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.

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 Retention in Glass Bead Columns: Further Evidence for the Importance of ADP

Blood ◽  
1974 ◽  
Vol 44 (3) ◽  
pp. 411-425 ◽  
Author(s):  
V. J. McPherson ◽  
M. B. Zucker ◽  
N. M. Friedberg ◽  
P. L. Rifkin
Keyword(s):  
Platelet Aggregation ◽  
Acetylsalicylic Acid ◽  
Glass Bead ◽  
Room Temperature ◽  
Creatine Phosphokinase ◽  
Platelet Rich Plasma ◽  
Creatine Phosphate ◽  
Inhibitory Effect ◽  
Normal Platelet ◽  
Normal Individuals

Abstract Plasma of normal heparinized blood contained 0.284 µM ± SD 0.097 (ADP + ATP) with an ATP:ADP ratio of 2.5:1. Plasma from thrombocytopenic blood contained only 0.106 µM ± 0.073 (ADP + ATP). Blood with normal platelet retention released 0.234 µM ± 0.187 (ADP + ATP) during passage through a glass bead column, with an ATP:ADP ratio of 1.6:1. Significantly less was released in blood with low retention, i.e., samples from patients with von Willebrand’s disease, thrombasthenia, or thrombocytopenia, and some samples from normal individuals. Thus, nucleotides in the plasma of pre- and postcolumn blood appear to be derived from platelets; their release within glass bead columns is closely associated with normal platelet retention. Since release occurred at room temperature and was not prevented by acetylsalicylic acid or accompanied by measurable release of 14C-serotonin, the classic release reaction may not have been responsible. The low retention in platelet-rich plasma was variably increased by adding 0.5 µM ADP, an increase at least partly due to trapping of preformed aggregates. Retention in undisturbed blood was markedly inhibited by creatine phosphokinase with creatine phosphate (CPK-CP) and moderately inhibited by apyrase I (ATPase:ADPase 0.8:1) at an ADP-removing activity between 1 and 5 U/ml, indicating that ADP is essential for retention. At less than 1 U/ml, both apyrase I and II (ATPase: ADPase 2.8:1) enhanced retention in undisturbed blood, but CPK-CP was still inhibitory. These results suggest that enhancement is due to conversion of released ATP to ADP, as shown to occur in studies of platelet aggregation with ATP and ADP. At less than 1 U/ml, all three enzymes protected against the inhibitory effect of disturbance; this protection was marked with apyrase II, moderate with apyrase I and slight with CPK-CP. These observations provide additional evidence that ADP is responsible for the low retention caused by disturbance of the blood.

Influence of Platelet Activation on Erythrocyte Deformability

1983 ◽  
Vol 49 (02) ◽  
pp. 084-086 ◽  
Author(s):  
W Palinski ◽  
A Torsellini ◽  
L Doni
Keyword(s):  
Arachidonic Acid ◽  
Acetylsalicylic Acid ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Plasma Viscosity ◽  
Erythrocyte Deformability ◽  
Direct Influence ◽  
Antagonistic Action ◽  
Malondialdehyde Formation ◽  
Autologous Platelet

SummaryErythrocyte deformability was demonstrated to be influenced by platelet activation. Deformability of erythrocytes suspended in autologous platelet poor plasma (PPP), obtained from platelet rich plasma (PRP), was significantly reduced when PRP had previously been incubated with a platelet activating substance (arachidonic acid, adrenaline or ADP). The possibility of a direct influence of the activating substance on erythrocyte deformability was examined and malondialdehyde formation was determined as an indicator of platelet activation. Erythrocyte deformability was not impaired when endoperoxide formation in platelets was blocked by an inhibitor of cyclooxigenase (acetylsalicylic acid). Plasma viscosity was not influenced by platelet activation as demonstrated by filtration and viscosimetry. Recent studies showed that prostacyclin (PGI2) increases erythrocyte deformability (1). The antagonistic action between prostacyclin released by vessel walls and products of platelet metabolism being well known, we discuss possible mechanisms of this effect and pathophysiological relevance of our results.

Effect Of Factor XIII On Platelet Aggregation. Study Of Platelet Function And Fibrin Stabilization Inhibitors

1981 ◽  
Author(s):  
M Maamer ◽  
O Demay ◽  
M Aurousseau
Keyword(s):  
Platelet Aggregation ◽  
Acetylsalicylic Acid ◽  
Platelet Function ◽  
Factor Xiii ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Photometric Method

