Sodium arachidonate can induce platelet shape change and aggregation which are independent of the release reaction.

Sodium arachidonate causes shape change and aggregation of rabbit or human platelets that have been washed and then degranulated by treatment with thrombin. Since these platelets do not contain releasable adenosine diphosphate (ADP) and the aggregation is not inhibited by the creatine phosphate-creatine phosphokinase system, sodium arachidonate must be able to cause aggregation that is independent of the release of ADP. Since aggregation of these platelets induced by sodium arachidonate is inhibited by acetylsalicylic acid or indomethacin, it seems likely that products (such as prostaglandin G2) formed from sodium arachidonate are responsible for aggregation. Thus, sodium arachidonate-induced shape change and aggregation of platelets may be caused (i) by the release of ADP by products of sodium arachidonate metabolism and (ii) directly by the products of sodium arachidonate metabolism, independently of released ADP.

Reversal of shortened platelet survival in rats by the antifibrinolytic agent, epsilon aminocaproic acid.

Platelet survival in rabbits and rats is shortened by placing indwelling catheters in the aorta; this shortening appears to be at least partly related to the extent of vessel wall injury and platelet interaction with the repeatedly damaged wall. Treatment of rabbit platelets with plasmin and other proteolytic enzymes in vitro shortens their survival when they are returned to the circulation. Because platelets may be exposed to plasmin and other proteolytic enzymes in rabbits and rats with indwelling aortic catheters, we examined the effect of epsilon-aminocaproic acid (EACA) on platelet survival in rats. At a dose of 1 g/kg every 4 h, EACA significantly reduced whole blood fibrinolytic activity and prolonged the shortened platelet survival in rats with indwelling aortic catheters. Mean platelet survival for untreated rats with indwelling aortic catheters was 38.6 +/- 1.9 h, and for rats treated with EACA, 53.8 +/- 3.8 h. Scanning electron microscopy showed that the injured vessel wall of these animals was mainly covered with platelets and fibrin, whereas in control animals that did not receive EACA, the injured surface was mainly covered with platelets and little fibrin was observed. Thus shortened platelet survival during continuous vessel wall injury may result from the local generation of plasmin or the release of proteolytic enzymes at sites where platelets (and possibly leukocytes) interact with the vessel wall.

Reduced membrane fluidity in platelets from diabetic patients.

Platelets from diabetic patients are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function, and reduced membrane fluidity in cholesterol-enriched platelets is associated with platelet hypersensitivity to agonists, including thrombin. Decreased membrane fluidity of these platelets is attributed to an increased cholesterol-phospholipid molar ratio in platelet membranes. We examined the response of platelets from diabetic subjects to thrombin, platelet membrane fluidity, and platelet cholesterol-phospholipid molar ratio. Twelve poorly controlled diabetic subjects were compared with 12 age- and sex-matched control subjects. In response to a low concentration of thrombin, mean values for release of [14C]serotonin from washed prelabeled platelets were not significantly different between diabetic and control subjects, but in 8 of 12 diabetic subjects, the release response was greater than in their paired control subjects. Mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5-hexatriene-labeled platelets prepared from diabetic subjects were significantly greater than in control subjects; this indicates a decreased membrane fluidity in platelets from diabetic subjects. Total or very-low-density (VLDL), low-density (LDL), or high-density (HDL2, HDL3) lipoprotein cholesterol concentrations in plasma were not significantly different between groups; however, the ratio of VLDL + LDL to HDL2 + HDL3 was significantly greater in diabetic than in control subjects. There was no difference in the total platelet cholesterol-phospholipid molar ratio between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dietary saturated fat and cholesterol on platelet function, platelet survival and response to continuous aortic injury in rats.

Induction of hypercholesterolemia in rats by diets containing milk fat, cholesterol and taurocholate caused increased sensitivity of platelets to thrombin-induced aggregation and release, but not to ADP- or collagen-induced aggregation or release. This hypersensitivity to thrombin persisted in the presence of CP/CPK to convert released ADP to ATP, and aspirin to block formation of thromboxane A2. The increased sensitivity of platelets to thrombin in hypercholesterolemic animals was associated with an increase in 18:1 omega 9, 18:2 omega 6 and 20:3 omega 6 and a decrease in 20:4 omega 6 and 22:4 omega 6 in their phospholipids. Hypercholesterolemic animals also had a shortened platelet survival that did not appear to be due to an alteration in the lipid composition of the platelets. The diet-induced changes in platelet function were not associated with enhanced thrombosis in animals with indwelling aortic catheters, but were associated with increased platelet accumulation on the exposed subendothelium.

