Human endothelial cells synthesize and release ADAMTS-13
2006; Elsevier BV; Volume: 4; Issue: 6 Linguagem: Inglês
10.1111/j.1538-7836.2006.01959.x
ISSN1538-7933
AutoresNA Turner, Leticia Nolasco, Zhenyin Tao, Jing Dong, Joel L. Moake,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoJournal of Thrombosis and HaemostasisVolume 4, Issue 6 p. 1396-1404 Free Access Human endothelial cells synthesize and release ADAMTS-13 N. TURNER, N. TURNER Department of Bioengineering, Rice University, Houston, TXSearch for more papers by this authorL. NOLASCO, L. NOLASCO Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorZ. TAO, Z. TAO Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorJ.-F. DONG, J.-F. DONG Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorJ. MOAKE, J. MOAKE Department of Bioengineering, Rice University, Houston, TX Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this author N. TURNER, N. TURNER Department of Bioengineering, Rice University, Houston, TXSearch for more papers by this authorL. NOLASCO, L. NOLASCO Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorZ. TAO, Z. TAO Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorJ.-F. DONG, J.-F. DONG Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorJ. MOAKE, J. MOAKE Department of Bioengineering, Rice University, Houston, TX Hematology and Thrombosis Research Sections, Department of Medicine, Baylor College of Medicine, Houston, TX, USASearch for more papers by this author First published: 17 May 2006 https://doi.org/10.1111/j.1538-7836.2006.01959.xCitations: 127 Joel Moake, Department of Bioengineering, Rice University, 6100 Main Street, Houston, TX 77005, USA.Tel.: +1 713 348 5357; fax: +1 713 348 5877; e-mail: jmoake@rice.edu AboutSections ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Summary. Hepatic stellate cells have been considered to be a primary source for human plasma ADAMTS-13, the von Willebrand factor (VWF)-cleaving metalloprotease. In this study, ADAMTS-13 antigen was detected by immunofluorescence in both venous (HUVECs) and arterial endothelial cells (HUAECs) using both polyclonal antibodies made against peptides found in various domains of human ADAMTS-13, as well as by a monoclonal antibody against the ADAMTS-13 metalloprotease domain. ADAMTS-13 antigen had an intra-cellular distribution in endothelial cells distinct from the Weibel–Palade body location of VWF, and was released from the cells during 48 h in culture. The mRNA for ADAMTS13 was detected in HUVECs and HUAECs using reverse transcription-polymerase chain reaction (RT-PCR), indicating that the enzyme is synthesized in these cells. The ADAMTS-13 protein was immunoprecipitated from HUVECs and had an approximate Mr of 170 kDa, similar to the molecular mass of recombinant ADAMTS-13. The ADAMTS-13 in HUVEC and HUAEC lysates had enzymatic activity using both static and flow assays. We conclude that ADAMTS-13 is synthesized in human endothelial cells, and released constitutively. The vast number of endothelial cells in the body may be an important source of ADAMTS-13. Introduction ADAMTS-13 is a Zn2+/Ca2+-dependent metalloprotease that cleaves von Willebrand factor (VWF) at the Tyr842–Met843 or (1605–6) peptide bond in the A2 domain of VWF monomeric subunits [1]. ADAMTS-13 (A Disintegrin And Metalloprotease with ThromboSpondin-1 domain) belongs to the 19-member ADAMTS enzyme family. ADAMTS-13 is composed of a series of domains (amino to carboxy terminal): metalloprotease; disintegrin; thrombospondin 1-like; cysteine-rich; spacer; seven additional thrombospondin 1-like domains; and two non-identical CUB domains. ‘CUB’ is another acronym for proteins containing this type of domain, which is involved in protein–protein interactions: Complement components (C1r/C1s); sea Urchin fibropellins; and Bone morphogenic protein 1. The 2 carboxy-terminal CUB domains are unique to ADAMTS-13 within the ADAMTS family [1-3]. The metalloprotease is inhibited by divalent chelating agents, including ethylenediamine tetra acetic acid (EDTA), ethylenebis(oxyethylenenitrilo) tetra acetic acid (EGTA) and 1,10-phenanthroline [4, 