Arachidonate metabolism, 5-hydroxytryptamine release and aggregation in human platelets activated by palmitaldehyde acetal phosphatidic acid

Palmitaldehyde acetal phosphatidic acid ( PGAP ) caused dose-dependent aggregation of human platelets resuspended in modified Tyrode medium, with a threshold concentration of 0.5-1 microM and an EC50 of 4 microM. Concentrations of PGAP which elicited biphasic irreversible aggregation concomitantly induced formation of 1.02 +/- 0.029 nmol (mean +/- s.e. mean) of malondialdehyde (MDA) per 10(9) platelets and caused release of 58 +/- 2.8% of platelet [14C]-5-hydroxytryptamine ([14C]-5-HT) from prelabelled platelets; no MDA formation or [14C]-5-HT release occurred at lower doses of PGAP which elicited only monophasic reversible aggregation. Adenosine 5'-pyrophosphate (ADP)-induced platelet activation resulted in formation of 0.344 +/- 0.004 nmol of MDA per 10(9) platelets in association with irreversible aggregation and 49.1 +/- 1% release of [14C]-5-HT. Mepacrine, a phospholipase A2 inhibitor, at 2.5 microM reduced PGAP -induced MDA formation and [14C]-5-HT release by the resuspended platelets without affecting irreversible aggregation; higher concentrations of mepacrine abolished all three responses. Chlorpromazine, a calmodulin antagonist, similarly inhibited PGAP -induced MDA formation and irreversible aggregation, and at 100 microM abolished monophasic aggregation. The cyclo-oxygenase inhibitor indomethacin caused a concentration-dependent reduction of PGAP -induced MDA formation by resuspended human platelets without significantly inhibiting [14C]-5-HT release or irreversible aggregation; concentrations (greater than or equal to 1.75 microM) which inhibited MDA formation by more than 94% abolished [14C]-5-HT release, and converted second phase irreversible aggregation to an extensive reversible response. 2-Methylthioadenosine 5'-phosphate (2 methylthio-AMP), an ADP antagonist, inhibited PGAP -induced MDA formation, [14C]-5-HT release and second phase aggregation in the human platelet suspensions in a parallel, concentration-dependent manner; at 9.4 microM 2-methylthio-AMP, both MDA formation and [14C]-5-HT release were abolished and monophasic, reversible aggregation remained. Albumin was required for aggregation of washed human platelets to PGAP . Irreversible PGAP -induced aggregation of washed [14C]-arachidonate-labelled platelets was accompanied by a low net loss of 14C from platelet phospholipids, an equivalent increase in 14C in free fatty acids, and the appearance of 14C in thromboxane (Tx)B2; mepacrine reduced the loss in 14C from phospholipids and inhibited aggregation and formation of [14C]-TxA2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acetal phosphatidic acids: novel platelet aggregating agents

1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.

2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 μM for human platelets and 0.25-0.5 μM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.

3 PGAP-induced irreversible aggregation of [¹⁴C]-5-hydroxytryptamine ([¹⁴C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0±2.4% (s.e.) of the platelet [¹⁴C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [¹⁴C]-5-HT-labelled sheep platelets was dose-dependent.

4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A₂ inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E₁ (PGE₁); these agents abolished the second, but not the first phase of ADP-induced aggregation.

5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 μM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 μM.

6 It is concluded that the acetal phosphatidic acids induce platelet aggregation per se by direct action at the platelet membrane, and that the acetal function is of primary importance in their potent platelet-stimulating activity. Moreover, as the acetal phosphatidic acids are the major components of the smooth muscle-contracting acidic phospholipid tissue extract `Darmstoff' (Vogt, 1949), their potent platelet-aggregating properties may be of physiological or pathological significance.

