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Thursday THROMBIN-INDUCED PLATELET ACTIVATION 1063 Thursday THROMBIN-INDUCED PLATELET ACTIVATION 1063 1064 DISSOCIATION BY EDTA OF THE HUMAN PLATELET GP IIb-IIIa COMPLEX PLATELETS OF A PATIENT LACKING GLYCOPROTEINS IIb AND PREVENTS ADP-INDUCED FIBRINOGEN DEPENDENT AGGREGATION BUT NOT Ilia AGGREGATE TO HIGH CONCENTRATIONS OF THROMBIN OR THROMBIN-INDUCED THROMBOSPONDIN DEPENDENT AGGREGATION. J.-P. COLLAGEN. J.L.McGregor, L.McGregor, M. Hans, A. Sayegh, ·cazenave, A. Bay-Sutter, C. Gachet, F. Lanza, and A. Stierle. M.C.Trzeciak.and M.Dechavanne. Fac. of Medecine Alexis INSERM U. 311, Centre Regional de Transfusion Sanguine, Strasbourg, Carrel, INSERM U63, Lyon, France. Stanford Medical France. School, California, USA. The aggregation of washed human platelets by ADP does not cause The aim of this study was to investigate the plat­ the release reaction and is dependent, following ADP binding to its elets of a patient having bleeding episodes that began receptor, on exposure of the membrane glycoprotein (GP) IIb-IIIa in infancy. The patient's platelets in citrated-PRP com~lex, platelet contact and presence in the external medium of did not aggregate when stimulated with ADP (5 and 10 Ca2 and fibrinogen. EDTA, which chelates Ca2+ can inhibit platelet uM), collagen (2.5 ug/ml), or sodium arachidonate aggregation by removal of Ca2+ from external medium or by (1 uM). However, washed patient platelets, in the pres­ dissociating the GPIIb-IIIa calcium dependent complex. We have ence of 2mM calcium, aggregated and secreted when stim­ studied the effects of prolonged incubation of human platelets with ulated with high concentrations of thrombin (0.36, 0.72 EDTA on the platelet ADP-fibrinogen dependent aggregation. Human and 1U/ml) or collagen (2, 4, 10 ug/ml). Monoclonal platelets were washed according to a modification of Mustard t s antibodies (Mab) LYP18 (directed against the IIb-IIIa method (Ann. Biol. Clin. 1983, ~· 167-179). Platelets in the second glycoprotein complex) and LYP8 (anti-thrombospondin) washing fluid were labeled with H-5HT and split in 2 parts : one part inhibited thrombin and collagen induced aggregation of (A) was incubated with 5 mM EDTA for 30 min at 37°C, the other (B) with control but not the patient platelets. Patient thrombin Tyrode buffer. A and B were centrifuged and the platelet pellet -stimulated platelets did not bind l25r-labelled fibri­ resuspended in Tyrode-0.3f %albumin buffer with apyrase at 37°C at nogen (40 to 320 ug/ml). Moreover, stimulating the 300,000 platelets per mm • In contrast to control platelets (B), washed patient's platelets with ADP (10-100 uM), in the EDTA-treated platelets (A) : i) did not aggregate to ADP (1-10~ ~M) presence of fibrinogen (2mg/ml), did not result in in the presence of added human fibrinogen, ii) did not bind 2:>I­ aggregation. Binding studies using Mab 125 I-LYP2 (dir­ fibrinogen, iii) had a dissociated GPIIb-IIIa on crossed ected against the IIb-IIIa glycoprotein complex) showed immunoelectrophoresis of 125r-labeled platelets (lactoperoxydase the absence of the complex on the patient's platelets. method) after migration against a rabbit anti-human platelet The absence of the IIb-IIIa complex on the patient's antibody, iv) did not aggregate to ZnC12 or monoclonal antibody platelets was also observed using crossed immunoelectro (Mab) 6C9. In response to thrombin (0.01-0.5 U/ml), EDTA-treated -phoresis and Mab 12 5I-LYP2 or 12 5I-LYP18. Individual platelets (A) released 3H-5HT and aggregated but to a lesser extent glycoproteins (Ilb or Ilia) were not detected on silver than B platelets. Thrombin induced aggregation of EDTA-treated stained two-dimensional (non-reduced/reduced) SDS-PAGE. platelets'was inhibited by 5 mM EDTA or anti-TSP antibody or by OKM5 Moreover, Western blots of fhe patients platelets used (Ortho), a Mabagainst the TSP receptor. In conclusion, dissociation in combination with anti-PLA or anti-LEK polyclonal of GPIIb-IIIa of human platelets by EDTA inhibits completely the antibodies failed to detect the presence of these two aggregation depending on fibrinogen, but platelets can aggregate glycoproteins. These results indicate that this pati­ when they are stimulated by thrombin through release of TSP which ent has