Plasma from a Patient with Thrombotic Thrombocytopenic Purpura Induces Endothelial Cell Apoptosis and Platelet Aggregation

Abstract

Thrombotic thrombocytopenic purpura (TTP) is characterized pathologically by platelet thrombi deposition and localized microvascular endothelial cell proliferation in arterioles and capillaries [1Kwaan H.C. The pathogenesis of thrombotic thrombocytopenic purpura.Semin Thromb Hemost. 1979; 5: 184-198PubMed Google Scholar]. The pathogenesis of this syndrome is still obscure. The disease process may be initiated by an abnormal interaction between plasma and platelets and/or vascular endothelium, leading to platelet thrombi formation with some fibrin in microvessels [2Lian E.C.-Y. Thrombotic thrombocytopenic purpura.Ann Rev Med. 1988; 39: 203-212Crossref PubMed Scopus (12) Google Scholar]. The presence of factors in plasmas from patients with TTP to induce platelet aggregation/agglutination has been reported [3Siddiqui F.A. Lian E.C.-Y. Novel platelet agglutinating protein isolated from a thrombotic thrombocytopenic purpura plasma.J Clin Invest. 1985; 76: 1330-1337Crossref PubMed Scopus (75) Google Scholar, 4Kelton J.G. Moore J. Santos A. Sheridan D. Detection of a platelet-agglutinating factor in thrombotic thrombocytopenic purpura.Ann Intern Med. 1984; 101: 589-593Crossref PubMed Scopus (75) Google Scholar, 5Consonni R. Falanga A. Barbui T. Further characterization of platelet-aggregating cysteine proteinase activity in thrombotic thrombocytopenic purpura.Br J Haematol. 1994; 87: 321-324Crossref PubMed Scopus (18) Google Scholar, 6Ahn Y.S. Jy W. Kolodny L. Horstman L.L. Mao W.W. Valant P.A. Duncan R.C. Activated platelet aggregates in thrombotic thrombocytopenic purpura Decrease with plasma infusions and normalization in remission.Br J Haematol. 1996; 95: 408-415Crossref PubMed Scopus (31) Google Scholar, 7Chen S.H. Lian E.C.-Y. Purification and some properties of 59KDa platelet aggregating protein from the plasma of a patient with thrombotic thrombocytopenic purpura.Blood. 1988; 72: 318aGoogle Scholar]. Endothelial cell damage has also been speculated to be a crucial feature in some serial events that proceed to develop microvascular thrombotic lesions [8Laurence J. Mitra D. Steiner M. Staiano-Coico L. Jaffe E. Plasma from patients with idiopathic and human immunodeficiency virus-associated thrombotic thrombocytopenic purpura induces apoptosis in microvascular endothelial cells.Blood. 1996; 87: 3245-3254PubMed Google Scholar, 9Takafuta T. Kawano H. Fujimura K. Kuramoto A. Vascular endothelial cell injury as the initial event in thrombotic thrombocytopenic purpura.Nippon Rinsho. 1993; 51: 138-141PubMed Google Scholar, 10Burns E.R. Zucker-Franklin D. Pathologic effects of plasma from patients with thrombotic thrombocytopenic purpura on platelets and cultured vascular endothelial cells.Blood. 1982; 60: 1030-1037Crossref PubMed Google Scholar]. In the present study, we demonstrate that substance(s) in the plasma from a patient with TTP induces both endothelial cell apoptosis and platelet aggregation. A TTP plasma sample was obtained from plasmapheresis during treatment through a blood bank. The diagnosis was based on clinical manifestations of thrombocytopenia, schizocytic hemolytic anemia, renal dysfunction, and fluctuating neurologic symptoms. Plasma controls were obtained from healthy volunteers through venepuncture as well as from two non-TTP patients undergoing plasmapheresis. Informed consents were obtained and the study approved by the Institutional Ethics Committee on Human Research. Human endothelial cell line ECV-304 from American Type Culture Collection (Rockville, MD) is a spontaneously transformed immortal endothelial cell line established from the vein of an apparently normal human umbilical cord. Cells were maintained in M-199 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO2. 