Effects of salicylic and acetylsalicylic acid alone and in combination on platelet aggregation and prostanoid synthesis in man.

The present study was designed to investigate the effects of salicylate on the antiplatelet action of acetylsalicylic acid as well as on in vivo prostanoid formation and platelet function in healthy volunteers. In the first study six female volunteers received 350 mg acetylsalicylic acid intravenously, with and without previous oral administration of sodium salicylate (1200 mg daily for 3 days). Urinary prostanoid excretion as well as platelet aggregation and thromboxane formation were measured before and during salicylate and after acetylsalicylic acid. In the second study seven female volunteers received sodium salicylate (52.6 mg kg-1) or acetylsalicylic acid (60.7 mg kg-1) for 8 days in a randomized cross-over protocol. Urinary prostanoid excretion, platelet aggregation and thromboxane formation as well as salicylate plasma concentrations were determined before, during and after administration of each drug. Sodium salicylate did not impair the complete suppression of arachidonic acid-induced platelet thromboxane formation and aggregation obtained by the single intravenous dose of acetylsalicylic acid in the first study. Sodium salicylate in the second study did not affect urinary excretion of prostaglandin E2, its major urinary metabolite (7 alpha-hydroxy-5,11-diketo-tetranor-prostane-1,16-dioic acid), and 2,3-dinor-6-keto-prostaglandin F1 alpha, the main urinary metabolite of epoprostenol (prostacyclin, PGI2). In contrast, acetylsalicylic acid significantly decreased excretion rates of these prostanoids by 64, 59 and 61%, respectively. In both studies platelet aggregation and thromboxane formation induced by collagen, thrombin or arachidonic acid were not significantly affected by salicylate administration, whereas acetylsalicylic acid inhibited platelet aggregation induced by all three agents as well as thrombin- and arachidonic acid induced thromboxane formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the inhibitory effects of cilostazol, acetylsalicylic acid and ticlopidine on platelet functions ex vivo. Randomized, double-blind cross-over study

A randomized double-blind cross-over study was conducted to determine the inhibitory effects of acetylsalicylic acid (ASA), ticlopidine (TP) and cilostazol (OPC-13013; in the following briefly called CS), a new antithrombotic agent on platelet functions ex vivo. Nine patients with cerebral thrombosis were enrolled in this study. Patients were given each of the three drugs for one week in a complete cross-over design according to a randomization schedule, followed by a wash-out period with a placebo for one week. It was found that CS and TP significantly inhibited platelet aggregation induced by ADP. Collagen- and arachidonic acid-induced platelet aggregation was all inhibited by CS, TP and ASA. Duncan's multiple range test to compare the anti-platelet effects of the three drugs revealed that: CS greater than ASA and TP greater than ASA in inhibiting ADP-induced platelet aggregation and CS greater than TP and ASA greater than TP in inhibiting arachidonic acid-induced platelet aggregation. These results may suggest that CS is superior to ASA and TP in inhibiting platelet aggregation ex vivo.     

Mechanisms of cell association of some non-steroidal anti-inflammatory drugs with isolated leucocytes

In the present study the degree and the mode of association of the radiolabelled drugs acetylsalicylic acid, sodium salicylate, and sodium benzoate with leucocytes were studied in view of the hypothesis that leucocytes are target cells for the anti-inflammatory activity of drugs. The overall association rate of acetylsalicylic acid is larger than that of sodium salicylate and sodium benzoate at 37 degrees, but smaller at 4 degrees. The ratio of the intracellular to the extracellular concentration varied between 1 and 2 for sodium salicylate and sodium benzoate, and between 3 and 6 for acetylsalicylic acid. The intracellular concentrations of these drugs were comparable in red blood cells and polymorphonuclear leucocytes, but lower in mononuclear leucocytes. The association of acetylsalicylic acid and sodium salicylate is markedly increased when the extracellular pH decreases. Lysis of cells decreases the association of acetylsalicylic acid and enhances the association of sodium salicylate and benzoate at 37 degrees twofold. It is suggested that the association of these drugs with leucocytes comprises binding to the membrane and uptake of undissociated species. Phorbol myristate acetate extensively inhibits the intracellular concentration of acetylsalicylic acid, while this inflammatory stimulus tends to increase the intracellular concentration of sodium salicylate. The major metabolites of salicylate enhance cell association of acetylsalicylic acid and salicylic acid. In conclusion, these findings indicate that the tested benzoic acid-like drugs associate with leucocytes in vitro to some extent and that environmental differences, e.g. pH, lysed cells, inflammatory stimuli and metabolites, may determine in vivo the degree of accumulation.     