5]. Severe ADAMTS-13 deficiency, caused either by mutations in the ADAMTS13 gene or acquired autoantibodies against the ADAMTS-13 enzyme, is associated with systemic microvascular thrombosis in the familial or acquired idiopathic types of thrombotic thrombocytopenic purpura (TTP) [3, 6-8]. The gene for ADAMTS-13 has been reported to be more actively expressed in the liver (stellate cells) than in other human tissues examined to date [1, 3, 9-12]. This observation, along with the decreased ADAMTS-13 levels in the plasma of patients with severe hepatic disease, suggest that the liver may be a source of plasma ADAMTS-13 [13]. ADAMTS-13 was also recently detected in human platelets and found to be secreted in response to intense stimulation by thrombin receptor-activating peptide (TRAP), but not to weaker stimulation by adenosine diphosphate (ADP) [14]. In this report, we have investigated the synthesis of ADAMTS-13 by human endothelial cells. Materials and methods Cell cultures Human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs) Endothelial cells were obtained from human umbilical veins and arteries using a collagenase method as previously described [15]. The endothelial cells were seeded on 35-mm culture dishes (flow studies), 35-mm glass coverslips (fluorescent microscopy), or into T-75 flasks coated with 1% gelatin and grown until confluent (5–7 days) in endothelial growth media (EGM; Cambrex Bio Science, Walkersville, MD, USA) supplemented with EGM-MV (complete EGM, containing bovine brain extract, heparin, hEGF, hydrocortisone, gentamicin, and 5% heat-inactivated fetal calf serum), 3% penicillin-streptomycin-neomycin (PSN) and 0.2 mm L-glutamine. The HUVECs and HUAECs used in most experiments were primary through passage 2. Human fibroblasts Fibroblasts were isolated from umbilical veins and arteries, after the endothelial cells were removed, by additional collagenase treatment. The cells were maintained in culture as with EGM with 20% fetal bovine serum, 3% PSN and 0.2 mm L-glutamine. Chinese hamster ovary cells (CHO) The full-length cDNA of human ADAMTS-13, cloned in the mammalian expression vector pSectag2 (Invitrogen, Carlsbad, CA, USA), was transfected into wild-type CHO cells using a lipid carrier (Lipofectamine; Invitrogen) producing the stable cell line CHO-13 [15]. Untransfected wild-type CHO and transfected CHO-13 cells were maintained in culture using D-MEM/F-12 (Invitrogen) supplemented with 10% FBS and 1% PSN. Preparation of cell lysates Confluent T-75 flasks of HUVECs, HUAECs, CHO-13 or wild-type CHO cells were washed thoroughly with phosphate-buffered saline (PBS) prior to being lysed with hypotonic saline containing either 2 mm of the serine protease inhibitor, Pefabloc (Roche Diagnostics, Indianapolis, IN, USA), or 1 mm EDTA. The cells were scraped, pelleted and re-suspended in 1 mL of either Pefabloc/saline or EDTA/saline and sonicated. Cellular debris was removed by centrifugation. Fluorescent microscopy Intra-cellular ADAMTS-13 was examined by fluorescent microscopy in two types of endothelial cells (HUVECs and HUAECs) and in the CHO-13, wild-type CHO cells and human fibroblasts. All cells were grown to confluence on glass coverslips coated with 1% gelatin. The cells were washed with PBS, fixed in 1% p-formaldehyde in PBS, and treated with Triton X-100 (0.1% in PBS). In some experiments, washed HUVECs were stimulated with 100 μm histamine (Sigma-Aldrich, St Louis, MO, USA) and not fixed or detergent-treated prior to immunostaining. The ADAMTS-13 protein was detected using: eight polyclonal antibodies (BL151 through to BL159) produced in goats against peptides from distinct regions of human ADAMTS-13; and a mouse monoclonal antibody, SZ-112, produced against the metalloprotease domain of a recombinant human ADAMTS-13 protein [16]. (The antibodies are described in the next section.) The polyclonal goat antibodies were visualized with a secondary Alexa-Fluor 594 donkey anti-goat IgG (Invitrogen). The mouse monoclonal antibody was visualized with Alexa Fluor 594 chicken anti-mouse IgG (Invitrogen). VWF was stained with polyclonal