Platelet function and survival in rats with genetically determined hypercholesterolaemia.

Platelets from rats with genetically determined hypercholesterolaemia are hypersensitive to aggregation induced by thrombin compared with platelets from their genetic controls without hypercholesterolaemia. Aggregation or release induced by thrombin of platelets from hypercholesterolaemic and control rats correlated significantly with plasma cholesterol concentrations. Platelet responses to ADP or collagen were not different between the groups. The hypersensitivity to thrombin-induced aggregation was independent of released ADP or products of arachidonic acid metabolism. The changes in platelet sensitivity occurred with only moderate increases in plasma cholesterol concentration and with no detectable changes in total platelet cholesterol. The hypersensitivity of platelets from hypercholesterolaemic rats was not associated with a reduction in platelet survival or any significant injury to the aortic endothelium in these animals. Platelets from hypercholesterolaemic rats were smaller than platelets from controls. Thus, platelets from rats with genetically determined hypercholesterolaemia have alterations in function similar to those found with platelets from rats with diet-induced hypercholesterolaemia indicating that this strain can be used to study the mechanisms by which cholesterol can change platelet function without the possible complicating effects of dietary factors. Since platelet hypersensitivity occurred in rats with genetically determined hypercholesterolaemia without a reduction in platelet survival, these studies are also consistent with the reduced platelet survival found in animals with diet-induced hypercholesterolaemia being independent of platelet changes.

Thrombin binding to platelets from hypercholesterolaemic rats.

Platelets from rats made hypercholesterolaemic with a diet enriched with milk fat and cholesterol and containing taurocholate to promote hypercholesterolaemia aggregated more extensively to a low concentration of thrombin than platelets from rats given a milk fat-enriched diet containing sitosterol. Total and specific binding of thrombin to platelets from hypercholesterolaemic rats was significantly greater than in controls when expressed per mg platelet protein, per mumol platelet cholesterol, or per unit relative surface area. Total and specific binding of thrombin per platelet were not different between the groups. However, platelets from hypercholesterolaemic rats had less protein and cholesterol, were smaller and had less surface area than control platelets; platelet cholesterol content expressed per mg platelet protein was not different. Thus, the increase in thrombin-binding to the smaller platelets from hypercholesterolaemic rats during the first 10 s after its addition may be responsible, at least in part, for the hypersensitivity of these platelets to thrombin.

Effect of the amount and type of dietary fat on platelet function, platelet survival and response to continuous aortic injury in rats.

The effect of giving diets containing 1.5 or 16% safflower or corn oil or 16% milk fat for 15 weeks on changes in the fatty acid composition of platelet phospholipids, in vitro platelet function, platelet survival and thrombosis was examined in rats. The mean plasma cholesterol concentration was not different among the groups. Diets containing 1.5% safflower or corn oil or 16% milk fat were associated with a decrease in 18:2n - 6 and an increase in 18:1n - 9 and the 20:4n - 6/18:2n - 6 ratio in the platelet phospholipids compared with the 16% safflower or corn oil diets. The 16% milk fat diet was associated with an increase in 14:0, 20:3n - 9, 22:3n - 9 and a decrease in 22:4n - 6 in platelet phospholipids compared with the other groups. There were no differences among the groups in the sensitivity of washed platelets to ADP-, thrombin- or collagen-induced aggregation, or thrombin- or collagen-induced release of granule contents or loss of arachidonate from platelet phospholipids. Platelet survival and turnover in rats given the diets were not different among the groups. In response to indwelling aortic catheters neither the percentage reduction in platelet survival nor the platelet accumulation on injured aortae and catheters were different among the groups. No macroscopic thrombi were seen in rats given any of the diets. The results of these studies provide no evidence that diet-induced alterations in fatty acid content (increases in 18:1n - 9, 20:3n - 9, 22:3n - 9, 20:3n - 6, and 20:4n - 6/18:2n - 6 ratio and a decrease in 22:4n - 6) of platelet phospholipids modify in vitro platelet function, platelet survival or turnover or influence thrombosis in rats.