     

Adenosine-3′,5′-diphosphate and coenzyme a—Effects on platelet function

The effects in vitro of adenosine-3′5′-diphosphate and coenzyme A on human platelet aggregation and [¹⁴C]hydroxytryptamine release were studied. Whereas coenzyme A, at concentrations between 0.06 and 0.24 mM, inhibited the aggregation of platelets induced by ADP, adenosine-3 ′, 5 ′-disphosphate, which is part of the coenzyme A molecule, blocked both ADP- and thrombin-induced platelet aggregation. The ADP-induced platelet aggregation was inhibited at a lower adenosine-3′, 5′-diphosphate (10–20μM) concentration than was thrombin-induced aggregation (60–200 μM). Adenosine-3′,5′-diphosphate also inhibited [¹⁴C]adenosine uptake by platelets in a concentration-dependent manner (20–200 μM), but only to a maximum of 40 per cent of total [¹⁴C]adenosine radioactivity incorporated into the platelets. The inhibitory effect of adenosine-3′,5′-diphosphate and coenzyme A on the release reaction was further documented by the decrease in aggregation-induced release of [¹⁴C]5-hydroxytryptamine from prelabeled platelets into the medium. The extent of inhibition caused by coenzyme A and adenosine-3′,5′-diphosphate was found to depend upon the concentration of inhibitor and incubation time. If these agents are indeed inhibitors of platelet aggregation, then they may serve as valuable tools to study platelet function.     

Effects of clofibrate and 6-substituted chroman analogs on human platelet function:: Mechanism of inhibitory action

The effects of cloflbrate (CPIB) and two related cyclic analogs, 6-chlorochroman-2-carboxylic acid (CCCA) and 6-phenylchroman-2-carboxylic acid (PCCA), on human platelet function were evaluated. CPIB, CCCA and PCCA all inhibited platelet activation, i.e. aggregation and secretion of [¹⁴C]serotonin induced by ADP, epinephrine, collagen and thrombin, in a concentration-dependent manner. PCCA was at least fifty-two times more effective as an inhibitor of ADP-, epinephrine- and collagen-induced platelet activation and only 2-fold more effective as an inhibitor of thrombin-induced platelet activation when compared with CPIB or CCCA. Only PCCA inhibited platelet aggregation and [¹⁴C]serotonin secretion induced by arachidonic acid (AA) in a concentration-dependent manner. CPIB and CCCA did not inhibit AA-induced platelet activation. In fact, both of these agents had a potentiating effect on the onset of platelet aggregation by AA. All three compounds inhibited thrombin-induced release of [³H]arachidonic acid ([³H]AA) from platelet phospholipids and thrombin-mediated malondialdehyde (MDA) production. Only PCCA, however, inhibited AA-induced MDA production. These results indicate that CPIB, CCCA and PCCA all inhibit platelet activation by inhibiting prostaglandin biosynthesis. PCCA blocked AA-induced platelet activation, and this additional inhibitory action of PCCA appears to be responsible for its comparatively higher inhibitory potency. A comparison of the structure-activity relationship of the inhibitors indicated that replacement of the chloro group by a phenyl group produced a compound (PCCA) that was a potent inhibitor of prostaglandin biosynthesis and was thereby a more effective antiaggregatory agent than either CPIB or CCCA.     

Effects of reserpine on rabbit platelet aggregation and adherence to collagen or injured rabbit aorta

