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International Immunology, Vol. 13, No. 2, 203-210, February 2001
© 2001 Japanese Society for Immunology

Anti-endothelial cell antibodies from patients with thrombotic thrombocytopenic purpura specifically activate small vessel endothelial cells

Sonja Praprotnik1,2, Miri Blank1, Yair Levy1, Sigal Tavor3, Marie-Claire Boffa4, Babette Weksler5, Amiram Eldor3 and Yehuda Shoenfeld1

1 Research Unit of Autoimmune Diseases and Department Medicine B, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel
2 Clinical center, Department of Rheumatology, Vodnikova 62, 1000 Ljubljana, Slovenia
3 Institute of Hematology, Sorasky Medical Center, Tel-Aviv 54145, Israel
4 INSERM U353, Hopital Saint-Louis, 75475 Paris, France
5 Weill Medical College of Cornell University, New York, NY 10021, USA

Correspondence to: Correspondence to: Y. Shoenfeld


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Thrombotic thrombocytopenic purpura (TTP) is an uncommon disease of an unknown etiology, characterized by consumptive thrombocytopenia, microangiopathic hemolytic anemia, fever and acute thrombotic complications, especially within the cerebral circulation. Although anti-endothelial cell antibodies (AECA) have occasionally been shown to be present in TTP, their role in the pathogenesis of the disease has never been ascertained. In the current study we demonstrated the pathogenic activity of affinity-purified anti-endothelial cell F(ab)2 antibodies (AECA/TTP) from four consecutive patients with active TTP. These AECA/TTP bound to and activated only microvascular endothelial cells (EC) and not large vessel EC. The specificity of AECA/TTP binding to microvascular EC was confirmed by competition assay employing membranes derived from small and large vessels EC. Activation included enhanced IL-6 and von Willebrand factor release from the EC followed by increased expression of adhesion molecules P-selectin, E-selectin and vascular cell adhesion molecule-1 on the EC, as evaluated by ELISA. Increased expression of adhesion molecules was followed by an increase in monocyte adhesion to EC. The level of soluble thrombomodulin (TM) also increased in the culture medium of activated microvascular EC upon exposure to AECA/TTP antibodies and was directly correlated to a decrease in cell-associated TM. Our data suggest that AECA/TTP directed against microvascular EC could play a pathogenic role in the development of endothelial injury in TTP that leads to thrombosis.

Keywords: adhesion molecules, anti-endothelial cell antibodies, autoimmunity, endothelial cell, thrombomodulin, thrombotic thrombocytopenic purpura


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Autoantibodies directed against endothelial cells (AECA) have been described in several diseases in which immune-mediated endothelial damage, either primary or secondary to systemic autoimmune processes, is present (1–5). Analysis of the antigens recognized by AECA showed that these antibodies are directed against a variety of both constitutive and inducible surface membrane proteins (2–5). The diversity of AECA-associated diseases suggests that the AECA are heterogeneous. Some of them have a pathogenic potential and may induce experimental autoimmune disease (6), whereas others may appear as a result of the inflammatory vascular injury rather than as a direct pathogenic factor. Recently, we (7,8) and others (9–11) have demonstrated that AECA from different sources [e.g. antiphospholipid syndrome, Wegener's granulomatosis, systemic sclerosis and Takayasu's arteritis (TA)] have the potential to activate endothelial cells (EC). Based on our previous observations that AECA from a large vessel disease like TA activate macrovascular human umbilical vein EC (HUVEC) and not microvascular EC, we hypothesize that AECA from patients with different diseases recognize different types of EC target molecules that are correlated to the disease origin. In the current study, TTP was chosen as a representative of a microvascular thrombotic condition. Thrombotic thrombocytopenic purpura (TTP) is an uncommon disease characterized by consumptive thrombocytopenia, microangiopathic hemolytic anemia, fever, renal dysfunction and acute thrombotic complications, especially within the cerebral circulation. The thrombotic complications result from occlusion of the arterioles and capillaries by platelet-fibrin thromboemboli. Endothelial proliferation may be seen in the older thrombi, although one report point to a limited perivascular inflammation in TTP (12). The hypotheses concerning the etiology of TTP are controversial and varied (12–19). An immune mechanism has also been suggested, and in this regard TTP has been associated with diseases characterized by abnormal immune responses and/or autoimmunity, including AIDS and systemic lupus erythematosus (12). The effectiveness of immunosuppressive therapy in some patients could reflect an underlying autoimmune pathogenesis (19,20). Complement-dependent antibodies cytotoxic for umbilical vein HUVEC have been reported in some TTP plasmas (21,22). Antibodies binding a 43 kDa protein derived from human renal microvascular EC were detected in patients with TTP (23). Additionally, antibodies against the endothelial cell membrane glycoprotein CD36 (GPIV) were identified in a large group of TTP patients (24,25) and shown to potentiate platelet activation (25). The authors anticipated that these antibodies could also interact with CD36 presented on the EC of the microvasculature. Although AECA have been shown to be present in TTP, their role in the pathogenesis of the disease has never been ascertained.