rabbit anti-human-VWF IgG and secondary Alexa Fluor 488 F(ab′)2 goat anti-rabbit IgG (Invitrogen). Cell nuclei were detected with 1 nm 4′6-di-amidino-2-phenylindole di-lactate (DAPI). Images were taken with a Nikon Diaphot TE300 microscope (Garden City, NY, USA), a SensiCamQE CCD camera (Cooke Corp., Romulus, MI, USA) and IP Lab software version 3.9.4r4 (Scanalytics, Inc., Fairfax, VA, USA) using a 60/1.4 aperture objective (600× magnification) with appropriate fluorescent filters. Antibodies used in fluorescent microscopy experiments: primary antibodies to ADAMTS-13 Principal antibodies: BL156, polyclonal goat IgG generated from the peptide CVPGADGLEAPVTEGPGSVDEKLPAPE located in fourth Tsp1-like domain of ADAMTS-13. SZ-112, monoclonal mouse antibody specific for ADAMTS-13 made against the metalloprotease domain [16]. Other polyclonal goat antibodies used in fluorescent microscopy experiments include three made to the metalloprotease region (BL151, VGPDVFQAHQED-TERYVLTNLNI; BL152, CGWSQTINPEDDTDPGHADLV; and BL153, GSAG-HPPDAQPGLYYSANEQCRVAFGPKAVA); one from the disintegrin region (BL154, TR/FDLELPDGNRQVRGVTQLGG); two from the Tsp1-like region (BL155, CVPGA-DGLEAPVTEGPGSVDEKLPAPE; and BL157, CARAHGEDDGEEILLDTQ); and two to the CUB regions (BL158, GRQHLEPTGTIDMRGPGQADCA; and BL159, TLQSWVPEMQDPQSWKGKEGT). The BL antibodies were produced at Bethyl Laboratories, Montgomery, TX, USA. Primary antibody to VWF VWF, polyclonal rabbit anti-human-VWF IgG against VWF purified from human cryoprecipitate. Secondary antibodies (detection) For goat polyclonal antibodies: Alexa Fluor 594 donkey anti-goat IgG (Invitrogen). For mouse monoclonal antibody: Alexa Fluor 594 chicken anti-mouse IgG (Invitrogen). For rabbit polyclonal antibody: Alexa Fluor 488 F(ab′)2 goat anti-rabbit IgG (Invitrogen). Constitutively secreted HUVEC supernatant Primary HUVECs were grown to confluency in T-75 gelatin-coated flasks with EGM supplemented with EGM-MV. The supernatant was collected 48 h after cell confluency and frozen at −80°C until use. Supernatant was then immobilized on glass coverslips for 30 min. The coverslips were washed twice with PBS, blocked with 1% bovine serum albumin (BSA) in PBS and immunostained with: BL156 (polyclonal goat antibody against the fourth Tsp1-like domain of ADAMTS-13) and secondary Alexa Fluor 594 donkey anti-goat IgG (red); and polyclonal rabbit anti-human VWF and secondary Alexa Fluor 488 F(ab′)2 goat anti-rabbit IgG (green). Some of the immobilized supernatant samples were stained only with the secondary fluorescent antibodies in the absence of primary antibodies (BL156 or anti-VWF). As controls for non-specific immunofluorescence, EGM, EGM supplemented with growth factors (EGM-MV) and undiluted fetal calf serum were immobilized onto glass coverslips and stained identically as the HUVEC 48-h supernatant. Platelet preparations Human blood was drawn from un-medicated healthy donors under a protocol approved by the Institutional Review Board of the Baylor College of Medicine. Informed consent was provided according to the Declaration of Helsinki. Washed platelets were obtained using a prostaglandin I2 method, as previously described [15], and re-suspended in a volume of Ca+2, Mg+2-free Tyrode's buffer [(Tyrode's buffer), 138 mm sodium chloride, 5.5 mm glucose, 12 mm sodium bicarbonate, 2.9 mm potassium chloride, and 0.36 mm dibasic sodium phosphate, pH 7.4] that was equivalent to the original platelet-rich-plasma volume. Parallel-plate perfusion assay of ULVWF-platelet string formation and cleavage Primary HUVECs were stimulated to secrete ULVWF for 3 min with 10 mm histamine. The formation and cleavage of ULVWF-platelet strings secreted from the stimulated HUVECs was studied under flow in a parallel-plate flow chamber system (GlycoTech, Rockville, MD, USA) and observed by phase-contrast microscopy using 20/0.45 aperture and 40/0.6 aperture objectives (200× and 400× magnification). Endothelial cells were grown on 35-mm culture dishes that became the bottom of each flow chamber. A syringe pump connected to the outlet port pulled washed platelets through the