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Deficiency of ADAMTS13 results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in the present study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (~5–70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor compared to the Adamts13−/− mice expressing eGFP. Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13−/− expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13−/− mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.
ADAMTS13, a member of A Disintegrin And Metalloprotease with ThromboSpondin type repeats (ADAMTS) family, [1, 2] controls the sizes of von Willebrand factor (vWF) by cleaving vWF at the Tyr1605-Met1606 bond in the central A2 domain.  ADAMTS13 protein consists of a metalloprotease domain, a disintegrin domain, first thrombospondin type 1 repeat (TSP1), a Cys-rich domain and a spacer domain in addition to seven TSP1 repeats and two CUB domains.  The amino terminal half of ADAMTS13 protease (up to the spacer domain) is found to be sufficient to recognize and cleave vWF under static and denatured conditions  or to cleave peptide substrates such as vWF73 derived from the central A2 domain of vWF.  The carboxyl terminal half of ADAMTS13, however, may be required for collaborative binding and cleavage of vWF under fluid shear stress in vitro. [6, 7] Deficiencies of plasma ADAMTS13 activity owing to hereditary mutations of ADAMTS13 gene  or acquired autoantibodies against ADAMTS13 protease  results in an accumulation of “unusually large” vWF multimers,  leading to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Profound thrombocytopenia, microangiopathic hemolytic anemia and organ ischemia are characteristic features of TTP syndrome.  If left untreated, the mortality rate is as high as 80~100%. [11, 12] Plasma infusion and/or plasma exchange is the only effective treatment available to date.
Besides developing a TTP-like syndrome,  mice lacking Adamts13 gene are prothrombotic, [14, 15] characterized by increased sizes of plasma vWF multimers and enhanced platelet adherence to activated endothelial cells in vivo. Intravenous infusion of recombinant human ADAMTS13 into Adamts13−/− mice reverses the prothrombotic phenotypes and protects mice against ferric chloride-induced arterial and venous thromboses.  However, the short half-life of infused human ADAMTS13 into humans (t1/2, 2–4 days)  or mice (t1/2, 15–20 minutes)  makes the intravenous infusion of recombinant ADAMTS13 a less desirable strategy for a life-long treatment of hereditary TTP.
Gene therapy may offer an attractive alternative strategy to recombinant ADAMTS13 or plasma infusion for prevention and treatment of TTP, because only approximately 5%~10% of ADAMTS13 activity (~0.05–0.1 mg/L protein) is required to induce and maintain remission of prothrombotic states in humans.  Based on our previous studies, [17, 18] we hypothesized that early gestational in utero gene transfer using self-inactivated lentiviral vectors would achieve this therapeutic level in plasma. The prenatal approach is an appealing therapeutic option for an inherited genetic disorder that has an early onset and is potentially fatal, because it may pre-empt manifestations of the disease. In addition, early gestational delivery of the gene of interest results in transduction of stem cell compartments that are less accessible for gene transfer after birth. [17, 18] With the use of an integrating vector, subsequent expansion of transduced stem and progenitor cells should amplify expression of the protein of interest and result in a life-long correction of the disease. Antigen processing by the naïve fetal immune system would be expected to result in specific tolerance to the therapeutic proteins. Prenatal gene transfer has been successfully applied in a mouse model of hemophilia B. 
In this study, we report the achievement of sustained therapeutic levels of human full-length ADAMTS13 and the carboxyl-terminal truncated variant (MDTCS) in Adamts13−/−mice by intra-amniotic and intravascular (via vitelline vein) gene transfer. The expressed ADAMTS13 and MDTCS proteases were both biologically active in reducing vWF multimer sizes and protecting against ferric chloride-induced arterial thromboses in the mouse model. Our data support the feasibility of developing a gene therapy-based approach for hereditary TTP in humans.
The survival rate of newborns in Adamts13−/− background without surgical and in utero manipulation was estimated to be 95%. The survival rates after intra-amniotic injection (E8) of lentiviral vectors encoding eGFP, ADAMTS13 and MDTCS into Adamts13−/− mice were approximately 59% (10/17), 63% (15/24), and 35% (10/29), respectively, suggesting various degrees of mortalities associated with the procedures. The survival rates after intravascular injection at E14 of vectors encoding eGFP, ADAMTS13 and MDTCS were approximately 79% (11/14), 55% (18/33) and 84% (16/19), respectively. The overall survival rate in mice injected at E14 was 76%, significantly higher than that in those injected at E8 (51%) (F value=18.0, p=0.002), suggesting that an administration of vectors at the later gestational stage (corresponding to early second trimester of human fetal development) may significantly reduce fetal losses. No gross structural abnormality was observed in any of the surviving newborns and adult mice, suggesting that administrations of lentiviral vectors encoding eGFP, ADAMTS13 and MDTCS either at E8 or E14 do not cause abnormality in prenatal and postnatal development. No spontaneous bleeding was observed in post-natal mice that received either the ADAMTS13 or MDTCS vectors.