Effects of reserpine in vivo and in vitro on rabbit platelets in citrated platelet-rich plasma and in suspensions of washed platelets have been studied. Administration of reserpine ($\text{5}\text{mg}\text{kg}$) intraperitoneally 18 hr before platelets were isolated caused inhibition of collagen-induced aggregation but not of aggregation induced by ADP or thrombin. Thrombin-induced aggregation was slightly enhanced. Platelets from reserpine-treated rabbits were less adherent than control platelets to collagen-coated glass surfaces or to the subendothelium of the rabbit thoracic aorta. Similar effects on aggregation were obtained when reserpine (0.2 to 10 μM) was added to suspensions of washed rabbit platelets as little as 2 sec before the addition of collagen. Collagen-induced release of nucleotides and [¹⁴C]serotonin from prelabeled washed rabbit platelets was not affected by the presence of reserpine, whereas thrombin-induced release was slightly enhanced. Inhibition by reserpine (2–10 μM) of platelet adherence to a collagen-coated surface or to the subendothelium was also observed within a time interval too short for the reserpine to have caused depletion of platelet granule contents. Thus, reserpine has an immediate effect on the plasma membrane of the platelets which is responsible for inhibition of platelet adherence to collagen and hence of collagen-induced aggregation. This inhibitory effect differs from a much slower effect of reserpine at the granule membrane which results in the depletion of the granule contents of serotonin and adenine nucleotides. The effect of reserpine is not abolished by washing and resuspending platelets that have been exposed to reserpine in vivo. By inhibiting the interaction of platelets with collagen, reserpine may interfere with one of the components of hemostatic plug and thrombus formation.     

Effects of two methylxanthines, pentoxifylline and propentofylline, on arachidonic acid metabolism in platelets stimulated by thrombin

[3H]Pentoxifylline and [3H]propentofylline were taken up by human platelets in a dose-dependent manner probably involving a passive diffusion through the plasma membrane. In vitro, the two drugs were able to inhibit platelet activation induced by thrombin. serotonin secretion was reduced from 57% to 38% and 28% in the presence of 1 mM pentoxifylline and 1 mM propentofylline, respectively. Platelet aggregation was inhibited in the same way. Modifications of [14C]arachidonic acid metabolism in human platelets stimulated by thrombin were then measured in the presence of drugs. Preincubation of platelets with 1 mM pentoxifylline or propentofylline inhibited the production of [14C]arachidonic acid metabolites, without any accumulation of free arachidonic acid, suggesting an action at a step preceding its conversion. Phosphatidylinositol and phosphatidylcholine hydrolysis measured upon thrombin treatment as well as phosphatidic acid production were reduced or suppressed in the presence of the drugs. A dose-dependence study showed that phosphatidylcholine hydrolysis was totally inhibited at 5.10(-4) M propentofylline, while phosphatidic acid formation was reduced by only 40%. Propentofylline was in general more efficient than pentoxifylline in inhibiting events occurring upon thrombin stimulation. Our results suggest that the two methylxanthines inhibit both phospholipase A2 and phospholipase C, the former displaying a greater sensitivity to the two drugs.     

Platelet alpha-granule secretion and its modification by SC-57101A,a GPIIb/IIIa antagonist

Platelet-agonist interaction results in aggregatory and secretory responses. While the activation of glycoprotein (GP) IIb/IIIa plays an essential role in platelet aggregation, its role in granule secretion is not clear. The present study was performed to examine the effect of 3-[[[[1-[4-(aminoiminomethyl) phenyl]-2-oxo-3S-pyrrolidinyl]amino]carbonyl]amino]-propanoate monohydrochloride salt (SC-57101A), a GPIIb/IIIa antagonist, on platelet alpha-granule secretion responses to collagen, ADP, and thrombin receptor activating peptide (TRAP). Both SC-57101A and prostaglandin E(1) (PGE(1)) inhibited collagen-, ADP-, and TRAP-induced platelet aggregation in a concentration-dependent manner. SC-57101A inhibited the collagen- and ADP-induced release of platelet-derived growth factor (PDGF) and beta-thromboglobulin (beta-TG) from platelets, but not TRAP-induced secretion of these granule contents. On the other hand, PGE(1) inhibited the release of PDGF and beta-TG from platelets activated with all the agonists used. ADP and TRAP elicited P-selectin expression in the absence of platelet aggregation, while collagen produced no such reaction. SC-57101A only moderately inhibited P-selectin expression induced by ADP and had no inhibitory effect on that induced by TRAP. The inhibition of ADP-induced secretion of alpha-granule contents by SC-57101A was abolished when platelets were pretreated with aspirin. These results suggest that GPIIb/IIIa activation plays a minor role, if any, in alpha-granule secretion in human platelets.     

Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.     

Mechanism of action of the platelet function inhibitor from Vipera russelli siamensis snake venom

Human platelet aggregation induced by ADP, adrenaline, collagen or thrombin was inhibited by the venom inhibitor. Heating reduced both its phospholipase A2 enzymatic and anti-aggregatory activities, although not in parallel. The inhibitor caused significant dose-related inhibitory effects on the clot retraction of rabbit platelet-rich plasma caused by thrombin, while platelet malondialdehyde formation stimulated by thrombin was not affected. Furthermore, the venom inhibitor increased basal cyclic AMP levels in platelets, while cyclic GMP content was slightly lowered, but not in a dose-dependent manner. In addition, microscopic study revealed that the cytoskeleton was disordered after treatment of platelets with the venom inhibitor. The platelets lost their discoid form, while the ultrastructural changes of platelet aggregation induced by ADP were blocked. It is concluded that increasing platelet cyclic AMP and the disorder of the cytoskeleton may be the mechanism of action of the venom inhibitor on platelet function.     

Interaction of local anaesthetics with blood platelet aggregation

The effect of three newly synthesized local anaesthetics on platelet aggregation, serotonin release and integrity of platelets was investigated. Pentacaine, heptacaine and carbisocaine were found to be about 10 times more active than "classic" local anaesthetics. They inhibited platelet aggregation stimulated by collagen, thrombin and ADP at the concentrations of 0.01, 0.1 and 1 mmol/l respectively. At millimolar concentrations the test local anaesthetics liberated serotonin from platelets and disintegrated platelet membranes. Pentacaine was the most effective, followed by heptacaine and carbisocaine. The authors suppose that the perturbation induced in platelet membranes by local anaesthetics could be responsible for inhibition of platelet aggregation and release of serotonin.     

Effects of brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets

Brazilin {7,11 b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol} inhibited thrombin-, collagen-and ADP-induced aggregation of washed rat platelets. Thrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane A₂ caused by thrombin, whereas it had no effect on the prostaglandin D₂ formation. Brazilin inhibited [³H]-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase (PLA₂) activity and [Ca²⁺]_i elevation might be at least a part of antiplatelet mechanism of brazilin.     

Effects of cryptolepine on collagen-induced aggregation and on the mobilization and metabolism of arachidonic acid by rabbit platelets

1. We examined the effect of cryptolepine on collagen-induced aggregation and on the mobilization, and metabolism of arachidonic acid in rabbit platelets.2. Preincubation of platelets with cryptolepine (50–100 μM) did not affect the primary wave of aggregation but resulted in a dose-dependent, surmountable inhibition of the secondary wave of aggregation induced by collagen (5 μg/ml). The inhibition by cryptolepine was greater when cryptolepine was incubated with the platelets after the peak of the primary wave of aggregation.3. Cryptolepine (50–100 μM) dose-dependently inhibited thrombin (1.5 U/ml) and A23187 (2.5 μM)-induced release of ¹⁴C[AA] from platelet membrane phospholipid pools. The percentage inhibition of A23187-induced ¹⁴C[AA] release was 31.3 ± 4.3% (50 μM) and 79.3 ± 5.4% (100 μM), while thrombin-induced release was inhibited by 39.2 ± 2.4% (50 μM) and 68.2 ± 3.6% (100 μM).4. At near maximal concentration (100 μM) which significantly inhibited secondary aggregatory response and ¹⁴C[AA] release, cryptolepine had no effect on the platelet metabolism of ¹⁴C[AA] to thromboxane B₂, HHT and 12 HETE.5. The present findings suggest that cryptolepine inhibited collagen-induced secondary aggregation through a selective antiphospholipase-like activity. There was not effect on platelet cyclooxygenase and lipoxygenase activities of platelets.     

