Inhibition of Platelet Function by an Aspirin-insensitive Endothelial Cell ADPase Thromboregulation by Endothelial Cells

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1 Inhibition of Platelet Funtion by an Aspirin-insensitive Endothelial Cell ADPase Thromboregulation by Endothelial Cells Aaron J. Marus, Lenore B. Safier, Katherine A. Haijar, Harris L. Ullman, Naziba Islam, M. Johan Broekman, and Ana M. Eiroa Divisions ofhematology-onology, Departments ofmediine and Pathology, Department of Veterans Affairs Medial Center, New York 11; and Cornell University Medial College, New York 121 Abstrat We previously reported that platelets beome unresponsive to agonists when stimulated in ombined suspension with aspirintreated human umbilial vein endothelial ells. Inhibition ourred onomitant with metabolism of platelet-derived endoperoxides to prostaylin by endothelial ells. We now demonstrate that if aspirin-treated platelets whih fully respond to appropriate doses of agonists are exposed to aspirin-treated endothelial ells, they remain unresponsive despite absene of prostaylin. Platelet inhibition is due in large part to eto- ADPase ativity on the endothelial ells. This was established by inubating aspirin-treated endothelial ells with '4C-ADP. Radio-thin layer hromatography and aggregometry demonstrated that '4C-ADP and indution of platelet ativation dereased rapidly and onurrently. AMP aumulated transiently, was further metabolized to adenosine, and deaminated to inosine. The apparent K, of the endothelial ell ADPase was 3342 ;M and the V nmol/min per 16 ells, values in the range of antithromboti potential. Thus, at least three omplementary systems in human endothelial ells ontrol platelet responsiveness: a ell-assoiated, aspirin-insensitive ADPase whih funtions in parallel with fluid phase autaoids suh as the aspirin-inhibitable eiosanoids, and the aspirin-insensitive endothelium-derived relaxing fator. (J. Clin. Invest : ) Key words: nuleotidases * ell-ell interations * platelet aggregation * platelet serotonin release * thrombosis Introdution Realization ofthe importane of vasular ell-ell interations and transellular metabolism has inreased in reent years (1). This is partiularly pertinent to the ase ofendothelial ells and platelets. Currently we hypothesize that endothelial ells ontrol platelet reativity via at least three mehanisms: a ell-assoiated ADPase system and two fluid phase reatants; eiosanoids suh as prostaylin (PGI2)'; and the endothelium-derived relaxing fator (EDRF). In this report we extend previous Address orrespondene to Aaron J. Marus, M.D., Thrombosis Researh Laboratory, Room 1328AW, Department of Veterans Affairs Medial Center, 423 E. 23rd Street, New York, NY 11. Reeivedfor publiation 17 Deember 199 and in revisedform 22 July Abbreviations used in this paper: ASA, aetylsaliyli aid, aspirin; EC, endothelial ell(s); EDRF, endothelium-derived relaxing fator; 5-HT, serotonin, 5-hydroxytryptamine; NO, nitri oxide; PGI2, prostaylin; PRP, platelet-rih plasma. The Journal of Clinial Investigation, In. Volume 88, November 1991, studies on platelet inhibition by PGI2 formed by aspirin-treated endothelial ells from platelet endoperoxides (2). Under experimental onditions in whih EDRF was not measurable, we found that platelet reativity was inhibited by endothelial ells even though both ell types were aspirin treated and PGI2 was absent. Biohemial and funtional data will be presented indiating that these aspirin-treated endothelial ells inhibit platelet funtion largely via a mehanism involving metabolism of ADP and onsequent