To determine sites of gene expression in mice after intra-amniotic administration of lentiviral vectors, mice were analyzed by stereoscopic fluorescent, immunofluorescent and immunperoxidase techniques described in more details previously.  When vectors were administered at E8, the kidneys and skin were the predominant organs expressing transgenes, which showed bright and punctuated green fluorescent signals of eGFP reporter under stereoscopic fluorescent microscope (Fig. 2A). Scattered fluorescent signals were also detectable in the brain but not in the liver (Fig. 2A). Immunofluorescent staining with rabbit anti–GFP IgG further demonstrated that the transgene was primarily localized to epidermis and bulge cells in the hair follicles of the skin (Fig. 2A–e), similar to what we have reported previously. [17, 18] In kidney, the transgene was predominantly detected in the tubular structures as well as in the glomeruli (Fig. 2A–f). No GFP signal was observed in the control (Adamts13−/−) mice that did not receive lentiviral vectors (data not shown). In contrast to the localization of the E8 intra-amniotic studies, an intravenous administration of vector at E14 resulted in primarily liver transduction (Fig. 2B) with minor gene delivery and expression evident as focal positive signals in the brain, heart and kidney by stereofluorescent microscopy (Fig. 2B). Immunoperoxidase staining further localized the transgene product to the muscle of the heart (Fig. 2B–e), and the hepatocytes in the liver (Fig. 2B–f). These data suggest that the tissue distribution of the transgene is highly dependent of the time when vector is administered.
To determine whether ADAMTS13 transduced in various tissues after intra-amniotic injection were secreted into blood circulation, the levels of ADAMTS13 protein were determined by an immunoprecipitation plus Western blot. As shown in Fig. 3, both ADAMTS13 and MDTCS were clearly detectable in mouse plasma at six weeks or longer after intra-amniotic administration of lentiviral vectors encoding ADAMTS13 and MDTCS, respectively. The similar protein bands were absent in the plasma of Adamts13−/− mice expressing eGFP alone, but present in normal human plasma and conditioned medium containing recombinant MDTCS after immunoprecipitation (Fig. 3), indicating the secretion and delivery of the transduced human full-length ADAMTS13 and MDTCS into the murine plasma.
Proteolytic activity toward the fluorescent peptide, FRETES-vWF73, was determined serially over several months in the plasmas from Adamts13−/− mice and the mice injected in utero with vectors encoding ADAMTS13 and MDTCS at E8 and E14. As demonstrated in Fig. 4, plasma from the mice receiving vectors at E8 and E14 encoding ADAMTS13 and MDTCS were able to cleave FRETS-vWF73 and GST-vWF73-H specifically at the Tyr1605-Met1606 bond (data not shown). After E8 injection, approximately 5–7 % and 60~70% of protease activity was detected in the plasma of these mice expressing ADAMTS13 and MDTCS, respectively (Fig. 4A). The plasma levels of proteolytic activity in mice receiving vectors at E14 encoding ADAMTS13 (5–7%) (Fig. 4B) were comparable with those receiving same vectors at E8. However, the plasma activity in E14 injected mice with the vector encoding MDTCS was approximately 4~6 fold lower than that in E8 injected mice (Fig. 4B). This difference in the plasma activity may reflect the relatively lower vector dose administered into mice at E14. No proteolytic cleavage FRETS-vWF73 (Fig. 4) and GST-vWF73 (data not shown) was detected in Adamts13−/− mice expressing eGFP alone, suggesting that these assays are highly specific for detecting ADAMTS13 activity in mouse plasma. The elevated proteolytic activity in plasma persisted for 12~24 weeks (the lengths of this study) in Adamts13−/− mice (Fig. 4) and up to 42 weeks in wild-type mice (Balb/c) (unpublished data) . The data demonstrate that a single injection of lentiviral vectors either at E8 or E14, encoding full-length ADAMTS13 and truncated variant into amniotic space or yolk sac vessels results in long-term expression of ADAMTS13 or variant protease at levels that may be therapeutic.