In the present study we demonstrate the ability of AECA from four TTP patients specifically to bind to microvascular but not to macrovascular EC and selectively to promote the activation of microvascular EC.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Origin of TTP antibodies
Plasmapheresis was collected from two TTP patients and sera from the other two TTP patients, admitted to the Institute of Hematology, by phlebotomy on the first plasma exchange before any other treatment was administered (these procedures were done under an institutionally approved protocol). Two patients had relapsing TTP and two had a single episode. All patients had thrombocytopenia, hemolytic anemia and neurologic events, and three-quarters had fever. Other causes of thrombocytopenia were excluded. All the plasmapheresis and sera from the TTP patients were inactivated (56°C, 1 h) and stored in aliquots at –70°C until use.

EC
Microvascular EC derived from human bone marrow were used in primary cultures (HBMEC) or as a cell line immortalized by SV-40 (TrHBMEC) (8,27). HUVEC primary cultures served as large vessel EC (8,28). In all the EC activation studies, the cells were placed for 8 h in the presence of 1% FCS, in order to avoid EC activation by 10% FCS, in which the EC are generally grown. Preliminary studies showed that this low serum pretreatment did not cause EC activation.

Preparation of affinity-purified AECA F(ab)2
AECA/TTP were affinity purified in a two-step procedure. (i) The total IgG fraction was isolated on Protein G–Sepharose columns (Pharmacia, Uppsala, Sweden). (ii) The purified total IgG was then incubated, with shaking for 4 h at 4°C on confluent monolayers of TrHBMEC which were then washed repeatedly. IgG bound to the EC during the incubation was eluted by glycine–HCl (0.2 M, pH 2.5), neutralized with Tris buffer, dialyzed extensively against PBS and concentrated. The percentage of AECA/TTP IgG in these patients' plasma varied between 0.5 and 2.5% of total IgG. We used two kinds of negative controls: (i) IgG from one of the TTP patients following removal of AECA/TTP and (ii) IgG from a patient with TA that binds large vessel EC (HUVEC) but not microvascular EC. No binding of the TTP patients' total IgG to HUVEC was detected. IgG from pooled normal plasma binds neither HUVEC nor TrHBMC (data not shown).

F(ab)2 preparation
F(ab)2 fragments were prepared from each patient's AECA IgG fraction as previously described by us (8). Affinity-purified AECA as an intact IgG molecule or its F(ab)2 fragment were used for the binding and functional studies presented here.

Detection of AECA
AECA/TTP IgG or F(ab)2 binding to EC (microvascular: HBMEC or TrBMEC; and macrovascular: HUVEC) was determined by cyto-ELISA on native EC, cultured in 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 48 h (8).

Determination of IL-6 secretion by EC
IL-6 secreted into the culture medium of EC during 24 h exposure to purified AECA/TTP was measured by commercial kits, according to the manufacturers' specifications (Duosets; R & D Systems, Minneapolis, MN).

Determination of von Willebrand factor (vWF) secretion by EC
vWF released into the culture medium by EC during exposure to purified AECA/TTP was determined by ELISA using plates coated with anti-vWF, preblocked with 3% BSA. EC culture media were added to the coated plates for 2 h, and probed with biotinylated rabbit anti-vWF and streptavidin–alkaline phosphatase.