loss of its proaggregatory ativity. Methods Preparation ofplatelet-rih plasma andplatelet suspensions. Blood was olleted from donors 12 h after they had ingested 65 mg aetylsaliyli aid, aspirin (ASA). Platelet-rih plasma (PRP) was prepared us- - ing aid itrate-dextrose (itri aid, 38 mm; sodium itrate, 75 mm; gluose, 135 mm) as antioagulant (3), with an initial whole blood entrifugation at 2 g, 15 min (25 C) and a seond entrifugation of the PRP (9 g, 1 min) to eliminate most of the residual erythroytes and leukoytes. PRP was maintained at room temperature under 5% C2-air. Platelet suspensions, when used, were prepared from PRP as desribed previously (3). Final resuspension was in old.15 M NaCI to a ount of 1 X 1' platelets/2 Ml. The suspension was kept in a losed ontainer at 4 C. Preparation ofendothelial ell suspensions. Cultured human endothelial ells (P2-P8) derived from umbilial ords (4) were treated with I mm ASA (1 Ml of I M ASA in ethanol/io ml) for 3 min at 37'C (2). Cells were washed in Hepes-buffered saline and detahed with ollagenase-edta-bsa solution (4). An equal volume ofhuman serum-ontaining medium was added, the ells entrifuged at 5 g for 1 min (22'C), and finally resuspended in ASA-free buffer (.25 ml/t-75 flask). Resuspension buffer was either Tris-Saline-Gluose (TSG) (Tris, 15 mm; NaCl, 134 mm; gluose, 5 mm, ph 7.4) or Hepes, 5 mm; NaCI, 14 mm; KCI, 5 mm; CaCI2, 1.29 mm; MgCI2, 1.2 mm, ph Indomethain was then added to a onentration of 1MOM. Suspensions were generally maintained at room temperature or at 4 C as speified. Endothelial ell (EC) ounts averaged 4,466 ells/ml. Aggregation studies with ombined suspensions of ASA-treated platelets andasa-treatedec. These experiments were arried out similarly to those previously reported in whih ASA-treated EC, but untreated platelets were studied (2). ASA-PRP ontaining 1 x 18 platelets or ASA-treated washed platelets (1 X 18) in buffer were preinubated (3 min, 37 C) in silionized uvettes ontaining stirring bars in an aggregometer. When used, SOD or other substanes of interest suh as hemoglobin, FeSO4, or nitroprusside were inluded with the platelets or during preinubation. ASA-treated EC were then added, followed in by the agonist. In the ase of TSG buffer, whih ontained no alium, Ca" (3 mm) was inluded 15 s before thrombin or ollagen. Total volumes were adjusted to 5 Ml with buffer. The aggregation response was reorded over a 5-min period in a Lumiaggregometer (Chrono-Log Corp., Havertown, PA). Control "platelet-poor" uvettes ontained equal numbers of EC to those in "platelet-rih" uvettes in order to orret for light absorption by the nonaggregating EC. 169 Marus et al.

2 Studies ofpossible inhibitory properties ofasa-ec supernatants. EC were tested for their inhibitory ativity as above, using ADP, thrombin, or ollagen as platelet agonist. Separate aliquots of EC were equilibrated at 37C in polypropylene mirofuge tubes with stirring (in the presene or absene of SOD, 6 U/ml). They were then inubated (15 s) with speifi EC stimuli suh as bradykinin (1 nm), aetylholine (2 MM), histamine (1MM), or thrombin (1.25 U/ml), followed by rapid entrifugation (1 s, 15,6 g; Eppendorf In., Fremont, CA). Supernatants were transferred to aggregometer uvettes ontaining 1 x 18 ASA-treated platelets (either washed or in PRP). Platelet agonists (ADP, ollagen, or thrombin; see below) were added 15-2 s later. Total volumes in the uvettes were adjusted to 5 Ml with buffer. Controls were arried out to evaluate effets of arry-over of EC agonists on platelet aggregation. In the ase of thrombin, the quantity