2×105 viable cells in 1 ml were seeded in 6-well plates and grown to a confluence. Before use, cells were rinsed with M-199 medium and then incubated with 1 ml of TTP or control plasmas in the incubator for 48 hours or for variable durations. In most of experiments, the plasmas were diluted to 80% with M-199 medium containing 10% FBS and 100 μg/ml of heparin. Propidium iodide (PI) was used to determine cells undergoing apoptosis as described by Nicoletti et al. [11Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunol Methods. 1991; 139: 271-279Crossref PubMed Scopus (4431) Google Scholar] with minor modifications. Briefly, cells were harvested by digestion with trypsin (0.25%) EDTA (0.03%) at 37°C for 10 minutes, washed in Hank’s balanced salt solution (HBSS), and fixed with 70% ethanol for 3 hours at 4°C. The cells were then washed and incubated with RNase (1 mg/ml) in HBSS, followed by addition of 0.1 ml of PI (100 μg/ml in HBSS). After gentle mixing cells were incubated for 45 minutes at 22°C in the dark. The PI-stained nuclei were measured in a flow cytometer for fluorescence intensity or viewed under fluorescence microscopy for morphology. After staining with PI, the cells were resuspended in 1 ml of HBSS and analyzed by flow cytometry [11Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunol Methods. 1991; 139: 271-279Crossref PubMed Scopus (4431) Google Scholar, 12Ormerod M.G. Collins M.K. Rodriguez-Tarduchy G. Robertson D. Apoptosis in interleukin-3 dependent haemopoietic cells Quantification by two flow cytometric methods.J Immunol Methods. 1992; 153: 57-65Crossref PubMed Scopus (190) Google Scholar]. Cell debris and clumps were excluded by gating for single cells under forward and side light scatters. A total of 5000 cells were analyzed, and the percentage of cells undergoing apoptosis with a characteristic hypodiploid sub-G0/G1 peak was determined. Cell morphology was observed under an inverted microscope with phase-contrast. The apoptotic features were directly visualized with a fluorescence microscope after staining with PI. DNA strand breaks were also detected by terminal deoxynucleotidyl transferase (TdT)–mediated deoxyuridine triphosphate (dUTP) nick-end labeling (Tunel) method by using ApopTag In Situ Apoptosis Detection kit-peroxidase (Oncor, Gaithersburg, MD) according to the manufacturer’s instruction. The DNA fragments were quantified using Cell Death Detection ELISAplus kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instruction. R-Phycoerythrin–conjugated MAb Apo2.7 (2.7A 6A3) (Immunotech, Westbrook, ME) was used to identify apoptosis [13Zhang C. Ao Z. Seth A. Schlossman S.F. A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells.J Immunology. 1996; 157: 3980-3987PubMed Google Scholar]. The binding of 2.7A6A3 to cells was studied by direct staining of cells and analyzed by flow cytometry. The effects of CD95 MAb or EDTA on TTP plasma induced apoptosis of ECV-304 were examined by incubation of cells with TTP plasma in the presence or absence of soluble MAb ZB4 against CD95 (1 ug/ml; Immunotech), which inhibits apoptosis, or EDTA (5mM). Apoptotic cells were quantified by determination of cytoplasmic histone-associated DNA fragments by using an ELISA method. The effects of TTP plasma and inhibitors on platelet aggregation were performed as previously described [3Siddiqui F.A. Lian E.C.-Y. Novel platelet agglutinating protein isolated from a thrombotic thrombocytopenic purpura plasma.J Clin Invest. 