Aspirin in the treatment of cardiovascular disease: a review.

Large-scale clinical trials of the use of aspirin in post-myocardial infarction patients were based on the assumption that inhibition of platelet activity would reduce thromboembolism associated with atherosclerosis, and that thromboembolism is a major cause of the clinical complications of atherosclerosis. However, spasm and occlusive thrombi may also contribute to this picture, and thus thromboembolism is probably only one of the mechanisms that cause the clinical complications. Aspirin inhibits thrombosis only if thromboxane A2 formation by platelets plays a major part in the growth of thrombi; aspirin has little effect on thrombosis when thrombin generation and fibrin formation are dominant factors. Nevertheless, analysis of the combined data from the six clinical trials indicates a highly significant (21 percent) reduction in reinfarction rate and a 16 percent reduction in cardiovascular mortality rate in patients treated with aspirin. Aspirin may be most useful in treating an as-yet-unidentified subgroup of patients.

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets.

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.

Prolonged expression of procoagulant activity of human platelets degranulated by thrombin.

Platelets are exposed to thrombin when they take part in arterial thrombus formation, and they may return to the circulation when they are freed by fibrinolysis and dislodged by flowing blood. Thrombin causes the expression of procoagulant activity on platelets, and if this activity persists, the recirculating platelets may contribute to subsequent thrombosis. We have developed techniques to degranulate human platelets by treatment with thrombin, and recover then as single, discrete platelets that aggregate in response to both weak and strong agonists. In the present study we examined the duration of procoagulant activity on the surface of thrombin-degranulated platelets by two methods: a prothrombinase assay, and the binding of 125I-labeled annexin. Control platelets generated 0.9 +/- 0.4 U thrombin per 10(7) platelets in 15 min. Suspensions of thrombin-degranulated platelets formed 5.4 +/- 0.1 U thrombin per 10(7) platelets in this time. Binding of 125I-annexin V was also greater with thrombin-treated platelets than with control platelets (controls: 1.7 +/- 0.1 ng annexin/10(7) platelets; thrombin-degranulated platelets: 6.8 +/- 0.2 ng annexin/10(7) platelets). With thrombin-degranulated platelets, increased procoagulant activity and annexin binding persisted for at least 4 h after degranulation and resuspension, indicating that the catalytic activity for the prothrombinase complex is not reversed during this time. These platelets maintained their ability to aggregate for 4 h, even in response to the weak agonist, ADP. Thus, platelets that have taken part in thrombus formation and returned to the circulation may contribute to the promotion of further thrombotic events because of the persistence of procoagulant activity on their surface.

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)

Contrasting effects of thrombin and the thrombin receptor peptide, SFLLRN, on aggregation and release of 14C-serotonin by human platelets pretreated with chymotrypsin or serratia marcescens protease.

Chymotrypsin cleaves glycoprotein Ib (GPIb) on platelets and reduces their responsiveness to thrombin; platelets from patients with the Bernard-Soulier syndrome, which lack GPIb, are also less responsive to thrombin than platelets from normal donors. However, Bernard-Soulier platelets respond normally to the thrombin receptor peptide SFLLRN (13). We compared responses of 14C-serotonin-labeled, chymotrypsin-treated platelets (and control platelets) to thrombin (0.25-2 U/ml) and SFLLRN (5-40 microM). Chymotrypsin treatment strongly inhibited thrombin-induced aggregation and release of 14C-serotonin when concentrations of thrombin of 0.5 U/ml or lower were used, even though these responses of control platelets remained near the maximum. In contrast, there was little difference between the responses of control and chymotrypsin-treated platelets to SFLLRN, even when the responses of control platelets were less than maximal. Thus, chymotrypsin treatment greatly inhibits the response to thrombin of the seven transmembrane domain thrombin receptor cloned by Coughlin's group (1, 2). Since Serratia marcescens protease also hydrolyses GPIb, but has less effect than chymotrypsin on other glycoproteins, we pretreated platelets with several concentrations of S. marcescens protease. Concentrations that abolished aggregation and release of 14C-serotonin in response to thrombin had little effect on these responses to SFLLRN. One interpretation of these findings would be that by cleaving GPIb, both proteases are affecting an interaction that may be important for activation of the cloned receptor by thrombin, but irrelevant to activation of this receptor by SFLLRN.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine-induced aggregation of rabbit platelets refractory to ADP.