arried over served as platelet agonist in the uvette. Time ourse ofec ADPase ativity. ASA-EC in a total volume of 4 Ml plus 15MuM ("C) ADP (41.7 mci/mmol; New England Nulear, Boston, MA), or buffer plus ("C) ADP for ontrols, were inubated as in the other entrifugation studies for varying periods of time (15 s-3 min). Stirring bars were rapidly removed and the tubes entrifuged as above. A 1-Ml aliquot of supernatant was transferred to the uvette ontaining platelets and the aggregation response reorded. In ontrol experiments ontaining no EC, the final ADP onentration in the uvettes was 3 MM. Immediately after removal of the initial 1,l of supernatant, an additional 2 Ml was plaed in a polypropylene mirofuge tube ontaining 1 Ml of "stop solution" (5). The stop solution stok onsisted of 16 mm disodium EDTA, neutralized to ph 7, plus 17 mm ADP in ie-old physiologial saline. The stop solution was added to prevent any possible further degradation of ADP. Tubes were then vortexed and kept on ie until the experiment was onluded. Sintillation ounting was performed on 5 Ml from eah sample. Tubes were stored at -7 C for thin-layer hromatography of ADP and its metabolites. Inubations ould also be arried out with endothelial ell monolayers in multiwell plates (well apaity 1.5 ml, growth area 2 m2; 3847; Falon Labware, Beton Dikinson & Co., Linoln Park, NJ), without stirring, using the same ADP onentration and total volume. Plates were seured by weights in a 37 C water bath. At indiated time intervals, supernatants were aspirated and entrifuged as above. The well system was also used to determine the ADPase ativity of fresh endothelial ells. Cells derived from two ords were pooled and divided between nine wells. Within 1-2 h of inubation, the endothelial ells were adherent. This permitted assay of EC ADPase ativity free from ontaminating erythroytes, whih were disarded in the supernatant. Aggregation studies using supernatants of either thrombin-stimulated platelets or thrombin-stimulated platelet-endothelial ell mixtures as platelet agonist. Washed platelets (1 x 18) from a donor who had ingested aspirin, or platelets plus 2 X 16 ASA-treated endothelial ells were preinubated for 3 min and then stimulated with thrombin (1 U/ml), in the presene of 3 mm Ca" and 2MgM hemoglobin. Total inubation volume was 4 Ml. The mirofuge tubes were inubated for 5 min after stimulation, stirring bars removed and entrifugation arried out for 1 s (Eppendorf). A ontrol tube ontaining thrombin but no ells was also inubated for 5 min. 1 Ml of supernatant was rapidly transferred to an aggregometer uvette ontaining 227 Ml of PRP from the same donor (1.3 X 18 platelets) and buffer (total volume ofthe assay uvette was 5 Ml). The aggregation response was reorded for 4 min. TLC studies of nuleotides, nuleosides, and bases. TLC was arried out on fluoresent silia gel 6 F254-oated plasti sheets (2 x 2 m; EM Separations, Gibbstown, NJ). 15 Ml ofradioative sample was applied and dried under a stream of air. At eah point of appliation, 2 ML of a mixture of standards was added and dried. The standard mixture onsisted of 2 mg eah of the following ompounds: ATP, ADP, AMP, inosine, hypoxanthine, adenosine, and adenine in distilled water (total vol = 1 ml) (6). The solvent system for nuleotides, nuleosides, and bases onsisted of isobutyl alohol/ I-pentanol/ethylene glyol monoethyl ether/nh4oh/h2 (9:6:18:9:12) (solvent 1) (6). For separation of hypoxanthine and adenosine, the solvent system onsisted of 1-butanol/ethyl aetate/methanol/nh4h (7:4:3:4) (solvent 2) (7). Solvent systems were prepared