Effects of Platelet Inhibitors on the Platelet Aggregation Induced by Plasma From Patients With Thrombotic Thrombocytopenic Purpura By Eric C-Y Lian and N. Savaraj Antiplatelet drugs have been used in the treatment of eisacotetraynoic acid, prostaglandin E,. prostaglandin I2, thrombotic thrombocytopenic purpura (TTP) but their in dBcAMP, apyrase. creatine phosphate/creatine phospho- vivo efficacy remains controversial. It has been shown kinase. antimycin. 2-deoxy-o-glucose. dipyridamole. clofi- that. in vitro. the plasmas obtained from patients with TTP brate, dextran 40. dextran 70. dibucaine, xylocaine. induced the aggregation of washed platelets from normal methylmaleimide. and ethylenediamine tetraacetic acid donors as well as patients in remission. The effects of had little or no effect at all. These data lead us to conclude platelet inhibitors on the TTP plasma-induced platelet that at least in certain cases. antiplatelet drugs probably aggregation were examined. It was found that aspirin. play a limited role in the treatment of patients with TTP. indomethacin. ibuprofen. sulfinpyrazone. 5. 8. 1 1 . 14- Downloaded from http://ashpublications.org/blood/article-pdf/58/2/354/586286/354.pdf by guest on 28 September 2021 A NTIPLATELET DRUGS, such as aspirin,’ “ plasmapheresis, was described by Dr. J. Ansell et al.’6 Approxi- mately 450 ml of blood was collected into iF-IS Blood-Pack unit dipyridamole,2 6.5 9 dextran,57”5’2#{176} 22 and sulfin- containing 63 ml of citrate phosphate dextrose (CPD) solution or pyrazone,t’6 have been used in the treatment of throm- Blood-Pack Unit containing 67.5 ml of anticoagulant citrate botic thrombocytopenic purpura (TTP), but its in vivo dextrose (ACD) solution (Fenwall). Blood was centrifuged at 2400 efficacy remains controversial.2324 Deficiency of PGI2 g for 20 mm at 4#{176}C,after which the supernatant was decanted and synthesis was postulated to contribute to the formation spun at 10.000 g for 10 mm at 4#{176}C.The collected platelet-poor of platelet thrombosis in the microcirculation.25’26 It plasma was divided into small aliquots and stored at -80#{176}C. The plasma of the second patient was a gift from Dr. J. Ansell. Both was thought that infusion of PGI2 into TTP patients plasmas were demonstrated to possess a platelet aggregating factor, might be clinically beneficial.26 Recently, we have which was inhibited by normal plasma.2729 demonstrated that in vitro plasmas obtained from patients with TTP induced the aggregation of washed platelets. This aggregation was inhibited by preincu- Chemicals bation with normal plasma but not by heparin, hiru- Aspirin, indomethacin, collagen type 11, apyrase grade II, N6-2-0 din, diisopropylfiuorophosphate, or prior adsorption dibutyryl cyclic 3’, 5’-adenosine monophosphate (dBcAMP), ethyl- enediaminetetraacetic acid ( E DTA ), creatine phosphate, antimy- with aluminum hydroxide.27 Using the same method, cm, and 2-deoxy-o-glucose were obtained from Sigma Chemical, St. we have studied the in vitro effects of antiplatelet Louis, Mo.; dipyridamole solution was provided by Dr. W.M. drugs and prostaglandins on platelet aggregation Benson, Boehringer Ingelheim. Rigefield. Conn.; sulfinpyrazone induced by plasmas obtained from two patients with and 0.25% dibucaine HCI were supplied by CIBA-GEIGY, classic TTP. The results of these studies are reported Summit, N.J. Clofibrate sodium salt was supplied by Imperial Chemical Industries, Macclesfield, Cheshire, England; N-ethylmal- here. eimide, Mallinckrodt Chemical, St. Louis, Mo.; I % xylocaine, Astra Pharmaceutical Products, Worcester, Mass.; absolute pure alcohol, MATERIALS AND METHODS U.S. Industrial Chemicals; creatine phosphokinase, C. F. Boehringer TTP Plasma and Sochne GS Mannheim, Germany; dextran 40 (10% W/V) and dextran 70 (6% W/V), Pharmacia, Piscataway, N.J. Prostacyclin -ri-P plasmas were obtained from two patients with classic TTP (PGI2) and prostaglandin E, (PGE, ) were gifts from Dr. John Pike, during active disease. One of the patients, who responded to plasma Upjohn, Kalamazoo, Mich.; 5, 8, 1 1, 14-eisacotetraynoic acid infusion, was reported from this Medical Center by Drs. J. Byrnes (ETYA) was supplied by Dr. WE. Scott, Hoffman-LaRoache, and M. Khurana.28 Another patient, who did not respond to plasma Nutley, N.J.; Ibuprofen was obtained from Upjohn, Kalamazoo, infusion initially and responded to exchange blood transfusion and Mich. Indomethacin, PGEI, antimycin, and ETYA were freshly From the Department of Medicine, Veterans Administration prepared in pure ethanol. Aspirin, ibuprofen, dBcAMP, PGI2 Medical Center and Center ftr Blood Diseases, University of sodium salt, clofIbrate sodium salt, apyrase. EDTA, sulfinpyrazone, Miami School ofMedicine. Miami. Fla. 2-deoxy-D-glucose, creatine phosphate, and creatine phosphokinase Supported in part by the Veterans Administration and the were freshly prepared and dissolved in Tris-saline buffer, pH 7.4, American Heart Association of Greater Miami. which contained 0. 