Glanzmann's thrombasthenia or a variant of this binds to its receptor. disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium. Thursday HEPARIN COFACTOR II; a 2-MACROGLOBULIN 1065 1066 THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN DETECTION OF INACTIVATED a 2-MACROGLOBULIN (a2M) IN HUMAN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES. PLASMA USING MO!iOCLONAL ANTIBODIES. J. Abbink, J. Nuijens, E. Delain 111 M. Barray 111 J. Tapon-Bretaudiere Ill F. Pochon 111 and F. Van C. Huijbregts and E. Hack. Central Laboratory of the Leuyen 121. lnstitut Gustave-Roussy 94805 Villejuif, France (1) and Dept. Human Netherlands Red Cross Blood Transfusion Service, Dept. of Genetics, Univ. Leuven, B-3000 Leuven, Belgium. Autoimmune Diseases, Amsterdam, The Netherlands. Electron microscopy is a very convenient method to localize the epitopes of Monoclonal antibodies (mAbs) were raised against human monoclonal antibodies (mAbs) at the surface of macromolecules for studying a2M. Five mAbs that bound to a2M in ELISA were further their tree-dimensional organization. analyzed by a radioimmunoassay (RIA) for their reaction We applied this immune-electron microscopic method to human with three types of a2M: native a2M, chemically inacti­ a2-macroglobulin (a2M). 29 anti-a2M mAbs have been tested with the four dif­ vated a 2M (ia2M) (methylamine treated), and proteolytically ferent forms of a2M : native and chymotrypsin-transformed tetramers, and the ia2M. One mAb reacted with all forms of a2M, while four corresponding dimers, obtained by dissociation with divalent cations. These mAbs bound both forms of ia2M but not native a2M. One of mAbs can be classified in three types : those wh1ch are specific for 1) the H-like these latter mAbs (Ml) was used to develop a RIA (the Ml­ transformed molecules, 2) the native molecules, and 3) those which can react assay) for the detection of ia2M in plasma: Ml coupled to with both forms of a2M. Sepharose is incubated with the plasma to be tested, and 1) Among the H-like a2M specific mAbs, several react with the 20 kD-domain bound ia2M is detected by a subsequent incubation with which is recognized by the cellular receptor of transformed a2M. This domain is polyclonal 125I-anti-a2M antibodies. As little as 5 ng located at the carboxyterminal end of each monomer. One lgG binds to the end of ia2M can be detected with this assay in the presence This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. of two adjacent tips of the H-I ike form. of an excess of native a 2M. This assay was then applied The other mAbs of this type bind to the a2M tips at non-terminal positions. to measure inactivation of a2M in vitro and in vivo. In Intermolecular connections built polymers of alternating cx2M and lgG mol­ vitro activation of the contact system in plasma by ecules. dextran sulfate results in the inactivation of ca 10% 2) Among the native a2M-specific mAbs some are able to inhibit the protease­ of a2M. When blood from normal donors was collected under induced transformation of the native a2M. The binding sites of these mAbs are optimal conditions, about 0.5% of the total a2M content demonstrated on the native half-molecules. One of these mAbs was also able to appeared to be ia2M. Longitudinal studies in patients react with transformed dimers, in a region corresponding very likely to an inac­ (a.o. with septicaemie, during cardiopulmunary bypass) cessible epitope in the tetrameric transformed a2M molecule. revealed that increased levels of ia2M occurred spora­ 3) Among the mAbs of this type, only two were able to inhibit the protease­ dically. The Ml-assay appears to be useful to monitor induced transformation of a2M. Obviously, their epitopes should be close to the the role of a 2M in human diseases. bait region of a2M. The other mAbs reacting with both a2M forms did not inhibit the a2M transformation. All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of a2M. The epitopes are more easily located on the dimers and on the H-like transformed a2M than on the native molecules. From these observations. we propose a new model of the tree-dimensional organization of the human a2M in its native and transformed configurations, and of its protease-induced transformation. 292 .
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