at least 48 h before use and added to the tank 1 h before insertion of plates (8). Development of plates was arried out for 5 h, 1 min in solvent 1, or 4 h in solvent 2. Plates were dried under a stream ofwarm air, the separated ompounds visualized under ultraviolet light (254 nm), and sanned for radioativity with an RTLC multi-sanner (Radiomati Instruments & Chemial Co., In., Tampa, FL). Substrate onentration urve ofec ADPase. Aspirin-treated EC (72,233/4 Ml total vol) in TSG buffer were inubated with stirring for 5 min (37C) with MM ADP ontaining 2.4 MCi of (3H) ADP (trisodium salt, 27.3 Ci/mmol; New England Nulear), in the presene of 1 MM dipyridamole. The latter was used to prevent reuptake of adenosine and onsequent resynthesis of ADP (7, 9). Ca" (1.22 mm) was added 3 s before ADP. As in the time ourse experiments desribed above, tubes were then entrifuged, supernatants treated with stop solution, aliquots ounted for total radioativity, and TLC performed. The dipyridamole stok solution was prepared in glyine-hcl buffer (.5 M, ph 2.8). During these experiments, EC stok suspensions were stored at 4VC. ('C) Serotonin, 5-hydroxytryptamine (5-HT) release. For platelet labeling,.2 nmol ("C) 5-HT reatinine sulfate (54 mci/mmol; Amersham Corp., Arlington Heights, IL) was added diretly to the antioagulant for eah ml of antioagulated blood. ("C) 5-HT uptake was determined 1 h after blood olletion by omparison of radioativity in 5-Ml aliquots of PRP and platelet-poor plasma. Assays ofthe effets of EC on platelet ("C) 5-HT release and aggregation were arried out in uvettes in the usual manner, using 1 x 1 o labeled platelets in suspension or PRP. To prevent reuptake of released 5-HT, imipramine (2.5 MM) was added 9 s before the agonist. Controls ontaining labeled platelets without agonist were arried out to measure any release of ("4C) 5-HT attributable to stirring alone. Reations were stopped 4 min after stimulation by plaing the uvettes on ie. Cuvette ontents were transferred to mirofuge tubes and entrifuged for 3 min (1, g, 4 C). 5-Ml aliquots ofeah supernatant were ounted in 4 ml Aquasol- 2 and ompared to total platelet ounts. Additional methods. Treatment ofplatelets with methylene blue, an inhibitor of soluble guanylate ylase, was essentially aording to Alheid et al. (1). Methylene blue was added to a washed platelet suspension at a onentration of 1 MM. The suspension was then left at room temperature for 3 min, entrifuged at 1,45 g (15 min 4C) and the pellet resuspended in old.15 M NaCl. Oxyhemoglobin was prepared from bovine hemoglobin (type 1; Sigma Chemial Co., St. Louis, MO) by the method of Martin et al. (1 1). Human hemoglobin yielded idential results. Briefly, a 1-mM solution of the ommerial mixture of oxyhemoglobin and methemoglobin was redued with a 1-fold molar exess of sodium dithionite, whih was then removed by dialysis. Purity was heked spetrophotometrially and aliquots frozen at -7 C. Adenosine-5'--(2-thiodiphosphate) trilithium salt (ADP-#-S) was obtained from Boehringer Mannheim, Indianapolis, IN. Prostaylin prodution was measured by RIA for 6-keto-PGF,a (DuPont-New England Nulear). Results Endothelial ells inhibit platelet reativity in totally aspirintreated systems. When ASA-treated washed platelets were stimulated by agonists in the presene of ASA-treated endothelial ells, platelet aggregation was inhibited (Fig. 1). This ourred under onditions where ontrol ASA-platelets alone were fully aggregated by the same quantity of stimulus (Fig. 1). RIA measurements verified that no PGI2 had formed in these experi- Human Endothelial Cell ADPase Inhibits Platelet Funtion 1691

