Inhibition of cyclooxygenase-independent platelet aggregation by sodium salicylate

The effect of acetylsalicylic acid (ASA) on platelet aggregation (PA) and thromboxane A₂ (TxA₂) formation was investigated in vitro and ex vivo after 1 g or 300 mg ASA administration to healthy subjects. 50–100 μM ASA inhibited PA by single aggregating agent such as platelet aggregating factor (PAF) or epinephrine and reduced to 5% of control platelet TxB₂ formation, but did not influence PA by epinephrine plus PAF. The latter was inhibited by increasing ASA concentration. In samples incubated with 100 μM ASA and stimulated with epinephrine plus PAF, PA could be inhibited by the addition of 100–300 μM sodium salicylate. After 300 mg-1 g ASA administration to healthy subjects, the inhibition of PA by epinephrine plus PAF was more marked by highest doses of ASA. This study suggests that aspirin inhibits PA with a cy clooxygenase-independent mechanism; this effect is mediated, at least in vitro, by salicylic acid.     

Mechanisms of vascular graft thrombosis: Role of altered canine platelet sensitivity to thromboxane

Using the standard turbidimetric method of platelet aggregation and quantitation of platelet secretion with ¹⁴C-Serotonin, we have examined the responsiveness of the platelets of mongrel dogs to arachidonic acid (AA), and the thromboxane agonist U46619 in the presence and absence of a subthreshold concentration of epinephrine. In response to stimulation with 750 μM AA, the platelets of 18 dogs produced irreversible aggregation (Group I), the platelets of 22 dogs showed, at most, reversible aggregation (Group II), while the platelets of 8 dogs demonstrated no aggregatory response (Group III). In the presence of AA and a subthreshold concentration of epinephrine (0.5 μM), the platelets of all three groups demonstrated enhanced aggregatory and secretory responses although the extent of ¹⁴C-Serotonin secretion differed significantly between all three groups. These differences in platelet aggregation correlate with the deposition of platelets onto synthetic vascular grafts and the maintenance of graft patency. When stimulated with 0.5 μM U46619 and a subthreshold concentration of epinephrine, the platelets of 97% Group I dogs and 75% of Group II dogs exhibited irreversible aggregation, while the platelets of all Group III dogs showed only reversible aggregation. In addition, significant differences in the extent of ¹⁴C-Serotonin secretion to this combination of agonists were observed between groups. Further examination of the specific effects of U46619 on canine platelets revealed that although the aggregatory and secretory responses to U46619 vary between the different canine platelet populations, the threshold concentration of U46619 required to produce platelet shape change is identical among all groups. Quantitation of the stable metabolite of AA produced via the cyclooxygenase pathway, thromboxane B₂(TxB₂), revealed no significant differences in the production of TxB₂ by the platelets of these different populations upon stimulation with AA. Our results suggest that the mechanisms underlying the differences in responsiveness of canine platelets to AA, are likely due to differences in sensitivity of canine platelets to TxA₂, and may be localized to the mechanism responsible for mediating platelet aggregation and secretion in response to TxA₂.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production

We studied the effects of picotamide (N,N′ bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B₂ (TxB₂) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/1) inhibited platelet aggregation, the release of ATP and TxB₂ production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/1) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB₂ production both under stirring and non-stirring conditions, whereas the pure thromboxane A₂ receptor antagonist BM13177 (0.5 mmol/1) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB₂ production, whereas BM13177 inhibits the potentiation of TxB₂ production due to TxA₂/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA₂/PE antagonism; (ii) inhibition of thromboxane synthase.     