chamber at 0.2 mL min−1 to generate a shear rate of approximately 60 s−1 (wall shear stress, ∼1–2 dyn cm−2). The assembled parallel-plate flow chamber was mounted onto an inverted-stage microscope (Eclipse TE300; Nikon) equipped with a high-speed digital camera (Model Quantix; Photometrics, Tucson, AZ, USA). Images were acquired using MetaMorph software version 5.0r7 (Universal Images, West Chester, PA, USA). The ULVWF-platelet strings were counted in 20 continuous view-fields at 400×. ADAMTS-13 cleavage of ULVWF Flowing conditions The disappearance of ULVWF multimeric strings secreted by stimulated HUVECs, and the platelets adherent to the strings, was visualized under phase contrast microscopy under flow. The disappearance of ULVWF-platelet strings in the presence of cell lysates indicates that the solutions contain ADAMTS-13 activity, as demonstrated by us in a series of reports during the past 3 years [15, 17-19]. Specific ADAMTS-13-mediated cleavage of the ULVWF-platelet strings was confirmed repeatedly throughout this study using EDTA in control runs. EDTA, which inhibits ADAMTS-13 activity, blocked the disappearance of ULVWF-platelet strings during the perfusion of solutions containing ADAMTS-13 activity. The endothelial cells were stimulated for 3 min with 10 mm histamine. The stimulated cells were then perfused with washed normal platelets suspended in: Tyrode's buffer alone; or Tyrode's buffer plus lysates from each cell type studied; or Tyrode's buffer + lysates + EDTA. During the initial 2 min after the addition of the washed platelets in buffer (±cell lysates) to the previously stimulated HUVECs, the platelets adhered to the ULVWF strings. The points of endothelial cell anchorage of the ULVWF-platelet strings, as well as the entire length of the ULVWF-platelet strings floating above the endothelial cells, were observed continuously during the experimental periods. During the next 2 min, the ULVWF strings were quantified in 20 continuous fields and/or photographed in a single field. Static conditions The ADAMTS-13 activity from lysates of HUVECs, HUAECs, CHO-13 or wild-type CHO cells was measured under static conditions using HUVEC supernatant containing VWF enriched in ULVWF as the substrate. This method was modified from Furlan et al. [20] The lysates were diluted (1 : 5) with low ion strength Tris [tris(hydroxymethyl)aminomethane]-saline containing 1 mm Pefabloc, activated for 5 min with 1 mm BaCl2 and mixed with the ULVWF substrate. The mixture was dialyzed in 1.5 m urea for 24 h at 37°C, and the reaction was stopped by the addition of 0.02 m EDTA. Some samples were incubated with anti-ADAMTS-13 autoantibody purified from a patient with acquired idiopathic TTP. The samples were electrophoresed into 1% agarose and transferred to a PVDF membrane. For display of VWF multimers, the membrane was overlaid with polyclonal goat anti-human-VWF antibody (Bethyl Laboratories), followed by secondary horseradish peroxidase (HRP)-labeled rabbit anti-goat IgG (Bethyl Laboratories) and chemiluminescent detection reagents (Perkin-Elmer, Fremont, CA, USA) before X-ray film exposure. Reverse transcription-polymerase chain reaction (RT-PCR) RNA from confluent T-75 flasks of P1 HUVECs, P1 HUAECs, transfected CHO-13 and wild-type CHO cells was isolated using TRIzol (Invitrogen), chloroform extraction and isopropanol precipitation. RNA integrity was verified by 260/280 optical density ratios and 1%-agarose-formaldeyde electrophoresis. The RNA was reverse transcribed into cDNAs with Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT; Promega, Madison, WI, USA). The cDNAs were then subjected to 34 cycles of PCR using two sets of oglionucleotide primers to human ADAMTS13. One set of primers (Tsp1) was selected from the thrombospondin 1-like domains 2–4 between nucleotides 2041–2700 of the mRNA of ADAMTS13 and the second set (CUB) was chosen from the two terminal CUB domains between nucleotides 3421–4320 (Table 1). The mRNA sequences selected for amplification did not complement any human gene other than ADAMTS13 according to the BLAST nucleotide program (http://www.ncbi.nlm.nih.gov/BLAST/). Table 1. Reverse transcriptase-polymerase chain reaction (RT-PCR) primers Sequence Product size (bp) Left primer ADAMTS13 Tsp1 gacagttacccccaacctga 246 Right primer ADAMTS13 Tsp1 tggatgtcagcatcttcctg Left primer ADAMTS13 CUB agccaacaggaaccattgac 172 Right primer ADAMTS13 CUB gcttcctgcacatcttcctc Left primer VWF aagccaatatagggcctcgt 109 Right primer VWF ctcagcaaatgggctttctc Left primer GAPDH aggggagattcagtgtggtg 202 Right primer GAPDH cgaccactttgtcaagctca PCR products were identified using ethidium bromide-stained 2% agarose gel electrophoresis and a KODAK EDAS imaging system (Eastman Kodak, Rochester, NY, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the ‘housekeeping’ gene for the endothelial cells. Immunoprecipitation and Western blotting Lysates from HUVECs, CHO-13, and wild-type CHO cells (500 ug protein) were incubated overnight at 4°C with a mouse monoclonal antibody produced against the N-terminal metalloprotease domain of human ADAMTS-13 (kindly provided by Drs F. Scheiflinger and B. Plaimauer of Baxter BioScience, Vienna, Austria). After incubation, the samples were mixed with goat anti-mouse IgG-Sepharose Beads (ZYMED, San Francisco, CA, USA) at 4°C for 1 h. The beads were washed three times with PBS and the eluted proteins were electrophoresed on NuPAGETM 10% Bis-Tris gels (Invitrogen) and transferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA). The membrane was blocked with milk and overlaid with polyclonal goat anti-human ADAMTS-13 antibody BL156 (made against the fourth Tsp1-like domain; Bethyl Laboratories) overnight at 4°C. The bands were visualized by incubation with HRP-labeled rabbit anti-goat IgG, followed by SuperSignal West Pico chemiluminescent reagents (Pierce Technologies, Rockford, IL, USA) and exposure to X-ray film. Statistical analysis All experimental data were presented as mean ± average deviation of the mean. Results HUVECs (Fig. 1A,B) and HUAECs (Fig. 1C,D) were studied using two distinct antibodies to human ADAMTS-13. One was a polyclonal antibody against the Tsp 1–4 region of ADAMTS-13 (BL156), and the other a monoclonal antibody made against the metalloprotease domain of ADAMTS-13 (SZ-112). In Fig. 1B,D, anti-human VWF antibody was also added. ADAMTS-13 antigen was detected in both venous and arterial endothelial cells. Endothelial cell ADAMTS-13 had an intra-cellular distribution that was distinct from the Weibel–Palade body location of VWF. The ADAMTS-13 staining varied in intensity, but was similar in pattern, using the seven other polyclonal antibodies generated from peptides in distinct regions of ADAMTS-13 described in the Materials and methods (fluorescent images not shown). HUVECs and HUAECs stained negatively with the addition of either secondary red fluorescent antibody (donkey anti-goat IgG or chicken anti-mouse IgG) alone or secondary green fluorescent antibody goat anti-rabbit IgG in the absence of primary antibody against either ADAMTS-13 (polyclonal goat or monoclonal mouse) or VWF (polyclonal rabbit; data not shown). Figure 1Open in figure viewerPowerPoint Fluorescent images of human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs) demonstrating intracellular ADAMTS-13 and von Willebrand factor (VWF) stored in granules. HUVECs (P1) were fixed and made permeable with Triton X-100. Cell nuclei were stained with 4’6-di-amidino-2-phenylindole di-lactate (DAPI) (blue). (A) P1 HUVECs stained with BL156 (polyclonal goat antibody against the fourth Tsp1-like domain of human ADAMTS-13), and secondary Alexa Fluor 594 donkey anti-goat IgG (red); (B) Overlay of the image in (A) and the same HUVECs stained with polyclonal rabbit anti-human VWF and secondary Alexa Fluor 488 F(ab’)2 goat anti-rabbit IgG (green); (C) HUAECs (P2) stained with monoclonal SZ-112 antibody against the ADAMTS-13 metalloprotease domain and secondary Alexa Fluor 594 chicken anti-mouse IgG (red); (D) Overlay of the image in (C) and the same HUAECs stained with polyclonal rabbit anti-human VWF antibody. Images are magnified 600× and are representative of four separate experiments. In control experiments, staining