Effects of salicylates and paracetamol compared to lidocaine on nerve conduction in vitro

The effects of acetylsalicylic acid, salicylic acid, sodium salicylate and paracetamol on the compound action potentials of the isolated left phrenic nerve of the rat were observed, and compared to that of lidocaine. All drugs depressed the compound action potential in a reversible, dose-dependent way. Lidocaine (inhibition between 0.05 and 10 mM) was more potent than the other drugs and the depression occurred faster. The inhibitory effects of acetylsalicylic acid and salicylic acid were similar to each other (between 1 and 20 mM). Sodium salicylate was less potent (inhibition between 5 and 70 mM). These concentrations cover the plasma levels reported after therapeutic and toxic doses for the salicylates. Inhibition of nerve conduction may therefore contribute to the adverse nervous effects seen after intoxication with salicylates. In contrast, the observed concentrations for inhibition of the compound action potential caused by paracetamol (between 6.7 and 53 mM) were far greater than the plasma concentrations reported after therapeutic doses or after intoxication with paracetamol. Thus, the inhibition of the compound action potential, caused by paracetamol does not contribute to the therapeutic analgesic effects of paracetamol.     

The influence of salicylate on platelets and whole blood adenine nucleotides

1. The addition of sodium salicylate to freshly withdrawn human blood or native platelet-rich plasma significantly delays platelet aggregation in vitro.2. The administration of acetylsalicylic acid to human subjects also significantly delays platelet aggregation in their whole blood and there is a short delay in the formation of fibrin but this is not statistically significant.3. Salicylate, whether added to human blood in vitro as sodium salicylate or given by mouth as acetylsalicylic acid, significantly reduces the platelet clumping activity of adenosine diphosphate (ADP) added to whole blood in vitro.4. The administration of aspirin in high doses for several days produces a marked increase in the total adenine nucleotide content of whole blood. The percentage of adenosine triphosphate (ATP) was increased, that of ADP decreased while there is an obvious increase in the ATP: ADP ratio.5. There is little correlation between the plasma salicylate level and the delay aspirin produces in platelet aggregation in vitro or the changes that occur in the levels of ADP or ATP in whole blood during administration of aspirin.6. Significant correlations do occur, however, between the delay in platelet aggregation in vitro and (i) the percentage increase in the ATP concentration, (ii) the percentage decrease in ADP concentration, (iii) the percentage change in the ATP: ADP ratio observed during aspirin administration.     

Dipyridamole alone or combined with low-dose acetylsalicylic acid inhibits platelet aggregation in human whole blood ex vivo.

1. In a randomized, double-blind trial we compared the inhibition of the platelet-vessel wall interactions in whole blood ex vivo. There were four groups of 24 healthy volunteers each of whom were treated orally for 3.5 days with either 200 mg dipyridamole (sustained release preparation), 25 mg acetylsalicylic acid, both drugs combined or placebo twice daily. 2. The mean area of all platelets/aggregates was reduced by 6.2% +/- 4.2% (+/- s.e. mean) by placebo (n = 23), 19.8% +/- 6.7% by dipyridamole (n = 22), 53.7% +/- 4.9% by acetylsalicylic acid (n = 23) and 71.4% +/- 3.7% by the combination of both drugs (n = 24), when compared with total inhibition of aggregation by EGTA. Thus, low-dose acetylsalicylic acid inhibited aggregation (P less than 0.001). 3. Dipyridamole reduced the size of platelet aggregates (P less than 0.01, two-fold analysis of variance). The reduction was correlated with the individual dipyridamole plasma levels (P less than 0.05, analysis of covariance). The subgroup of large and very large thrombi being formed was also reduced by dipyridamole (P less than 0.05). 4. This ex vivo study demonstrates that dipyridamole alone inhibits formation of thrombi on subendothelial matrix and enhances the inhibitory effect of low dose acetylsalicylic acid in this model of thrombosis.     

