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Inhibitors of "spontaneous" platelet aggregation in whole blood.

In vitro 'spontaneous' platelet aggregation has been studied in whole blood. The spectrum of activity of materials known to influence platelet aggregation in platelet-rich plasma proved different in whole blood. Thus dipyridamole and one of its analogues SH1242 had a striking effect in whole blood whilst aspirin, chlorpromazine and K3920 had little or no effect. The combination of aspirin and dipyridamole as currently employed in clinical practice had no greater inhibitory effect than dipyridamole alone. The possible clinical relevance of these findings is discussed.

Aspirin↗

Plasminogen activator induction and platelet aggregation by phorbol and some of its derivatives: correlation with skin irritancy and tumor-promoting activity.

Phorbol and eight of its derivatives were investigated for their ability to stimulate the synthesis of the enzyme plasminogen activator in cultured chick embryo fibroblasts and to aggregate human blood platelets and have been assayed for tumor, promoting and skin, irritant activities. Over a range of concentrations, elevation in the levels of plasminogen activator activity induced by phorbol derivatives correlates well with their promoting and irritant properties. In the platelet aggregation assay however, the parallelism between the activities measured in different biological assays was less complete. While strong promoters, such as TPA, are potent aggregating agents, and weak promoters, such as PDA, are poor or ineffective inducers of aggregation, two derivatives, PDD and PDB, deviate from this general result. Platelets must be exposed to PDD in relatively high concentrations before they will aggregate, and PDB was found to be the most potent aggregating agent of all the derivatives tested.

Animals↗

Evaluation of platelet hyperfunction in aged subjects using spontaneous platelet aggregation in whole blood.

To evaluate the changes in platelet function in connection with aging, spontaneous platelet aggregation in whole blood (SPAWB) from healthy aged people was measured and compared to that in the whole blood of young subjects as well as patients with old cerebral infarction. SPAWB was determined by the fall in non-aggregated single platelet count by stirring without adding platelet aggregating agents to whole blood. SPAWB in the aged subjects was significantly higher than that in the young volunteers. In patients with old cerebral infarction taking no platelet suppressive agents, SPAWB was also high but was suppressed to the level of that in young subjects by prescription of ticlopidine. Although when aspirin or a mixture of phosphocreatine and creatine phosphokinase (CP/CPK) was added in vitro to whole blood obtained from the aged subjects, SPAWB was inhibited to 50%, addition of the specific PAF (platelet activating factor) inhibitor TCV-309 induced only partial inhibition of SPAWB. Only CP/CPK showed more than 50% inhibition of SPAWB in the young volunteers. These findings suggest that platelet function in aged subjects is in a hyperaggregable state in vivo due to increased thromboxane A(2).

Journal Article↗

Effect of D-003 on intravascular platelet aggregation induced with collagen in rats.

D-003 is a mixture of higher aliphatic primary acids purified from sugar-cane (Saccharum officinarum L.) wax that inhibits platelet aggregation induced ex vivo by addition of agonists to platelet-rich plasma (PRP) of rats, guinea pigs, and healthy human volunteers. Because the ex vivo platelet aggregation model does not mimic properly platelet aggregation occurring inside the arteries, since all blood factors regulating the formation of a platelet aggregate or thrombus are not present in PRP, this work was undertaken in order to investigate the effects of different oral doses of D-003 on platelet aggregation induced by collagen in vivo in rats. Effects of single (5, 25, 100, and 200 mg/kg) or repeated doses (1, 5, 25, 100, and 200 mg/kg during 10 days) of D-003 on in vivo platelet aggregation in rats were studied. D-003 (5-200 mg/kg) orally administered as single or repeated doses inhibited significantly and dose-dependently collagen-induced platelet aggregation in rats. The minimal dose investigated effective in both single and repeated administration schemes was 5 mg/kg. The highest dose assessed in both cases was 200 mg/kg, causing inhibitions of 61.5% (single doses) and 74.4% (repeated doses). Thus, the effects of repeated doses were more pronounced than those obtained with single administration. The mean 50% effective dose of D-003 in both schemes was 2.3 mg/kg, which indicates a promising anti-thrombotic potential of D-003.