The mechanisms involved in platelet aggregation induced by epinephrine are unclear. Although epinephrine does not aggregate washed rabbit platelets, platelets made refractory to ADP will aggregate in response to epinephrine in the presence of ADP. We have examined whether the mechanism(s) by which epinephrine induces aggregation of refractory platelets involves fibrinogen binding and Ca2+ association. With normal platelets, ADP causes aggregation, fibrinogen binding and Ca2+ association in a medium containing 0.2 mM 45Ca2+. After 3 min of incubation with ADP, fibrinogen dissociates from platelets, but 45Ca2+ does not. Epinephrine alone does not cause aggregation, fibrinogen binding or 45Ca2+ association. Platelets that are refractory to ADP do not aggregate and bind fibrinogen upon addition of ADP, but aggregate and bind fibrinogen in response to epinephrine, provided ADP is still present. These effects of epinephrine are mediated by the alpha-adrenergic receptor since they are blocked by phentolamine or verapamil and potentiated by propranolol. However, epinephrine-induced aggregation of platelets refractory to ADP does not involve further detectable increase in the amount of 45Ca2+ associated with the platelets.

The effect of phospholipase inhibitor mepacrine on platelet aggregation, the platelet release reaction and fibrinogen binding to the platelet surface.

We have examined whether inhibition by mepacrine or freeing of arachidonic acid from platelet phospholipids inhibits platelet aggregation to collagen, thrombin or ADP, and the release reaction induced by thrombin or collagen. Loss of arachidonic acid was monitored by measuring the amount of 14C feed from platelets prelabelled with 14C-arachidonic acid. Mepacrine inhibited 14C loss by more than 80% but did not inhibit thrombin-induced platelet aggregation and had a small effect on release. ADP-induced platelet aggregation did not cause 14C loss. Mepacrine inhibited ADP-induced platelet aggregation by inhibiting the association of fibrinogen with platelets during aggregation. The effect of mepacrine on fibrinogen binding could be considerably decreased by washing the platelets but the inhibition of 14C loss persisted. Platelets pretreated with mepacrine and then washed show restoration of aggregation to collagen. Thus, mepacrine has two effects; 1. it inhibits phospholipases, 2. it inhibits fibrinogen binding. Freeing of arachidonic acid is not necessary for platelet aggregation or the release reaction.

Membrane fluidity is related to the extent of glycation of proteins, but not to alterations in the cholesterol to phospholipid molar ratio in isolated platelet membranes from diabetic and control subjects.

Platelets from diabetic subjects are hypersensitive to aggregating agents in vitro. Membrane fluidity modulates cell function and we previously reported reduced membrane fluidity associated with hypersensitivity to thrombin in intact platelets from diabetic subjects. Reduced membrane fluidity and hypersensitivity to agonists has also been reported in platelets from non-diabetic subjects whose platelets have an increased cholesterol/phospholipid molar ratio. Glycation of platelet membrane proteins is enhanced in diabetic subjects, and could contribute to the decreased membrane fluidity in these platelets. We examined the relation among fluidity, cholesterol/phospholipid molar ratio, and glycation of proteins in isolated platelet membranes from diabetic and control subjects. Seven poorly controlled diabetic subjects were compared with 7 age- and sex-matched control subjects. The mean steady-state fluorescence polarization value in 1,6-diphenyl-1,3,5-hexatriene-labeled isolated platelet membranes from diabetic subjects (0.184 +/- 0.004) was significantly greater than from control subjects (0.171 +/- 0.004, p less than 0.01); thus, fluidity in platelet membranes from diabetic subjects is decreased. Reduced fluidity in platelet membranes from diabetic subjects could not be attributed to changes in the cholesterol/phospholipid molar ratio. Total or very low density (VLDL), low density (LDL), or high density (HDL3) lipoprotein cholesterol concentration in plasma was not significantly different between groups, but the ratio of VLDL+LDL to HDL2 + HDL3 cholesterol was significantly greater in diabetic subjects (4.79 +/- 0.73) than in control subjects (2.54 +/- 0.30, p less than 0.02). Proteins were glycated significantly more extensively in platelet membranes from diabetic subjects (25.5 +/- 0.9 nmol glucose/mg protein) than those from control subjects (21.0 +/- 0.6 nmol glucose/mg protein, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of amino sugars on platelet aggregation and on fibrinogen binding.