Comparison of the effects of drugs on the aggregation of hamster platelets in vivo and in vitro

1. A method is described for measuring the inhibitory effectiveness of drugs on the aggregation by ADP of hamster platelets in vivo.2. The method was used to compare the effects of several drugs, viz. adenosine, imipramine, desmethylimipramine and aspirin, on platelet aggregation in vivo with their in vitro effects measured photometrically.3. The concentrations of adenosine and imipramine present in the cheek pouch after 10 min infusions were measured using radioactively labelled drugs.4. The results show that adenosine (0.4 muM) inhibited platelet aggregation in vivo by 43%, whereas several times this concentration was required to produce the same inhibition in vitro.5. Imipramine and desmethylimipramine (0.4 muM) did not inhibit platelet aggregation in vivo; in vitro, however, desmethylimipramine caused up to 34% inhibition at concentrations as low at 0.25 muM.6. Aspirin (estimated 0.2 mM) inhibited platelet aggregation in vivo by 37% whereas similar inhibition in vitro required about 1 mM aspirin. Sodium salicylate was several times less potent than aspirin in vivo.     

Plasma levels of aspirin following effervescent and enteric coated tablets,and their effect on platelet function

Summary Oxcarbazepine (oxcarb) 600 and 900 mg (2360 and 3540 μmol) was taken by 3 volunteers (2 ♀, 1 ♂; 45–67 kg; age 22–34 years) after an overnight fast. Blood, saliva and urine were collected for the next 72 h for assay of oxcarb, 10,11-dihydro-10-hydroxy-carbamazepine (OHcarb), and 10,11-dihydrotrans-10,11-dihydroxy-carbamazepine (diol). Oxcarb reached a maximum level of about 1 μg/ml (3.93 μmol/l) within 1 h and dropped below the detection limit (0.1 μg/ml=0.39 μmol/l) within 3 h. The active metabolite OHcarb appeared in the blood before oxcarb and reached the higher maximum level of 7.4 μg/ml (29 μmol/l) after 7 h. Thereafter serum levels decreased with a t_1/2 of about 25 h, and after 40 h with a t_1/2 of 9 h, the latter agreeing with the renal excretory t_1/2 calculated from the urine data (10 h). The ratio of OHcarb concentration in saliva to that in plasma varied considerably (0.3–1.7; median 1; r>0.9), whereas that of blood to plasma was 1.25 with only small variation (r>0.98); OHcarb concentrations in erythrocytes were 50% higher than in plasma. Diol was detected in blood (maximum level 0.5 μg/ml=1.84 μmol/l) in 2 volunteers. 45% of the dose could be recovered in urine (Oxcarb 5%, OH-carb 36%, Diol 4%). Whereas Oxcarb was completely conjugated, only 25% of OHcarb was conjugated and diol was unconjugated.     

Effect of curcumin on platelet aggregation and vascular prostacyclin synthesis

In vitro and ex vivo effects of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (diferuloylmethane, curcumin) and acetylsalicylic acid (ASA) on the synthesis of prostacyclin (PGI2) and on platelet aggregation has been studied in rat. Both drugs inhibited adenosine diphosphate (ADP)-, epinephrine (adrenaline)- and collagen-induced platelet aggregation in monkey plasma. Pretreatment with ASA (25-100 mg/kg), but not curcumin (100-300 mg/kg), inhibited PGI2 synthesis in rat aorta. In the in vitro system, too, curcumin caused a slight increase in the synthesis of PGI2, while ASA inhibited it. Curcumin may, therefore, be preferable in patients prone to vascular thrombosis and requiring antiarthritic therapy.     

Effects of propranolol in vitro and in vivo on platelet function and thromboxane formation in normal volunteers

In vitro and ex vivo effects of propranolol on platelet aggregation, formation of thromboxane B2 (TXB2) and platelet sensitivity to prostacyclin were studied in healthy men. Propranolol, added in vitro to platelet rich plasma (PRP) inhibited platelet aggregation and TXB2 formation induced by ADP, 1-epinephrine, collagen and arachidonic acid. Concentrations of 20-100 microM propranolol were effective when ADP, 1-epinephrine and collagen were used as stimuli. Higher concentrations (250-500 microM) were needed to inhibit aggregation induced by arachidonic acid. Oral administration of propranolol either as a single dose (120 mg) or for one week (3 x 40 mg/day) did, however, not affect platelet aggregation, thromboxane formation and platelet sensitivity to prostacyclin. In addition, withdrawal of propranolol was without effect on these parameters. Although propranolol has potent effects on platelet function in vitro, it seems that the blood levels achievable by oral administration of propranolol are too low to affect platelet aggregation and TXB2 formation.     