There is little information on the participation of Factor XIII in platelet aggregation. Using BORN’s photometric method to study platelet aggregation induced by ADP in vitro on platelet rich plasma (PRP) of rabbit; clot solubility in 1 % monochloracetic acid and incorporation of dansylcadaverin into casein (LORAND L. et al.) to measure plasma FXIII concentration ; we showed that addition of activated F.XIII (F.XIIIa) to a PRP, aggregating power of platelets was significantly increased (+ 30.4 %, p<0.00l). Addition of inactive F.XIII or thrombin + Ca++ in concentrations used to activate F.XIII, had no significant effect on platelet aggregation induced by ADP.When F.XIIIa was added to plasma in presence of F.XIII inhibitors as 3178 AQ (a new synthetic benzothiophen keton derivative) or monodansylcadaverin (DC) in concentrations of (3.27 × 10-4 M and 9.31 × 10-4 m respectively), the platelet aggregation was significantly inhibited (- 48.8 % and - 35.4 % respectively, p<0.001). This inhibitory effect was not seen when dipyridamole or Acetylsalicylic Acid (ASA) in concentrations of (6.18 × 10-4 M and 17.3 × 10-4 M respectively) ware added in PRP in presence of F.XIIIa When platelet aggregation was performed without addition of F.XIIIa the inhibitory effect of 3178 AQ and DC was respectively (- 76.6 % and - 65.1 %, p<0.001), dipyridamole (- 37.6 %, p<0.00l) and ASA (-4.1%, no significant)These results suggest that F.XIIIa increased the platelet aggregation induced by ADP and compounds which are both inhibitors of platelet aggregation and F.XIII would be more potent antithrombotic by acting on platelets and fibrin stabilization, than drugs which are inhibitors of platelet aggregation only.

Enhanced Platelet Aggregation And Arachidonic Acid Metabolism Associated To Reduced Sensitivity To Exogenous Prostacyclin In Hypercholesterolemic Subjects

1981 ◽  
Author(s):  
E Tremoli ◽  
P Maderna ◽  
S Colli ◽  
M Sirtori ◽  
C R Sirtori
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Arachidonic Acid Metabolism ◽  
Type Ii ◽  
Thrombotic Risk ◽  
Cholesterol Levels ◽  
Clinical Series ◽  
Age And Sex

The platelet sensitivity to aggregating agents and the production of arachidonic acid (AA) metabolites were studied in 97 hypercholesterolemic patients (WHO types IIA and IIB;serum cholesterol levels >250 mg/dl, LDL>170 mg/dl,triglycerides >180 mg/dl). The findings were compared with those of 55 age and sex matched normocholesterolemic subjects.Platelet aggregation was studied in platelet rich plasma (PRP) with ADP, collagen, epinephrine and thrombofax* as aggregating agents. Malondialdehyde (MDA) production was determined according to Smith et al.. Thromboxane B2 (TXB2) was measured in washed platelets stimulated with AA by a specific RIA. The platelet sensitivity to the inhibitory effect of synthetic prostacyclin (PGI2) was also assessed in 40 of the patients and in 20 age and sex matched control subjects, using fixed concentrations of aggregating agents.Platelets from patients required to aggregate significantly lower concentrations of collagen, epinephrine and thrombofax* (p< 0.001) and of ADP ( p<O.05); MDA production by thrombin and collagen and TXB formation were significantly raised (p<0.001) in the patient group. Higher concentrations of PGI2 were required to inhibit the aggregation of platelets in the type II patients.The reported findings confirm in a large clinical series the potential role of platelet hyperreactivity in the thrombotic risk of type II hypercholesterolemia.

Mechanism of platelet plug formation and role of adenosine diphosphate

1964 ◽  
Vol 206 (6) ◽  
pp. 1267-1274 ◽  
Author(s):  
Theodore H. Spaet ◽  
Marjorie B. Zucker
Keyword(s):  
Connective Tissue ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Suitable Model ◽  
Plug Formation ◽  
Rat Platelets

Traumatized rat omentum was used to demonstrate the development of "platelet plugs" following agitation in platelet-rich plasma. In the absence of divalent cation there was only platelet adhesion to connective tissue fibers; in the presence of divalent cation masses of platelets formed (cohesion) even in plasma adequately anticoagulated with heparin. Exposure of these platelet masses to thrombin produced greater compactness and stability. Human and rat platelets behaved alike with the traumatized rat omentum; platelets from two patients with von Willebrand's disease gave normal reactions whereas platelets from a patient with thrombasthenia showed adhesion only. Exposure of human platelets to washed connective-tissue fragments or to thrombin elicited clumping accompanied by release of serotonin and of adenine nucleotides (AN) of which about one-third was adenosine diphosphate. Intermediate concentrations of connective tissue and thrombin also caused clumping but no liberation of AN or serotonin. ADP caused intense clumping but failed to liberate serotonin or additional ADP. It is suggested that cohesion reaction is mediated by release of ADP. The traumatized omentum appears to be a suitable model for studying the hemostatic process.

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.

