Platelet aggregation in human whole blood after chronic administration of aspirin

We have used the impedance aggregometer to study the "ex vivo" effect of acetylsalicylic acid (ASA) in whole blood (WB) versus platelet-rich plasma (PRP) in 35 male healthy volunteers after 10 days of treatment with 25, 50, 125, 250, and 500 mg/day of ASA. Percent of inhibition of platelet aggregation was determinated at the end of treatment. A greater inhibition of platelet aggregation was observed in WB than in PRP when ASA was administrated at almost all doses. Maximal differences were at 25, 50, and 125 mg/day of ASA on adrenaline, collagen and arachidonic acid induced aggregation, and with 250 and 500 mg/day of ASA when ADP was used as aggregating agent. In the "in vitro" trials, IC-50 values of ASA on ADP and collagen induced aggregation were determined in platelet aggregation by the impedance method in both WB and PRP. ASA shows a lower IC-50 in WB than in PRP. When leucocytes were incubated in PRP samples, it effect was similar to the percent of inhibition in WB.     

Why single daily dose of aspirin may not prevent platelet aggregation

The effect of different doses of aspirin on the synergistic activity of sodium arachidonate plus platelet activating factor (paf) ADP or collagen in platelet aggregation was studied in human volunteers. Aggregation studies in platelet rich plasma (PRP) showed that aspirinated platelets, unresponsive to arachidonate, when stirred with threshold concentrations of paf, ADP or collagen, reacted differently according to the dose of aspirin and the time elapsed since ingestion. After a single or daily 50 mg dose for 7-10 days independent of elapsed time until blood withdrawal, a complete synergistic activity was obtained. In PRP samples obtained 24 hours after the last aspirin intake, a complete synergistic aggregation was achieved after a single dose or after 7-10 days of 500 mg aspirin ingestion; synergistic effect did not appear when blood was drawn 2.5 hours after intake. The thromboxane B2 concentrations were very low in all samples after PRP stimulation with sodium arachidonate or paf or both. As rationale is that platelet activation in vivo occurs in response to several stimuli, the therapeutic implications of our results is that aspirin may not prevent the agonist potentiation effect when low dose or daily high dose (500mg) are administrated. This may explain the erratic results of most aspirin trials in which this drug was used to suppress platelet function.     

Arachidonate-induced platelet aggregation in the dog.

The platelet rich plasma (PRP) from most dogs aggregates in response to ADP, collagen and thrombin. Dog PRP generally does not aggregate in response to epinephrine, and previous studies found that dog PRP uniformly failed to aggregate when exposed to arachidonic acid prepared in an ethanol-sodium carbonate medium. Our studies demonstrate that 30% of randomly selected mongrel dogs have PRP which aggregates when exposed to sodium arachidonate dissolved in modified Tyrode's buffer, PRP from these dogs also aggregates with lower concentrations of ADP and collagen than PRP which is unresponsive to arachidonate. Pre-incubation of PRP with epinephrine uniformly transforms PRP which does not aggregate on exposure to arachidonate alone into arachidonate-aggregating PRP. Dog PRP which aggregates with arachidonate release ¹⁴C-serotonin, while non-aggregating PRP does not. However, arachidonate stimulates malondialdehyde production in both aggregating and non-aggregating PRP.The results of this study indicate that dogs are heterogeneous in regard to their aggregation response to arachidonate. The mechanism of this heterogeneity is unknown: however, since prostaglandin metabolism is intact and the platelets of some dogs respond to arachidonate alone, it appears to be the result of variable sensitivity to endoperoxides and thromboxane A₂.     

Increased in vitro platelet aggregation in hypertriglyceridemias

Platelet aggregation induced by ADP and collagen was monitored in 25 patients with increased serum levels of triglycerides (7 HLPs type IIb, 14 type IV, 4 type V patients) and compared with a group of 10 normolipidaemic control persons. Platelet aggregation was studied simultaneously in platelet rich plasma (PRP) by both turbidometric and impedance technique and also in whole blood (WB) by the impedance method. Whereas platelet aggregation testing by turbidometry was limited by the optical density of the plasma samples in hypertriglyceridemia, the aggregation process could easily be registered in PRP and WB using the impedance method. Threshold aggregatory concentrations with both agonists were significantly lower for all three groups of HLP.     

