A23187-induced Platelet Aggregation Research Articles

The Direct Thrombin Inhibitors Dabigatran and Lepirudin Inhibit GPIbα-Mediated Platelet Aggregation.

The direct thrombin inhibitor (DTI) dabigatran is a non-vitamin K antagonist oral anticoagulant for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. In addition to its anti-thrombotic efficacy, dabigatran has been suggested to exert some pro-thrombotic effect due to fostering the ligation of thrombin to its high affinity platelet receptor glycoprotein (GP) Ibα in patients with atrial fibrillation. On the other hand, we provided evidence that a member of another class of DTIs, lepirudin, stimulates the inhibitory cyclic guanosine monophosphate (cGMP)/soluble guanylate cyclase pathway in human platelets. Here, we investigated the effect of lepirudin and dabigatran spiked to platelets from healthy volunteers on GPIbα-mediated platelet aggregation and agglutination. Ristocetin/von Willebrand factor (vWF)-induced aggregation of platelets in the presence or absence of plasma was significantly inhibited by lepirudin, dabigatran and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK). However, ristocetin/vWF-mediated platelet agglutination and binding of vWF to platelets were not affected by the DTIs. The anti-aggregatory effect was confirmed by using the GPIbα-specific agonist echicetin beads for human and murine platelets. DTIs diminished echicetin beads-induced Syk Y352 phosphorylation (used here as readout for an early signal occurring during echicetin-induced platelet aggregation), but did not inhibit adenosine diphosphate- or thromboxane A2-induced platelet aggregation. Thrombin was not generated in response to ristocetin/vWF or echicetin beads and therefore did not explain the inhibitory effect of the DTIs. Therapeutic concentration of lepirudin and dabigatran did not affect significantly platelet vasodilator-stimulated phosphoprotein S239 phosphorylation or cGMP and cyclic adenosine monophosphate levels. These data suggest that the DTIs, lepirudin and dabigatran, impair platelet activation measured during platelet aggregation induced by ristocetin/vWF or echicetin beads.

Aspirin resistance: basic and clinic

Aspirin,playing antiplatelet effect through inhibitting thromboxane A2-induced platelet aggregation,is commonly clinical anti-platelet drugs.It is observed that platelet aggregation could not be inhibited and still arterial thromboembolic events occurr in some patients administered aspirin,which is called aspirin resistance.Related research progress is summarized as follows. Key words: Aspirin; Antiplatelet; Resistance

Biological properties of a specific Gαq/11 inhibitor, YM‐254890, on platelet functions and thrombus formation under high‐shear stress

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).

Platelet Thromboxane A2Receptor Blockade by Losartan in Hypertensive Patients

It has been shown that losartan inhibits vasoconstriction through binding to the thromboxane A2 receptors. We investigated whether Thromboxane A2-induced platelet aggregation can be inhibited by losartan and if this effect is present in losartan treated hypertensive patients. Platelet aggregation was measured in platelet rich plasma from healthy volunteers and hypertensive patients, with the use of an aggregometer. Losartan inhibited the effect of Thomboxane A2 agonist (U46619)-induced platelet aggregation, with a pA2 value of 48.9 μM and inhibited both second and first phases of Adenosine diphosphate-induced aggregation, from 109 ± 9 to 65 ± 10 mm and from 62± 8 to 0 mm. U46619's and Adenosine diphosphate's concentration required to produce half-maximal response were higher in normotensive volunteers (EC50 2.01 ± .3 μM and 2.6 ± .4 μM) compared to the hypertensive patients without treatment (EC50 0.6 ± .2 μM and 2.0 ± .3 μM). The concentration required to produce half-maximal response in losartan treated...

Comparative effects of AT1-antagonism and angiotensin-converting enzyme inhibition on markers of inflammation and platelet aggregation in patients with coronary artery disease

