Is Spontaneous Platelet Aggregation Present in Whole Blood In Vitro?

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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Factors that Contribute to Spontaneous Platelet Aggregation and Streptokinase-Induced Aggregation in Whole Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653768 ◽

1995 ◽

Vol 73 (02) ◽

pp. 297-303 ◽

Cited By ~ 11

Author(s):

Ruth Armstrong ◽

Jane A May ◽

Wolfgang Lösche ◽

Stan Heptinstall

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Divalent Cations ◽

High Concentration ◽

No Donor ◽

Rgd Sequence ◽

Spontaneous Platelet Aggregation ◽

Adhesive Proteins ◽

Induced Aggregation

SummaryWhen whole blood is stirred there is a “spontaneous” platelet aggregation (SPA) which is presumed to be caused by proaggregatory factors released from platelets and other blood cells. Adding streptokinase (SK) to stirred whole blood frequently increases the rate and extent of the platelet aggregation that occurs; this is likely to be via immune complex formation between SK and natural anti-SK antibodies leading to increased release of pro-aggregatory factors.In this investigation we have examined the effects of several inhibitors and antagonists in an attempt to identify the proaggregatory factors that contribute to both SPA and SK-induced aggregation (SKA) and to evaluate different means of inhibiting both processes. The effects of the inhibitors/antagonists were determined in vitro after adding them to citrated whole blood obtained from healthy volunteers. Platelet aggregation was measured using a platelet counting technique.Inhibition of both SPA and SKA by apyrase and by FPL 66096 (a P2T receptor antagonist) demonstrated the involvement of ADP in both processes. Inhibition by chlorpromazine indicated that the most likely source of the ADP is red cells. The effects of sulotroban (a TXA2 antagonist) indicated involvement of TXA2 in SKA but not in SPA. The lack of effect of specific antagonists at S2, α2 and PAF receptors suggested lack of involvement of serotonin, catecholamines and plateletactivating factor in either SPA or SKA. Both SPA and SKA were potently inhibited by low concentrations of iloprost (a PGI2 analogue), but a high concentration of SIN-1 (a NO donor) was much less effective. SPA and SKA were prevented by EDTA and by RGDS indicating the importance of divalent cations and of the RGD sequence in adhesive proteins in mediating the platelet aggregation that occurred.We also determined the effects on SPA and SKA of adding MgCl2 to whole blood. In this case we used blood containing hirudin as anticoagulant. MgCl2 (1 mM) appeared to delay the onset of SPA and markedly inhibited SKA.

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Dipyridamole Inhibits Platelet Aggregation in Whole Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665327 ◽

1983 ◽

Vol 50 (04) ◽

pp. 852-856 ◽

Cited By ~ 104

Author(s):

P Gresele ◽

C Zoja ◽

H Deckmyn ◽

J Arnout ◽

J Vermylen ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Significant Negative Correlation ◽

Significant Inhibition ◽

Oral Drug ◽

Human Blood Samples ◽

Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

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Inhibition of Spontaneous Platelet Aggregation by Sulfinpyrazone

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666900 ◽

1979 ◽

Vol 42 (02) ◽

pp. 621-625 ◽

Cited By ~ 1

Author(s):

G G Nenci ◽

G Agnelli ◽

M Berrettini ◽

P Parise ◽

E Ballatori

Keyword(s):

Platelet Aggregation ◽

Crossover Study ◽

Double Blind ◽

Platelet Aggregability ◽

Initiation Of Therapy ◽

Spontaneous Platelet Aggregation ◽

Blind Crossover Study ◽

Double Blind Crossover Study

SummaryIn a randomized double-blind crossover study in 16 patients with enhanced in vitro spontaneous platelet aggregation, sulfinpyrazone proved to be effective in normalizing platelet aggregability within 4 days after initiation of therapy.