Adhesion of monocytes and detection of adhesion molecule expression by EC
The monocyte adhesion assay was performed as previously described (8,9). Briefly, U937 (a monocyte macrophage-like cell line) cells were pretreated with heat-aggregated {gamma}-globulin for 30 min at 37°C (to block Fc receptor binding) and labeled with 0.5 µCi/ml of [3H]thymidine (Amersham, Little Chalfont, UK) for 24 h. Adhesion assays were performed on TrHBMEC monolayers, 24 h preincubated with AECA/TTP affinity-purified IgG F(ab)2 fractions at concentrations ranging from 0 to 50 µg/ml. The EC monolayers were extensively washed and radiolabled U937 cells were added to each well in RPMI 1640 medium containing 0.2% BSA for 30 min at 37°C. The non-adherent cells were removed by washing and the cells were lysed with formic acid. Radioactivity associated with adherence was quantified by ß-scintillation spectroscopy. The results were expressed as percent of added U937 cells that adhered and are presented as mean ± SD % adhesion from three separate experiments. As 100% adhesion we calculated the same concentration of monocytes added into a studied well, lysed with formic acid.

A separate set of experiments was designed to correlate between adhesion molecules expression and U937 adhesion mediated by the AECA/TTP. Accordingly, a mixture of monoclonal anti-intercellular adhesion molecule (ICAM)-1, anti-vascular cell adhesion molecule (VCAM)-1 and anti-E-selectin antibodies (PharMingen, San Diego, CA) (20 µg/ml, each) was added to the TrHBMEC following preincubation with each of the mAb (25 µg/ml), and the assay was continued as described above.

Expression of adhesion molecules VCAM-1, E-selectin and P-selectin on EC was detected on EC permeabilized by Triton X-100, and probed by specific biotinylated antibody, as detailed elsewhere (8,29). The expression of ICAM-1 was performed according to the same protocol employing non-permeabilized cells. In part of the experiment Polymixin B was added to the assay in order to confirm the specific activation of adhesion molecule expression caused by AECA/TTP and not by enterotoxin contamination.

Detection of TM in the culture medium and in lysed TrHBMEC
Soluble TM levels in the cell culture supernatants or lysed EC were determined by a capture ELISA (Asserachrom TM ELISA kit; Diagnostica Stago, Asnieres, France). The ELISA was performed with anti-TM-precoated plates, according to the manufacturer's instructions. The dilutions used were 1:3 for cell culture supernatants and 1:6 for cell lysates.

Statistical analysis
Groups of observations were analyzed using ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Anti-endothelial cell binding properties of TTP patient plasmas
Affinity-purified AECA/TTP from all four patients with TTP (TTP-1–4) bound microvascular EC, including both primary HBMEC and transformed TrHBMEC (Table 1Go). When HUVEC were exposed to the same AECA/TTP, no significant binding above the background with control IgG from pooled normal plasma was observed (Table 1Go). For a positive control to demonstrate AECA binding to HUVEC, we used AECA IgG from a patient with TA which targets specifically macrovascular HUVEC (8).


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  		Table 1. Binding of AECA from TTP patients to ECs
 
In order to clarify which part of the Ig molecule binds EC, we compared binding of F(ab)2 and of Fc fragments of AECA/TTP to EC. AECA/TTP bind EC via the F(ab)2 portion of the Ig and not via the Fc [e.g. binding of AECA F(ab)2 from patient TTP-2 to TrHBMEC resulted in an OD (405 nm) of 1.303 ± 0.172 while the AECA Fc resulted in only 0.232 ± 0.071 OD, P > 0.001] (Table 1Go). Moreover, AECA IgG F(ab)2 binding to microvascular EC increased in a concentration-dependent manner (Fig. 1Go).



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  		Fig. 1. Kinetic study of AECA/TTP binding to TrHBMEC. Microvascular EC were exposed for 16 h to different concentrations of AECA/TTP F(ab)2. Data are presented as OD 405 nm, mean ± SD of three separate experiments.

 
AECA/TTP from all four TTP patients bound microvascular EC with high specificity, evidenced by increasing inhibition of AECA/TTP binding to TrHBMEC by increasing concentrations of TrHBMEC membranes (P < 0.001) (data not shown). In contrast, no inhibition of binding of AECA/TTP to microvascular EC was detected in the presence of membranes prepared from HUVEC, representing macrovascular EC (P > 0.05) (data not shown).

Effect of AECA/TTP on IL-6 and vWF secretion by TrHBMEC
IL-6 secretion by the TrHBMEC upon exposure to affinity-purified AECA/TTP was used as evidence for EC activation. As demonstrated in Fig. 2Go, confluent TrHBMEC exposed to 25 µg/ml AECA/TTP-1 to AECA/TTP-4 for 16 h secreted high levels of IL-6 during that time (P < 0.001 as compared to TrHBMEC exposed to control IgG and IgG from a patient with TA).