3 Thrombin (.5 U/ml) lonophore (1 um) 1 i E Ca 1 \8XE 2 jb a.c 6-~ ~ ~ ~ ~ ~ ~~.~'1.84 ASA - Platelets ASA - Platelets ASA- Platelets + A5A - Endothelial Cells Figure 1. Inhibition of platelet aggregation by endothelial ell suspensions. The upper urves (ontrols) represent the response of washed, aspirin-treated platelets to thrombin and ionophore, respetively. The lower urves depit inhibited platelet responsiveness when aspirin-treated endothelial ells were present. Aspirin treatment of both ell types prevented transellular metabolism of platelet endoperoxide to prostaylin by the endothelial ells (2). ments. As shown in Fig. 2 A, PRP from a donor who had ingested aspirin was fully aggregated by 1 gm ADP. In ontrast, when this PRP was stimulated in the presene of 1 X 16 ASA- EC, the inhibited aggregation urve was haraterized by a brief asending limb followed by reversal. The pattern of reversibility was reminisent of previous experiments in this laboratory wherein ADP released from platelets by PGH2 was intentionally removed by enzymati means (apyrase) (Fig. 2 B). Conurrent metabolism ofadp by endothelial ells and loss of its potential as a platelet agonist. The hypothesis that an ADPase ativity was present on these human endothelial ells and ould possibly aount for their platelet-inhibitory properties was tested biohemially and funtionally. (14C) ADP (15 MiM) was inubated with ASA-EC for inreasing periods of time. The supernatants were then examined for their ontent of residual (14C) ADP and its metabolites, as well as for the platelet aggregating potential of the unmetabolized (14C) ADP. Re- Z 2.6 E~~~~~~~~~.I.. I I. *. I. a Preinubation (min) Preinubation (min) Figure 3. Time ourse of metabolism of (14C) ADP by aspirin-treated endothelial ell suspensions. The derease in (14C) ADP onentration as measured by thin-layer radiohromatography (-o ), was aompanied by loss ofplatelet proaggregatory ativity (-a-) of supernatants derived from inubation of endothelial ells with ('4C) ADP. This also ourred when endothelial ell monolayers were used (inset). Results shown are from representative experiments in whih 72,215 suspended EC or 84,625 EC in monolayers were assayed. suits are depited in Figs. 3 and 4. When ompared to (14C) ADP ontrols whih had been inubated with buffer alone, the presene of EC resulted in a progressive derease in (14C) ADP onentration. This was paralleled by loss of supernatant proaggregatory ativity as measured by the derease in maximum height of the platelet aggregation urves. Comparable results were obtained whether endothelial ell suspensions (Fig. 3) or monolayers (Fig. 3, inset) were employed. ADPase ativity was also present on freshly adherent, but unultured endo- CONTROL (No EC) AMP 72,215 EC + 151,M I'4C] ADP (5min) C E -C 1 A ADP (1pM) ASA- P B I PGh2 (18pUM) ASA- PRP 72,215 EC + I5jLM [14C]ADP (1 min) ASA- PRP ApVrase t*h Figure 2. Comparison of the inhibition of ADP-indued aggregation in PRP by endothelial ells (A), with that due to enzymati removal by apyrase of platelet ADP released by prostaglandin endoperoxide (PGH2) stimulation (B). Similarities in the shapes of the urves of inhibited aggregation (lower urves) suggest enzymati removal of ADP in A as well Marus et al. ADO J V V HYPA _ P V 72,215 EC+ 15MM [14C]ADP (3 min) Figure 4. Time ourse of formation of metabolites of (14C) ADP by aspirin-treated endothelial ell suspensions. Cell-free supernatants derived from inubations of endothelial ells with (14C) ADP were analyzed for (14C) ADP and its metabolites by TLC.