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Mechanism of the Antiplatelet Action of Dipyridamole in Whole Blood: Modulation of Adenosine Concentration and Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661437 ◽

1986 ◽

Vol 55 (01) ◽

pp. 012-018 ◽

Cited By ~ 86

Author(s):

Paolo Gresele ◽

Jef Arnout ◽

Hans Deckmyn ◽

Jos Vermylen

Keyword(s):

Platelet Aggregation ◽

Adenosine Receptor ◽

Whole Blood ◽

Blood Cells ◽

Inhibitory Action ◽

Phosphodiesterase Inhibitor ◽

Inhibitory Effect ◽

Adenosine Concentration ◽

Pharmacological Studies

SummaryDipyridamole inhibits platelet aggregation in whole blood at lower concentrations than in plasma. The blood cells responsible for increased effectiveness in blood are the erythrocytes. Using the impedance aggregometer we have carried out a series of pharmacological studies in vitro to elucidate the mechanism of action of dipyridamole in whole blood. Adenosine deaminase, an enzyme breaking down adenosine, reverses the inhibitory action of dipyridamole. Two different adenosine receptor antagonists, 5’-deoxy-5’-methylthioadenosine and theophylline, also partially neutralize the activity of dipyridamole in blood. Enprofylline, a phosphodiesterase inhibitor with almost no adenosine receptor antagonistic properties, potentiates the inhibition of platelet aggregation by dipyridamole. An inhibitory effect similar to that of dipyridamole can be obtained combining a pure adenosine uptake inhibitor (RE 102 BS) with a pure phosphodiesterase inhibitor (MX-MB 82 or enprofylline). Mixing the blood during preincubation with dipyridamole increases the degree of inhibition. Lowering the haematocrit slightly reduces the effectiveness.Although we did not carry out direct measurements of adenosine levels, the results of our pharmacological studies clearly show that dipyridamole inhibits platelet aggregation in whole blood by blocking the reuptake of adenosine formed from precursors released by red blood cells following microtrauma. Its slight phosphodiesterase inhibitory action potentiates the effects of adenosine on platelets.

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

Thrombosis and Haemostasis ◽

10.1160/th06-05-0242 ◽

2006 ◽

Vol 96 (12) ◽

pp. 781-788 ◽

Cited By ~ 288

Author(s):

Andreas Calatzis ◽

Sandra Penz ◽

Hajna Losonczy ◽

Wolfgang Siess ◽

Orsolya Tóth

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Blood Platelet ◽

Platelet Inhibition ◽

New Device ◽

Platelet Aggregometry ◽

Spontaneous Platelet Aggregation ◽

Induced Aggregation ◽

Blood Platelet Aggregation ◽

Multiple Electrode

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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EFFECTS OF BLOOD CELLS ON PLATELET AGGREGATION BY IMPEDANCE METHOD

10.1055/s-0038-1644870 ◽

1987 ◽

Author(s):

L Mannucci ◽

R Redaelli ◽

E Tremoll

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Blood Cells ◽

White Blood Cells ◽

Normal Subjects ◽

Impedance Method ◽

Induced Aggregation ◽

Vitro Data ◽

In Vitro Data

To evaluate the effects of blood cells on the response of platelets to aggregating agents using whole blood impedance aggregometer, studies were carried out on whole blood (WB) of normal subjects and of patients with: polycythemia vera (PV), iatrogenic anemia (IA), primary thrombocytosis (PT), idiopathic thrombotic purpura (ITP), myeloid chronic leukemia (MCL), iatrogenic leukopenia (IL). The in vitro effects of red blood cells (RBC) and of white blood cells (WBC) on platelet rich plasma (PRP) aggregation were also evaluated. WB, PRP, WBC and RBC were prepared by conventional methods. Aggregation was performed using the impedance aggregometer (mod. 540, Chrono Log Corp). In normal subjects the concentration of collagen giving 50 % aggregation (AC50 ) found in PRP did not differ from that of WB, indicating that hematocrit values within the normal range did not appreciably affect platelet aggregation. The results obtained in WB of patients are summarized in the table: In vitro data showed that aggregation in prp in wb of normal subjects was related to the number of platelets present in the sample. RBC added to PRP significant reduced aggregation only when the RBC number was greater than 4.101 cells. No effect of WBC on collagen induced aggregation of PRP was observed, whereas significant inhibition was detected after ADP. It is concluded that the aggregation evaluated in WB with impedance method is dependent on the platelet number. Also, in vitro data and studies in WB of patients indicate that aggregation is significantly affected by the presence of cells other than platelets only in conditions of changes of the ratio between platelets and leukocytes and/or red cells.

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Endotoxin-Induced Platelet Activation in Human Whole Blood In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647499 ◽

1988 ◽

Vol 59 (03) ◽

pp. 378-382 ◽

Cited By ~ 11

Author(s):

Gyorgy Csako ◽

Eva A Suba ◽

Ronald J Elin

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Atp Release ◽

Human Platelets ◽

Lag Phase ◽

Human Whole Blood ◽

Dose Dependent

SummaryThe effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 μg/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand, endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by “solubilized” endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

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