Administration, Oral↗

Verapamil is a potent inhibitor of 5-HT-induced platelet aggregation.

We have studied the effects of verapamil, diltiazem and amlodipine on 5-HT-induced platelet aggregation and compared the results with those obtained for other platelet aggregating agents. Experiments were carried out using both human whole blood and platelet-rich plasma (PRP). Verapamil (but not diltiazem or amlodipine) inhibited 5-HT-induced platelet aggregation at much lower concentrations (IC50 = about 1 microM) than were required for inhibition of aggregation induced by other aggregating agents. Like some other selective inhibitors of 5-HT-induced platelet aggregation, it was not possible to completely overcome the inhibition by increasing the concentration of 5-HT. The antiaggregatory effects of verapamil were similar, but not identical, in whole blood and PRP. These results show that the Ca2+ channel blocker verapamil has some selectivity as an inhibitor of 5-HT-induced platelet aggregation and that this behaviour as a 5-HT antagonist should be taken into account when interpreting any therapeutic benefit ascribed to this drug.

Adenosine Diphosphate↗

Platelet aggregation: involvement of thrombin and fibrin(ogen).

Platelets play a key role in hemostasis and thrombosis. The formation of a platelet plug is accompanied by the generation of thrombin, which results in the generation of fibrin required for stabilization of the platelet plug. Platelet plug formation and coagulation are closely linked processes. Thrombin is a potent platelet activator, which proceeds through proteolysis of the protease activated receptors (PARs). Furthermore, thrombin binds glycoprotein Ib(alpha), which amplifies platelet activation by accelerating PAR-1 activation, and possibly also by direct signaling events through glycoprotein Ib(alpha). Moreover, thrombin's specificity towards other substrates changes after binding glycoprotein Ib(alpha). Fibrinogen and fibrin, the end product of the coagulation cascade, are also involved in platelet aggregation. Both fibrinogen and fibrin bind the integrin alpha(IIb)beta(3), and another fibrin receptor involved in platelet aggregation has been postulated. This review will discuss the role of thrombin and fibrin(ogen) in platelet functioning, and will highlight pathways at the crossroad of coagulation and platelet functioning, which are potential targets for antithrombotic therapy.

Fibrin↗

Inhibition of platelet aggregation by abnormal high density lipoprotein particles in plasma from patients with hepatic cirrhosis.

ADP-induced aggregation of normal washed platelets was measured by nephelometry in the presence of plasma high density lipoprotein (HDL) from normal subjects and from 30 patients with hepatic cirrhosis. HDL, at one-eighth of its plasma concentration, inhibited platelet aggregation; the effect of cirrhotic HDL (40% [SD 29%] inhibition) was significantly greater than that of normal HDL (16% [11%]). The mean apolipoprotein E content of cirrhotic HDL was significantly higher than that of normal HDL, and strongly inhibitory HDL contained twice as many apolipoprotein-E-rich particles as weakly inhibitory HDL. Inhibition of platelet aggregation was correlated with the apolipoprotein E content of HDL from patients with cirrhosis.

Adenosine Diphosphate↗

Platelet aggregation induced by strains of various species of coagulase-negative staphylococci.

Major species of coagulase-negative staphylococci (CNS) were tested for their ability to induce platelet aggregation in rabbit platelet-rich plasma (PRP). Among 11 species of CNS tested, a majority of the strains of 10 species of CNS (S. epidermidis, S. simulans, S. capitis, S. hyicus, S. sciuri, S. cohnii, S. xylosus, S. hominis, S. haemolyticus, S. warneri) caused induction of the platelet aggregation and serotonin release, while S. saprophyticus did not show such activity. The addition of aspirin (10 mM) or quinacrine (1 mM) to PRP resulted in no remarkable effect on the platelet aggregation induced by these strains and it was shown that the platelet aggregation did not require arachidonate pathways. Complement system components were shown to be one of the plasma factors required for platelet aggregation by ten strains of each species of CNS. The bacterial substance participating in the platelet aggregation by ten species of CNS tested was indicated to be heat-stable and trypsin-resistant, while the activity of a strain of S. epidermidis was susceptible to trypsin.