1985; 76: 1330-1337Crossref PubMed Scopus (75) Google Scholar]. The effects of TTP plasma on ECV-304 cells were first examined by phase-contrast microscopy for cell morphology. Compared to normal plasmas, incubation of cells with TTP plasma for 48 hours caused cells to round up from typical cobble stone shape, loss of cell connections, and increase of intracytoplasmic granules and vacuoles (Figure 1).Fig. 1Morphologic change of ECV-304 cells caused by TTP plasma. (original ×100). The cells were incubated with normal (A) or TTP (B) plasma at 37°C with 5% CO2 for 24 hoursView Large Image Figure ViewerDownload (PPT) It has been recognized that apoptotic cells have reduced DNA stainability after staining with a variety of fluorochrome [14Darzynkiewicz Z. Bruno G. Del Bino G. Gorczyca W. Hotz M.A. Lassota P. Traganos F. Features of apoptotic cells measured by flow cytometry.Cytometry. 1992; 13: 795-808Crossref PubMed Scopus (1913) Google Scholar]. Therefore, the appearance of cells with reduced DNA stainability, lower than that of G0/G1 cells (termed as sub-G0/G1 cell) in cultures treated with various reagents, has been considered as a marker of apoptosis [12Ormerod M.G. Collins M.K. Rodriguez-Tarduchy G. Robertson D. Apoptosis in interleukin-3 dependent haemopoietic cells Quantification by two flow cytometric methods.J Immunol Methods. 1992; 153: 57-65Crossref PubMed Scopus (190) Google Scholar]. When this method was applied to the cultured ECV-304 cells exposed to plasma from a TTP patient or control subjects, TTP plasma treated ECV-304 cells showed marked apoptosis as manifested by a sub-G0/G1 peak with reduced DNA content in flow cytometry (Figure 2). When PI-stained ECV-304 cells were examined under fluorescence microscope, cell nuclei in normal control plasma exhibited regular shape and no apoptotic cells were found (Figure 3A). In contrast, cells treated with TTP plasma displayed the presence of apoptotic characteristics, such as nuclear fragmentation or condensation and dispersion of chromatin (Figure 3B).Fig. 3Direct microscopic visualization of plasma-treated ECV-304 cells stained with PI. ECV-304 cells were exposed to 80% normal plasma (A) or TTP plasma (B) for 48 hours. Cells were fixed with 70% ethanol, preincubated with RNase, and then stained with PIView Large Image Figure ViewerDownload (PPT) MAb Apo2.7 reacts with a 38-kDa mitochondrial membrane protein that is expressed on cells undergoing apoptosis. It has been suggested that Apo2.7 protein is involved in the molecular cascade of apoptosis and its expression represents an early event of apoptosis rather than a final product of apoptotic cells [13Zhang C. Ao Z. Seth A. Schlossman S.F. A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells.J Immunology. 1996; 157: 3980-3987PubMed Google Scholar]. To quantify the apoptotic cells after incubation with TTP plasma, cells were stained with PE-conjugated Apo2.7 without permeation and analyzed by flow cytometry. As shown in Figure 4, in the presence of TTP plasma, 22.0± 2.5% cells had increased expression of Apo2.7, which is consistent with the results from DNA content study. To further confirm the apoptosis of ECV-304 cells after incubation with TTP plasma, TdT-mediated digoxigenin-dUTP/anti-digoxigenin-peroxidase nick-end labeling using ApopTag In Situ Apoptosis Detection kit-peroxidase was studied. In control plasma treated ECV-304 cells, there was no suggestive apoptotic stain (Figure 5A). In contrast, TTP plasma treated ECV-304 cells showed about 25% cells with positive staining (Figure 5B). A time-dependent effect of TTP plasma or control plasma on ECV-304 cells was studied for up to 72 hours. The change of cell morphology was determined directly under a phase-contrast microscope within 1 hour after addition of TTP plasma in the culture. However, apoptosis was detected by flow cytometry showing an increased percentage of cells in the sub-G0/G1 peak only after 24 hours of incubation with TTP plasma (Figure 6). To determine the effect of plasma concentrations on TTP plasma– induced apoptosis of ECV-304 cells, the TTP plasma or normal plasma controls were diluted with M199 medium containing 10% FBS and 100 ug/ml of heparin. The minimal concentration of plasma required to induce cell apoptosis in this experimental setting is 60% of plasma (Figure 7). The CD95 (FAS/APO-1) antigen is a 40–50-kDa transmembrane glycoprotein belonging to the nerve growth factor/tumor necrosis factors receptor superfamily. It mediates apoptosis in various cell types [15Nagata S. Golstein P. The Fas death factor.Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3980) Google Scholar]. To define whether this mechanism is applicable in TTP plasma–induced ECV-304 cell apoptosis, a MAb ZB4 (to CD95) with inhibitory effect was used. ZB4 (1 ug/mL) had no effect when compared with control. This indicates that CD95 may not play an important role in this process. Ca++ is an important cofactor for many proteins or enzymes, such as endonuclease. To clarify the effects of Ca++ on the TTP plasma induced apoptosis of ECV-304 cells, we added EDTA to the incubation mixture of TTP-plasma and ECV-304 cells. In the presence of 5 mM EDTA, TTP plasma induced apoptosis of ECV-304 was completely inhibited. After addition of 0.1 ml washed platelet suspensions into the mixture of 0.2 ml TTP plasma and 0.2 ml buffer that was preincubated at 37°C for 1 hour, platelet aggregation was detected using a Chronolog aggregometer. When 0.2 ml TTP plasma was preincubated with 0.2 ml normal plasma at 37°C for 1 hour, platelet aggregation was inhibited (Figure 8). Preincubation of TTP plasma with 2 mM of aspirin, 2 U/ml of heparin, or 2 U/ml of hirudin at 37°C for 10 minutes did not inhibit TTP plasma–induced platelet aggregation. TTP occurs idiopathically or in association with infections, organ transplantation, cancer, drugs, and collagen vascular diseases. The pathogenesis of TTP remains unknown. Our previous studies have demonstrated that TTP plasma is capable of inducing platelet aggregation [16Lian E.C.Y. Harkness D.R. Byrnes J.J. Wallach H. Nunez R. Presence of a platelet aggregating factor in the plasma of patients with thrombotic thrombocytopenic purpura (TTP) and its inhibition by normal plasma.Blood. 1979; 53: 333-338PubMed Google Scholar] that also has been confirmed by other investigators [6Ahn Y.S. Jy W. Kolodny L. Horstman L.L. Mao W.W. Valant P.A. Duncan R.C. Activated platelet aggregates in thrombotic thrombocytopenic purpura Decrease with plasma infusions and normalization in remission.Br J Haematol. 1996; 95: 408-415Crossref PubMed Scopus (31) Google Scholar, 17Ansell J.E. Slepchuk Jr, N.I. Pechet L. Platelet-aggregating factor in thrombotic thrombocytopenic purpura plasma.Blood. 1979; 54: 959-960PubMed Google Scholar, 18Brandt J.T. Kennedy M.S. Senhausen D.A. Platelet aggregating factor in thrombocytopenic purpura.Lancet. 1979; 2: 463-464Abstract PubMed Scopus (13) Google Scholar]. Several hypotheses to explain the activation of platelets in TTP, including platelet clumping factors [3Siddiqui F.A. Lian E.C.-Y. Novel platelet agglutinating protein isolated from a thrombotic thrombocytopenic purpura plasma.J Clin Invest. 1985; 76: 1330-1337Crossref PubMed Scopus (75) Google Scholar, 7Chen S.H. Lian E.C.-Y. Purification and some properties of 59KDa platelet aggregating protein from the plasma of a patient with thrombotic thrombocytopenic purpura.Blood. 1988; 72: 318aGoogle Scholar], unusually large vWF multimers due to lack of depolymerase [19Moake J.L. Rudy C.K. Troll J.H. Weinstein M.J. Colannino N.M. Azocar J. Seder R.H. Hong S.L. Deykin D. Unusually large plasma factor VIII Von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura.N Engl J Med. 1982; 307: 1432-1435Crossref PubMed Scopus (930) Google Scholar, 20Furlan M. Robles R. Solenthaler M, Wassmer M, Sandoz P, Lammle B. Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1997; 89: 3097-3103Crossref PubMed Google Scholar], calpain [4Kelton J.G. Moore J. Santos A. Sheridan D. Detection of a platelet-agglutinating factor in thrombotic thrombocytopenic purpura.Ann Intern Med. 1984; 101: 589-593Crossref PubMed Scopus (75) Google Scholar], antibodies [10Burns E.R. Zucker-Franklin D. Pathologic effects of plasma from patients with thrombotic thrombocytopenic purpura on platelets and cultured vascular endothelial cells.Blood. 1982; 60: 1030-1037Crossref PubMed Google Scholar, 21Tandon N.N. Rock G. Jamieson G.A. Anti-CD36 antibodies in thrombotic thrombocytopenic purpura.Br J Haematol. 1994; 88: 816-825Crossref PubMed Scopus (102) Google Scholar], and decreased PGI2 synthesis and stability [22Remuzzi G. Misiani R. Mecca G. de Gaetano G. Donati M.B. Thrombotic thrombocytopenic purpura—a deficiency of plasma factors regulating platelet-vessel-wall interaction?.N Engl J Med. 1978; 299: 311PubMed Google Scholar, 23Chen Y.C. McLeod R. Hall E.R. Wu K.K. Accelerated prostacyclin degradation in thrombotic thrombocytopenic purpura.Lancet. 1981; 2: 267-269Abstract PubMed Scopus (85) Google Scholar], or secondary to endothelial cell damage [8Laurence J. Mitra D. Steiner M. Staiano-Coico L. Jaffe E. Plasma from patients with idiopathic and human immunodeficiency virus-associated thrombotic thrombocytopenic purpura induces apoptosis in microvascular endothelial cells.Blood. 1996; 87: 3245-3254PubMed Google Scholar, 9Takafuta T. Kawano H. Fujimura K. Kuramoto A. Vascular endothelial cell injury as the initial event in thrombotic thrombocytopenic purpura.Nippon Rinsho. 1993; 51: 138-141PubMed Google Scholar, 10Burns E.R. Zucker-Franklin D. Pathologic effects of plasma from patients with thrombotic thrombocytopenic purpura on platelets and cultured vascular endothelial cells.Blood. 1982; 60: 1030-1037Crossref PubMed Google Scholar], have been postulated. We have characterized several platelet clumping factors in TTP plasma and found that they are heterogeneous [3Siddiqui F.A. Lian E.C.-Y. Novel platelet agglutinating protein isolated from a thrombotic thrombocytopenic purpura plasma.J Clin Invest. 1985; 76: 1330-1337Crossref PubMed Scopus (75) Google Scholar, 7Chen S.H. Lian E.C.-Y. Purification and some properties of 59KDa platelet aggregating protein from the plasma of a patient with thrombotic thrombocytopenic purpura.Blood. 1988; 72: 318aGoogle Scholar, 24Lian E.C.-Y. The role of increased platelet aggregation in TTP.Semin Thromb Hemostasis. 1980; 6: 401-415Crossref PubMed Scopus (27) Google Scholar]. This indicates that TTP is caused by different etiology. It is not clear whether the stimuli that activate platelets might also cause endothelial cell damage. In the present study, plasma from a TTP patient not only induced platelet aggregation but also caused endothelial cell apoptosis. The pathological lesion of TTP consists of platelet thrombi deposition, endothelial cell swelling and proliferation in affected microvasculature [25Baehr G. Kelmperer P. Schifrin A. An acute febrile anemia and thrombocytopenic purpura with diffuse platelet thrombosis of capillaries and arterioles.Trans Assoc Am Physicians. 1936; 51: 43Google Scholar]. Endothelial cells appear in the peripheral blood of patients with TTP [26Lefevre P. George F. Durand J.M. Sampol J. Detection of circulating endothelial cells in thrombotic thrombocytopenic purpura.Thromb Haemost. 