ObjectivesWe evaluated whether renin-angiotensin system (RAS) blockade attenuates cardiovascular events. BackgroundBecause inflammation and enhanced thrombogenesis are hallmarks of atherosclerosis, we assessed whether RAS inhibition elicits anti-inflammatory and anti-aggregatory effects. MethodsInterleukin 6 (IL-6), high-sensitivity C-reactive protein (hsCRP), metalloprotease 9 (MMP-9), and interleukin 10 (IL-10) were determined in patients with coronary artery disease (CAD) and arterial hypertension six to eight weeks after coronary angioplasty (low-density lipoprotein serum levels <150 mg/dl). Patients were randomized double-blind to either 20 mg enalapril (ENAL, n = 27) or 300 mg irbesartan (IRB, n = 21) for 3 months. Blood samples were drawn at baseline and at three months. Thromboxane A2-induced platelet aggregation was determined turbidimetrically; urine bicyclo-prostaglandin E2 (PGE2) and inflammatory markers were measured by enzyme-linked immunosorbent assay technique. ResultsBoth treatment regimens enhanced serum IL-10 levels (IRB p < 0.001, ENAL p < 0.03) and reduced serum MMP-9 protein (IRB p < 0.001, ENAL p < 0.05) and MMP-9 activity (IRB p < 0.005, ENAL p < 0.05). Only IRB reduced serum IL-6 and hsCRP levels significantly compared with baseline (p < 0.01), whereas ENAL did not (hsCRP p < 0.02 IRB vs. ENAL, p < 0.01 IRB vs. ENAL). Platelet aggregation was only reduced by IRB (p < 0.001, ENAL p < 0.06, IRB vs. ENAL p < 0.001) while urine PGE2levels remained unchanged. ConclusionsAngiotensin-converting enzyme (ACE) inhibition and angiotensin II type 1 receptor (AT1) blockade reduced serum MMP-9 protein/activity to a similar extent, and only AT1 blockade reduced hsCRP, IL-6, and platelet aggregation in patients with CAD. Thus, AT1-blockade appears to exert stronger systemic anti-inflammatory and anti-aggregatory effects compared with ACE inhibition.

Involvement of thromboxane A2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation.

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 mM) was inhibited by PAF receptor blocker (WEB 2086, IC50 = 0.65 mM) and calcium channel blockers, diltiazem (IC50 = 13 mM) and verapamil (IC50 = 18 mM). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 mM), a cyclooxygenase inhibitor (indomethacin; IC50 = 0.2 mM) and inhibitor of TLCK, herbimycin A with IC50 value of 5 mM. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 mM. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.

Inhibitory Effects of J78, a Newly Synthesized 1,4-Naphthoquinone Derivative, on Experimental Thrombosis and Platelet Aggregation

Several compounds with the backbone of 1,4-naphthoquinone chemical structure have been reported to display antiplatelet and antithrombotic activities, indicating that this congener compound may be a new source in the antithrombotic drug development. In the present study, the possible antiplatelet activity and antithrombotic efficacy of J78 (2-chloro-3-[2′-bromo, 4′-fluoro- phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, were examined. Orally administered J78 (50, 100 mg/kg) dose dependently protected mice against the collagen + epinephrine-induced thromboembolic death. Orally administered J78 also significantly inhibited the ADP- and collagen-induced rat platelet aggregation ex vivo, with inhibition values of 44 and 40%, respectively. J78 inhibited the collagen-, arachidonic acid- and thrombin-induced human platelet aggregation concentration dependently in vitro, with IC₅₀ values of 7.8 ± 0.4, 10.1 ± 0.4 and 18.4 ± 2.0 µmol/l, respectively. It was also active in inhibiting Ca²⁺ ionophore, A23187-induced platelet aggregation, suggesting that J78 may have an inhibitory effect on Ca²⁺ mobilization. J78, however, did not alter coagulation parameters such as activated partial thromboplastin time and prothrombin time in human plasma. Taken together, these results suggest that J78 may be a promising antithrombotic agent, and its antithrombotic activity may be due to antiplatelet rather than anticoagulation activity.

Potentiation of Thromboxane A2-induced Platelet Secretion by Gi Signaling through the Phosphoinositide-3 Kinase Pathway

Platelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3'phosphate-5'-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T(AC) receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T(AC) receptor or the alpha2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2T(AC) receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.

Relationship between intracellular calcium-dependent process and protein-tyrosine phosphorylation in human platelets: studies of platelets from a patient with defective A23187-induced platelet aggregation.

We had postulated that in a patient with defective calcium ionophore (A23187)-induced platelet aggregation, whose platelets showed normal intracellular Ca2+ mobilization in either the presence or absence of extracellular Ca2+ in response to A23187. A defect was present in an intracellular calcium-dependent process. We have now investigated whether the agonist-induced protein-tyrosine phosphorylation (PTP) was altered. Protein-tyrosine phosphorylation (PTP)-induced by A23187 in the patient's platelets was greatly diminished but that induced by thrombin was almost normal. These results suggest that an intracellular calcium-dependent process plays a fundamental role in A23187-induced PTP, whereas it does not in thrombin-induced PTP.