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  		Fig. 2. IL-6 secretion by TrHBMEC upon exposure to AECA/TTP. The amount of IL-6 detected in the culture fluid of TrHBMEC cultured in the presence of AECA/TTP during 16 h. Data are presented as ng/ml, mean ± SD of three separate experiments.

 
Release of vWF into the EC culture medium by EC exposed to AECA was also studied by capture ELISA. Elevated levels of vWF were detected in the culture fluid of TrHBMEC exposed to AECA/TTP-1 to AECA/TTP-4 (range 1.324 ± 0.243 to 0.793 ± 0.078 OD at 405 nm, P < 0.001 for TTP-1–3 and P < 0.03 for TTP-4 when compared to AECA/TA or IgG control).

Effect of AECA/TTP on adhesion molecule expression by EC
As an additional parameter of EC activation, we studied the capacity of all four affinity-purified AECA/TTP to increase the expression of adhesion molecules by microvascular EC. As shown in Fig. 3Go, AECA F(ab)2 induced an increase in E-selectin, P-selectin and VCAM-1 expression in HBMEC, whereas AECA F(ab)2 from a patient with TA and normal human IgG F(ab)2 did not induce expression of these adhesion molecules. AECA/TTP had different effects upon expression of particular adhesion molecules, e.g. they did not induce ICAM-1 in HBMEC, although incubation of HBMEC with tumor necrosis factor-{alpha} did (P > 0.05) (data not shown). Induced expression of E-selectin and P-selectin expression by TrHBMEC followed a concentration-dependent pattern of response to AECA/TTP (data not shown). The elevated expression of adhesion molecules was not related to endotoxin contamination because not all of the adhesion molecules tested showed enhanced expression (ICAM-1 not) and addition of Polymixin B did not showed any effect on the level of adhesion molecules expression (data not shown).



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  		Fig. 3. Adhesion molecule expression by HBMEC exposed to AECA/TTP: E-selectin, P-selectin, VCAM-1 and ICAM-1 expression on HBMEC upon exposure to 25 µg/ml AECA/TTP F(ab)2 for 16 h (TTP-1, dashed bars; TTP-2, dot bars; TTP-3, ladder bars; TTP-4, white bars; AECA/TA, solid bars; control IgG, square bars). Data are presented as OD 405 nm, mean ± SD of three separate experiments.

 
Effect of AECA/TTP on monocyte adhesion onto microvascular EC (TrHBMEC)
Adhesion of monocytes to EC is considered a marker of EC activation. Figure 4Go illustrates that U937 cells increase adhesion following exposure of EC to AECA. The AECA/TTP that best induced U937 adhesion to microvascular EC also induced the most expression of E-selectin (cf. Fig. 3Go). Adhesion of U937 cells to the EC was dose dependent, reaching maximal values of 93 ± 7, 82 ± 6, 68 ± 4 and 53 ± 7% after incubation with 50 µg/ml of purified AECA from TTP-2, -3, -1 and -4 respectively. In comparison, AECA from TA and control human IgG produced <15% adhesion (P < 0.01 for TTP-1–3 and P < 0.03 for TTP-4). Prior incubation of microvascular EC with mAb against E-selectin, P-selectin and VCAM-1, using the same assays, significantly decreased the effect of AECA induction U937 adherence to EC (Fig. 4A and BGo) (P > 0.01). Kinetic studies revealed that AECA/TTP-1 to AECA/TTP-3 already showed significant adhesion by 8 h of incubation of the microvascular cells with the Ig (P < 0.04), while AECA/TTP-4 induced monocyte adhesion after 12 h of incubation. All AECA/TTP-1–4 further increased monocyte adhesion after 16 h of pretreatment of the EC (P < 0.001) (Fig. 5Go).




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  		Fig. 4. Adhesion of U937 to TrHBMEC exposed to AECA/TTP: TrHBMEC were incubated with AECA/TTP at a concentration of 25 µg/ml for 16 h. Adhesion of [3H]thymidine-labeled U937 cells was measured by scintillation spectroscopy. (A) Percent of monocyte adhesion to TrHBMEC exposed to different concentrations of AECA/TTP. Data are presented as OD 405 nm, mean ± SD of three separate experiments. (B) Percent of monocyte adhesion to TrHBMEC, preincubated with a cocktail of mAb directed to E-selectin, P-selectin, VCAM-1 and ICAM-1 during 4 h, and then exposed to AECA/TTP. Data are presented as OD 405 nm, mean ± SD of three separate experiments.