Platelet aggregation following exposure of phospholipase-treated platelet rich plasma to hydrogen peroxide

Hydrogen peroxide at micromolar concentrations (250–500 μM) can induce platelet aggregation of phospholipase-treated PRP. This effect, which occurs independently of the release of ADP, is blocked by aspirin, furosemide, catalase and 2-mercaptoethanol. PRP preincubated with H₂O₂ for 2–5 min. does not respond to Phl-H₂O₂ or collagen. This inhibitory effect is abolished with longer preincubations. In the presence of ADP or epinephrine, H₂O₂ enhances aggregation, if added to PRP with the inducers, and decreases the platelet response to the inducers, if preincubated with PRP for 2 min. The data suggest that micromolar concentrations of H₂O₂, which could be generated at sites of platelet plug formation by granulocytes, could influence the processes of hemostasis and thrombosis.     

Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of ³H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.     

Epinephrine—Via Activation of P38-Mapk—Abolishes the Effect of Aspirin on Platelet Deposition to Collagen

The mechanism by which epinephrine enhances experimental thrombosis in the presence of aspirin is poorly understood. In this study, we set to explore, in aspirinised platelet-rich plasma (PRP), the effect of epinephrine (100 nmol/l) on platelet deposition to immobilised collagen and the subsequent involvement of several intracellular pathways. Under these experimental conditions, which allow platelet aggregation on top of the collagen-adherent platelets, epinephrine increased platelet deposition by 55–86%. This enhancement could be specifically prohibited by the _2A-adrenoceptor antagonist, atipamezole, the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, and the cytosolic phospholipase A₂ (cPLA₂) inhibitor, mepacrine. The effect of epinephrine coincided with increased phosphorylation of p38MAPK and cPLA₂ and with arachidonic acid (AA) release from platelet membrane. We conclude that epinephrine enhanced platelet deposition on collagen in aspirinised PRP via a mechanism dependent on both free AA in platelet cytosol (released by cPLA₂) and p38MAPK.     

Involvement of calmodulin in platelet reaction

Penothiazines, known as selective inhibitors of calmodulin, completely inhibited platelet aggregation and secretion induced by ADP, collagen, epinephrine, thrombin or calcium ionophore. They also completely inhibited aggregation induced by exogenous arachidonate (AA) or a mixture of thromboxane A₂ and prostaglandin endoperoxides (TxA₂/PG G₂,H₂). Also, in the presence of these calmodulin inhibitors, the release of AA from platelet phospholipids (PL) was dosedependently inhibited in stimulated platelets. These observations suggest that in platelet reaction, calmodulin is involved in at least two different steps of the reaction: activation of phospholipases and contraction of platelet actomyosin after the formation of TxA₂.     

Phospholipid-induced human platelet activation: Effects of calcium channel blockers and calcium chelators

Human platelet activation (aggregation, [¹⁴C]-5HT release and TxB₂ production) induced by the phospholipids, PAF and lysophosphatidic acid (LPA) was inhibited by EGTA, TMB-8 (an intracellular calcium antagonist) and by phenylalkylamine (Class II) but not 1,4-dihydropyridine (Class I) calcium channel blockers. Primary aggregation induced by PAF was selectively inhibited by phenylalkylamine (verapamil, methoxyverapamil) calcium channel blockers. Phospholipid-induced human platelet activation depends predominantly on the influx of extracellular calcium, possibly via specific receptor-operated calcium channels.     