Losartan inhibits thromboxane A2-induced platelet aggregation and vascular constriction in spontaneously hypertensive rats.

Our recent studies have shown that the nonpeptide angiotensin II (Ang II) antagonist losartan interacts with thromboxane A2/prostaglandin H2 receptors and inhibits the thromboxane A2 (TxA2) analog U46619-induced vasoconstriction in canine coronary arteries. In this study, we further investigated whether losartan prevents TxA2-induced platelet aggregation and vasoconstriction in spontaneously hypertensive rats (SHRs). Pretreatment with losartan (10 microM) significantly reduced U46619-induced, concentration-dependent washed platelet aggregation. The inhibition is specific for losartan, because another Ang II AT1-receptor antagonist, CV11974 (10 microM), an active metabolite of TCV116, did not block the platelet aggregation caused by U46619. In addition, losartan (10 microM) augmented acetylcholine (ACH)-induced nitric oxide (NO)-dependent vasodilation and abolished the ACH-induced endothelium-derived contracting factor (EDCF)-mediated vasoconstriction in the aortic rings from adult SHRs. U46619 produced dose-dependent vasoconstriction in aortic vessels of SHRs, which was demonstrated to be blocked by the potent, selective TxA2/PGH2 receptor antagonist SQ29,548. Pretreatment with losartan (10(-6)-10(-5) M) inhibited the contractile response of U46619 and shifted the concentration-response curve to the right in a dose-dependent manner. The effective concentration at half maximal contraction (EC50) of U46619 was increased 2.5- and 7.6-fold in the presence of 1 and 10 microM losartan, respectively, without changes in maximal contraction. The active metabolite of losartan, EXP3174, at 1 microM also competitively inhibited U46619-induced contractions in aortic rings of SHRs. In contrast, neither the AT1-receptor antagonist CV11974, the AT2 antagonist PD123319, nor the angiotensin-converting enzyme inhibitor lisinopril, each at concentrations of 1 microM, had any effect on the U46619-induced constriction in aortic rings. In conclusion, losartan, acting as both AT1- and TxA2/PGH2-receptor antagonists, may enhance its therapeutic profile in the treatment of hypertension and cardiovascular disease.

On the effect of 2-deuterium- and 2-methyl-eicosapentaenoic acid derivatives on triglycerides, peroxisomal β-oxidation and platelet aggregation in rats

A series of 2-substituted eicosapentaenoic acid (EPA) derivatives (as ethyl esters) have been synthesized and evaluated as hypolipidemic and antithrombotic agents in feeding experiments in rats. Repeated administration of purified 2-methyl-eicosapentaenoic acid and its deuterium analogues (all as ethyl esters) to rats resulted in a decrease in plasma triglycerides and high density lipoprotein cholesterol. The 2-methyl-EPA analogues were, apparently, four times more potent than EPA in inducing the triglyceride lowering effect. The 2-deuterium-2-methyl-EPA decreased plasma cholesterol level to ∼40%. A moderate enlargement of the liver was observed in 2-methyl-EPA treated rats. This was accompanied with an acute reduction in the liver content of triglycerides and a stimulation of peroxisomal β-oxidation and fatty acyl-CoA oxidase activity. The results suggest that the triglyceride-lowering effect of 2-methyl-EPA may be due to a reduced supply of fatty acids for hepatic triglyceride biosynthesis because of increased fatty acid oxidation. Platelet aggregation with ADP and A23187 was performed ex vivo in platelet-rich plasma, after administration of different doses of the EPA-derivatives for five days. EPA and 2,2-dideuterium EPA had no effect on ADP-induced aggregation, while 2-deuterium-, 2-methyl- and 2-deuterium-2-methyl EPA produced a biphasic effect, i.e. potentiation and inhibition at low (250 mg/day kg body weight) and higher doses (600–1300 mg/day kg body weight), respectively. A23187-induced platelet aggregation was affected in a similar way by feeding the 2-substituted EPA derivatives, except that 2-deuterium-2-methyl EPA had no effect relative to EPA itself and that the inhibition was far greater than that for ADP-induced aggregation (∼100% inhibition with 600 mg 2-methyl-EPA/day kg body weight). The ranking order of the EPA-derivatives to affect platelet aggregation and to cause hypolipidemia was different, suggesting different mechanisms. Our observations suggest that the effects of the EPA derivatives on platelet aggregation could be related to the degree of bulkiness around C 2 and that an asymmetric substitution at C 2 caused inhibition of platelet aggregation while a symmetric substitution did not. It is suggested that the bulky, asymmetric derivatives inhibit platelet aggregation by altering platelet membrane phospholipid packing.