Animals↗

Inhibition of human platelet aggregation by a novel S-nitrosothiol is abolished by haemoglobin and red blood cells in vitro: implications for anti-thrombotic therapy.

1. S-Nitrosothiols are nitric oxide (NO) donor drugs that have been shown to inhibit platelet aggregation in platelet rich plasma (PRP) in vitro and to inhibit platelet activation in vivo. The aim of this study was to compare the platelet effects of a novel S-nitrosated glyco-amino acid, RIG200, with an established S-nitrosothiol, S-nitrosoglutathione (GSNO) in PRP, and to investigate the effects of cell-free haemoglobin and red blood cells on S-nitrosothiol-mediated inhibition of platelet aggregation. 2. The effects of GSNO and RIG200 in collagen (2.5 microg ml(-1))-induced platelet aggregation in PRP and whole blood were investigated in vitro. Both compounds were found to be powerful inhibitors of aggregation in PRP, and RIG200 was significantly more potent (IC(50)=2.0 microM for GSNO and 0.8 microM for RIG200; P=0.04). 3. Neither compound inhibited aggregation in whole blood, even at concentrations of 100 microM. Red blood cell concentrations as low as 1% of the haematocrit, and cell-free haemoglobin (> or = 2.5 microM), significantly reduced their inhibitory effects on platelets. 4. Experiments involving measurement of cyclic GMP levels, electrochemical detection of NO and electron paramagnetic resonance of haemoglobin in red blood cells, indicated that scavenging of NO generated from S-nitrosothiols by haemoglobin was responsible for the lack of effect of S-nitrosothiols on platelets in whole blood. 5. These studies suggest that scavenging of NO by haemoglobin in blood might limit the therapeutic application of S-nitrosothiols as anti-platelet agents.

Adult↗

Inhibition of ADP-induced platelet aggregation by adenosine tetraphosphate.

In contrast to previous reports, highly purified adenosine tetraphosphate (AP4) does not induce the aggregation of platelets but inhibits the aggregation and release reaction in platelet-rich plasma promoted by ADP. The inhibitory action of AP4 on the aggregation by ADP is compared with that of AMP and ATP. The data presented suggest a competitive manner of inhibition of the ADP-induced aggregation by AP4.

Adenine Nucleotides↗

Correlation between chemical stimulation and filtration flow resistance in human platelet aggregates.

Human platelet-rich plasma (PRP) was aggregated with ADP to produce varying degrees of reversibility and platelet release, and then the chemical stimulation (as evidenced by optical transmittance) was compared with the flow resistance of physiologically-significant microaggregates, determined by constant-pressure filtration. The aggregate flow resistance was attributed to "weak" aggregates (which occluded the micropore filter at 20 mm Hg but which passed through the filter at 80 mm Hg) and "strong" aggregates (which occluded the filter at 80 mm Hg). As the ADP concentration was increased from 1 microM to 10 microM, the flow resistance of both the "weak" and "strong" aggregates generally increased, as did the maximum transmittance. No consistent trend was seen as the time after ADP addition increased from 5 to 30 minutes. Pretreatment of the PRP with epinephrine increased the maximum transmittance during ADP aggregation, produced less-reversible aggregates (less subject to deaggregation by PGE1), and increased the aggregate flow resistance over the range of filtration pressures tested. The addition of PGE1 to the aggregated sample before filtration nearly eliminated the flow resistance of the aggregates at all filtration pressures tested and partially reversed the optically-apparent aggregation. Pretreatment of PRP with PGE1 before epinephrine or ADP addition inhibited both the optically-detected aggregation and the development of flow resistance. The physical (hydrodynamic) strength of platelet aggregates is apparently related to the chemical stimulation of the release reaction and to aggregation irreversibility, possibly through the mediation of adenylate cyclase and cyclic AMP.