Some properties of mouse platelets

Mouse platelets were aggregated by arachidonate, thrombin, collagen and ADP. In general they were, like rat platelets, more aggregable in heparinized PRP than in citrated (3.8%) PRP. Mouse platelets underwent the release reaction when aggregated by arachidonate, collagen and thrombin, but not when stimulated by ADP. The aggregation of the platelets to arachidonate was inhibited by cyclooxygenase inhibitors and by prostacyclin. Studies with tritiated arachidonate showed that mouse platelets possess the lipoxygenase and cyclooxygenase pathways found in other mammalian platelets and produce thromboxane and 12-HETE. The mouse provides a convenient model for the study of many conditions known to affect platelet aggregation. The similarity of mouse platelets to the platelets of other mammals together with the ability to study large numbers of animals at low cost, should encourage further use of mouse platelets.     

Synergism between platelet aggregating agents: the role of the arachidonate pathway.

Aggregation of platelets by low concentrations of ADP is augmented by non-aggregating concentrations of collagen, thrombin, arachidonate or the divalent cation ionophore A23,187. Release-inducing agents act synergistically with ADP and with each other. Both collagen and thrombin cause aggregation by releasing ADP and by freeing platelet arachidonate to form prostaglandin endoperoxides which give rise to thromboxane A₂. In these experiments the role of the arachidonate pathway in the synergism between pairs of aggregating and release-inducing agents was examined. Indomethacin was used to inhibit conversion of arachidonate to prostaglandin endoperoxides and thromboxane A₂ and creatine phosphate/creatine phosphokinase (CP/CPK) was used in some experiments to convert released ADP to ATP. Synergism of collagen with ADP, arachidonate or thrombin was inhibited by indomethacin indicating that the arachidonate pathway plays a major role in the synergistic effects to which collagen contributes. Synergism of thrombin with collagen or arachidonate was inhibited by indomethacin but synergism of thrombin with ADP was only slightly affected. Indomethacin had little influence on the combined effects of these two agents on platelet aggregation. Thus it appears that the conversion of platelet arachidonate to prostaglandin endoperoxides and thromboxane A₂ plays a minor part in the synergistic effects in which thrombin or A23,187 are involved. Thus, the non-steroidal anti-inflammatory drugs may have only limited use in inhibiting the contribution of thrombin and ADP to the formation of platelet thrombi at sites of vessel injury.     

Effect of acetylsalicylic acid on platelet aggregation and thromboxane B2 production in flowing aortic blood in the rat studied with a filter loop technique

A method of measuring $\text{in vivo}$ platelet function using a filter loop technique has been used to study platelet aggregation in response to Adenosine Diphosphate (ADP) and Sodium Arachidonate infusion in flowing aortic blood in the rat. ADP infusion produced reversible platelet aggregation $\text{in vivo}$ with no change in thromboxane B₂ (TXB₂) levels whereas sodium arachidonate infusion resulted in virtually irreversible aggregation with a rise in TXB₂ levels. Oral Acetylsalicylic Acid (ASA) in doses of 1–100 mg/kg had no effect on ADP induced aggregation but prevented platelet aggregation $\text{in vivo}$ induced by sodium arachidonate and the concomitant rise in TXB₂ levels.     

Synergistic effects of platelet-activating factor and other platelet agonists in human platelet aggregation and release: The role of adp and products of the cyclooxygenase pathway

The synergistic effects of platelet-activating factor (PAF) with ADP, collagen, thrombin, A23187, adrenaline, sodium arachidonate and ristocetin in human platelet aggregation and ß-thromboglobulin (ß-TG) release were investigated in citrated platelet-rich plasma (PRP). Synergism in both aggregation and release was present with all agonists except ristocetin.Upon oral intake of aspirin (ASA) the PAF-induced irreversible aggregation as well as the synergistic irreversible aggregation became reversible. Both prior to and after ASA ingestion ADP removal by creatine phosphate/creatine phosphokinase (CP/CPK) resulted in a reduced, reversible platelet aggregation induced by PAF alone or in combination with the other agonists. The ADP-removal and ASA-ingestion also strongly inhibited the ß-TG release. The synergistic aggregation and release were also inhibited by ASA and indomethacin in vitro as well as by the competitive ADP-inhibitor ATP.It is concluded that not only the activation of human platelets by low doses of PAF itself, but also the synergism of PAF and other platelet agonists is highly dependent upon ADP and products of the cyclooxygenase pathway.     

Testosterone potentiation of ionophore and ADP induced platelet aggregation: relationship to arachidonic acid metabolism

The 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 (less than 1 micro M ) 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 form increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.     

Malondialdehyde formation by blood platelets: a diagnostic test to assess acetylsalicylic acid induced thrombocytopathy?

