Multiple electrode aggregometry: A new device to measure platelet aggregation in whole blood

SummarySeveral methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p<0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.

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EFFECT OF COLLAGEN AND CITRATE ON HEPARIN-HEDIATED PLATELET ANTIAGGREGATORY ACTIVITY

10.1055/s-0038-1642869 ◽

1987 ◽

Author(s):

S R Saba ◽

H I Saba ◽

G A Morelli

Keyword(s):

Platelet Aggregation ◽

Inhibitory Activity ◽

Whole Blood ◽

Sodium Citrate ◽

Blood Platelet ◽

Inhibit Platelet Aggregation ◽

Agonist Activity ◽

Antiaggregatory Activity ◽

Induced Aggregation ◽

Blood Platelet Aggregation

Heparin has been reported to inhibit platelet aggregation. Our studies show that this activity is easily demonstrable in washed platelet systems, but fails to occur in citrated platelet-rich plasma (PRP) in the presence of a variety of agonists, except collagen. Studies were performed to answer the following questions: (1) Why does heparin inhibit the aggregation of washed platelets but not of citrated PRP, which is the system commonly used for platelet aggregation studies? (2) What is the effect of heparin on platelet aggregation occurring in whole blood, where it can be examined both with and without the presence of sodium citrate? (3) Why does heparin consistently inhibit the collagen-induced aggregation even in citrated PRP, while it fails to inhibit aggregation caused by other agonists? Results of the studies clearly demonstrated that heparin has the ability to directly react with sodium citrate, causing loss of its inhibitory activity on platelets. The antiaggregatory activity of heparin in the presence of collagen as the agonists appears to be directly related to the blocking of collagen’s agonist activity by heparin. Small concentrations of heparin which were unable to inhibit aggregation per se, effectively blocked the collagen agonist activity on platelet aggregation when heparin was directly added to collagen. Further studies showed that heparin, in a native whole blood platelet aggregation system (in the absence of any anticoagulant), exhibited significant inhibitory activity. This activity was lost when citrate was present in the whole blood preparation. These studies, therefore, indicate that failure of heparin to inhibit platelet aggregation in citrated PRP does not negate the importance of this inhibitory activity. Reactivity of heparin with sodium citrate renders citrated systems unsuitable for studying heparin's effect upon platelets. The whole blood platelet aggregation system without the presence of anticoagulants appears to be a more suitable system for the study of heparin and platelet aggregation, and is closer to the physiological system. Heparin exhibits marked inhibitory activity on platelet aggregation in this system, and this suggests it may be an important activity which deserves further attention.

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ADP-Induced Whole Blood Aggregometry and Platelet-Associated Thrombin Generation (TG) In Essential Thrombocythemia (ET) and Polycythemia Vera (PV) Patients

Blood ◽

10.1182/blood.v116.21.1981.1981 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1981-1981

Author(s):

Marina Panova-Noeva ◽

Marina Marchetti ◽

Annamaria Leuzzi ◽

Laura Russo ◽

Guido Finazzi ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Thrombin Generation ◽