To determine whether the expressed ADAMTS13 and MDTCS in mice were able to alter the distribution of circulating vWF multimers in mice, we assessed the plasma multimers by agarose gel electrophoresis followed by Western blot. In addition, we also determined the ratio of vWF collagen-binding activity to antigen and multimer analyses. The binding affinity to immobilized human type III collagen is proportional to the size of vWF multimers.  The high molecular weight vWF multimers were present in plasma of Adamts13−/− mice or Adamts13−/− mice expressing GFP, but markedly reduced in plasma of mice expressing full-length ADAMTS13 and MDTCS (both injected at E8 and E14) (Fig. 5A). These results were consistent with the significantly reduced mean ratios of plasma vWF-collagen binding activity to antigen by the quantitative measurement in mice expressing full-length ADAMTS13 and MDTCS were detected regardless of the time vectors administered (Fig. 5B). These data suggest a substantial alteration of vWF multimer distribution has occurred in the murine plasma containing even low levels of the expressed recombinant ADAMTS13 and MDTCS proteases in circulation.
To determine whether the expressed ADAMTS13 and MDTCS proteases were biologically functional in preventing arterial thrombosis, we performed the ferric chloride-induced carotid arterial thrombosis assay. This assay was performed in a blind fashion (the operator was blinded to the genotype of the mice). The carotid arterial occlusion times in the mice receiving vector at E8 and expressing ADAMTS13 and MDTCS were 9.0 ± 0.6, and 25.2 ± 3.2 minutes respectively, which were significantly prolonged compared to those of the Adamts13−/− mice expressing eGFP alone (5.6 ± 0.5 minutes) (p<0.01) (Fig. 6A). Similar results were obtained in mice receiving vectors at E14 and expressing ADAMTS13 and MDTCS (Fig. 6C). The markedly prolonged carotid arterial occlusion times in mice expressing MDTCS were consistent with higher levels of proteolytic activity (Fig. 4). No statistical difference in carotid arterial occlusion times was observed between the mice expressing human ADAMTS13 and wild-type (C57BL/6 strain) mice (8.3 ± 0.4 minutes) (p>0.05) (Figs. 6A and 6C). In addition, the numbers of occluded arteries at 30 minutes in the mice expressing MDTCS (at E8 and at E14) were approximately 30%, significantly lower than that in the wild-type mice and the mice expressing ADAMTS13, consistent with the possibility of over correction of Adamts13 deficiency by human MDTCS in these mice or the ectopic expression of the transgene that may be more efficacious for anti-thrombosis. Nevertheless, these results demonstrate that the expressed human full-length ADAMTS13 (although only ~5–7%) and truncated variant (MDTCS) (~10–70%) are biologically functional in vivo.
This study reports the successful long-term correction of the prothrombotic phenotypes of Adamts13−/− mice by early gestational intra-amniotic gene transfer. Recombinant human ADAMTS13 has been shown to be able to cleave murine vWF in vivo and offers systemic protection against ferric chloride induced arterial and venous thrombosis after tail vein injection.  To develop gene therapy-based treatment for hereditary TTP and gain more insight into the structure-function relationship of human ADAMTS13 protease in vivo, human full-length ADAMTS13 and the carboxyl-terminal truncated variant (MDTCS) well characterized previously [5, 22, 4] were expressed in Adamts13-null mice by in utero administration of lentiviral vectors. The transgene product (eGFP) after intra-amniotic injection (at E8) of lentiviral vectors was primarily localized to the parenchyma of the kidneys, large vessels of the heart, and skin (Fig. 2A). This is in contrast with the localization of endogenous ADAMTS13 which is detected in hepatic stellate cells, [23, 24, 25] endothelial cells, [26, 27] megakaryocytes/platelets. [28, 29] The predominant expression of ADAMTS13 in renal parenchyma (tubules and glomeruli) by early embryonic injection of lentiviral vector may be important for preventing renal failure in patients with TTP due to thrombosis in kidney microcirculation. The distribution of the transgene expression is largely related to the timing of vector administration and the contact of specific tissues with the amniotic fluid. The amniotic space primarily contacts epithelial tissues at this early gestational time point,  but is not entirely restricted to epithelium due to exposure of some mesodermal tissue prior to completion of gastrulation in the primitive streak. There may also be leakage of vector to the extracoelomic cavity (such as the expression of transgenes in pancreas) (data not shown), which contacts mesodermal progenitors. When the same vectors were injected via a yolk sac vessel at E14, however, the transgene expression was rather limited to the hepatocytes in the liver (Fig. 2B). Thus, the distribution of transgenes observed in this study is consistent with the developmental stage at which the injections were performed and those that we have previously reported. [17, 18]
Despite the cell origin, the synthesized human full-length ADAMTS13 and MDTCS proteins were secreted into the blood stream as demonstrated by the presence of ADAMTS13 (~195K) and MDTCS (~95K) proteins, respectively, in mouse plasma with their expected molecular masses on a SDS-polyacrylamide gel (Fig. 3A and 3B). Although it remains to be determined how such a large protein synthesized from various cells gets into the blood stream, the secreted recombinant full-length ADAMTS13 and MDTCS in murine plasma were proteolytically active toward two highly specific peptide substrates FRETS-vWF73 (Fig. 4) and GST-vWF73 (data not shown). This elevated protease activity in murine plasma persisted at least for the length of this study, i.e. 12–24 weeks in the Adamts13−/− mice (Fig. 4) and 42 weeks in the wild-type mice observed (our unpublished data). 