We investigated whether the measurement of N-ethylmaleimide stimulated malondialdehyde (MDA) formation by blood platelets from normal subjects is equally sensitive to acetylsalicylic acid intake as are platelet aggregation studies. MDA production and platelet aggregation by collagen and arachidonate were assayed in ten healthy volunteers before and up to ten days after a single oral dose of 500 mg aspirin. Discordant results of the two tests were seen in several subjects 4 to 6 days after aspirin intake. In three cases with still suppressed MDA values on day 4, collagen or arachidonate induced aggregation was normalized. However, on day 6, when MDA was normalized in all subjects, the aggregation response to arachidonate was still pathologic in 5 of the ten volunteers. In case of a patient with abnormal aggregation response to arachidonate and/or collagen, therefore, a normal MDA value does not permit to exclude aspirin as the cause of the platelet dysfunction.     

The effects of argon laser on in vitro aggregation of platelets in platelet rich plasma and whole blood

The effects of an Argon laser on platelet aggregation were studied, since platelets may be exposed to laser energy when used intravascularly. Various preparations of platelets in platelet rich plasma (PRP) and whole blood, with or without aspirin, were tested with the aggregating agents ADP, collagen, thrombin, and epinephrine. Simultaneous release of ATP was also measured in PRP. At relatively low levels of irradiation, platelet aggregation was potentiated. Enhancement was evidenced by an increase in percent aggregation, earlier onset of the reaction, and reduction in the amount of aggregating agent required. In PRP, the mechanism of laser potentiation appeared to be the release of endogenous ATP from platelets. At relatively high levels of irradiation, platelets were destroyed and aggregation abolished. In whole blood, the mechanism was somewhat more complicated since release of ATP occurred from RBCs as well as platelets. Spontaneous aggregation following laser treatment occurred in isolated instances in PRP and in every trial in whole blood preparations. Aspirin ingestion inhibited the laser's effects in PRP but not in whole blood. These results may have important clinical implications for laser angioplasty, and the potentiated aggregation response may prove useful in laboratory studies of platelet function.     

Platelet aggregation studies in whole human blood

Since the development of the photometric aggregometer, platelet aggregation studies are routinely performed on platelet-rich plasma (PRP). We have studied platelet aggregation in fresh citrated whole human blood using the recently developed Ultra Flo 100 Whole Blood Platelet Counter. Aggregation of platelets in whole blood was induced with adenosine 5′-diphosphate (ADP; 0.25–10μM), collagen (0.25–1.0μg/ml) and thrombin (0.05–0.2U/ml). Platelet aggregation induced by ADP and thrombin was maximum within 1 min, and that of collagen within 3 mins. Aggregation responses to low concentration of ADP and thrombin were rapidly reversible, whereas responses to collagen and high concentrations of ADP and thrombin were virtually irreversible. The aggregation of platelets was indicated by a fall in platelet count; confirmed by scanning electron micrographs which revealed the presence of large aggregates of platelets, and was prevented when blood was treated with EDTA as anticoagulant. The present technique appears to be rapid, sensitive and reliable; and allows direct measurement of platelet aggregation and disaggregation in whole blood in vitro and ex-vivo.     

Effect of amino sugars on platelet aggregation and on fibrinogen binding

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.     

Effects of tris on responses of human and rabbit platelets to aggregating agents

Despite reports that Tris [tris (hydroxymethyl)aminomethane] affects platelets, it is often used to buffer suspending media. Human or rabbit platelets were washed and resuspended in Tyrode solution containing apyrase and 0.35% albumin. Addition of 15 mM Tris partially inhibited primary aggregation induced by 10 microM ADP and inhibited aggregation and release of 14C-serotonin from prelabelled platelets stimulated with low concentrations of thrombin (0.05-0.2 U/mL), or collagen. Platelets resuspended in 15 mM Tris, 0.15 M NaCl, 0.35% albumin, pH 7.5, did not aggregate in response to 10 microM ADP whereas platelets in Tyrode-albumin aggregated extensively. Ca2+ (5 mM) did not overcome the inhibition of thrombin-induced aggregation. Tris (15 or 1.5 mM) potentiated aggregation and release induced by sodium arachidonate (20-50 microM) or the ionophore A23187 (0.6-1 microM). Pretreatment of platelets with aspirin did not prevent potentiation by A23187, indicating that it is not mediated through activation of the arachidonate pathway. The inhibitory and potentiating effects of Tris are similar to those of amino sugars, lysine, arginine and primary amines such as methylamine and cadaverine, and may represent general effects of amines on platelets. Potentiation of the effects of some aggregating agents and inhibition of others re-emphasizes the concept that there are several different mechanisms through which aggregation can occur. Tris-based buffers are unsuitable for platelet suspending media and their use as solvents for aggregating agents or inhibitors should be limited.     