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca 2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied ion Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43±0.08 μmol/l. The stimulatory of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1mM EGTA was established by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca 2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-brorno cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 μg/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A 2 formation. 8brcGMP (10–1000 μM) inhibited collagen-induced platelet aggregation and TXA 2; formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [ 3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [ 32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na +-H 4 exchange, but could not in the absence of extracellular Ca 2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca 2+ -dependent phospholipase A 2. However, cyclic GMP seems to inhibit the Ca 2+-activated phospholipase A 2 indirectly, since 8brcGMP had no effect on Ca 2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca 2+ which is responsible for phospholipase A 2 activation in rabbit platelets.

Characterization of the inhibition of U46619-mediated human platelet activation by the trimetoquinol isomers: Evidence for endoperoxide/thromboxane A 2 receptor blockade

Sites of inhibition for the trimetoquinol (TMQ) isomers on 15S-hydroxy-11α,9α-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619)-, 12- O-tetradecanoylphorbol 13-acetate (TPA)- and A23187-induced human platelet activation were investigated. Experiments using washed human platelets were designed to characterize relationships among functional (aggregation, secretion) and biochemical (protein phosphorylation, metabolism of inositol phospholipids and radioligand displacement analysis) processes of platelet activation by U46619 and the specificity of inhibition by the TMQ isomers. Thromboxane A 2 receptor stimulation by U46619 in human platelets resulted in a time- and concentration-dependent breakdown of inositol phospholipids [phosphatidylinositol 4,5-bisphosphate (PIP 2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI)], phosphatidic acid (PA) accumulation, phosphorylation of 20 and 45 kD proteins, aggregation and serotonin secretion. The TMQ isomers stereoselectively inhibited all U46619-mediated platelet activation processes. R(+)TMQ was 40- and 22-fold more potent than S(−)-TMQ as an inhibitor of U46619-induced platelet aggregation and serotonin secretion respectively. In addition, R(+)-TMQ blocked U46619-induced 20 kD protein phosphorylation, 45 kD protein phosphorylation, PIP 2, PIP and PI breakdown, and PA accumulation with a potency which was 8-, 13-, 45-, 37-, 33- and 33-fold greater than the S(−)-isomer respectively. In contrast to S(−)-TMQ, R(+)-TMQ produced a concentration-dependent inhibition of specific [ 3H]U46619 binding to endoperoxide/thromboxane A 2 receptor sites in washed platelets. In other experiments, S(−)-TMQ was more potent than R(+)-TMQ as an inhibitor of TPA- and A23187-induced platelet aggregation and serotonin secretion, and of TPA-induced phosphorylation of 45 and 20 kD proteins. The inhibitory potencies of S(−)-TMQ against TPA- or A23187-induced responses were similar to those needed for antagonism of U46619-mediated platelet activation. In contrast, much higher concentrations of R(+)-TMQ were required for blockade of TPA or A23187 versus U46619-mediated responses in human platelets. Taken collectively, the data show that the TMQ isomers interfered with the endoperoxide/thromboxane A 2 receptor-mediated phospholipase C-signal cascade of inositol phospholipid hydrolysis, calcium mobilization, and protein phosphorylation leading to platelet aggregation and secretion. R(+)-TMQ acted as a pharmacologically selective and highly stereospecific [ R(+)-TMQ S(−)-TMQ] antagonist of endoperoxide/thromboxane A 2 receptor sites in platelets. Additionally, S(−)-TMQ was more potent than R(+)-TMQ as an antagonist of platelet activation by inducers of protein kinase C activity and calcium mobilization. Thus, the isomers of TMQ may also interfere by a yet undefined mechanism with calcium-regulated cytosolic processes in platelets.