Adenosine Diphosphate↗

Increased platelet aggregate formation in patients with chronic airflow obstruction and hypoxaemia.

Platelet aggregate formation in vivo was assessed by means of the platelet aggregate ratio and from platelet release products (beta thromboglobulin, platelet factor 4, thromboxane B2) in 23 patients with chronic airflow obstruction with and without hypoxaemia and in 10 control subjects without respiratory disease. Eight of the 11 hypoxaemic patients were having long term oxygen therapy. The platelet aggregate ratio was lower in the hypoxaemic patients (0.88 (SE 0.03] than in the non-hypoxaemic (0.97 (0.01] and control groups (1.00 (0.02], and there was a trend to lower aggregate ratios in the more hypoxaemic patients. Platelet release products in the peripheral venous blood were not increased in the patients or control subjects. Platelet behaviour is altered in chronic hypoxaemia and this enhanced platelet activity could contribute to the pulmonary vascular damage found in these patients through direct effects or mediator release.

Aged↗

Effect of ciprofibrate on platelet aggregation in patients with combined hyperlipidemia.

This study was designed to investigate the effect of ciprofibrate on platelet aggregation in patients with combined hyperlipidaemia. Platelet aggregation measurements in platelet-rich plasma were carried out in 30 patients using the Biola 230 LA platelet aggregation analyser before treatment, 4 weeks and 8 weeks after ciprofibrate (100 mg/day) therapy. A control group consisted of 37 healthy subjects. We found that spontaneous and 0.25 microM ADP-induced platelet aggregation were significantly decreased after 4-weeks of therapy, from 4.6 to 3.2% and from 11.3 to 7.6% , respectively. However, there was no difference 8 weeks after the treatment onset. Platelet aggregation induced by adrenaline was unchanged during the ciprofibrate therapy.

Journal Article↗

The effect of Craviten, a new antiarrhythmic drug, its 2R, 2'R isomer, quinidine and procaine amide on human platelet aggregation.

Among the optical isomers of N, N'-dimethyl-N, N'-bis[1-(3', 4', 5-trimethoxybenzoyloxy)-butyl-2]-ethylenediamine dihydrochloride only the isomer with the (2S, 2'S) configuration (Craviten) in a concentration of 0.2 microgram/ml inhibits the ADP-induced aggregation of human platelets. The (2R, 2'R) isomer and quinidine exert a similar effect but only in a concentration ten times higher. Procaine amide (2 micrograms/ml) has an opposite effect, stimulating human platelet aggregation.

Adenosine Diphosphate↗

Factors affecting the size of platelet aggregates in blood.

Factors affecting the size distribution of platelet aggregates formed in whole blood were measured using an electronic particle counter. Lysis of red cells after dilution of blood for analysis allows detection of smaller aggregates without altering aggregate measurement. Differences in platelet response to increasing concentrations of ADP are detected by an increase in the mean size of platelet aggregates while the total volume of aggregates varies linearly with the volume of available platelets (r = .99). The mean aggregate size varies directly with the total volume of aggregates formed (r = .99). Mean aggregate size is inversely related to the packed red cell volume. As the reactivity of platelets is reduced by incubation with aggregation inhibitors at increasing concentrations, the mean size of aggregates becomes smaller, followed by a decrease in the total volume of aggregates. The total volume and mean size of aggregates formed after incubation with collagen and epinephrine, but not ADP, increases with time after venepuncture.

Adenosine Diphosphate↗

Effects of 11-week increases in dietary eicosapentaenoic acid on bleeding time, lipids, and platelet aggregation.