Platelet aggregation in whole blood determined using the Ultra-Flo 100 Platelet Counter

The Ultra-Flo 100 Whole Blood Platelet Counter has proved a useful tool for measuring platelet aggregation in whole blood, the extent of aggregation being deduced from the number of single platelets that remain. The technique has allowed us to show that platelets aggregate spontaneously in citrated blood and in heparinized blood but not in whole blood collected into EDTA. The aggregation occurs during storage but its rate is enhanced by stirring and it occurs more readily when the whole blood has been exposed to plastic rather than glass. It occurs much more readily in whole blood from some individuals than from others and the process may involve adenosine diphosphate (ADP). The rate of aggregation in whole blood is enhanced by several aggregating agents including collagen, ADP and sodium arachidonate which are more usually studied in platelet-rich plasma.     

Increase of platelet thromboxane A2 formation and of its plasmatic half-life in diabetes mellitus

Platelet-rich plasmas (PRP) of diabetic subjects exhibited a hyperaggregability to either collagen or sodium arachidonate. These platelets, isolated from their plasma, only showed a normal response to sodium arachidonate. Moreover, the biosynthesis of prostaglandins and related compounds from endogenous arachidonate (thrombin as inducer) was enhanced in diabetic platelets whereas the biosynthesis from exogenous arachidonate was normal. On the other hand, the half-life of thromboxane A₂ was longer in diabetic platelet-poor plasma than in the control. It is concluded that a part of the hyperaggregability of diabetic PRP to sodium arachidonate could be due to the increase of the half-life of thromboxane A₂. Otherwise, hypersensitivity of isolated diabetic platelets to either collagen or thrombin shows a greater availability of phospholipidic arachidonate to the prostaglandin synthetase system.     

Impairment of phosphatidylinositol metabolism in a patient with a bleeding disorder associated with defects of initial platelet responses

Phosphoinositide/polyphosphoinositide (PI/PPI) metabolism, measured by the increase of 3H-phosphatidic acid (PA) and the decrease of 3H-phosphatidylinositol (PI) in 3H-arachidonate-labeled platelet suspensions, was assessed in five patients whose platelet functional defects included impaired initial rates of ADP, epinephrine and U44069 aggregation in platelet-rich plasma (PRP). In one patient, 3H-PA formation induced by collagen and thrombin was reduced or absent on two of three occasions, and the decrease in 3H-PI was reduced on one of these two occasions in response to collagen and A23187, and on all 3 occasions in response to thrombin. The variations in the formation of 3H-PA in this patient on different occasions broadly paralleled the variations in the initial rates of ADP and U44069 aggregation and in epinephrine aggregation seen in PRP. No such abnormalities of PI metabolism were found in four other patients with similar, but not identical, functional defects. These results suggest an impairment affecting metabolism of PI/PPI via the PI/PPI cycle in this patient's platelets. The association of abnormalities of PI metabolism with defects of initial platelet responses provides further support for a physiological role of phosphoinositide metabolism in the early activation mechanism of platelets.     

Unexpected effects of aurin tricarboxylic acid on human platelets.

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exercise-induced changes in platelet aggregation; a comparison of whole blood and platelet rich plasma techniques

Studies have been performed to assess the effect of exercise on spontaneous platelet aggregation in shaken whole blood, and on agonist-induced platelet aggregation in whole blood and platelet rich plasma (PRP). Spontaneous platelet aggregation in shaken whole blood was increased following exercise compared to pre-exercise values. The increase in spontaneous aggregation after exercise correlated inversely with the increase in white cell count in whole blood. Platelet sensitivity in whole blood to adrenaline, collagen and adenosine diphosphate (ADP) was increased following exercise. Changes in platelet sensitivity to adrenaline following exercise correlated with increases in plasma noradrenaline levels but not with changes in blood cell counts. In PRP, platelet sensitivity to ADP and to collagen was increased following exercise when the pre and post-exercise PRP platelet counts were not corrected to allow for the increase in platelet count which occurred with exercise. When the PRP platelet counts were corrected, no changes in platelet sensitivity to any agonist after exercise were observed.     

Sex differences in mouse platelet aggregation

The role of platelets in the sex difference observed in mouse thrombosis models was evaluated by examining platelet diminution in vivo after thrombotic challenge, and aggregation of mouse platelets in PRP. A fall in platelet count was observed in both sexes after i.v. injection of either arachidonic acid or the thromboxane agonist, U46619. Platelet diminution induced by high dose arachidonate (50 mg/kg) was significantly greater in males compared to female mice. Responses to U46619 were similar in both sexes. In PRP, male platelets exhibited a greater response than female platelets to both ADP (15 uM) and arachidonate (0.3 mM), but not to U46619 (4.6 and 6.9 uM). These results suggest that the gender difference in arachidonate-induced sudden death, in which males are more susceptible than females, is related to a sex difference in mouse platelet function.