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- MATERIALS AND METHODS
The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.
The human CD36 is a single-chain integral membrane polypeptide (also known as GPIV; IIIb) expressed by platelets as well as many other cells, cell lines and tissues ( Okumura & Jamieson, 1976; Asch et al, 1987 ; Tandon et al, 1989b ; Greenwalt et al, 1990 , 1992; Daviet & McGregor, 1997). Numerous functions have been described for CD36, including a role as a cell surface receptor interactive with a large number of ligands, in cytoadhesion reactions, and implication in intracellular signal transduction ( Jaffe et al, 1982 ; Leung, 1984; Silverstein et al, 1984 , 1989; Majack et al, 1988 ; Kieffer et al, 1989 ; Tandon et al, 1989a , b; Goodet al, 1990 ; Murphy-Ullrich et al, 1992 ; Savill et al, 1992 ; Greenwalt et al, 1992 ; Endemann et al, 1993 ; Abumrad et al, 1993 ; Daviet et al, 1997 ; Kehrel et al, 1998 ). The molecular mass of CD36 on different cells is variable; for example, Mr values of 88 000, 85 000 and 78 000 have been reported for human platelets, human mammary epithelial cells, and human erythroblasts, respectively ( Tandon et al, 1989b ; Greenwalt et al, 1990 ; Kieffer et al, 1989 ).
A cDNA clone encoding CD36 was isolated from a human placenta cDNA library and expressed in COS cells by Oquendo et al (1989 ), resulting in a polypeptide with 471 residues and a predicted Mr of approximately 53 kD. The identification of 10 potential N-linked glycosylation sites accounted for the difference in Mr between the polypeptide and the 83 kD species found by immunoprecipitation with a pool of mouse monoclonal anti-CD36 antibodies. Tandon et al (1989b ) purified CD36 from solubilized Triton X-114 extracts of human platelet membranes, and reported the 88 kD protein as having 26% carbohydrate, of which approximately two-thirds were in alkali-labile O-glycosidic linkages. Treatment of human platelet CD36 with endoglycosidase F resulted in a protein with an apparent Mr of 57 000 after removal of N-linked oligosaccharides ( Greenwalt et al, 1990 ).
Platelet membrane CD36 may be important immunologically in the aetiology of thrombosis ( Vermylen et al, 1997 ). Burns & Zucker-Franklin (1982) showed that an IgG in TTP plasma mediated time-dependent immune destruction of human cultured endothelial cells. Pfueller et al (1990 ) described a patient with thrombosis and thrombocytopenia whose plasma had potent platelet aggregating activity caused by an IgG directed against platelet CD36. Further support for these observations came from a report by Tandon et al (1994 ) which showed a high frequency of anti-CD36 antibodies in patients with TTP. Specifically, they showed, using plasma from TTP patients as the source of autoantibodies and radio- or enzyme-labelled protein A as the reporter molecule for the patient IgG, that 85% were CD36 positive by immunoprecipitation, 75% were positive by protein blotting, and 60% were positive by dot blots, using purified CD36 as the antigen. Furthermore, platelets from a Naka-negative donor (constitutively lacking CD36) ( Yamamoto et al, 1990 ) were tested by Tandon et al (1994 ). Approximately 50% of their TTP plasma samples that caused 70% release of 14C-serotonin from control platelets failed to induce release from Naka-negative platelets, indicating that CD36 was the major target for antibodies in those TTP plasmas. In direct contrast to the data of Tandon et al (1994 ), in a recent report by Raife et al (1997 ) antibodies to CD36 were not found in 49 acute-phase plasma samples from TTP patients by an ELISA and flow cytometric assays.
In order to clarify this important aspect of the possible aetiology of TTP we have examined plasmas from TTP patients for antibodies reactive with platelet membrane CD36. Furthermore, these studies were expanded to test for anti-CD36 antibodies in patients with idiopathic thrombocytopenic purpura (ITP) and heparin-induced thrombocytopenia without and with thrombosis (HIT, HITT). Additionally, a lighter glycoform of CD36 (85 kD) was separated from the classic 88 kD form chromatographically and shown to react predominantly with autoantibodies from most TTP patients as well as patients with ITP, HIT and HITT.
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- MATERIALS AND METHODS
Our findings indicated that >70% of the TTP patients investigated in this study had plasma antibodies reactive with the platelet membrane glycoprotein CD36. There was a close correlation between the two methods used to test the plasmas, the PAICA and immunoblots (Western). We routinely use the PAICA as a rapid screening assay because it is sensitive enough to detect changes shown by immunoblots. Previously, a Triton X-100 extract of pooled normal platelets was used as a source of CD36. This source, as was shown in this study, is a mixture of the 85 and 88 kD CD36. It is now recommended that the antigen should be enriched with the 85 kD CD36 for immunoassays. These results with patients' plasmas confirmed the study of Tandon et al (1994 ), and refuted the report of Raife et al (1997 ).
In addition to TTP plasmas, a close correlation was also found between the PAICA and immunoblots for detecting anti-CD36 antibodies in plasmas of patients with ITP: 20% were detected with PAICA and 19% with immunoblots. He et al (1994 ), using mAb FA6-152 and antigen capture methods, found antibodies to GPIV (CD36) in 38% of 47 patients with chronic ITP. The form of CD36 was not investigated.
A disparity was found in PAICA and immunoblots when testing 10 plasmas from HIT patients, with 30% anti-CD36 positive by PAICA and 60% by immunoblots. In tests of patients with HITT, the plasmas of only four cases were available, and 50% were positive by PAICA and 100% by immunoblots. Currently we are collecting plasmas from patients with HIT and HITT for additional tests.
