory species. In the guinea pig it was active at 0.5 mg/kg. The LD50 in mice was greater than 1000 mg/kg (i.p.). This compound, which was designed through a systematic drug development program, may have high potential as an antithrombotic agent.

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