Comparison of in vitro and ex vivo antiplatelet effects of 1,2-benzisothiazolin-3-one and its 2-amino derivative

The in vitro and ex vivo antiplatelet effects of 2-amino-1,2-benzisothiazolin-3-one (1) are compared with those of its parent compound 1,2-benzisothiazolin-3-one (2) and with acetylsalicylic acid (ASA) against different agonists. 2-Amino-1,2-benzisothiazolin-3-one inhibits adenosine diphosphate (ADP)-, arachidonic acid (AA)- and collagen-induced human platelet aggregation in vitro, with IC50 values of 8.90 x 10(-5), 1.50 x 10(-6) and 5.11 x 10(-8) mol/l, respectively. The strong inhibitory activity is significant not only for collagen but also for AA-induced aggregation. The same compound inhibits ex vivo collagen- and particularly AA-induced rabbit platelet aggregation at the tested dose of 10 mg/kg i.m. In view of the potential use of 2-amino-1,2-benzisothiazolin-3-one as antithrombotic agent, the log P values for both 1,2-benzisothiazolin-3-one derivatives 1 and 2 are determined, to gain an understanding of the significance of the 2-amino group in the 1,2-benzisothiazolin-3-one moiety with respect to the biological activity under study.     

Effect of arachidonic acid on some inhibitors of the human platelet release reaction.

The inhibitory action of many agents on platelet aggregation and 5-hydroxy-tryptamine release inducible by epinephrine, comprising several non-steroidal anti-inflammatory drugs (NSAA)—indomethacin, acetylsalicylic acid, sodium salicylate, phenylbutazone and sulfinpyrazone—and other non-anti-inflammatory agents (NAA)—prostaglandin E₁, adenosine, dipyridamole, methysergide and cyproheptadine—can be prevented, and under special circumstanees reversed, by the prior addition to human platelet rich plasma of the essential fatty acid, 5,8,11,14 eicosatetraenoic (arachidonic) acid. Although this agent, and only its all cis stereosiomeric form, can per se induce aggregation and release, its protective action against the platelet-toxic drugs is attributed to its role as the key substance essential for prostaglandin enzymatic biosynthesis. the mechanism of NSAA action is evidently different from that of the non-anti-inflammatory agents.     

The mode for the manifestation of the inhibitory effects of ifenprodil tartrate on platelet aggregation in vivo and ex vivo

The mode for the manifestation of the inhibitory effect of ifenprodil tartrate on platelet aggregation in vivo and ex vivo was studied in mice and men, respectively. The ifenprodil level in plasma reached the maximum in 20 min after oral administration of 30 mg ifenprodil tartrate/kg in mice, and it decreased over a 3 hr period after the administration. On the other hand, the maximal inhibitory effect was observed 60 min after the administration. Thus ifenprodil tartrate manifested its inhibitory effect on platelet aggregation only after the maximum plasma concentration of ifenprodil was reached. The same phenomenon was observed with the inhibitory effects of ifenprodil tartrate on platelet aggregation ex vivo in man. To clarify the reason for the delay in the manifestation of the inhibitory effects of ifenprodil, the ifenprodil contents in mouse platelets after the oral administration of the drug was measured. The pattern of change in the ifenprodil contents in platelets was found to resemble closely the pattern of the change in its inhibitory effects, suggesting that the manifestation of the inhibitory effects on platelet aggregation by oral administration of ifenprodil tartrate was directly related to the ifenprodil contents in platelets rather than the ifenprodil level in plasma.     

Levamisole inhibits in vivo rat platelet aggregation by a release of prostacyclin-like factor

1. The anti-thrombotic effect of levamisole (LMS) and acetylsalicylic acid (ASA) were examined in vitro and in vivo models. 2. LMS inhibits rat platelet aggregation induced by either adenosine 5'-diphosphate (ADP) or collagen (CLG) in vitro and in vivo. 3. LMS is more active in vivo than in vitro while acetylsalicylic acid (ASA) is more active in vitro than in vivo. It seems that in vivo LMS does not act by blocking thromboxane A2 formation only, but via participation of an endogenous factor. 4. The release of LMS-induced anti-thrombotic factor is inhibited by ASA pretreatment, indicating to be a cyclooxygenase metabolite of arachidonic acid. 5. The LMS-induced anti-thrombotic factor has a t1/2 of 3.6 +/- 0.8 min that is similar to the t1/2 of synthetic prostacyclin (PGI2) tested in our system (3.9 +/- 0.5 min; P = NS). 6. The release of PGI2-like substance from vascular tissue is LMS dose-dependent.     