1993; 69: 522PubMed Google Scholar], suggesting endothelial cell injury. In this study, we found that ECV-304 endothelial cells underwent an apoptotic process upon exposure to TTP plasma. This was demonstrated by the following observations: (1) after stimulation with TTP, plasma cells became rounded-up, detached from each other, and showed increased intracytoplasmic granules and vacuoles; (2) TTP plasma–treated cells exhibited a DNA content typical of apoptosis in flow cytometry after staining with PI; (3) PI-stained nuclei from TTP plasma–treated ECV-304 cells displayed the presence of apoptotic characteristics; (4) There was an increased binding of MAb against Apo2.7 to the cells exposed to TTP plasma; and (5) terminal deoxynucleotidyl transferase–mediated digoxigenin-dUTP/antidigoxigenin-peroxidase nick-end labeling showed positive stain in cells after TTP plasma treatment. The in vascular endothelial cell injury by TTP plasma or serum has been demonstrated et al. endothelial cell injury in the pathogenesis of thrombotic thrombocytopenic purpura Scholar] and and P.A. of plasma from patients with thrombotic thrombocytopenic purpura (TTP) on cultured human endothelial cells.Blood. 1979; 54: Scholar] showed a factor for endothelial cells in the serum or plasma from some TTP and Zucker-Franklin [10Burns E.R. Zucker-Franklin D. Pathologic effects of plasma from patients with thrombotic thrombocytopenic purpura on platelets and cultured vascular endothelial cells.Blood. 1982; 60: 1030-1037Crossref PubMed Google Scholar] demonstrated that serum and of TTP patients caused the of endothelial cells and that plasmas induced aggregation of normal human platelets in However, other investigators to demonstrate the activity against endothelial Thrombotic thrombocytopenic purpura results of 1980; PubMed Scopus (3) Google Scholar]. Human antibodies against have been shown to with cultured endothelial cells and cause H. T. E. of human endothelial cell by 1979; PubMed Scopus Google Scholar]. plasma from patients with TTP was shown to induce apoptosis in microvascular endothelial cells of [8Laurence J. Mitra D. Steiner M. Staiano-Coico L. Jaffe E. Plasma from patients with idiopathic and human immunodeficiency virus-associated thrombotic thrombocytopenic purpura induces apoptosis in microvascular endothelial cells.Blood. 1996; 87: 3245-3254PubMed Google Scholar, D. Jaffe B. C. J. Thrombotic thrombocytopenic purpura and syndrome plasmas induce apoptosis in of human microvascular endothelial cells.Blood. 1997; 89: PubMed Google Scholar]. Apoptosis is a process through various of Fas or of apoptosis by an antibody in cells is of the [15Nagata S. Golstein P. The Fas death factor.Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3980) Google Scholar, S. A. M. M. A. S. The by the for human cell antigen Fas 1991; PubMed Scopus Google Scholar]. It has been reported that the endothelial cells exposed to TTP plasma We used a soluble CD95 MAb that inhibits the apoptosis to study the mechanism of TTP plasma–induced endothelial cell apoptosis. Our results showed that CD95 MAb did not inhibit the apoptosis, suggesting that Fas may not be involved in this process. This is different from a previous that plasma from patients with idiopathic and human immunodeficiency apoptosis in microvascular endothelial cells, which was to the rapid of CD95 on the cells, and soluble antibody was capable of TTP endothelial cell apoptosis [8Laurence J. Mitra D. Steiner M. Staiano-Coico L. Jaffe E. Plasma from patients with idiopathic and human immunodeficiency virus-associated thrombotic thrombocytopenic purpura induces apoptosis in microvascular endothelial cells.Blood. 1996; 87: 3245-3254PubMed Google Scholar]. The of 5 mM EDTA to inhibit apoptosis the of in TTP plasma. In the results of the present study substance(s) in TTP plasma may induce endothelial cell damage by apoptosis and platelet The properties of substance(s) in this TTP plasma that induces platelet aggregation and endothelial cell apoptosis are under The is in by the of and the