4 E ADP (2uM) ADP-P-S (1OM) o am (a) +EC (b) () (d) +EC Figure 5. Studies with ADP-(i-S, a nonmetabolizable ADP analogue that ativates platelets. (a) ADP eliited a full aggregation response in aspirin-treated platelets. (b) Inhibition of the ADP response by aspirin-treated endothelial ells. () Aggregation response ofaspirin-treated platelets to ADP-#-S. (d) Aggregation indued by ADP- (l-s was not appreiably influened by the presene of endothelial ells, whih were unable to metabolize the ADP-,B-S. thelial ells. There was only a slight derease in ADPase ativity onomitant with ell passage. Fig. 4 depits radio-tlc sans of the metabolites of('4c) ADP after 5, 1, and 3 min inubation with ASA-EC suspensions. At the 5-min time point, ADP had dereased to 51% of total nuleotides, nuleosides, and bases. In addition, AMP (28%) and inosine (13%) were identified, together with trae quantities ofadenosine, hypoxanthine, and adenine. By 1 min, onentrations of ADP, AMP, and inosine averaged 3%, 29%, and 29%, respetively. Inosine (85%) was the major (14C) ADP metabolite at the 3-min interval, and ADP itself was virtually absent. Chromatography with solvent system 2 indiated that hypoxanthine (8%) had been synthesized. As an be seen in Fig. 3, the absene ofadp orrelated with total loss of aggregatory ativity of the ASA-EC supernatant. The presene or absene of endothelial ells did not affet the reovery of total radioativity in supernatants. This indiated that nonspeifi adsorption of ADP to the endothelial ells was not the ause ofthe derease in supernatant proaggregatory ativity. Endothelial ell inhibition ofadp-indued platelet reativity requires ADP hydrolysis. Experiments were arried out with ADP-,3-S, a strutural analogue ofadp that ativates platelets, but is not metabolized by ADPases (12). As shown in Fig. 5, ASA-platelets stimulated alone with 2 MM ADP demonstrated a full aggregation response (a). In the presene of 1 x 16 ASA- EC, this response was markedly attenuated and reversed (b). ADP-(-S (1 MM) indued a reversible aggregation response of lesser amplitude than ADP (2 AM) with ASA-platelets alone (). However, in ontrast to results with ADP, ASA-EC did not exert an appreiable inhibitory effet on ADP-,3-S-stimulated ASA-platelets (d). These results provided further evidene for ADP hydrolysis by EC as a major mehanism underlying their inhibitory effet on stimulated platelets. Kineti parameters ofhuman endothelial ell ADPase. We measured the rate of hydrolysis of (3H) ADP by ASA-EC as a funtion of substrate onentration. Apparent Km and Vat values were determined from Lineweaver-Burk plots of the data (Fig. 6). In two experiments, the Km ranged from 33 to 42 MM and the V., from 17 to 43 nmol/min per 16 ells. These = 2% V3.1, / Figure 6. Lineweaver-Burk plot relating the rate of (14C) I * Ị *. *.1 *2 i i ADP hydrolysis by aspirin-treated endothelial ells to ADP onentration. The Km alulated from these data was 33.M and the V. was 42.5 nmol/min per 16 ells. (ADP PM)-' Results depited are representative of two separate experiments. Human Endothelial Cell ADPase Inhibits Platelet Funtion 1693

5 kineti parameters are ompared in Table I with values reported in the literature for porine aorti endothelial ells (13, 14). Endothelial elladpase inhibits platelet reativity independent ofedrf. ASA-endothelial ells (whih did not produe PGI2), inhibited platelet aggregation by an aspirin-insensitive mehanism. This inhibition ould have been due in part to a fluid phase reatant suh as EDRF in ombination with the EC ADPase ativity desribed above. To test this possibility, known modulators of EDRF ativity were added to the platelet-ec inubations. Inhibition of platelet aggregation by endothelial ells was not affeted by: (a) hemoglobin or ferrous ion, whih inativate EDRF/nitri oxide (NO); (b) methylene blue, whih prevents the effets of EDRF/NO by inhibiting soluble guanylate ylase; or () superoxide dismutase, a potentiator of EDRF/NO via removal of superoxide radials. Supernatants ofec that had been previously stimulated (15 s) with bradykinin, aetylholine, or histamine were rapidly transferred to uvettes ontaining platelets. There was little (< 1%) if any inhibition of ADP, thrombin, or ollagenstimulated platelet aggregation. This indiated that the platelet inhibitory ativity observed in the presene ofasa-endothelial ells was not transferable, and therefore was mainly surfae assoiated. When endothelial ells were prestimulated