Although low levels of plasma protease activity were transduced in mice with human full-length ADAMTS13 (5–7% of NHP), these levels of expression were sufficient to correct the prothrombotic phenotypes. This was indicated by a marked decrease in the ratio of plasma vWF collagen binding activity to antigen (Fig. 5) which is indicative of altered multimer distribution, and a significant prolongation of ferric chloride-induced carotid occlusion times compared to Adamts13 null mice expressing eGFP alone (Fig. 6), comparable to the occlusion times of the wild-type mice. These results are also consistent with the clinical data showing that approximately 5–10% of plasma protease activity may be sufficient to induce clinical remission and maintain patients with TTP symptom-free.  The lower levels of plasma proteolytic activity in mice expressing ADAMTS13 than in those expressing MDTCS, as determined by cleavage of the peptide substrate may be in part due to lower transduction efficiency (due to longer construct) and perhaps greater instability of ADAMTS13 than MDTCS in murine plasma. The half-life of recombinant human full-length ADAMTS13 and MDTCS in mice was determined to be 15 and 22 minutes (Suppl. Fig. 1), respectively, similar to what has been reported for full-length ADAMTS13 in the literature.  Consistent with the notion of lower transduction efficiency was that the positive immunofluorescent signals in various tissues of the mice expressing full-length ADAMTS13 were much fewer than those expressing MDTCS as determined by fluorescent microscopy (data not shown).
An unanticipated finding was our observation that the truncated MDTCS variant that was previously found to be significantly impaired in proteolytic activity toward plasma-derived vWF under vortex-induced fluid shear stress in vitro was capable of cleaving murine vWF efficiently in vivo (Fig. 5) and protecting mice from the ferric chloride-induced arterial thrombosis (Fig. 6). The explanation for why the truncated MDTCS remained highly active in processing UL-vWF in vivo is not known. Dong and his colleague using a parallel flow chamber assay also observed such “hyperactivity” of human MDTCS in proteolytic processing of cell bound UL-vWF.  It is possible that the carboxyl terminus of ADAMTS13 that is required for proteolytic cleavage of soluble vWF may not necessarily important for cleavage of newly secreted UL-vWF bound on endothelial cells in vivo. Alternatively, the proteolytic activity of MDTCS may be rescued by the constant arterial shear stress and potential cofactors present in plasma such as factor VIII,  platelets, [32, 33] and possibly endothelial cells. Nevertheless, the detection of biological activity of MDTCS effectively reducing vWF multimer sizes in vivo may be important for a rational design of an adeno-associated viral vector (AAV) for a better safety profile because AAV has a limited capacity of approximately 4.3 kb including promoter and other regulatory elements. The MDTCS (~2.4 kb) will fit into the AAV cassette.
It will be important in the future to demonstrate the efficacy of this approach in mice susceptible to developing TTP-like syndrome. Complete null mutations of Adamts13 in other strains of mice such as those used in this study exhibit prothrombotic phenotypes as evidenced by an accumulation of high molecular weight vWF multimers in plasma upon stimulation [14, 10] and an enhanced platelet aggregation and thrombus formation induced by ferric chloride (Fig. 6) and by calcium ionophore in vivo. [14, 15] Therefore, our model should provide reasonable experimental evidence of therapeutic efficacy of the exogenous human ADAMTS13 and MDTCS proteins if the plasma vWF multimer sizes are reduced and the formation of arterial thrombosis are attenuated or prevented. The success in correcting the prothrombotic phenotype in mice by intra-amniotic gene transfer of lentiviral vectors encoding either wild-type ADAMTS13 or MDTCS variant supports the feasibility of developing a novel gene therapy-based treatment for hereditary TTP in humans.