 


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  		Fig. 5. Kinetics of U937 monocyte adhesion to TrHBMEC exposed to AECA/TTP: TrHBMEC were incubated for 4, 8, 12 and 16 h with AECA/TTP 25 µg/ml, monocyte adhesion to the EC was studied thereafter (TTP-1, dashed bars; TTP-2, dot bars, TTP-3; ladder bars; TTP-4, white bars; AECA/TA, solid bars; control IgG, square bars). Data are presented as OD 405 nm, mean ± SD of three separate experiments.

 
Effect of AECA/TTP on TM expression by microvascular TrHBMEC
TM levels increased in culture medium of TrHBMEC exposed to AECA/TTP, when AECA/ TTP-1 and -2 were tested at concentrations that produced 38 and 59% increase in monocyte adhesion respectively (Fig. 4Go). TM expression was significantly increased at all time points from 4 to 24 h incubation compared to control IgG (P < 0.001) (Fig. 6Go). Furthermore, increased TM concentration in the culture medium was correlated with decreased cell-associated TM expression (e.g. 132 ± 6 ng/5x105 cells at time 0 compared to 22 ± 3 ng/5x105 cells at 16 h for AECA/TTP-2) (P < 0.001) (Fig. 6Go). TM antigen was not detected in culture medium of EC incubated with control IgG from pooled normal plasma or without any IgG at 4, 8, 16 and 24 h of incubation.



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  		Fig. 6. TM expression by TrHBMEC exposed to AECA/TTP. TM levels in culture medium (med.) and TrHBMEC cells upon exposure to 20 µg/ml AECA/TTP-1 and AECA/TTP-2 for 4, 8, 16 and 24 h. Data are presented as mean of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that plasma from four consecutive patients with active TTP contains IgG antibodies directed against microvascular EC (AECA). Although the hypotheses concerning the etiology of TTP are controversial (12), increasing evidence suggests that TTP involves disordered immunoregulation and that AECA may be involved in the pathogenesis of vascular injury in this disease (14–16,20–25). Antibodies binding a 43 kDa protein derived from human renal microvascular EC were detected in TTP patients but their biological role has not been elucidated (23). In a study, recently published, it has been observed that TTP sera exhibited reactivity to both cultured human microvascular EC and to HUVEC, but the reactivity to cultured human microvascular EC was significantly stronger (26). Furthermore, employing cellular immunofluorescence assay on fixed cells, fluorescence observed with HUVEC was primarily cytoplasmic, whereas surface or membrane fluorescence was seen with cultured human microvascular EC (26). Cell fixation may give rise to false positivities due to the binding of antibodies reacting with cytoplasmic antigens (4). It is also worth noting that the functional consequences of the reported increased binding to micro or macrovascular EC was not investigated.

Our results demonstrate not only that AECA from all active TTP patients, specifically the F(ab)2 portion of the Ig, selectively bind to, but also activate microvascular EC. The specificity of AECA/TTP binding to microvascular EC but not to HUVEC was confirmed by competition assays using membranes derived from EC from small or large vessels. AECA from all our TTP patients increased IL-6 and vWF secretion by microvascular EC, up-regulated adhesion molecule expression (E-selectin, P-selectin and VCAM-1), and increased adhesion of monocytes onto EC. The increased production of IL-6 in response to AECA might contribute to adhesion molecule up-regulation, since IL-6 has been shown to enhance ICAM-1, VCAM-1 and E-selectin expression on EC (30). It should be noted that preincubation of EC with AECA/TTP does not modulate ICAM-1 expression, although stimulation of EC with tumor necrosis factor-{alpha} induces ICAM-1 expression. According to some reports (30,31), ICAM-1 is essential for transendothelial migration of leukocytes. However, a characteristic pathologic feature of TTP is the absence of perivascular cellular infiltration. Thus, in TTP, leukocytes may not migrate into the tissues, but may cause direct vessel wall injury by secretion of oxygen radicals, enzymes and other cytotoxic molecules (31). The susceptibility of the studied EC to AECA-mediated monocyte adhesion was attenuated by mAb directed against E-selectin, P-selectin and VCAM-1. However, as E-selectin, P-selectin and VCAM-1 blockade did not completely abrogate the hyperadhesive state, we believe that there are other ligands, yet to be defined, which are involved in-concert with the others, to the endothelial susceptibility to AECA/TTP. The EC activation by AECA/TTP can cause elevation of in leukocyte chemoattractants and thus may be able to initiate or amplify inflammatory injury (9).