with the same antibodies to ADAMTS-13 (BL156) and VWF, ADAMTS-13 antigen was detected in cultured CHO-13 cells that had been transfected with human ADAMTS13 DNA (Fig. 2A), but not in wild-type (un-transfected) CHO cells (Fig. 2B). Human fibroblasts also stained negatively with these same antibodies against ADAMTS-13 (BL156) or VWF (Fig. 2C). Figure 2Open in figure viewerPowerPoint Fluorescent images of intracellular ADAMTS-13 in Chinese hamster ovary (CHO) cells transfected with human ADAMTS-13 DNA (CHO-13), untransfected wild-type CHO cells (CHO) and human fibroblasts. CHO cells and human fibroblasts were fixed and made permeable with Triton X-100. ADAMTS-13 was detected by staining with BL156 (polyclonal goat antibody against the fourth Tsp1-like domain of ADAMTS-13) and secondary Alexa Fluor 594 donkey anti-goat IgG (red). (A) CHO-13 cells stain positively for the ADAMTS-13 Tsp1-like region, whereas (B) wild-type, untransfected CHO cells and (C) fibroblasts both stain negatively. (C) Fibroblasts also stain negatively for von Willebrand factor (VWF). The image shown is the overlay of the red and green channels of fibroblasts stained with BL156 and polyclonal rabbit anti-human VWF and secondary Alexa Fluor 488 F(ab’)2 goat anti-rabbit IgG (green). Cell nuclei were stained with 4’6-di-amidino-2-phenylindole di-lactate (DAPI) (blue). Images are magnified 600×, and are representative of three separate experiments. HUVECs contained ADAMTS-13 activity, as demonstrated by the capacity of endothelial cell lysates to cleave (within 2 min) the ULVWF multimeric strings secreted from intact, histamine-stimulated HUVECs under flowing conditions (Fig. 3A). The ULVWF strings were outlined (and visualized by phase contrast microscopy) by the adherence of perfused washed normal platelets as the ULVWF strings were exposed to suspensions of platelets suspended in buffer ± cell lysate (and ± EDTA). The ADAMTS-13 activity from the HUVEC lysates was comparable to the activity from the human ADAMTS13-transfected CHO-13 cell lysates (Fig. 3A,B,D). Lysates were prepared from confluent culture flasks (T-75) of equal size. There were two to three times fewer HUVECs per flask than CHO-13 or wild-type CHO cells because the CHO cells are smaller. The ADAMTS-13 in lysates prepared from HUVECs, nevertheless, cleaved 55% of the secreted ULVWF strings, an enzymatic activity comparable to the 61% activity in lysates from three times as many CHO-13 cells. This ULVWF-platelet string cleavage activity in lysates from both HUVECs and CHO-13 cells was inhibited by the ADAMTS-13 inhibitor, EDTA (Fig. 3A,C,E), but not by the serine protease inhibitor, Pefabloc. Figure 3Open in figure viewerPowerPoint Human umbilical vein endothelial cells (HUVECs) contain ADAMTS-13-cleaving activity (flowing conditions). HUVECs were grown on culture dishes that became the bottom of the flow chamber, and stimulated for 3 min with 10 mm histamine. (A) The stimulated HUVECs were perfused with washed normal platelets (suspended in Tyrodes buffer) that had been mixed with equal volumes of lysates from either HUVECs, Chinese hamster ovary (CHO)-13 or untransfected wild-type CHO [in the presence or absence of 1 mm ethylenediamine tetra acetic acid (EDTA)]. After 2 min of perfusion, the number of ULVWF-platelet strings was counted in 20 contiguous fields (magnification = 400×). The number of ULVWF-platelet strings after perfusion with each platelet-cell lysate mixture (±EDTA) was normalized to the ULVWF-platelet string number after perfusion with platelet-buffer controls for each experiment. Each experiment was repeated three to ten times. Photographs of the microscope fields (magnification = 200×) were taken after 2 min of perfusion with washed platelets that had been mixed either with: (B) HUVEC lysates; (C) HUVEC lysates + EDTA; (D) CHO-13 lysates; or (E) CHO-13 lysates + EDTA. The black arrows indicate several of the ULVWF-platelet strings. Photographs are representative of six to ten separate experiments. The lysates prepared from the untransfected wild-type CHO cells did not cleave ULVWF-platelet strings, and