Effects of Ethanol on Platelet Responses Associated with Adhesion to Collagen

Adhesion of platelets to collagen in damaged blood vessels or ruptured atherosclerotic plaques is important in hemostasis and arterial thrombosis. Adhesion to collagen results in secretion of granule contents and formation of thromboxane A₂; thromboxane A₂ and released ADP synergistically promote aggregation around platelets adherent to collagen. Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A₂ formation but does not inhibit platelet adhesion to de-endothelialized rabbit aortae. We investigated whether ethanol affects the initial signalling events and responses of platelets adherent to collagen, independent of the actions of secondary agonists. Suspensions of washed human platelets, labelled by incorporation of [³H]oleate into phospholipids, were used to measure platelet adhesion to collagen by a filtration method; studies were done in the presence of an ADP-removing system and blockers of receptors for thromboxane A₂, platelet-activating factor, serotonin, and fibrinogen. Ethanol (87 mM) did not affect the rate or extent of platelet adhesion to collagen or secretion of [¹⁴C]serotonin from prelabelled platelets adherent to collagen, but ethanol did inhibit thromboxane A₂ formation. Previous studies showed that ethanol does not affect platelet stimulation by arachidonate, leading to the suggestion that reduced mobilization of arachidonate, rather than inhibition of its conversion to thromboxane A₂, is responsible for inhibition by ethanol of thromboxane A₂ formation. Here, we show by a gel mobility shift assay and immunoblotting, that ethanol delays the collagen-induced increase in the phosphorylation of cytosolic phospholipase A₂, the enzyme responsible for arachidonate mobilization. However, ethanol has no effect on collagen-induced tyrosine phosphorylation of phospholipase Cγ2, determined by immunoprecipitation and immunoblotting. Thus, ethanol's effect on signal transduction in collagen-adherent platelets occurs distal to phosphorylation of phospholipase Cγ2 but proximal to phosphorylation of cytosolic phospholipase A₂.     

Paired analysis of urinary thromboxane B2 metabolites in humans

11-Dehydro-TxB₂ and 2,3-dinor-TxB₂ are products of the two major pathways of thromboxane metabolism in man. In this study we compared urinary excretion of 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ as indices of Tx biosynthesis in vivo.

We performed three studies to assess i) the relative abundance of these two metabolites in the urine of healthy subjects, ii) their cellular origin under physiological conditions and iii) their relative formation during platelet activation. In healthy normal volunteers urinary 11-dehydro-TxB₂ is more abundant than 2,3-dinor-TxB₂ (792 ± 119 pg/mg creatinine vs 106 ± 21 pg/mg creatinine). Administration of a dose of aspirin selective for platelet cyclooxygenase (20 mg/day for 10 days) caused substantial and comparable suppression of both 11-dehydro-TxB₂ (mean 82±4.9%) and 2,3-dinor-TxB₂ (mean 79±6.9%). Recovery of excretion of both metabolites after a nonselective aspirin regimen (325 mg BID for 3 days) corresponded to platelet life-span. Furthermore, excretion of both metabolites was increased in patients with severe atherosclerosis consistent with the known increase in platelet activation in this setting.

Quantitative analysis of both urinary 11-dehydro-TxB₂ and 2,3-dinor-TxB₂ by GC-MS established that, in contrast to previous assumptions, 11-dehydro-TxB₂ is the most abundant urinary metabolite of TxB₂. The aspirin study demonstrates that platelets are the major source of both metabolites in urine, consistent with their increased excretion in severe atherosclerosis. Combined analysis of both metabolites will distinguish altered metabolism from increased biosynthesis of thromboxane A₂.     

Evidence that the rat is not an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation

Abnormal platelet aggregation seen in experimentally induced diabetic, hypercholesterolemic and spontaneously hypertensive rats (SHR) has been linked with increased prostaglandin synthesis. The present study was conducted to examine the role of prostaglandins in rat platelet activation using normal Wistar Kyoto (WKY) and SHR rats. Up to 30 microM ADP did not induce secondary phase of platelet aggregation in rat PRP and up to 30 microM epinephrine did not produce any response in rat PRP. In other experiments ADP (1.0 microM) and epinephrine (2.0 microM) induced typical biphasic aggregation responses in human PRP. Up to 20 microM U46619, a stable analog of prostaglandin H2, did not induce platelet aggregation in rat PRP or washed rat platelets. In contrast 2.0 microM U46619 caused maximal aggregation in human PRP and washed human platelets. Arachidonic acid (1.5-2.0 mM) induced aggregation in washed rat platelets. However, this was associated with excessive (67% and 94%) loss of cytoplasmic LDH. The low concentrations of thrombin (0.04 and 0.05 U/ml), induced two to three-fold increase in aggregation response in SHR platelets as compared to WKY platelets. Higher concentrations of thrombin (0.1 and 0.3 U/ml) induced similar aggregation responses in SHR and WKY platelets. Thrombin (0.04-0.3 U/ml) induced serotonin secretion in a concentration dependent manner. The extent of secretion was the same in SHR and WKY platelets at all concentrations. Thrombin-induced synthesis of thromboxane A2 (TXA2) in WKY and SHR platelets was quantified using a radioimmunoassay for TXB2. Thrombin (0.04-0.3 U/ml) produced TXB2 in WKY and SHR platelets in a concentration dependent manner. The SHR platelets produced significantly larger amounts of TXB2 as compared to WKY platelets. In other experiments aspirin (500 microM) inhibited thrombin (0.05 U/ml) induced TXB2 synthesis by 75% in both WKY and SHR platelets but failed to inhibit aggregation or secretion in either WKY or SHR platelets. Based on these data it is suggested that: (a) rat platelets inspite of their ability to synthesize TXA2 do not require TXA2 for aggregation; and (b) the rat may not be an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation.     

