Snake Venom Constituents that Affect Platelet Function

Snake venoms contain complex mixtures of proteins with biological activities. These venom proteins affect blood coagulation and platelet function in various ways. Many inducers and inhibitors of platelet aggregation have been isolated from snake venoms, especially from the Crotalidae and Viperidae families. According to their biochemical properties and modes of action, they can be classified into ten groups: (1) thrombin-like enzymes which show higher activity towards platelets than towards fibrinogen; (2) procoagulant enzymes which generate thrombin and then activate platelets indirectly; (3) noncoagulant glycoproteins which activate platelets independently of ADP release or thromboxane formation; (4) lectin-like peptides which cause agglutination of platelets through binding to a sugar moiety; (5) coagglutinins whose activities are dependent on the presence of von Willebrand factor (vWF); (6) membrane-active polypeptides which potentiate the aggregating action of other inducers by activation of platelet endogenous phospholipase A(2); (7) phospholipase A(2) enzymes which show biphasic aggregating and inhibitory effects on platelets; (8) α-fibrinogenases which degrade the α (A) chain of fibrinogen; (9) 5'-nucleotidase or ADPase which act on the ADP released from platelet dense bodies; and (10) fibrinogen-receptor antagonists which interfere with the interaction of fibrinogen and glycoprotein IIb/IIIa on activated platelets. These venom proteins are unique research tools for study of the haemostatic process and some of them are potential antithrombotic agents.     

Characterization of the effect of plasma lipoproteins on platelet function in vitro

Thrombin-induced platelet activation is enhanced by very low and low density lipoproteins but decreased by high density lipoprotein. Plasma lipoproteins maximally affect platelet aggregation and 14C-serotonin release in a gel-filtered platelet preparation within 10 min of incubation at 37 degrees C. This effect is saturable and physiologic concentrations of lipoproteins are required in order to attain this saturation. When no aggregating agent is added to the incubation medium, the lipoproteins alone did not alter platelet aggregation. However, 14C-serotonin release is increased by very-low- and low-density lipoproteins alone more than by high density lipoprotein. On removal of the lipoproteins after incubation with the platelets, and subsequent testing of platelet function, minimal influence of these lipoproteins on the platelet function remains. Arachidonic acid causes similar results to thrombin when added to the platelet suspension after incubation with the lipoprotein. Our results further emphasize the 'opposing effects' of very low and low density lipoproteins as compared to high density lipoproteins on platelets and/or platelet-thrombin interaction.     

Hematin: unique effects of hemostasis

Hematin is clinically useful in the treatment of acute intermittent porphyria. Recently, hematin-induced coagulopathy has been reported, and a patient we treated bled during hematin therapy. On 3 separate occasions, infusions of hematin (4 mg/kg) induced thrombocytopenia, prolongation of the prothrombin time, partial thromboplastin time. Reptilase time, and apparent decreases in fibrinogen and increases in fibrin(ogen) degradation products (FDP). However, fibrinogen assayed by heat precipitation was unchanged, the protamine paracoagulation test was negative, there was no red blood cell fragmentation, and plasminogen and antithrombin III remained normal, excluding the presence of disseminated intravascular coagulation. Furthermore, premedication with heparin, 5000 U i.v., failed to prevent the lengthening of the Reptilase time and exacerbated the thrombocytopenia. In vitro studies revealed that hematin, 0.1 mg/ml, aggregated platelets and induced the release of 14C-serotonin and adenosine triphosphate (ATP). Hematin also aggregated washed or gel-filtered platelets but had no effect on formalin-fixed platelets. Aggregation was inhibited by aspirin (0.12 mg/ml), adenosine triphosphate, and apyrase, suggesting that hematin aggregated platelets by inducing adenosine diphosphate (ADP) release. Hematin (0.07 mg/ml) progressively inactivated thrombin and 0.1 mg/ml prolonged the Reptilase time. Thus, hematin is unique in that it both induces platelet aggregation and inhibits coagulation.     