Effects of salicylic acid derivatives on red blood cell membranes

Salicylamide, sodium salicylate and acetylsalicylic acid are salicylic acid derivates. They differ in their substitution on the benzene ring and may have different effects on membranes. Red blood cells were used as a prototypical cellular system regarding drug mediated plasma bilayer effects. Established photometric methods sensing tiny changes of red blood cell morphology at rest (red blood cell shape) and at very low shear forces (red blood cell stiffness, red blood cell relaxation time) were applied. The derivative induced effects were detected in a time- and dose-dependent manner. Salicylamide induced a most pronounced echinocytic shape at 5 mM. The shape effect was smaller above as well as below 5 mM. Sodium salicylate induced echinocytes with increasing concentrations showing a saturation above 10 mM. In contrast, the shape was not affected by acetylsalicylic acid. All shape changes occurred within 2 min, and were reversible. The above tendencies were in parallel to a slight red blood cell stiffening. The relaxation time continuously increased with increasing concentrations in both salicylamide and sodium salicylate, with salicylamide always acting stronger. Acetylsalicylic acid again showed no effect. We hypothesize that the observed effects of sodium salicylate and salicylamide are due to their phenolic character mediating a molecular hydrophobicity. According to the bilayer couple hypothesis this would lead to an insertion into the red blood cells outer plasma bilayer leaflet. The extension induced here would cause a positive membrane bending leading to echinocytic shapes and the observed loss of red blood cell fluidity. In contrast, the hydrophilic aspirin would penetrate and thus not affect the red blood cell plasma membrane.     

Ex vivo assay to determine the cyclooxygenase selectivity of non-steroidal anti-inflammatory drugs.

1. In this study we describe experiments to establish ex vivo the selectivity of non-steroidal anti-inflammatory drugs (NSAIDs) given in vivo. 2. Anaesthetised (Inactin, 120 mg kg(-1)) male Wistar rats (220-250 g) received an i.v. dose of one of the following compounds (dose mg kg(-1)): aspirin (20), diclofenac (3), L-745,337 (30), nimesulide (15), salicylate (20), sulindac (10). Blood samples were taken before and up to 6 h after dosing and the plasma obtained from it was tested for its ability to inhibit prostanoid formation in IL-1beta-treated A549 cells (COX-2 system) and human washed platelets (COX-1 system). For control the same compounds were also added directly to the assay systems. 3. All drugs, except sodium salicylate, inhibited COX-1 and COX-2 when added directly to the test systems. Plasma from aspirin-treated rats was without effect on either COX-1 or COX-2, consistent with the rapid in vivo metabolism to salicylate. Conversely, plasma from sulindac-treated rats inhibited COX-1 and COX-2 with potencies according with in vivo metabolism to sulindac sulphide. Diclofenac was COX-1/2 non-selective when tested in vitro, but a slightly preferential inhibitor of COX-2 when tested ex vivo. Nimesulide was confirmed as preferential inhibitor of COX-2 both in vitro and ex vivo. L-745,337 was a selective COX-2 inhibitor when tested in vitro or ex vivo. 4. In conclusion, our experiments show clearly (a) NSAIDs inactivation, (b) activation of prodrugs, and (c) NSAIDs selectivity. Our assay provides useful information about the selectivity of NSAIDs that could be extended by the analysis of plasma samples taken from humans similarly treated with test drugs.     