there was no appreciable difference in ADAMTS-13 activity in the wild-type CHO lysates in the presence or absence of EDTA (Fig. 3A). Endothelial cell lysates (HUVECs and HUAECs) also cleaved ULVWF and VWF forms in the soluble VWF supernatant released constitutively over 24 h from HUVECs. Cleavage occurred under these static conditions if the soluble VWF/ULVWF forms were incubated for a prolonged period (24 h) in the presence of BaCl2 and urea. ADAMTS-13 activity is indicated by the disappearance of VWF forms, including ULVWF, from gel lanes (Fig. 4A,B) No cleavage occurred in the presence of EDTA (Fig. 4A,B), or in the presence of an anti-ADAMTS-13 autoantibody from a patient with acquired idiopathic TTP (Fig. 4B). The wild-type CHO lysates had no ADAMTS-13 activity (Fig. 4A). Figure 4Open in figure viewerPowerPoint Human umbilical vein endothelial cells (HUVECs) contain ADAMTS-13-cleaving activity (static conditions). Lysates from one group of (A) HUVECs, human umbilical artery endothelial cells (HUAECs), Chinese hamster ovarian (CHO)-13 or wild-type CHO cells or (B) a second group of CHO-13 cells and HUVECs were prepared in the presence or absence of 1 mm ethylenediamine tetra acetic acid (EDTA), activated with BaCl2, and incubated with substrate. In (B) the CHO-13 cells and HUVEC lysates were also incubated with an autoantibody against ADAMTS-13 isolated from a patient with acquired idiopathic thrombotic thrombocytopenic purpura (TTP). The substrate in (A) and (B) was soluble HUVEC supernatant containing von Willebrand factor (VWF) enriched in ULVWF forms. The reaction products were separated by SDS/1% agarose electrophoresis. Absence of VWF multimeric regions and bands indicate ADAMTS-13 activity. The gels are representative of three to five separate experiments. ADAMTS-13 was immunoprecipitated from HUVEC, CHO-13 and wild-type CHO lysates with a mouse monoclonal antibody made against the N-terminal metalloprotease portion of human ADAMTS-13, and immunologically attached to beads. The immunoprecipitated protein was analyzed by Western blotting using BL156, the goat polyclonal anti-human ADAMTS-13 antibody. The single immuno-reactive band eluted from the HUVEC lysates had an approximate molecular weight of 170 kDa, and migrated with the band immunoprecipitated from the transfected CHO-13 cells. No similar band was detected in the immunoprecipitated wild-type CHO lysates (Fig. 5). Figure 5Open in figure viewerPowerPoint Detection of ADAMTS-13 protein in human umbilical vein endothelial cells (HUVECs) by Western blotting. Lysates from HUVECs (P1), Chinese hamster ovarian (CHO)-13, and wild-type CHO cells were incubated with a mouse monoclonal antibody against the N-terminal metalloprotease portion of human ADAMTS-13. The immunoprecipitated proteins were separated by 10% Bis-Tris gel electrophoresis, transferred to PVDF membrane and detected using polyclonal goat anti-human ADAMTS-13 antibody BL156 (reacts with the fourth Tsp1-like domain of human ADAMTS-13) followed by HRP-secondary rabbit anti-goat IgG and chemiluminescence. The arrow indicates the ADAMTS-13 band. The figure is representative of five separate experiments. The mRNA for ADAMTS13 was detected in HUVECs and HUAECs using RT-PCR (Fig. 6A), indicating that the enzyme is synthesized in these types of endothelial cells. Two sets of primers, chosen from the mRNA, translate into two distinct domains of the mature protein. One set spanned the second through fourth Tsp1-like domains, and the second primer set bridged both CUB domains. Neither selection of mRNA sequence complemented any known human gene other than ADAMTS13. Resulting PCR products were identical to those produced from the reverse-transcribed RNA extracted from the human ADAMTS13-transfected CHO-13 cells. The cDNA from wild-type, un-transfected CHO cells did not result in PCR products similar in size to those identified as ADAMTS-13 (data not shown). Figure 6Open in figure viewerPowerPoint ADAMTS-13 is synthesized in human umbilical vein endothelial cells (HUVECs) and human umbil
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