Effect of op 1206, a prostaglandin E1 derivative, on guinea-pig platelet functions

OP 1206, 17(S)-methyl-ω-homo-trans-Δ²-prostaglandin E₁, inhibited guinea-pig platelet aggregation induced by ADP, collagen, A 23187, arachidonic acid, labile aggregation stimulating substances (LASS) and thromboxane A₂ (TXA₂)-like substance, and platelet adhesiveness to a glass bead column. The potency of inhibition was 10–16 times stronger than that of prostaglandin E₁ (PGE₁). ADP-induced platelet aggregation was notably disaggregated by the addition of OP 1206 after induction of aggregation. The release of ATP and ADP from guinea-pig platelets induced by collagen was suppressed by OP 1206, of which potency was 9–10 times stronger than that of PGE₁. OP 1206 and PGE₁ increased guinea-pig platelet cyclic AMP levels, and the increased levels were augmented by the pretreatment with theophylline. OP 1206 and PGE₁ inhibited synthesis of guinea-pig platelet malondialdehyde (MDA) induced by thrombin but not by arachidonic acid. This inhibition was released by exogenous calcium. OP 1206 and PGE₁ showed no influence on synthesis of radioactive TXA₂ (measured as a stable form, TXB₂) from [¹⁴C] arachidonic acid. From these results, increased levels of platelet cyclic AMP by OP 1206 as well as PGE₁ may exert their action on platelet functions.     

Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of ¹⁴C-serotonin from ¹⁴C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation.     

Effects of phenacetin and its metabolite p-phenetidine on COX-1 and COX-2 activities and expression in vitro

The present study was aimed to test the possible cyclooxygenase (COX)-1/COX-2 selectivity of the old analgesic drug phenacetin and its metabolite p-phenetidine, which exhibits high renal toxicity. Paracetamol (acetaminophen), the main metabolite of phenacetin with low renal toxicity, and indomethacin were selected as reference compounds. Collagen-stimulated platelet thromboxane B₂ (TxB₂) production and phorbol 12-myristate-13-acetate (PMA)-induced neutrophil prostaglandin E₂ (PGE₂) synthesis were used as indicators for COX-1 and COX-2 activity, respectively. Phenacetin was even less potent than paracetamol to reduce the production of both TxB₂ and PGE₂, and no clear preference for either of the COX-enzymes was seen. p-Phenetidine was a more potent inhibitor, already at nanomolar level, of the synthesis of these prostanoids than indomethacin and showed some preference to COX-2 inhibition. Somewhat higher, micromolar, concentrations of p-phenetidine also reduced COX-2 expression in neutrophils. We suggest that the very potent inhibitory activity of p-phenetidine on PGE₂ synthesis combined with the reduction of COX-2 expression could explain the renal papillary necrosis in phenacetin kidney.     