Effects of Salicylic Acid Derivatives on Red Blood Cell Membranes

Abstract Salicylamide, sodium salicylate and acetylsalicylic acid are salicylic acid derivates. They differ in their substitution on the benzene ring and may have different effects on membranes. Red blood cells were used as a prototypical cellular system regarding drug mediated plasma bilayer effects. Established photometric methods sensing tiny changes of red blood cell morphology at rest (red blood cell shape) and at very low shear forces (red blood cell stiffness, red blood cell relaxation time) were applied. The derivative induced effects were detected in a time- and dose-dependent manner. Salicylamide induced a most pronounced echinocytic shape at 5 mM. The shape effect was smaller above as well as below 5 mM. Sodium salicylate induced echinocytes with increasing concentrations showing a saturation above 10 mM. In contrast, the shape was not affected by acetylsalicylic acid. All shape changes occurred within 2 min. and were reversible. The above tendencies were in parallel to a slight red blood cell stiffening. The relaxation time continuously increased with increasing concentrations in both salicylamide and sodium salicylate, with salicylamide always acting stronger. Acetylsalicylic acid again showed no effect. We hypothesize that the observed effects of sodium salicylate and salicylamide are due to their phenolic character mediating a molecular hydrophobicity. According to the bilayer couple hypothesis this would lead to an insertion into the red blood cells outer plasma bilayer leaflet. The extension induced here would cause a positive membrane bending leading to echinocytic shapes and the observed loss of red blood cell fluidity. In contrast, the hydrophilic aspirin would penetrate and thus not affect the red blood cell plasma membrane.     

Effect of triflusal and acetylsalicylic acid on platelet aggregation in human whole blood: influence of red blood cells and leukocytes

A study has been made on the in vitro effect of triflusal, acetylsalicylic acid (ASA and their major metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB), and salicylic acid (SA), on platelet aggregation in human whole blood. SA exhibited no significant antiplatelet effects (IC50 greater than 2mM) against several inducers; the IC50 values for the other compounds were: triflusal, 140 microM against ADP and 63.2 microM against collagen; HTB, 100 microM against ADP and 260 microM against collagen; ASA 687 microM against ADP and 9.3 microM against collagen. Red blood cells potentiate the antiaggregant effect of HTB and of triflusal, and to a lesser extent, that of ASA; leukocytes primarily potentiate the effect of ASA and, to a lesser extent, that of triflusal.     

Inhibitory mechanism of ifenprodil tartrate on rabbit platelet aggregation

The effects of dl-erythro-4-benzyl-alpha-(4-hydroxyphenyl)-beta-methyl-l-piperidine-eth anol tartrate (ifenprodil tartrate) on rabbit platelet aggregation in vitro and ex vivo were studied. Ifenprodil tartrate inhibited platelet aggregation in vitro induced by ADP, collagen and epinephrine. It also inhibited 5-hydroxytryptamine (5-HT) uptake into platelets and 5-HT release from platelets. Since these inhibitory effects of ifenprodil tartrate on the functions of rabbit platelets were similar to the effects of imipramine, the effects of ifenprodil tartrate may be due to the stabilizing action of ifenprodil tartrate on the platelet membrane. The platelet aggregation by ADP was significantly inhibited in rabbits after oral administration of ifenprodil tartrate, the maximal plasma level of ifenprodil being reached at 20 ng/ml ex vivo, while the maximal level was only 1/40 of the minimal concentration of ifenprodil tartrate necessary to inhibit platelet aggregation in vitro. These results indicate that factors other than ifenprodil tartrate acting directly on the platelets (e.g., PGI2 which is an endogenous inhibitor of platelet aggregation) are involved in inducing the inhibitory effects of ifenprodil tartrate on platelet aggregation ex vivo. The effects of ifenprodil tartrate on both PGI2 release from the aorta and the inhibitory effects of PGI2 on platelet aggregation in vitro were investigated: PGI2 was found to intensify the inhibitory effects of ifenprodil tartrate on platelet aggregation in vitro, but there was little effect, if any, on PGI2 release. Therefore, it is considered that the ex vivo effects of ifenprodil tartrate might be due to its interaction with endogenous PGI2 in the blood.     

In vivo platelet aggregation in the rat: dependence on extracellular divalent cation and inhibition by nonsteroidal anti-inflammatory drugs.

Using the Technicon Autocounter, the mechanisms involved in collagen-induced platelet aggregation in vivo have been studied without the interference of an anticoagulant. Extracellular divalent cation was essential for in vivo platelet aggregation. Non-steroidal anti-inflammatory drugs completely inhibited the aggregation induced by collagen in platelet-rich plasma in in vitro or ex vivo studies. In vivo only a maximum of 50% inhibition was achieved when release of thromboxane A2 (TXA2) was completely inhibited. Therefore in vivo, collagen causes aggregation through more than one pathway which operate independently of each other and which are all dependent on extracellular divalent cation. In vivo, when different doses of collagen were compared, aggregation produced by low doses of collagen was more dependent upon prostaglandin endoperoxide/TXA2 formation.