Platelet aggregation: The use of optical density fluctuations to study microaggregate formation in platelet suspension

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.

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Mg++ - and Ca++-Induced Platelet Aggregation and ADP

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654252 ◽

1970 ◽

Vol 24 (03/04) ◽

pp. 432-437 ◽

Cited By ~ 5

Author(s):

S Cronberg ◽

J. P Caen

Keyword(s):

Platelet Aggregation ◽

Pyruvate Kinase ◽

Pyruvic Acid ◽

Active Principle ◽

Platelet Suspension ◽

Edta Plasma ◽

Phosphoenol Pyruvic Acid

SummaryReports on platelet aggregation after addition of calcium or magnesium to EDTA- PRP or platelet suspensions were confirmed. An aggregating principle was found in the EDTA-plasma and the supernatant of the platelet suspensions. Aggregation by magnesium in a platelet suspension was inhibited by adenosine and phosphoenol- pyruvic acid and pyruvate kinase, which suggested that the active principle was identical with ADP. Degradation of ADP in EDTA plasma was blocked.It thus appears that aggregation induced by calcium or magnesium in EDTA-PRP and platelet suspension was due to accumulation of spontaneously liberated ADP, which was not degraded.

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New Method for Assessment of Plasma Von Willebrand Factor

10.1055/s-0039-1680819 ◽

1977 ◽

Cited By ~ 1

Author(s):

T. Sano ◽

T. Motomiya ◽

N. Mashimo ◽

H. Yamazaki

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Normal Subjects ◽

Induce Platelet Aggregation ◽

Platelet Suspension ◽

Maximal Plasma ◽

Von Willebrand ◽

Willebrand Factor ◽

Diabetes Melitus

As much interests have been focused on von Willebrand factor (vWF) in diabetes melitus and atherosclerosis, request to determine vWF has been increasing recently. Two methods for assessment of plasma vWF level, without platelet aggregometer, were devised. 1) Platelet-rich plasma (PRP) sensitivity to ristocetin-induced platelet aggregation (RIPA): PRP was separated without centrifugation from citrated blood. Serially two-fold diluted restocetin (16 to 16x2-10 mg/ml) was prepared in a Cooke Microtiter tray and PRP (25 μl each) was added to each concentration of ristocetin. Then the ristocetin-PRP mixture was agitated for 15 seconds using a Kowa Kizai Micromixer and the minimum effective final concentration of ristocetin to give platelet aggregation was obtained microscopically and this was defined as PRP sensitivity to RIPA. This method is convenient for screening test. 2) vWF assay:Serially two-fold diluted plasma (2 to 1024 times, in Tris-salin pH 7.2 containing 12 mg/ml bovine serum albumin), fixed and washed platelet suspension (6x105 /μl, Macfarlane et al.1975) and 3 mg/ml ristocetin were mixed (25 μl each) in a microtiter tray and agitated for 15 seconds. The maximal plasma dilution to induce platelet aggregation was obtained microscopically and defined as the titer of plasma vWF. In normal subjects, minimum effective ristocetin concentration (PRP sensitivity to RIPA) was around 1 to 0.5 mg/ml and maximal plasma dilution to give platelet aggregation (vWF titer) was around 16 to 32 times. The present methods have a good reproducibility and are performed easily without aggregometer and thought to be useful clinically.

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A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom

Biochemical Journal ◽

10.1042/bj2770351 ◽

1991 ◽

Vol 277 (2) ◽

pp. 351-357 ◽

Cited By ~ 53

Author(s):

T F Huang ◽

J R Sheu ◽

C M Teng

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Ionic Exchange ◽

Dependent Manner ◽

Single Chain ◽

Platelet Suspension ◽

Trimeresurus Flavoviridis ◽

Platelet Membranes ◽

Fibrinogen Binding ◽

Induced Aggregation

The interaction of fibrinogen with its receptors on platelet surfaces leads to platelet aggregation. A snake-venom peptide, trigramin, has previously been demonstrated to inhibit platelet aggregation by acting as a fibrinogen-receptor antagonist. By means of gel filtration, ionic-exchange chromatography and reverse-phase h.p.l.c., a potent platelet-aggregation inhibitor, triflavin, has now been purified from the venom of Trimeresurus flavoviridis. The purified triflavin is a single-chain polypeptide, consisting of about 71 amino acid residues with a molecular mass of 7600 Da, and its N-terminal sequence is Gly-Glu-Glu-Cys-Asp. Triflavin dose-dependently inhibited human platelet aggregation stimulated by ADP, adrenaline, collagen, thrombin or prostaglandin endoperoxide analogue U46619 in preparations of platelet-rich plasma, platelet suspension and whole blood. Its IC50 ranged from 38 to 84 nM, depending on the aggregation inducer used and the platelet preparation. However, triflavin apparently did not affect the platelet shape change and ATP-release reactions caused by these agonists. Triflavin inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner, indicating that it directly interferes with the binding of fibrinogen to its receptors on platelet membranes exposed by elastase treatment. Additionally, triflavin dose-dependently blocked 125I-labelled fibrinogen binding to ADP-activated platelets. In conclusion, triflavin inhibits platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors on platelet membranes.