Although our study is proof-in-principle of this strategy, there are a number of hurdles that would need to be overcome prior to any clinical application of this approach. First, there are obvious concerns about the fetal loss, the potential for insertional mutagenesis,  developmental effects, and the potential for germ line alteration  that exists for lentiviral vector based approaches. While greater tissue specificity and safety can likely be accomplished by the use of tissue specific promoters, or regulated transgene expression, safer gene transfer techniques will need to be developed to alleviate these concerns. The second major impediment is that stage for stage, the timing of our early gestational injections around E8 in the mouse, corresponds to 21– 35 days gestation in human development,  a time in pregnancy that precedes current capabilities for prenatal diagnosis. On the other hand E14 corresponds to early second trimester that is well within the window for prenatal diagnosis and intervention. In the foreseeable future, prenatal diagnosis may allow diagnosis of genetic disorders within an earlier timeframe. Finally, this model system may be useful for experimental screening of various constructs for efficacy in treatment of TTP associated with ADAMTS13 deficiency, for instance, if postnatal epidermal based secretory strategies are contemplated. 
The ZHK construct is a self-inactivating, replication incompetent HIV-1 based lentiviral vector that has previously been described.  The transgene cassette was composed of the human cytomegalovirus (CMV) immediate early promoter driving enhanced green fluorescent protein (eGFP) expression and human ADAMTS13 or the variant truncated after the spacer domain (MDTCS) (Fig. 1). Bicistronic expression was accomplished by inserting the therapeutic gene downstream and in frame with the eGFP cDNA and TaV sequence, a cis-acting hydrolase element derived from the Thosea asigna virus. 
Vesicular stomatitis virus (VSV)-G pseudotyped vectors were produced by calcium phosphate-mediated transient transfection of HEK293T cells.  Briefly, cells cultured in DMEM (GIBCO Invitrogen) containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 100 U/ml of penicillin and 100 mg/ml streptomycin (GIBCO-Invitrogen) were cotransfected with appropriate amounts of the lentiviral vector plasmid, the packaging plasmid, pCMVΔ8.91, and the VSV-G expression plasmid, pHCMVG.  Approximately 16 hours prior to virus isolation, the media were replaced with similar media minus phenol red. High-titer vectors were prepared by concentrating viral supernatants with ultracentrifugation, then aliquoted and stored at −80 °C. The virus stocks were titered on HEK293T cells by quantifying eGFP positive cells, ranging from 108 to 1010 transducing units (TU)/ml.
Adamts13−/− mice (C57BL/6J/129Sv) were generated by crossing Adamts13B/129+/− mice  with C57BL/6 mice (Taconic, Rensselaer, NY) to generate Adamts13+/− and Adamts13−/− mice. The mice were crossed for at least 4 generations prior to experimentation. Time-dated pregnant mice at post-coital days 8 (E8) and 14 (E14) were anesthetized by isoflurane inhalation (3.5% [vol/vol] for induction and 2% [vol/vol] for maintenance). The intra-amniotic [17, 18] and intravascular injections [41, 19] were performed at E8 and E14, respectively, as previously described.
Newborn mice were sacrificed and various organs were directly placed under a stereoscopic fluorescent microscope (MZ16FA; Leica, Heerburg, Switzerland) to visualize the reporter eGFP expression.
Various mouse tissues were sectioned with a cryostat after being frozen in TissueTek OCT embedding medium (Miles Inc. Elkhart, Indiana). The cryosections were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). After being permeabilized with 1% Triton X-100, the sections were blocked with 2 % fetal bovine serum (FBS) (Invitrogen) in PBS, the sections were incubated anti-GFP IgG (1:100) in 2 % FBS in PBS at 4 °C overnight. The bound antibody was detected by incubation with Cy2-conjugated anti-rabbit (1:200) (Jackson ImmunoResearch, West Grove, PA). The nuclei were stained with 4′, 6-diamidin-2-phenylindole (DAPI) in the mounting medium (Vector Laboratories). The images were taken under a Leica Inverted fluorescent microscope (Wetzlar, Germany).