丹酚酸B镁盐对兔洗涤血小板聚集和5-HT释放反应的影响(英文)

AIM: To study the effects and mechanism of magnesium lithospermate B(MLB) on rabbit platelet aggregation and 5-HT release. METHODS: The platelet aggregation was determined by Born's method. Release of serotonin (5-HT) and formation of thromboxane A2 (TXA2) were measured by fluorophotometry and radioimmunoassay (RIA) respectively. Cytoplasmic free Ca2+ concentration ([Ca2+]i) in platelets was measured by Fura 2-AM fluorescence technique. RESULTS: In washed platelets, thrombin (200 U/L) or arachidonic acid (AA) (30 mumol/L)-induced aggregation was inhibited by MLB 50-800 mg/L in a concentration-dependent manner. In addition, MLB had more inhibitory effects on platelet aggregation in the absence of extracellular calcium with IC50 of 102 mg/L than in the presence of CaCl2 1 mmol/L with IC50 of 194 mg/L. MLB concentration-dependently decreased the thrombin-activated release of 5-HT, whereas it did not affect the formation of TXA2 in platelets. Furthermore, MLB not only inhibited the rise of [Ca2+]i in thrombin stimulated platelets, but decreased the [Ca2+]i in resting platelets. CONCLUSION: MLB inhibited the aggregation and 5-HT release in rabbit platelets and it is probably by attenuating intracellular calcium concentration.     

Induction of the platelet release reaction by concanavalin A—Relation of con A binding to release and modification of release by ATP,PGE1 and amantadine

The binding of concanavalin A (con A) to washed human platelets was demonstrated with [⁶³Ni] and [³H] labeled preparations. When con A binding was inhibited by α-methyl-d-mannoside, the con A-induced platelet release reaction was inhibited. PGE₁ markedly inhibited the release of nucleotides, serotonin and α-mannosidase (α-man) produced by both con A and thrombin but caused only a moderate decrease in con A binding. ATP, which inhibited release of serotonin by con A and thrombin, also caused a moderate decrease in con A binding. Amantadine, 5 mM, potentiated release of serotonin and most glycosidases from thrombin-treated platelets, but did not affect nucleotide or α-man release. Although 1 mM amantadine also enhanced serotonin release by con A-treated platelets without affecting release of nucleotides, 5 mM amantadine inhibited con A-stimulated release of serotonin, nucleotides and α-man and produced a 30 per cent reduction in con A binding. These data suggest that: (1) con A and thrombin activate release receptors by different mechanisms, (2) activation by con A may require binding to the receptors, (3) the mechanism for release after activation is similar for both, and (4) receptors governing the release of serotonin are different from those involved in release of nucleotides and α-man.     

Potentiating effects of clofibrate on prostaglandin-dependent and -independent pathways of human platelet activation: evidence for involvement of cyclic AMP

Although clofibrate has been shown to inhibit platelet aggregation that is caused by thrombin, ADP and epinephrine, by blocking the release of arachidonic acid from platelet phospholipids [8], here we have demonstrated that clofibrate enhanced platelet aggregation by arachidonic acid and PLC and reversed the effects of PGE1 on platelet cAMP concentration and on PLC-induced secretion of [14C]-5HT in similar, concentration-dependent manners. Taken together, these findings strongly suggest that the proaggregatory effect of clofibrate is mediated by a lowering of cAMP in platelets.     

Mechanism of Action of p-Chlorobiphenyl on the Inhibition of Platelet Aggregation

p-Chlorobiphenyl (1–50 μm ) concentration-dependently inhibited the aggregation and release reaction of rabbit washed platelets induced by arachidonic acid and collagen, but not those induced by platelet-activating factor (PAF), U46619 and thrombin. The IC50 values of p-chlorobiphenyl on the arachidonic acid and collagen-induced platelet aggregation were 2.9 ± 0.5 and 12.8 ± 2.3 μm , respectively. The formation of both platelet thromboxane B₂ and prostaglandin D₂ caused by arachidonic acid was inhibited by p-chlorobiphenyl concentration-dependently. In myo-[³H]inositol-labeled and fura-2-loaded platelets, [³H]inositol monophosphate generation and the rise in intracellular Ca²⁺ stimulated by arachidonic acid were inhibited by p-chlorobiphenyl. In human platelet-rich plasma, p-chlorobiphenyl and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by adenosine 5′-diphosphate and adrenaline without affecting the primary aggregation. It is concluded that p-chlorobiphenyl may be a cyclo-oxygenase inhibitor and its antiplatelet action is mainly due to the inhibition of thromboxane formation.     

Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets.

Dicentrine is an antiplatelet agent isolated from the Chinese herb Lindera megaphylla. We examined the in vitro effects of dicentrine on various aspects of platelet reactivity. Dicentrine inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA), collagen, ADP, platelet-activating factor (PAF), thrombin and U46619. Dicentrine also inhibited the thromboxane B2 formation caused by AA, collagen and thrombin in washed intact platelets or that induced by AA in lysed platelet homogenate, while prostaglandin D2 formation caused by AA was not increased. The generation of inositol monophosphates (in the presence of indomethacin) caused by thrombin, collagen and PAF was not suppressed significantly, nor did dicentrine suppress fibrinogen-induced aggregation of elastase-treated platelets. Dicentrine inhibited the intracellular Ca2+ increase in quin-2/AM-loaded platelets caused by thrombin, PAF, collagen and AA. The cyclic AMP level was elevated by dicentrine in a concentration-dependent manner. These data indicate that the inhibitory effect of dicentrine on platelet aggregation and ATP release was due to the inhibition of thromboxane formation and the elevation of the level of cyclic AMP.     

Pharmacological characteristics of solid-phase von Willebrand factor in human platelets.

1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.     

Regulation of human platelet activation--analysis of cyclooxygenase and cyclic AMP-dependent pathways

We have studied the regulation of human platelet activation by cyclic AMP (cAMP), and the cyclooxygenase products by examining the effect of prostacyclin (PGI2) and indomethacin on platelet aggregation, release reaction and thromboxane B2 (TxB2) generation induced by the full dose range of common platelet agonists in both platelet-rich plasma and washed platelets. Platelet aggregation, [14C]-5HT and TxB2 release induced by "threshold" and "supramaximal" concentrations of ADP, adrenaline, platelet-activating factor (PAF) and U46619 were totally abolished by low concentrations of PGI2 (3-6 nM). In contrast, platelet activation induced by submaximal concentrations of collagen, thrombin and the calcium ionophore A23187 was only partially inhibited by PGI2 (3-3000 nM). PAF-induced release reaction like that induced by ADP and adrenaline was totally dependent on the cyclooxygenase products and aggregation, while U46619-induced release reaction was only partially dependent on aggregation and the cyclooxygenase products. While both PGI2 (18-3000 nM) and indomethacin (10 microM) abolished collagen-induced aggregation and the aggregation-mediated release reaction, neither inhibitor significantly inhibited platelet adhesion or the adhesion-mediated release reaction. Maximal thrombin-induced aggregation and release reaction was also not significantly inhibited by PGI2 (300 nM) or indomethacin (10 microM). Thromboxane (TxB2) generation induced by sub-maximal to maximal concentrations of collagen, thrombin and A23187 was, although significantly inhibited, not abolished by PGI2. These results demonstrate that PAF is a "weak" agonist similar to ADP and adrenaline, U46619 is an agonist intermediate between weak and strong which induces a release reaction that is only partially dependent on aggregation, but unlike the strong agonists, is totally susceptible to inhibition by PGI2, PGI2 is an indirect inhibitor of phospholipase activation, which does not significantly inhibit non-aggregation-mediated arachidonate mobilization, induced by the strong agonists, and the so-called third pathway in the collagen and thrombin-induced release reaction, which is insensitive to indomethacin, is also insensitive to elevators of cAMP such as PGI2.     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.