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Use of increased concentrations of adenosinediphosphate for high residual platelet reactivity in patients with coronary heart disease

10.18411/lj-02-2021-33 ◽

2021 ◽

Vol 70 (1) ◽

pp. 129-132

Author(s):

O.A. Trubacheva ◽

S.N. Belyaeva ◽

T.E. Suslova ◽

I.V. Petrova

Keyword(s):

Coronary Heart Disease ◽

Heart Disease ◽

Platelet Aggregation ◽

Antiplatelet Therapy ◽

Light Transmission ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

Transmission Curve ◽

Platelet Suspension

Detection of a tendency to increased thrombosis in patients with coronary heart disease (CHD) is of important prognostic value in the selection of drugs aimed at achieving a persistent antithrombotic effect. The aim of the study was to evaluate the use of elevated ADP inducer concentrations to improve the accuracy of ADP-induced platelet aggregation in patients with coronary heart disease. Material and method. Material and method. We studied 48 patients with CHD who were on continuous double antiplatelet therapy for 6 months (aspirin 75mg and clopidogrel 75mg per day). The aggregation activity of the platelet suspension was studied using the Born method G. in the modification of Gabbasov Z. A. Platelet activity was evaluated by the degree of aggregation of platelet-rich plasma along the light transmission curve under the influence of the inducer adenosine diphosphate (ADP) at a concentration of 2 mmol/l and by its own patented method against the background of additional ADP application. Results. In patients, platelet aggregation decreased to 5-35% (p<0.005) compared to the standard values, which are 50-60%. The values of platelet aggregation with the additional introduction of the inducer of aggregation ADP in a ratio of 2:1 to 2 µmol/l for 1, 2, 3, and 4-minute registration of platelet aggregation, resulted in increased aggregation from 55% to 75% (p<0.001), indicating high residual platelet reactivity on the background of double antiplatelet therapy. Correlations of the degree of aggregation for elevated ADP concentrations with multivessel arterial lesion and dyslipidemia were also found, r=0.86 and r=0.92, respectively. Conclusion. The use of elevated concentrations of adenosine diphosphate in platelet aggregation in patients with ischemic heart disease increases the accuracy of assessing ADP-induced platelet aggregation against the background of dual antiplatelet therapy and contributes to the detection of high residual platelet reactivity.

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Enhancement of ADP-Induced Platelet Aggregation by Exercise Test in Coronary Patients and Its Prevention by Pyridinolcarbamate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654253 ◽

1970 ◽

Vol 24 (03/04) ◽

pp. 438-449 ◽

Cited By ~ 14

Author(s):

H Yamazaki ◽

I Kobayashi ◽

T Shimamoto

Keyword(s):

Platelet Aggregation ◽

Optical Density ◽

Exercise Test ◽

Platelet Rich Plasma ◽

Statistical Significance ◽

Parallel Line ◽

Inhibitory Effect ◽

Repeated Measurements ◽

Step Test ◽

Coronary Patients

SummaryADP-induced platelet aggregation in citrated platelet rich plasma (CPRP) was examined using a photoelectric system. To exclude a variation in intensities of platelet aggregation in repeated measurements and to compare intensities of different samples, the maximum deflection of the optical density of CPRP induced by adding ADP solution was divided by a deflection of the optical density of the platelet free plasma and its value, shown as a percentage, was defined as an intensity of ADP-induced platelet aggregation. In this method, the linearity was found in the dose response curve of the platelet aggregation induced by 10−6, 3 × 10−6 and 10−5 molar of ADP with statistical significance. These variation coefficients were less than 5% in the responses induced by the higher doses of ADP. Changes in the ADP-induced platelet aggregation after a Master’s two step test were examined in 13 patients with angina pectoris 3 h after oral administration of placebo or 1 g of pyridinolcarbamate. Under placebo pretreatment, an enhancement of platelet aggregation was observed 1 min after the exercise test with statistical significance (P < 0.01 ∼ 0.05). In the cases of the same subjects pretreated with pyridinolcarbamate, such change was not observed at any time. Using a parallel line assay, an inhibitory effect of pyridinolcarbamate against enhancement of ADP-induced platelet aggregation after the exercise was also recognized with statistical significance (P < 0.01). In the 10 healthy volunteers, there was no statistically significant enhancement of ADP-induced platelet aggregation using any concentration of ADP 1 to 10 min after the exercise test.