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Osthole from Angelica pubescens

1989 ◽  
Vol 62 (03) ◽  
pp. 996-999 ◽  
Author(s):  
Feng-Nien Ko ◽  
Tian-Shung Wu ◽  
Meei-Jen Liou ◽  
Tur-Fu Huang ◽  
Che-Ming Teng
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Inositol Phosphates ◽  
Platelet Rich Plasma ◽  
Chinese Herb ◽  
Atp Release ◽  
Inhibitory Effect ◽  
Thromboxane B2 ◽  
Ionophore A23187 ◽  
Thromboxane Formation

SummaryOsthole, isolated from Chinese herb Angelica pubescens, inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin in washed rabbit platelets. It showed a weak activity in platelet-rich plasma. Osthole inhibited the thromboxane B2 formation caused by arachidonic acid, collagen, ionophore A23187 and thrombin in washed platelets, and also the thromboxane B2 formation caused by the incubation of lysed platelet homogenate with arachidonic acid. The generation of inositol phosphates in washed platelets caused by collagen, PAF and thrombin was suppressed by osthole. These data indicate that the inhibitory effect of osthole on platelet aggregation and release reaction was due to the inhibition of thromboxane formation and phosphoinositides breakdown.

Recovery Of Platelet And Endothelial Cyclooxygenase After Aspirin Measured By Radioimmunoassay For Thromboxane B2 And 6-Keto-Pg-F1α

1981 ◽  
Author(s):  
M Greaves ◽  
Helen Bull ◽  
P A Castaldi
Keyword(s):  
Arachidonic Acid ◽  
Platelet Rich Plasma ◽  
Platelet Inhibition ◽  
Thromboxane B2 ◽  
Synthetic Activity ◽  
Indirect Methods ◽  
Platelet Recovery ◽  
Before And After ◽  
Prostacyclin Production ◽  
Rat Platelets

Recovery of platelet and endothelial prostaglandin synthetic activity by membrane cyclooxygenase after aspirin suppression of the enzyme is relevant to attempts to influence these pathways in platelet and microvascular disorders. Recovery from platelet inhibition has previously been measured by a colorimetric technique and proposed as a method for measurement of platelet turnover. Indirect methods have been used to determine prostacyclin production after similar inhibition and have not clarified the precise recovery rates. In the present study recovery in human and rat platelets was measured using a radioimmunoassay (RIA) for thromboxane B2 (TXB2) and in rat aortic endothelium with a RIA for 6-keto-prostaglandin- F1α (6KPGF1α).TXB2 was measured in human platelet rich plasma stimulated with collagen and arachidonic acid before and after ingestion of 900 mg of aspirin. TXB2 returned to preaspirin levels in 8.5 ± 0.6 (S.D.) and 8.25 ± 0.5 days respectively with collagen and arachidonic acid, the recovery corresponding with platelet survival time of 51Cr autologous platelets. The recovery curve was linear with collagen but not with arachidonic acid where a late rise occurred.Rats were treated with a single dose of aspirin 20 or 100 mg/kg and sacrificed at intervals for measurement of collagen stimulated platelet TXB2 aortic endothelial 6KPGF1α- Recovery of both of these products was linear and reached pretreatment levels after 120 hrs in platelets and 48 hrs in endothelium after 20 mgAg but 100 mg/kg delayed endothelial recovery to 96 hrs.These results indicate that the endothelial and platelet enzyme are equally susceptible to aspirin but endothelial recovery is more rapid at low doses. Collagen stimulated platelet recovery may prove a useful method for measuring platelet production.

scholarly journals Relationship Between Aggregation and Prostaglandin (PG)-Biosynthesis in Rat Platelet Rich Plasma (PRP)

1977 ◽  
Author(s):  
H. Bult ◽  
P. C. Bragt ◽  
I. L. Bonta
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Essential Fatty Acid ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Essential Fatty Acid Deficient ◽  
Normal Rat ◽  
Threshold Doses ◽  
High Doses ◽  
Rat Platelets

The importance of PG biosynthesis in aggregation has been studied by comparing PRP from normal and essential fatty acid deficient (EFAD) rats. The latter show a marked lack of the PG precursor, arachidonic acid (AA).No differences were observed in aggregation with collagen (from 10 μg/ml), ADP (from 0.8 μM) or the PG-endoperoxide PGH2 (1 μg/ml). PGH2 induced only a small, transient aggregation. However, with a collagen dose that produced a 50% response in normal PRP, the aggregation of EFAD PRP was 94% reduced. This correlated with reduced (84–89%) biosynthesis of thromboxane A2 and PGE-like material (PGE), both assessed by bioassay. The PG synthetase of EFAD PRP was unaltered, since PGE and malondialdehyde production were normal when exogenous AA was added. AA, up to 0.33 mM, did not aggregate normal PRP, but high amounts of PGE were formed.These results indicate that internal PG biosynthesis is only essential for aggregation with threshold doses of collagen. At higher collagen doses endogenous PG production does not seem to be important for aggregation. It is unlikely that residual formation of PG-endoperoxides in EFAD PRP, at high doses of collagen, is sufficient for a normal aggregation, since normal rat platelets form PG-endoperoxides from exogenous AA without aggregation.

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