with thrombin, the quantity present in the transferred supernatant served as agonist in the aggregometry uvette. Sine the same degree of aggregation was obtained with this supernatant (even after a 3-min inubation of thrombin with EC) as with thrombin alone, nonspeifi adsorption of agonist to the EC ould again be ruled out as the possible etiology ofplatelet inhibition. Release of ("4C) 5-HT from stimulated platelets was used as another parameter to gauge the effets of ASA-endothelial ells on platelet reativity. ASA-endothelial ells inhibited 5-HT release from ativated platelets as well as their aggregation (Table II). Hemoglobin did not reverse the inhibitory effet of the ASA-endothelial ells on either 5-HT release or aggregation. Endothelial ells reverse the apaity of thrombin-eliited platelet releasates to enhane platelet aggregation. To more losely relate the loss of thrombin-indued proaggregatory a- Table I. Kineti Parameters for Endothelial Cell ADPases from Different Soures Km V. V,,,/Km A1M nmol/min per 16 ells Human umbilial vein endothelial ells Porine aorti endothelial ells (13) Porine aorti endothelial ells (14) Aspirin-treated endothelial ells were inubated with (3H) ADP. After 5 min the tubes were entrifuged and thin-layer hromatography performed on the supernatants. The rate of hydrolysis of ADP was measured as a funtion of substrate onentration. Apparent Km and Vn, values were determined from Lineweaver-Burk plots as shown in Fig. 6. Values for human umbilial vein endothelial ells represent averages from two separate experiments. These parameters are ompared with values reported in the literature. Table II. Inhibition ofserotonin Releasefrom Stimulated Aspirin-treated Platelets by Aspirin-treated Endothelial ells Collagen Serotonin release (%) Thrombin 1 zg/ml.3 U/ml Platelets alone X 16 endothelial ells X 16 endothelial ells + hemoglobin (2 MM) x 16 endothelial ells x 16 endothelial ells + hemoglobin (2 um) 31 Values represent radioative serotonin released into the supernatant, as perent of total radioative serotonin in the labeled platelets. Hemoglobin was used as an inhibitor of EDRF/NO ativity. If the inhibition of serotonin release were due to EDRF/NO, the inhibition of release would have been reversed in the presene of hemoglobin. These results are representative of four separate experiments. tivity to EC ADPase ation, the following experiments were performed. Supernatants from platelets that had been stimulated with thrombin (1 U/ml) in the absene or presene of endothelial ells were used as agonists for platelet aggregation. Curve (a) in Fig. 7 was generated by the threshold level of thrombin (.2 U/ml) transferred from the inubation tube to the aggregometry assay uvette. The enhaned aggregation response in urve () was evoked by the ombined presene of transferred thrombin-indued platelet releasate in addition to transferred thrombin in the supernatant. Comparable synergy was demonstrated in ontrol experiments in whih thrombin (.2 U/ml) plus onentrations ofadp expeted to be released during the initial inubation were added diretly to the aggregometer uvette (data not shown). The presumed onentration of ADP released into the inubation tube, alulated from values reported in the literature (15) was 6.25 MM. The kineti parameters we derived for the human endothelial ell ADPase under study indiate that this quantity of ADP would have been hydrolyzed by the 2 X 16 EC present during our 5-mi inubation. It an be seen in urve (b) Fig. 7 that the presene of EC did indeed reverse the enhaned aggregation. Disussion In our earlier studies of platelet responsiveness to agonists when in ombined suspension with human endothelial ells, we orrelated the observed inhibition of platelet aggregation with prostaylin formation (2). Under those experimental onditions, the endothelial ells had been pretreated with aspirin, but the platelets were not. Prostaylin formation was demonstrated to result from metabolism ofplatelet-derived endoperoxides by endothelial ells in apposition. In the last deade, at least two endothelial ell-assoiated platelet inhibitory substanes in addition to PGI2 have been identified. These are endothelial ell eto-adpases (9, 16) and the endothelium-derived relaxing fator (EDRF/NO) (17-2). Prodution of EDRF and the ation of endothelial ell ADPases are insensitive to aspirin and ould have ontributed at least in part to results observed in earlier studies (2) Marus et al.