Thromboxane synthase inhibition causes re-direction of prostaglandin endoperoxides to prostaglandin D2 during collagen stimulated aggregation of human platelet rich plasma

Prostanoid synthesis and release during collagen-induced aggregation of human platelet rich plasma (PRP) was studied using a novel gas chromatography/mass spectrometry assay technique. Aggregation was associated with the production of mainly thromboxane A₂ (TXA₂), measured as TXB₂, and smaller amounts of the prostaglandins (PGs) D₂, E₂ and F₂. UK 37,248 inhibited T₁XB₂ formation by >95% and increased the production of PGD₂, PGE₂ and PGF₂ twenty-fold. The relative amounts of these three prostanoids were not changed by UK 37,248. Even though high concentrations of PGD₂ were formed, aggregation was not inhibited. In contrast, flurbiprofen inhibited aggregation, demonstrating that platelet aggregation produced by this concentration of collagen is cyclooxygenase dependent. These results support the proposal that the prostaglandin endoperoxides can induce aggregation alone, irrespective of the amount of PGD₂ that is produced.     

The primary wave of epinephrine-induced platelet aggregation represents alpha 2-adrenoceptor status

We examined relationships between epinephrine-induced slope of primary wave of aggregation and the alpha₂-adrenoceptor status on platelets. A concentration (10^-9 to 10^-6 )-dependent increase in slope of primary wave with EC50 of epinephrine at 4.5 ± 0.4 × 10^-7 was observed. In studies on epinephrine binding to alpha₂adrenoceptors in competition with ³H-yohimbine to platelets, (IC₅₀) of epinephrine was 4.8 ± 3.4 x 10^-7 M. There was a significant (P<0.02) correlation between EC₅₀ of epinephrine to evoke biological response and IC₅₀ of epinephrine to bind to alpha₂-adrenoceptors (r-0.75). There was no relationship between number of receptor sites or dissociation constant of ³H-yohimbine binding and primary wave of platelet aggregation. These data show that the slope of primary wave in response to epinephrine reflects alpha₂-adrenoceptor binding of the agonist.     

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.     

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)     

Effect of submaximal and incremental upper extremity exercise on platelet function and the role of blood shear stress

Introduction: Platelets are involved in the pathogenesis of atherosclerosis. Although physical exercise is recommended to prevent atherosclerosis, the effect of exercise on platelet function and the underlying mechanisms of these effects are not completely understood. Accordingly, we aimed to examine the effect of different intensities acute arm exercises on platelet function. In addition, we evaluated the effect of lipid peroxidation and fluid shear rate on platelet response. Materials and methods: Twenty four healthy sedentary male volunteers aged 18–24 years performed submaximal and incremental exercises by upper extremity ergometer. The shear rate in the right artery was measured by Power Doppler Ultrasound (US) at rest and immediately after exercise. Pre and postexercise maximum intensities of ADP and collagen-induced platelet aggregation were measured using the impedance technique. Bioluminescent detection of thrombin-induced platelet ATP release and measurement of thromboxane B₂ (TxB₂) levels (as a marker of thromboxane A₂ (TxA₂) formation) by enzyme-linked immunoassay were performed before and after exercise. Results and conclusion: Shear rate increased after both submaximal and incremental exercise. Collagen-induced platelet aggregation increased after submaximal exercise, while ADP-induced aggregation and thromboxane B₂ levels did not alter with this protocol. Incremental exercise caused increased collagen and ADP-induced platelet aggregation and thromboxane B₂ levels. Neither of the protocols altered platelet ATP release. It was shown that acute upper extremity exercise increased platelet aggregation, without an increase in platelet release. Collagen-induced signalling pathways were more sensitive than those induced by ADP. The increase in thromboxane B₂ after incremental exercise implied increase in thromboxane A₂ formation and lipid peroxidation. Despite a significant correlation between platelet aggregation and thromboxane B₂ levels at rest, we found no clear-cut relationship between thromboxane A₂ formation, blood shear rate and platelet response to exercise.