The effect of a diet rich in eicosapentaenoic acid (EPA) on platelet phospholipid fatty acid composition, platelet aggregation, and bleeding time was studied in 10 healthy men, whose usual diet was partly replaced by fish for 11 weeks. This diet provided 2-3 g EPA per day. Two doses (3.5 and 10 mg/kg body-weight) of acetylsalicylic acid (ASA) were given before and during the diet. The fish diet prolonged bleeding time (by 42%) and decreased platelet aggregability. The changes in platelet phospholipid fatty acid composition consisted of increases in the omega-3 series (C20:5 and C22:6) and decreases in the omega-6 series (C18:2 and C20:3). The reduction in platelet aggregation induced by collagen and ADP did not parallel the changes in platelet membrane phospholipids and bleeding times. Diminished platelet aggregation induced by collagen lasted only 3 weeks (while subject was still on the diet), whereas the decreased sensitivity to ADP persisted for at least 11 weeks after the volunteers had resumed their normal diet. ASA taken before the diet prolonged bleeding time by as much as did the diet itself. ASA taken during the diet prolonged bleeding time by more than the sum of the increases in bleeding time caused by ASA and by the EPA diet separately, but the synergism was not significantly more than additive. The findings suggest that a diet rich in omega-3 polyunsaturated fatty acids reduces tha interaction between platelets and the vessel wall by mechanisms which are more complex than just a reduction in susceptibility of platelets to the naturally occurring agents collagen and ADP, or an imbalance between proaggregatory and anti-aggregatory prostaglandin derivatives.

Adult↗

Effect of indobufen on whole blood platelet aggregation recorded in the morning in patients with ischaemic heart disease.

Platelets are involved in the progression of coronary atherosclerosis as well as in the development of the acute precipitating event. Recently, it has been shown that normal subjects present increased platelet aggregation between 6.00 a.m. and 9.00 a.m.; epidemiological studies have shown a higher incidence of myocardial infarction between these times. This study evaluated, by an impedence method using whole blood, platelet aggregation induced by ADP (3 microM) and collagen (2 microM/ml). Measurements were made at 6.00 a.m., 9.00 a.m. and 12.00 noon, the day before and the day after evening administration of 200 mg indobufen, a platelet aggregation inhibitor, in 12 patients with ischaemic heart disease. Patients showed a significant increase of platelet aggregation between 6.00 a.m. and 9.00 a.m. which was inhibited by the prior evening administration of indobufen.

Adenosine Diphosphate↗

Inhibition of prostanoid-mediated platelet aggregation in vivo and in vitro by 3-hydroxymethyl-dibenzo(b,f)thiepin 5,5-dioxide (L-640,035).

Platelet aggregation was studied in vitro with human platelets and in vivo in guinea pigs and rabbits. L-640,035 and its acetic acid metabolite, L-636,499, significantly inhibited human platelet aggregation induced by arachidonic acid, collagen and a prostaglandin endoperoxide analog (U44069) but not ADP. In guinea pigs, circulating platelets labeled with 111indium were monitored with probes in the lung and abdominal regions. Platelet aggregation was indicated by an increase in the ratio of gamma-radiation of the lung vs. the abdominal region, as aggregates of platelets accumulate in the microvascular network in the lung. L-640,035 (1 and 3 mg/kg i.v.) inhibited platelet aggregation induced in this model by U-44069 but not by ADP. In rabbits acute thrombosis was induced in the carotid artery by local electrical stimulation and platelet accumulation at the stimulus site was quantitated using 111indium-labeled autologous platelets. L-640,035 (1 and 3 mg/kg i.v.) significantly reduced electrically evoked platelet accumulation. It is concluded that L-640,035 is a novel and selective antagonist of platelet aggregation induced by thromboxane A2 or prostaglandin endoperoxides and that it may have utility as an antiplatelet drug.

Adenosine Diphosphate↗