Blood Platelet ◽

Jak2v617f Mutation ◽

Mutation Carriers ◽

Impedance Aggregometry ◽

Platelet Aggregometry ◽

V617f Mutation ◽

Blood Platelet Aggregation

Abstract Abstract 1981 Patients with ET and PV are characterized by an increased rate of thrombotic complications and by several abnormalities of platelets, more pronounced in JAK2V617F mutation carriers. Platelet function inhibitors are widely utilized for thromboprophylaxis in ET and PV patients. An increased reactivity to ADP of platelets from these patients has previously been shown by light transmission platelet aggregometry studies, which might explain the failure of aspirin to fully protect from thrombosis these patients, mainly those at high risk. While ADP activation pathway is critical for platelet aggregation, less is known about its role to the initiation of blood coagulation by platelets. In this study we wanted to evaluate in a group of patients with ET and PV, the platelet reactivity to ADP in terms of both proaggregating and procoagulant responses. ADP-induced platelet aggregation and platelet procoagulant potential (i.e. thrombin generation capacity) were measured by the whole blood impedance aggregometry (Multiplate analyser) and by the Calibrated Automated Thrombogram (CAT) assay, respectively. Whole blood platelet aggregation was induced by 6.5 μM ADP. Whereas thrombin generation (TG) was measured in platelet rich plasma (PRP: 150,000 platelets/μl) both in resting conditions and after stimulation by 1.6 μM and 8.3 μM ADP. Fifty patients with ET (58% V617F mutation carriers) and 40 patients with PV (95% V617F mutation carriers) were enrolled into the study. The results show that ET and PV patients presented with significantly higher ADP-induced whole blood platelet aggregation compared to control subjects (786±70 AU*min, and 777±54 AU*min, respectively, versus 465±70 AU*min, both p<0.05), the highest values being observed in ET and PV patients carriers of the JAK2V617F mutation (806±75 AU*min; p<0.001 vs controls). In resting conditions, TG in PRP from ET (133±38 nM) and PV (144±54 nM) patients was significantly greater compared to control PRP (103±21 nM; p<0.05). Similarly, in ADP-stimulated conditions, platelet TG was significantly higher (p<0.05) in ET and PV patients at both 1.6 μM ADP (140±32 nM and 154±59 nM; respectively) and 8.3 μM ADP (161±38 nM and 181±65 nM; respectively) compared to controls (1.6 μM ADP: 112±19 nM; 8.3 μM ADP: 132±26 nM). The analysis according to the JAK2 mutational status showed that platelets from JAK2V617F carriers induced significantly higher TG when stimulated by 8.3 μM ADP (185±47nM) compared to 1.6 μM ADP (146±45nM; p<0.001). Differently, this difference was not observed in JAK2 wild type patients. While in resting conditions patients on hydroxyurea (HU) generated less thrombin compared to non-HU treated patients, no effects of patients’ therapy on TG (i.e. aspirin, HU, aspirin+HU) was observed in the ADP-stimulated conditions. In conclusion, for the first time we demonstrated that platelets from ET and PV patients are more reactive to ADP, not only in terms of increased platelet aggregation in a whole blood system, but also as an enhanced thrombin generation, particularly in those carriers of JAK2V617F mutation. These data support the hypothesis that the use of ADP receptor-inhibitors, in addition to aspirin, might be of help in the prevention of thrombosis in these conditions, by allowing a more complete inhibition of platelet functions. Disclosures: No relevant conflicts of interest to declare.

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THE EFFECT OF ADP RECEPTOR ANTAGONISTS AND ASPIRIN ON WHOLE BLOOD PLATELET AGGREGATION MEASURED BY MULTIPLE ELECTRODE AGGREGOMETRY

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2007.tb02117.x ◽

2007 ◽

Vol 5 ◽

pp. P-T-360-P-T-360

Author(s):

O. Toth ◽

S. Penz ◽

A. Calatzis ◽

H. Losonczy ◽

W. Siess

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Blood Platelet ◽

Receptor Antagonists ◽

Multiple Electrode Aggregometry ◽

Adp Receptor ◽

Adp Receptor Antagonists ◽

Blood Platelet Aggregation ◽

Multiple Electrode

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Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645978 ◽

1987 ◽

Vol 58 (02) ◽

pp. 744-748 ◽

Cited By ~ 11

Author(s):

A R Saniabadi ◽

G D O Lowe ◽

J C Barbenel ◽

C D Forbes

Keyword(s):

Platelet Aggregation ◽

Correlation Coefficient ◽

Whole Blood ◽

Blood Platelet ◽

Inhibitory Effect ◽

Time Interval ◽

Adp Receptor ◽

Spontaneous Platelet Aggregation

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

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The Effect of Calcium and Magnesium on Rabbit Blood Platelet Aggregation in Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655584 ◽

1964 ◽

Vol 12 (01) ◽

pp. 179-200 ◽

Cited By ~ 17

Author(s):

Torstein Hovig

Keyword(s):

Platelet Aggregation ◽

Cation Exchange ◽

Calcium Concentration ◽

Blood Platelets ◽

Blood Platelet ◽

Rabbit Blood ◽

Calcium And Magnesium ◽

Induced Aggregation ◽

Blood Platelet Aggregation

SummaryThe effect of calcium and magnesium on the aggregation of rabbit blood platelets in vitro was studied, with the following results:1. Platelet aggregation induced by ADP or collagen could be prevented by EGTA or EDTA. The aggregating effect was restored by recalcification. The effect was also restored by addition of magnesium in EDTA-PRP, but not in EGTA-PRP unless a surplus of calcium was present.2. Calcium remained in concentrations of the order of 0.15–0.25 mM after dialysis or cation exchange of plasma. Aggregation of washed platelets resuspended in such plasma could not be produced with ADP or collagen, unless the calcium concentration was increased or that magnesium was added.3. The adhesiveness of blood platelets to collagen was reduced in EGTA-PRP and EDTA-PRP. Release of ADP from platelets influenced by collagen could not be demonstrated either in EGTA-PRP (presence of magnesium) or in EDTA-PRP.4. It is concluded that calcium is a necessary factor both for the reaction leading to release of ADP and for the the aggregation produced by ADP.5. Thrombin induced aggregation of washed platelets suspended in tris-buffered saline in the presence of calcium. No effect of magnesium could be observed unless small quantities of calcium were present.

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