Concentration effects of platelets, fibrinogen and thrombin on platelet aggregation and fibrin clotting

The effects of varying concentrations of platelets, fibrinogen and thrombin on platelet aggregation and on fibrin clotting were investigated. The results indicated that a threshold thrombin to platelet concentration ratio may be required to cause platelet activation. Above the threshold ratio, platelets exhibited properties which enhanced thrombin action in causing aggregation and fibrin clotting. At T/P ratios below the threshold level, the presence of platelets reduced thrombin activity, in other words, platelets exerted an antithrombin action. Fibrinogen at low concentrations (0.02-1.5 mg/ml) enhanced platelet aggregation induced by thrombin; whereas, at high concentrations of fibrinogen (2.0-4.0 mg/ml), aggregation was markedly inhibited. Continuous mixing of samples of paltelets and fibrinogen at physiological concentrations with thrombin at low concentrations (less than 2.0 U/ml) resulted in platelet aggregation. On the other hand, fibrin clots formed in samples without mixing or with high thrombin concentrations (greater than or equal to 5.0 U/ml). These results suggested that the quantitative relationships between platelets, fibrinogen and thrombin, and the presence or absence of cell contact may be important factors in determining the overall hemostasis.     

Thrombin interaction with human platelets. Potentiation of thrombin-induced aggregation and release by inactivated thrombin

The possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombin-induced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.     

Disaggregatory effects of prostaglandin E1, amrinone and milrinone on platelet aggregation in human whole blood

Prostaglandin E1 (PGE1), amrinone and milrinone not only inhibit platelet aggregation, but also potentially induce disaggregation. We examined the disaggregatory effects of PGE1, amrinone and milrinone on platelet aggregation, and measured the platelet cyclic adenosine monophosphate (cAMP) levels using a standard radioimmunoassay. Platelet aggregation was induced by 10 microM adenosine diphosphate (ADP) or 0.625 microg/ml collagen that induces a secondary aggregation, and measured by whole blood aggregometry. PGE1 (0.7-10.0 microM), amrinone (106.8-801.3 microM), or milrinone (9.5-190.0 microM) was added when aggregation reached 5 mohm of impedance, and the percentage of disaggregation was measured 5 min after initiating disaggregation. Disaggregatory effects were also examined using a light transmission aggregometer and gel-filtered platelets. In platelet aggregation induced by ADP, the drug concentrations required to cause 50% disaggregation (EC50) were PGE1; 3.1+/-1.2 microM, amrinone; 313.1+/-128.8 microM, milrinone; 57.1+/-20.8 microM. In platelet aggregation induced by collagen, the maximum disaggregation percentages were 15.6+/-5.0% for 10 microM PGE1, 7.8+/-2.4% for 801.3 microM amrinone, and 5.9+/-2.3% for 190.0 microM milrinone. The EC50 concentrations of platelet cAMP that caused 50% disaggregation in ADP-induced platelet aggregation were 67+/-13 pmoles/10(8) platelets for PGE1, 54+/-12 pmoles/10(8) platelets for amrinone, and 52+/-12 pmoles/10(8) platelets for milrinone. In gel-filtered platelets, the percentages of disaggregation in ADP- or collagen-induced platelet aggregation were 33.1+/-5.2% or 11.2+/-3.1% for PGE1 (10 microM), 22.3+/-4.1% or 15.2+/-3.2% for amrinone (801.3 microM), and 23.5+/-4.3% or 14.6+/-3.5% for milrinone (190.0 microM).     

Disaggregatory effects of prostaglandin E 1 , amrinone and milrinone on platelet aggregation in human whole blood