Also in agreement with the report of Tandon et al (1994 ), our data indicated that antibodies directed against CD36 may be involved in initiating thrombotic complications because the target CD36 antigen is present on both platelets and endothelial cells. As we ( Valant et al, 1998 ) and others ( Tandon et al, 1994 ) have shown, TTP plasma causes platelet activation. In addition, TTP plasmas have platelet-neutrophil aggregate promoting activity ( Valant et al, 1998 ). It was possible to test for these activities in TTP plasma because of the development of sensitive flow cytometric methods ( Valant et al, 1998 ).
A valuable source of autoantibodies for this study was the plasma of TTP patient 1. A detailed description of the clinical and laboratory findings of this patient are found in Materials and Methods. During the course of purifying platelet membrane glycoprotein CD36 from Triton X-100 extracts of pooled normal outdated platelets, a second molecular weight form of CD36 was discovered. In addition to the classic 88 kD form purified and characterized by Tandon et al (1989b ) and others ( Oquendo et al, 1989 ; McGregor et al, 1989 ), a lighter 85 kD form of CD36 was highly purified by FPLC technology. Without the use of the autoantibodies, the 85 kD form would have been much more difficult to distinguish from the classic 88 kD form of CD36. The one polyclonal and two monoclonal antisera produced privately (American Red Cross, Rockville, Md.) and the two monoclonal antisera produced commercially reacted strongly with the 88 kD form. All of these antisera reacted weakly or required modifications to produce more sensitive reactions (e.g. biotin-ExtrAvidinTM technology) with the 85 kD form. As we have shown, all patients' autoantibodies collected for this study reacted principally with the 85 kD form.
Clinically, it may be useful to test for autoantibodies to the 85 kD CD36, and this was shown in Fig 3. There appeared to be a correlation between platelet count, haemoglobin, haematocrit, and the detection of antibodies to the 85 kD form of CD36 by immunoblots. Further characterization of this 85 kD form of CD36 is currently under investigation.
The concept that thrombosis is an immune event mediated by specific antibodies has been reviewed by Vermylen et al (1997 ). From our investigation of platelet CD36, two questions relate to antibody-mediated thrombosis: (1) what is the origin of the platelet 85 kD CD36, and (2) are elevated levels of autoantibodies reactive with this form important in the pathophysiology of TTP, ITP and HIT/HITT? It is well documented that native platelet CD36 is 88 kD ( Tandon et al, 1989b ; Oquendo et al, 1989 ; McGregor et al, 1989 ), and is heavily glycosylated, containing 26% carbohydrate ( Tandon et al, 1989b ). Approximately two-thirds were in alkali-labile O-glycosidic linkages ( Tandon et al, 1989b ), and 10 potential N-linked glycosylation sites were located in the extracelluar portion ( Oquendo et al, 1989 ). Sialic acid was identified as a substantial part of the carbohydrate composition ( Tandon et al, 1989b ). The 85 kD form of CD36 may arise as an epiphenomenon which accompanies the pathologic events causing the thrombocytopenia characteristic of the three different conditions. Partially lysed and/or aggregated platelets may not be completely cleared, allowing indigenous sialidases to desialylate oligosaccharides of CD36. Terminal sialic acid (S.A.) removal may enhance accessibility of the immune system to CD36. There are many reports showing that S.A.s mask sequences recognized by autoimmune antibodies (reviewed by Pilatte et al, 1993 ). S.A.s are capable of reducing or preventing accessibility of penultimate recognition sites to the immunosurveillance system ( Schauer, 1985). For example, the T antigen on human erythrocytes treated with neuraminidase renders these cells polyagglutinable because of the anti-T antibody in sera of most adults ( Bird, 1977). Compared to the 88 kD CD36, we have shown that the 85 kD CD36 is deficient in S.A. (unpublished observations), which could possibly explain why this form is the optimal target of the autoantibodies.
For the second question, the concept that antibodies mediate thrombogenic activity is now well appreciated ( Vermylen et al, 1997 ). Binding of antibodies to specific epitopes of cell membranes may cause activation of platelets, leading to activation of prothrombotic events via FcγRII receptors and complement pathway activation. Firm binding of immune complexes to the FcγRII of platelets leads to signal transduction, thromboxane A2 production and granule release ( Vermylen et al, 1997 ). The findings of a higher incidence of anti-CD36 antibodies in TTP and HITT and lower incidence in HIT and ITP ( Table I) fit well with this concept of antibody-mediated thrombosis. Anti-CD36 antibodies may be the crucial cofactor that works in conjunction with other thrombogenic factors to potentiate platelet activation, endothelial cell damage, and enhance prothrombogenic activity. In HITT and HIT, heparin-dependent antibodies interact with platelet factor 4 to form immune complexes that may bind FcγRII, leading to activation of platelets. The presence of additional antibodies such as anti-CD36 may potentiate prothrombotic activity prior to thrombosis. Anti-CD36 antibodies alone or in conjunction with other platelet-activating factors may play a role in the pathogenesis of TTP and HITT. In contrast, in ITP, most anti-platelet antibodies are directed to GPIIb, IIIa ( Karpatkin, 1997), and they seldom activate platelets. The presence of anti-CD36 antibodies in the small percentages of ITP patients may fail to induce thrombogenic activity. In only one of our cases of ITP were recurrent thrombotic complications reported, and anti-CD36 antibodies were detected in this patient.