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.

Degranulation of human platelets by the thrombin receptor peptide SFLLRN: comparison with degranulation by thrombin.

A new, simplified method of degranulating human platelets using the thrombin receptor peptide SFLLRN (20 microM) is described; released fibrinogen cannot be converted to fibrin, and the platelets are not exposed to a proteolytic enzyme, as they are when thrombin is used for degranulation. The peptide-degranulated platelets regain their disc shape and are recovered as single platelets which have released approximately 90% of the contents of their dense granules. Their procoagulant activity is greater than that of control platelets, but somewhat less than that of thrombin-degranulated platelets. Without added fibrinogen, the peptide-degranulated platelets aggregate slightly in response to 50 microM SFLLRN, and to collagen, arachidonic acid, the thromboxane A2 mimetic U46619, platelet activating factor, ADP, and the divalent cation ionophore A23187; added fibrinogen enhances aggregation caused by these agonists. Extensive aggregation of peptide-degranulated platelets is caused by thrombin in the absence of added fibrinogen; it may be that the alternative thrombin receptor that is not activated by SFLLRN is responsible for the strong response to thrombin. Aggregation responses to most of the agonists are greater than those observed previously with thrombin-degranulated platelets. By this method, platelets are obtained that have been degranulated in a way that is similar to in vivo degranulation. They are useful for studies of platelet responses without the complicating effects of released granule contents, and for investigation of the characteristics and functions of platelets that have come in contact with release-inducing agents in vivo.

The effect of antithrombin III-independent thrombin inhibitors and heparin on fibrin accretion onto fibrin-coated polyethylene.

Prosthetic vascular grafts become coated with a layer of fibrin that contributes to graft thrombosis and occlusion. We compared the effect of antithrombin III-independent inhibitors of thrombin with heparin for their ability to prevent fibrin accretion onto a model of a vascular graft formed in vitro by coating polyethylene tubing with thrombin bound to a layer of polymerized fibrin. Equivalent antithrombin concentrations of heparin, D-Phe-Pro-Arg CH2Cl (PPACK), recombinant hirudin (r-hirudin), and Hirulog-1 were added to barium chloride-absorbed plasma containing radiolabelled fibrinogen. Whereas, PPACK and r-hirudin persistently inhibited fibrin accretion, the inhibition by heparin was transient. Hirulog-1 had no effect on early fibrin accretion and was actually associated with enhanced accretion at 30 min (control 11.7 +/- 2.0 micrograms fibrin/cm2; Hirulog-1, 18.4 +/- 3.5 micrograms fibrin/cm2, p < 0.001). Both Hirulog-1 and r-hirudin displaced radiolabelled thrombin from the fibrin surface. Whereas hirudin-thrombin complexes are stable, Hirulog-1 produces only transient inhibition of the displaced thrombin thereby accounting for the enhanced fibrin accretion with this anticoagulant. These studies show that the antithrombin III-independent inhibitors, r-hirudin and PPACK, are more effective inhibitors of fibrin accretion onto fibrin-coated polyethylene than heparin or Hirulog-1. In addition, they emphasize the importance of determining the ability of anticoagulants to displace thrombin from fibrin and to form stable thrombin-inhibitor complexes; lack of stability of thrombin-inhibitor complexes must be countered by levels of anticoagulant that are adequate to maintain its effectiveness.

Ticlopidine facilitates the deaggregation of human platelets aggregated by thrombin.

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80-90% 14C-serotonin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5x the activity of thrombin) and PGE1 (10 mumol/l) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 mumol/l) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Pretreatment of human platelets with plasmin inhibits responses to thrombin, but potentiates responses to low concentrations of aggregating agents, including the thrombin receptor activating peptide, SFLLRN.

Effects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37 degrees C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2Cl (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.