Prostaglandin E 1 (PGE 1 ), amrinone and milrinone not only inhibit platelet aggregation, but also potentially induce disaggregation. We examined the disaggregatory effects of PGE 1 , amrinone and milrinone on platelet aggregation, and measured the platelet cyclic adenosine monophosphate (cAMP) levels using a standard radioimmunoassay. Platelet aggregation was induced by 10 w M adenosine diphosphate (ADP) or 0.625 w g/ml collagen that induces a secondary aggregation, and measured by whole blood aggregometry. PGE 1 (0.7-10.0 w M), amrinone (106.8-801.3 w M), or milrinone (9.5-190.0 w M) was added when aggregation reached 5 m z of impedance, and the percentage of disaggregation was measured 5 min after initiating disaggregation. Disaggregatory effects were also examined using a light transmission aggregometer and gel-filtered platelets. In platelet aggregation induced by ADP, the drug concentrations required to cause 50% disaggregation (EC 50 ) were PGE 1 ; 3.1 - 1.2 w M, amrinone; 313.1 - 128.8 w M, milrinone; 57.1 - 20.8 w M. In platelet aggregation induced by collagen, the maximum disaggregation percentages were 15.6 - 5.0% for 10 w M PGE 1 , 7.8 - 2.4% for 801.3 w M amrinone, and 5.9 - 2.3% for 190.0 w M milrinone. The EC 50 concentrations of platelet cAMP that caused 50% disaggregation in ADP-induced platelet aggregation were 67 - 13 pmoles/10 8 platelets for PGE 1 , 54 - 12 pmoles/10 8 platelets for amrinone, and 52 - 12 pmoles/10 8 platelets for milrinone. In gel-filtered platelets, the percentages of disaggregation in ADP- or collagen-induced platelet aggregation were 33.1 - 5.2% or 11.2 - 3.1% for PGE 1 (10 w M), 22.3 - 4.1% or 15.2 - 3.2% for amrinone (801.3 w M), and 23.5 - 4.3% or 14.6 - 3.5% for milrinone (190.0 w M).     

Platelet Aggregation and Release by ADP and Thrombin: Evidence for Two Separate Effects of ADP on Platelets, Involvement of Fibrinogen in Release, and Mechanism of Inhibitory Action of Acetylsalicylic Acid

S ummary . The previously demonstrated roles of thrombin and fibrinogen in platelet interaction (Ardlie & Han, 1974) suggested a possible relationship between platelet aggregation and the release reaction. The present results show that a product of the action of thrombin on fibrinogen induced the release of platelet constituents. This activity was not inhibited by heparin. The thrombin-fibrinogen reaction which occurs when platelets come into contact could be the cause of the subsequent release reaction. ADP was shown to be essential for the thrombin-induced aggregation, but not the alteration in platelet morphology. Evidence of involvement of ADP in cell contact was obtained and it is possible that its role may be to facilitate aggregation through an effect on platelet surface charge. Thus, ADP induces aggregation through two distinct actions: (1) alteration of platelet morphology; and (2) reduction in net negative surface charge of platelets. Incubation of thrombin with aspirin inhibited its action on platelets and it is suggested that acetylation of thrombin (or prothrombin) by aspirin may account for the action of this compound on platelets. The significance of these observations and a possible mechanism for the release reaction is discussed.     

Blood Clotting Activity of Reptilase and Bovine Thrombin in Vitro: A Comparative Study on Seven Different Species

S ummary . The clotting activities of Reptilase and purified bovine thrombin towards platelet poor plasma from seven species and purified human and rabbit fibrinogen were studied. Bovine thrombin exhibited particularly low activity towards plasma from rat, guinea-pig and cow compared with the other species tested. Reptilase showed a relatively larger species dependent variation in activity than thrombin, and rabbit plasma was particularly resistant to Reptilase action. This was due to a low activity of Reptilase towards rabbit fibrinogen and not to inhibition of Reptilase in rabbit plasma.
The presence of 1% (w/v) dextran (70 000 mol wt) in the clotting mixtures significantly decreased both the thrombin and Reptilase clotting times in plasmas from all species.     

ADP and epinephrine-induced release of platelet fibrinogen

Human platelets gel-filtered into Tyrode's buffer containing 1 mM Mg++ and 0.35% bovine serum albumin were studied to determine whether they would undergo biphasic aggregation and release of alpha-granule proteins in response to adenosine diphosphate (ADP) or epinephrine without addition of exogenous fibrinogen. Fibrinogen concentration in the supernatant of unaggregated gel-filtered platelets was less than 1 pmole/ml. With addition of ADP or epinephrine, biphasic aggregation was seen, with release of platelet fibrinogen, beta-thromboglobulin, and platelet factor 4. Fibrinogen concentration in the supernatant after aggregation ranged from 15 to 70 pmole/ml. Release of the alpha-granule proteins by epinephrine was coincidental with release of the dense granule adenine nucleotides. Aggregation and alpha-granule protein release by both ADP and epinephrine were inhibited by added Ca++ at 1-- 2 mM. The ability of gel-filtered platelets to undergo ADP- and epinephrine-induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen may be able to fulfill the requirement for fibrinogen in ADP- and epinephrine-induced platelet aggregation and release.     