1 33 M NaCI, 0.0 1 5 M Tris-CI, 0.005 M KCI, and Presented in part at the Southern Society for Clinical investiga- 0.001 M MgCI2. The pH ofthe reagent solution was adjusted to 7.4 lion, New Orleans, January 1980. before being added to the platelet suspension. Collagen was Submitted December 30. 1 980; accepted April /3. 1981. prepared as described by Hovig.’#{176} Address reprint requests to Eric C-Y Lian. M.D.. Division of Hematology (1 1 ID), V.A. Medical Center, 1201 N. W. /6th Street. Preparation ofNormal Plasma and Platelets Miami, Fla. 33125. (t) 1981 by Grune & Stratton, Inc. Nine parts of whole blood were drawn from the antecubital vein 0006-4971/81/5802--0025$OI.00/0 into polyethylene tubes containing one part 3.8% sodium citrate 354 Blood, Vol. 58. No. 2 (August). 1981 EFFECTS OF PLATELET INHIBITORS IN UP 355 using double plastic syringe technique. The platelet-poor plasma TIP Plasma (PPP) used as a blank in the collagen-induced platelet aggregation was prepared by centrifugation at 2400 g for 20 mm at 4#{176}C. Platelet-rich plasma (PRP) was prepared by centrifugation at I 80 g for 10 mm at 22#{176}C.For collagen studies, the platelet concentration z of PRP was adjusted to 250 x 109/Iiter with PPP. Platelet washing 0 was performed according to the method of Walsh et al.” with slight modification. After 1/25 volume of 25% human albumin (Armour) (D was introduced at the bottom of conical plastic tubes, the PRP was w centrifuged at 22#{176}Cfor I 5 mm at 1650 g. The supernatant PPP was (D removed by siliconized Pasteur pipette. The platelets and the albu- 0 mm were suspended in the same volume (as that oforiginal PRP) of 1.8mM Tris-saline buffer, pH 7.4, containing 0.133 M NaCI, 0.015 M Tris-CI, 0.005 M KCI, and 0.001 M MgCl2. The platelets were washed twice with the same technique and then suspended in the Downloaded from http://ashpublications.org/blood/article-pdf/58/2/354/586286/354.pdf by guest on 28 September 2021 same buffer. Platelet concentrations were adjusted to about 750 x l09/liter for the platelet aggregation studies. Method to Study the EJfrct ofinhibitors on Platelet Aggregation a MINUTES Platelet aggregation was performed in a Chrono-log platelet aggregometer using a 609 m red filter. A 0. 1 5 ml of Tris-saline buffer solution containing proper concentration of inhibitors was added to 0.15 ml of washed platelet suspension and incubated at 37#{176}Cfor 3 mm, then, 0.2 ml of the mixture was transferred to the cell warmed up to 37#{176}Cin the aggregometer. which contained 0.3 ml of TTP plasma, undiluted or partially diluted with Tris-saline buffer, pH 7.4. The percentage decrease of optical density resulting z from platelet aggregation was recorded. Appropriate controls with- 0 out inhibitor and blanks with all reaction mixtures except platelets were always included. The concentration of inhibitors was expressed 0 as that in the final 0.5 ml reaction mixture. w The effect of platelet inhibitors on the collagen-induced platelet 0 aggregation was performed by addition of inhibitors to the platelet- 0 rich plasma in the aggregometer to make a final concentration as that in the TTP plasma-induced platelet aggregation 3 mm before 3 pg ofcollagen was added. It was shown that, at the same concentra- tion, aspirin, ibuprofen, indomethacin, sullmnpyrazone, ETYA. PGE, PGI2, dBcAMP, apyrase, and CP/CPK inhibited at least 80% of collagen-induced platelet aggregation. Contml RESULTS Effects ofinhihitors ofArachidonic Acid Oxygenation 1 2 3 4 5 6 Aspirin (from 0.1 mM to 3.6 mM) and ibuprofen b0 (0.2 mg/mI), inhibitors of cyclooxygenase,3233 inter- MINUTES fered with collagen-induced platelet aggregation in Fig. 1 . Lack of the inhibitory effect of aspirin and ibuprofen on platelet-rich plasma but not with TTP plasma-induced the TTP plasma-induced platelet aggregation. platelet aggregation (Fig. I and Table 1 ). Other cyclooxygenase inhibitors, indomethacin (8 .tM ),32 (2 mM), an analogue ofcAMP, also did not affect the sulfinpyrazone (0.3 mg/mI),34 and ETYA (3 .iM and aggregation of platelets by TTP plasma (Table 2). 60 sM)32 also had little or no effect on TTP plasma- induced platelet aggregation. Effects ofAgents That Remove ADP Effects ofSubstances That Increase Platelet cAMP Apyrase ( I 7 U/mI), an inhibitor of aggregation due Incubation of PGE, (0.3-3 zM)35 or PGI2 (60 to ADP,35 had virtually no effect on the TTP plasma- sM)36 with platelet suspension failed to inhibit the induced platelet aggregation. CP (2 pM)/CPK (4 platelet aggregation caused by TTP plasma. dBcAMP mg/mI), which removes ADP through different 356 LIAN AND SAVARAJ Table 1 . Effects of Inhibitors of Arachidonic Acid Oxygenation Patient 1 Patent 2 Final Maximal Inhibition Maximal inhibition Concentration Aggregation (%) (%) Aggregation (%) (%) Aspirin None 47.2 26.6 0.1 mM 51.2 No 27.0 No 1.0mM 50.5 No 26.6 No 1.8mM 41.1 13 - - 3.6mM 44.4 6 - - Ibuprofen None 47.2 - 0.2mg/mI 47.8 No - - Indomethacin None 39.8 3 1 .