6 a o._ GMN s i C SUPERNATANT OBTAINED FROM: Platelets + Thrombin Vb Platelets+EC + Thrombin a t Supernatant added Buffer + Thrombin Figure 7. Aggregation response of platelets in PRP to supernatants derived from thrombin-stimulated platelets or thrombin-stimulated platelet-endothelial ell mixtures. (a) Aggregation due to thrombin arried over in the supernatant ofa buffer ontrol whih ontained no ells. In () the presene of thrombin-indued platelet releasate in the supernatant produed an enhaned aggregation response. The enhanement of supernatant aggregatory poteny as shown in () was reversed by the interation ofendothelial ell ADPase with the platelet releasate as shown in (b). We therefore performed experiments using both aspirintreated platelets and aspirin-treated endothelial ells, thereby eliminating prodution of any ylooxygenase-derived eiosanoids. As shown in Figs. 1 and 2 and Table II, endothelial ells inhibited platelet responsiveness to all agonists tested even when the entire system was aspirin-treated. The endothelial ell-indued inhibition was ell assoiated sine the inhibitory ativity was not present in supernatants from endothelial ells even when they had been stimulated with known agonists for EDRF/NO. Substanes known to reverse or enhane EDRF/ NO ativity had no influene on the inhibitory effets ofendothelial ells on the platelet responsiveness observed in these experiments. This is shown for hemoglobin in Table II. When ADP-indued platelet aggregation was reversed in the presene of endothelial ells, the shape ofthe reorded pattern of reversal suggested that ADP had been enzymatially removed (Fig. 2). In this regard, it was possible to biohemially and funtionally orrelate metabolism of ADP by ASA- EC with disappearane of its properties as an agonist for platelet aggregation (Figs. 3 and 4). The fat that similar ativity was demonstrable with either endothelial ell suspensions or adherent monolayers suggests that the ADPase ativity is loated on the luminal surfae of the vessel and would interfae diretly with platelets. Comparable results have been reported by Cruthley and assoiates with bovine pulmonary arterial endothelial ells (7) and Glasgow et al. with human endothelial ell monolayers (2 1). As shown in Table I, values obtained with our human endothelial ell preparations for the apparent Km and atalyti effiieny (V. /Km) with ADP as substrate, ompared favorably with those reported for porine aorti endothelial ells (13, 14). Calulations using these figures indiate that the quantity of ADP hydrolyzable by our endothelial ell suspensions would be suffiient to result in inhibition of platelet aggregation. For example,.5 X 16 EC would metabolize 2,M ADP to.8,m in 3 s. In the ase of agonists other than ADP, suh as thrombin, ollagen, and ionophore, hydrolysis of released platelet ADP may also be involved in the loss ofplatelet responsiveness (Fig. 1, Table II). Calulations based on results depited in Fig. 7 indiated that enhanement ofthreshold thrombin aggregation was attributable to thrombin-released platelet ADP. Removal ofthis ADP by endothelial eto-adpase reversed the enhanement, and aggregation reverted to the original threshold level. This ourred in the total absene of endothelial ell PGI2 and EDRF and emphasizes the role of endothelial ell ADPases in ontrol of platelet reruitment. A major reason for eluidating biohemial and funtional properties of endothelial ell-assoiated eto-adpases is that they an exert a signifiant effet on platelet reativity independent ofthe ation- of other known endothelium derived inhibitors. For evaluation of the entire known spetrum of EC ontrol of platelet reativity ("thromboregulation"), ell preparations whih simultaneously produe PGI2, EDRF/NO, and possess ADPase ativity will require additional assessment. It is also possible that as yet undefined endothelial ell thromboregulators ould have ontributed to results obtained by ourselves and others. Aknowledaments We thank Drs. Juana Valles and M. Teresa Santos for helpful disussions and suggestions, and Ms. Evelyn M. Ludwig for her expert editorial ontributions. This work was supported by grants from the Department of Veterans Affairs Medial Center, National Institutes of Health grants HL SCOR (Drs. Marus and Broekman), HL , HL (Drs. Marus, Hajjar, and Broekman), HL-2934 (Dr. Broekman), HL (Dr. Hajjar), the Edward Gruenstein Fund, the S. M. Louis Fund, and the Sallie Wihman Fund (Dr. Marus). Dr. Hajjar is an Established Investigator of the Amerian Heart Assoiation and a Syntex Sholar (1989). Referenes 1. Marus, A. 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