Crotalocytin: characterization of the timber rattlesnake platelet activating protein

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.     

Aggregation of Human Platelets by Bovine Platelet Fibrinogen

Aggregation of human platelets by addition of purified bovine platelet fibrinogen is described. Bovine plasma fibrinogen showed the same but much weaker effect. Human fibrinogen produced no aggregation. No absolute requirement for divalent cations or plasma proteins could be demonstrated. The aggregation of washed platelets appeared monophasic whereas in platelet-rich plasma it was usually biphasic. The use of inhibitors of ADP-induced platelet aggregation, inhibition of intracellular ATP-production, enzyme-catalyzed removal of ADP, and direct determinations of ADP in the medium showed the second phase to be mediated by ADP released from the platelets whereas the first phase was nearly independant of ADP. While the ability of platelet fibrinogen to aggregate platelets and to clot with thrombin were otherwise intimately interconnected, some aggregation activity remained after heat-denaturation of the platelet fibrinogen.     

Effect of fibrinogen concentration on the velocity of platelet aggregation

The relationship between fibrinogen binding to its receptor and platelet aggregation has been investigated by comparing 125I-fibrinogen binding and aggregation velocities of gel-filtered platelets in the presence of adenosine diphosphate (ADP). Aggregometric responses at various fibrinogen concentrations are found to be bell-shaped and show a maximum at fibrinogen concentrations (Fmax) similar to the 125I-fibrinogen hemisaturating doses. At higher and lower fibrinogen concentrations, platelet aggregation velocities decrease in a parallel manner. Lowering ADP concentration increases Fmax, in agreement with the modulatory effect of ADP on fibrinogen binding to platelets. Variations of fibrinogen in the range of physiopathologic plasma concentrations affect platelet aggregation induced by any ADP dose. These results clarify the relationship between the fibrinogen binding process and aggregation and demonstrate that plasma fibrinogen concentration has a major influence on the velocity of platelet aggregation.     

The effect of plasmin on platelet function

There is controversy in the literature regarding the effects of plasmin on human platelets. We have studied the effects of plasmin on platelet glycoproteins, aggregation, shape change and secretion and found them to be dependent on experimental conditions: (a) Plasmin's effects on human platelets are only seen in gel-filtered platelets (GFP), presumably because in platelet rich plasma plasmin is bound to a2-antiplasmin; (b) in GFP to which fibrinogen has been added, platelet function remains intact; and (c) in the absence of fibrinogen, the effect of plasmin on GFP depends on whether stirring is performed or not. With stirring, platelets undergo shape change, secretion and aggregation in response to added plasmin. Aggregation is much stronger when CaCl(z) 1 mM is added. Without stirring, preincubation of GFP with plasmin leads to inhibition of platelet aggregation induced by subsequent platelet stimuli (thrombin, collagen, ristocetin or U46619). We have demonstrated that plasmin is a true platelet activating agent, in the sense that it induces platelet shape change and secretion. Plasmin will induce aggregation when added to stirred GFP. This may be because stirring protects glycoprotein (GP) IIbIIIa bound fibrinogen from being degradated by plasmin. When added to unstirred GFP, GP IIbIIIa bound fibrinogen may be readily accessible to degradation by plasmin, which may then behave like a platelet inhibitor.     

The inhibition of platelet aggregation and blood coagulation by Micropechis ikaheka venom

Uncoagulable blood and life-threatening bleeding can result from the action of some snake venom toxins on haemostatic components of blood and vessel walls. Although envenoming by Micropechis ikaheka primarily affects neurones and muscle cells causing post-synaptic neuromuscular blockade and rhabdomyolysis, disturbances of haemostasis also occur. Therefore, the present study explored the effects of M. ikaheka venom on platelets and endothelium, which are important components of the haemostatic mechanism. The venom inhibited platelet aggregation in response to ADP and collagen, and also delayed clotting dependent on platelet activation or endothelial cell tissue factor expression. Some of these effects were reduced by the incubation of venom with a phospholipase A2 (PLA2) inhibitor and could be reproduced by a 17 kDa venom fraction containing a PLA2. In addition, an 11 kDa fraction containing a long-chain neurotoxin reduced ADP-induced aggregation. The venom was also found to reduce endothelial cell adherence to vitronectin-, fibronectin- and collagen-coated surfaces. These results suggest that, by inhibiting procoagulant activities of platelets and endothelial cells, a 17 kDa PLA2 plays an important role in the anticoagulant action of M. ikaheka venom.     