The predilection of AECA/TTP recognizing target epitopes on microvascular EC is compatible with the pathological characteristic of TTP, which exclusively affects capillaries and precapillary arterioles in certain organs (12). The current results support the concept that AECA in some cases may have specific pathogenic biological function. Accordingly, examples for such specificity have been described in other models: (i) anti-PF4/heparin antibodies from patients with heparin-induced thrombocytopenia, a microvascular disease, bind macrovascular and microvascular EC but activate only microvascular EC (32 and our unpublished data), and (ii) AECA mAb derived from a patient with TA (a large vessel disease) activate specifically macrovascular EC (8). Previously, it was well documented that EC upon exposure to AECA from different sources can lead to differential adhesion molecules expression associated with enhanced monocyte adhesion, lytic activity, tissue factor released by EC and ability to mediate EC apoptosis (7–9,21,22,33–38). It is known that EC comprise a heterogeneous population which carry out similar basic functions and share many common characteristics; however, multiple antigenic determinants are usually recognized by AECA (39–42). Thus, it may explain the variability of biological functions of AECA from different sources, on different EC.

Determining the initial event in TTP has always been a very difficult problem. Two major possibilities exist: initial endothelial damage inducing platelet deposition versus primary platelet activation and subsequent vascular deposition of platelet thrombi (15). Plasma of patients with TTP has been shown to contain unusually large vWF multimers that may cause platelet agglutination in vivo (12). It was suggested that the presence of unusually large vWF multimers, associated with lack of the vWF-cleaving protease activity, was depleted by an autoimmune mechanism (13). Although platelet activation has been shown to occur in TTP, some data are most consistent with minimal platelet activation early in the disease (i.e. the first 12–24 h) followed by more extensive activation later in the patient's course of treatment. For example, P-selectin expression was not increased on circulating platelets in patients with TTP very early in the disease according to one recent study (15). In contrast, markers of endothelial damage such as TM and tissue-type plasminogen (t-PA) activator are often elevated at early stages, suggesting that it is the endothelium in TTP that is first affected (15).

The literature reports a solid correlation between soluble TM, vWF and t-PA, and EC activation and injury, pointing to these factors as useful markers for disease activity (43–45). TM is a transmembrane proteoglycan receptor for thrombin which exerts powerful antithrombogenic properties at the luminal surface of vascular EC (46,47). Increased levels of soluble TM are generally considered to reflect EC damage (45,46), e.g. in TTP, diabetic microangiopathy, disseminated intravascular coagulation, systemic lupus erythematosus, adult respiratory distress syndrome and malignancy (46–52). In our study, microvascular EC activation induced by exposure to AECA/TTP was associated with increased release of soluble TM into the culture medium and decreased levels of EC-associated TM.

Activation of microvascular EC by AECA followed by release of soluble TM could be an initial event leading to endothelial injury and/or induction of apoptosis. We hypothesize a series of events leading to TTP as follows: AECA activate EC and thus initiate or amplify inflammatory injury. The injured EC release unusually large vWF multimers and TM, which promote thrombosis together with deficiency of vWF cleaving protease activity, which may also be antibody mediated, favoring thrombosis.


   Acknowledgments
 
This work was supported by the Israel Science Foundation funded by the Israeli Academy of Sciences and Humanities (no. 736/96).


   Abbreviations
 
AECA anti-endothelial cell antibody
EC endothelial cell
HBMEC human bone marrow endothelial cell
HUVEC human umbilical vein endothelial cell
ICAM intercellular adhesion molecule
TA Takayasu arteritis
TM thrombomodulin
t-PA tissue-type plasminogen activator
TrHBMEC SV-40 immortalized NBMEC
TTP thrombotic thrombocytopenic purpura
VCAM vascular cell adhesion molecule
vWF von Willebrand factor

   Notes
 
The first two authors contributed equally to this work

Transmitting editor: I. Pecht

Received 26 June 2000, accepted 1 November 2000.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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