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Evaluation of Highly Purified Bovine F. VIII and Bovine Plasma as Reagents to Induce Platelet Aggregation

10.1055/s-0039-1680433 ◽

1977 ◽

Author(s):

M. A. Lazzari ◽

C. Simonetti ◽

G. Casillas ◽

M. Pavlovsky

Keyword(s):

Platelet Aggregation ◽

Optical Density ◽

Sodium Citrate ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Induce Platelet Aggregation ◽

Bovine Plasma ◽

Aggregation Factor ◽

High Concentrations

Purified bovine F. VIII is a known reagent for human platelets aggregation used for the study of thrombopaties. As bovine plasma (BP)is easier to prepare and standardize it was studied as an alternative reagent, comparing it with purified bovine F. VIII. Platelet aggregation was studied by a turbidimetric technique(Aggregometer Bryston AG 1). The substrates were platelet rich plasma (PRP)and gel filtered platelets using between 150,000 and 200,000 platelets/mm3 to standardize the platelets surface. 1/10 dilution of bovine plasma was found to be the best for PRP and 1/1 for filtered platelets. Incubation of the substrates with bovine plasma at 37°C for 5 min seems to enhance PAF activity of the platelet aggregation factor (PAF). Primary aggregation was obtained in PRP treated with 2% tetrasodic EDTA, 3. 4% sodium citrate and in plasma with aspirin. No secondary aggregation was observed in EDTA or aspirin plasma. Unlike platelets treated with ADP, their shape did not change since the optical density was not modified. No synergism or competition between PAF and ADP or adrenalin were found. High concentrations of NaCl or urea interfere with PAF, and esposure to 56°C inactivates it. In 50 normal PRP the percentage of total aggregation were: BP 1/10 42% ± 12 ; BP 1/1 79% ± 13 ;purified bovine F. VIII 1/10 86% ± 6 and in 10 batches of filtered platelets bp 1/1 82% ± 7. We consider an advantage to use bovine plasma for platelets aggregation studies.

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PLATELET FUNCTION IN VASOSPASTIC DISORDERS

10.1055/s-0038-1643476 ◽

1987 ◽

Author(s):

D Wilkinson ◽

P Vowden ◽

L Gilka ◽

S M Rajah ◽

R C Kester

Keyword(s):

Platelet Aggregation ◽

Optical Density ◽

Platelet Function ◽

Normal Subjects ◽

Lag Phase ◽

Vibration White Finger ◽

Significant Difference ◽

Eicosanoid Metabolism ◽

Platelet Aggregate ◽

Vibration White Finger Disease

If vasospastic disorders are associated with abnormal eicosanoid metabolism then disturbed platelet function may be observed. We have measured platelet aggregation (PAG) at 37°C to ADP (8 1:1 sequential dilutions from 10μM), collagen (2mg/l) and adrenaline (5μM) in 37 patients with Raynaud's syndrome (21 primary (RD), 9 secondary to scleroderma (RP) and 7 vibration white finger disease (VWF)) and 10 normal subjects (N). In addition we have determined the platelet aggregate ratio (PAR). PAG was expressed as the percentage fall in optical density after the addition of the aggregant. We also recorded the duration of the collagen lag phase and the concentration of ADP at which disaggregation occurred (DAC).Patients with RP showed enhanced PAG compared to normals. This reached significance for collagen lag phase (p<0.001), percentage PAG at 3 minutes to ADP 1.25μM (p<0.05) and the DAC (p<0.01). Similar differences were observed between patients with RP and VWF. Patients with RP also showed significantly enhanced PAG with regard to ADP 1.25μM (p<0.01) and the DAC, (p<0.05) compared to patients with RD. There was no significant difference in platelet function between normal subjects and patients with VWF or between normal subjects and patients with RD. Only with regard to DAC did patients with RD differ from patients with VWF (p<0.05). The differences observed in PAG were not reflected by the PAR. Patients with RP have significantly enhanced PAG when compared to all other groups. This may relate to eicosanoid metabolism and provides a rationale for treatment.

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Testing of the Aggregating Activity from Various Batches of a Platelet Specific Collagen Preparation

10.1055/s-0039-1682370 ◽

1977 ◽

Author(s):

F. Schulte

Keyword(s):

Platelet Aggregation ◽

Optical Density ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Blood Samples ◽

Whole Blood Samples ◽

Aggregation Of Platelets ◽

Free Plasma ◽

Single Batch ◽

Platelet Functions

Producing of a collagen preparation for determining platelet functions requires the testing of the activity of each single batch to make possible a standardization. For this purpose methods using PRP (platelet rich plasma) to measure the platelet aggregation are not very appropriate. Since four years a collagen centrifugation test proves to be suitable for determining the activity of various batches of Collagenreagent “Horm”. In this test the platelets of citrated whole blood are mixed with graduated doses of collagen directly by the withdrawal of blood. The turbidimetrical evaluation of the PRP obtained from the collagen treated whole blood samples shows 15% (SD ± 3. 5 %) of the O.D. (optical density) of the PFP (platelet free plasma) after a dose of 1 meg and 80% (SD ± 2 %) after a dose of 2 meg collagen per ml whole blood. The aggregation of platelets in citrated whole blood simultaneously with graduated doses of collagen allows to standardize different batches of platelet specific collagen preparations.

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PLATELET PHAGOCYTOSIS AND AGGREGATION

The Journal of Cell Biology ◽

10.1083/jcb.27.3.531 ◽

1965 ◽

Vol 27 (3) ◽

pp. 531-543 ◽

Cited By ~ 103

Author(s):

Henry Z. Movat ◽

William J. Weiser ◽

Michael F. Glynn ◽

James F. Mustard

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Chelating Agent ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Inhibit Platelet Aggregation ◽

Platelet Suspension ◽

Latex Particles ◽

Washed Platelet

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.

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