The effect of green pit viper (Trimeresurus albolabris) venom on platelet morphology by electron microscopy

The incidence of venomous snake bites increases every year in Thailand, especially due to green pit viper. After the bite, there is bleeding due to thrombin-like property of the venom. The mean platelet volume has been reported to be decreased in those who have been bitten by this snake. In this study we investigate the effect of green pit viper venom (Trimeresurus albolabris) on platelet volume (MPV), number and morphology of platelets in vitro. The test was carried out by washing platelets in phosphate buffer at pH 7.2 to remove fibrinogen, then the washed platelets were mixed with green pit viper venom. Platelet morphology was examined by scanning electron microscope (SEM).The morphology of platelets was smaller than normal which ranges from 1.1- 1.2 microm. Green pit viper venom can directly effect platelet morphology, decreasing platelet volume.     

Fibrin monomer induces binding of endogenous platelet von Willebrand factor to the glycocalicin portion of platelet glycoprotein IB

This study demonstrates that when platelets are stimulated by thrombin in the presence of low concentrations of purified human fibrinogen (10 to 20 micrograms/mL, final concentration) binding of released platelet von Willebrand factor (plt-vWF) to the platelet membrane is enhanced. This effect appears to be mediated by fibrin monomer produced by the action of thrombin on the fibrinogen in the incubation suspension. When fibrin polymerization is inhibited, the binding of released plt-vWF to the platelets is markedly increased. This enhanced binding is dependent on platelet glycoprotein Ib (GPIb) as shown by a decreased response with Bernard-Soulier platelets and inhibition by both monoclonal and polyclonal antibodies against glycocalicin. The binding of fibrin to thrombin-activated platelets preincubated with monoclonal antibody against GPIIb/IIIa is increased when the predominant form of fibrin is fibrin monomer. The fibrin binding is also decreased in the presence of antibody against glycocalicin. Our data demonstrate that fibrin monomer facilitates plt-vWF binding to the glycocalicin portion of platelet GPIb on thrombin-stimulated platelets and that binding of fibrin monomer to glycocalicin is necessary for this response to occur.     

Platelets interact with fibrin only after activation

Interactions between platelets and fibrin have been visualized by phase contrast, epifluorescence, and scanning electron microscope examination of clots formed with dansylcadaverine-labeled fibrin and gel-filtered platelets. After thrombin activation, the platelets appeared as fluorescent aggregates with bridging strands of fibrin; formaldehyde- fixed platelets were not fluorescent under the same experimental conditions. Scanning electron micrographs demonstrated that thrombin- activated cells had numerous pseudopods to which the fibrin strands adhered; fixed platelets exhibited a smooth discoid appearance and did not interact with the clot. Platelets trapped in clots formed with Batroxobin (Pentapharm) (platelets are not activated by Batroxobin as confirmed by light-scattering aggregometry measurements) remained as nonfluorescent, discoid cells, whereas platelets first activated by adenosine diphosphate formed brightly fluorescent aggregates. Light- scattering data of thrombin activation (0.2 U/mL) indicated that preincubation of platelets with 0.1 mmol/L prostaglandin E1 (PGE1) prior to addition of thrombin decreased the extent and rate of platelet shape change and resulted in 100-fold slower aggregation. Clots formed in the presence of PGE1 revealed decreased fluorescence intensity and fewer platelet-fibrin contacts. Gly-Pro-Arg-Pro, which blocks fibrinogen binding and fibrin assembly, was also effective in blocking platelet-fibrin interactions. These results indicate that platelet activation is a prerequisite for attachment of platelets to fibrin.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.