抗血小板剤の４β‐ｐｈｏｒｂｏｌ‐１２β ｍｙｒｉｓｔａｔｅ‐１３α ａｃｅｔａｔｅ（ＰＭＡ）凝集に及ぼす影響と先天性血小板機能異常症および慢性骨髄増殖性疾患におけるＰＭＡ凝集の検討

The phorbol ester 4β-phorbol-12β myriste-13α acetate (PMA), a potent tumor promoter, is known to be a agent which causes platelet aggregation and release reaction. But the exact mechanism of PMA induced platelet aggregation remains unclear. In this paper, to search for a possible mechanism of PMA induced platelet aggregation, the following anti-platelet agents were applied; acetylsalicylic acid (ASA), phenidone, OP. 3708, verapamil, TMB-8, cilostazol, monoclonal antibody (Ab) to GPIIb/IIIa, W-7, H-7 and PGI2. Futhermore in congenital platelet disorders (platelet cyclooxygenase (PCO) deficiency, Bernard-Soulier syndrome, Hermansky-Pudlak syndrome, thrombasthenia, familiar defect of A23187 induced platelet aggregation), PMA induced platelet aggregation was studied.In order to clarify the usefullness of PMA induced platelet aggregation in clinical cases, aggregation study at various PMA doses was performed in chronic myeloproliferative disorders.Among anti-platelet agents, TMB-8, verapamil, PGI2, W-7 and H-7 had inhibitory effects on PMA induced platelet aggregation. In congenital platelet disorders, PMA induced platelet aggregation was markedly decreased only in thrombasthenia. These data need further confirmation, but they suggest that PMA (especially at low concentrations) induced platelet aggregation was not mediated by cyclooxygenase and lipoxygenase products, release of dense granules and GPIb. In most patients with chronic myeloproliferative disorders, PMA induced platelet aggregation was decreased at low PMA concentrations. And it was significantly decreased at low PMA concentrations (<0.1μg/ml) in essential thrombocythemia. This may be related to bleeding tendency in essential thrombocythemia.Platelet aggregation study induced by PMA, as well as other platelet agonists, may be a useful method for the elucidation of the mechanism of various bleeding and/or thrombus formation tendency in clinical cases.

Inhibition of Human Platelet Functions by Verapamil

The effects of verapamil, a coronary vasodilator, on platelet functions was studied. Platelet aggregation induced by ADP, epinephrine or collagen was inhibited by verapamil in vitro. Calcium ionophore A23187-induced platelet aggregation was also inhibited by verapamil in a concentration dependent manner. In washed platelets, verapamil caused a dose-dependent inhibition of serotonin release induced either by thrombin or A23187 in the absence of extracellular calcium. Addition of 1 mM CaCl2 with A23187 or thrombin partially overcame this inhibition. Addition of 1 mM CaCl2 in the absence of verapamil had no effect on thrombin- or A23187-induced secretion. When verapamil was administered to the healthy volunteers at the dosage commonly used, inhibition of platelet aggregation was observed 2 hrs after the drug ingestion. It is of great interest that verapamil potentiated the anti-aggregating activity of prostacyclin in vitro. Our results may suggest a potential role for verapamil in the treatment of thrombotic disorders.

Inhibition of platelet aggregation and secretion by prostacyclin

The effect of prostacyclin (PGI2) on platelet functions was investigated using synthesized PGI2 sodium, which was a gift of Dr. Scholkens in Hoechst Research Institute in West Germany. Platelet aggregation induced by ADP (4μM), epinephrine (2μg/ml) and arachidonic acid (2mM) was markedly inhibited by PGI2 at a concentration of 10nM. Minimal concentration of PGI2 to inhibit platelet aggregation induced by collagen (2μg/ml) or A23187 (10μM) was 2.5nM and 20nM respectively. The inhibitory activity of PGI2 was found to be five times potent than that of PGE1. PGI2 was less potent in medium than in plasma and its potency appeared to be dependent of plasma concentration. Plasma factor to stabilize PGI2 activity was not determined, but not albumin, fibrinogen nor γ-globulins. Minimal concentration of PGI2 to inhibit A23187-induced platelet aggregation was 20nM, while that of TMB-8, intracellular calcium antagonist, was 1mM. However, the addition of 0.1mM TMB-8 and 5nM PGI2 markedly inhibited platelet aggregation induced by A23187, demonstrating the synergistic effect of PGI2 and TMB-8 on platelet aggregation. 14C-serotonin release induced by thrombin (1u/ml) in tris-buffered saline containing 2mM EDTA, was inhibited by 20-50nM PGI2, while that by A23187 (1μM) was not inhibited even by 500nM PGI2. These results gave additional informations to characterize the inhibitory action of PGI2.