Tissue specimens were fixed in 10% buffered formalin solution and embedded in paraffin. Sections were prepared using a paraffin microtome and deparaffinized by a standard protocol. After being blocked with goat serum (1:10 dilution), the slides were incubated with rabbit anti-GFP IgG (1:200) (Molecular Probes, Eugene, OR), followed by an incubation with biotinylated goat anti-rabbit IgG, avidin–peroxidase and peroxidase substrate (Vector Lab, Burlingame, CA) as described previously.  The cell nuclei were lightly stained with Harris hematoxylin.
Blood was collected from retinal sinus via capillary tube using 3.2% sodium citrate as anticoagulant at 4, 8, 12, 16, 20 and 24 weeks after the injection of lentiviral vectors. After centrifugation at 1,000 g for 5 minutes in a micro centrifuge, plasma was aspirated, aliquoted and frozen at −80 °C until assay.
The proteolytic activity in mouse plasma was determined by FRETS-vWF73 assay [42, 23] as described previously. To estimate the concentration of recombinant ADAMTS13 and variant in murine plasma, pooled normal human plasma (NHP) was used as a reference and defined as 100% of activity.
To detect full-length ADAMTS13 in mouse plasma, 100 μl of mouse plasma pooled from 5 mice expressing eGFP (controls) and ADAMTS13 at 5~6 weeks of age or normal human plasma was diluted 1:2 with 100 μl of PBS and incubated with 20 μl each of protein G/A Sepharose 4B (BD Biosciences) at room temperature for 1 hour. After mouse IgG was removed, 100 μg of purified human IgG autoantibody against ADAMTS13 isolated from a patient with acquired TTP that recognizes both N- and C-termini of human ADAMTS13 (unpublished data) was added and incubated at 25 °C for 30 minutes. The antibody-antigen complexes were pulled-down by incubation with 15 μl of protein G/A Sepharose 4B for 60 minutes. After washing the Sepharose-4B beads once with PBS, the bound immune complexes were eluted from the beads by boiling them at 100 °C for 5 minutes and detected by Western blot with rabbit anti-ADAMTS13 IgG (kindly provided by Dr. Fritz Scheiflinger, Baxter Biosciences, Orth, Austria) as described previously. [26, 27]
Alternatively, to detect MDTCS in mouse plasma, the immunoprecipitation was modified in such to avoid non-specific protein bands obscuring visualization of the protein of interest (~97 kDa). Pooled plasma (100 μl) from 5 mice expressing eGFP and MDTCS was incubated with 50 μl of the same patient IgG autoantibody against ADAMTS13 (~100 μg IgG) that was covalently immobilized onto Affi-gel10 (Bio-Rad) (2 mg/ml beads) at 4 °C overnight. The bound recombinant human MDTCS was eluted from the beads by adding 40 μl of 0.1 M glycine-HCl, pH 2.5. The MDTCS protein was then detected by Western blot with mouse anti-Dis IgG (1:1,250), followed by incubation with IRDye CW800-labeled anti-mouse IgG (1:12,500) and imaging on an Odyssey Imaging System (LI-COR, Lincoln, Nebraska).
Plasma vWF antigen was determined by an ELISA assay using two different rabbit anti-vWF antibodies.  Briefly, plasma vWF was captured by rabbit anti-vWF IgG (DAKO) (10 tg/ml) immobilized on microtiter plate and detected by peroxidase-conjugated rabbit anti-vWF IgG (1: 5,000) (DAKO), followed by incubation with o-phenylenediamine, (OPD, Sigma)-H2O2. To stop the reaction, 100 tl of 1.5 N H2SO4 was added. The plate was read at 492/620 nm on SpectraMax 190 ELISA reader (Mol. Devices, Union City, CA).
Citrated plasma (10 tl) was diluted 1:20 (vol/vol) with PBS and 100 tl was added to the wells coated with human collagen type III (10 tg/ml) in duplicate. After incubation at 25 C for 1 hour, the bound vWF was detected by peroxidase-conjugated rabbit anti-vWF IgG (1:3,000) and substrate OPD-H2O2 as described previously. [44, 45] Pooled normal murine plasma (PNP) was used in as a reference.
The vWF multimers were performed according to a method described by Banno et al  with modifications. 10 μl of citrated mouse plasma was denatured by heating at 60 °C for 20 minutes in 190 μl of 50 mM Tris-HCl, pH 6.5 containing 10% (wt/vol) SDS, 42% (wt/vol) glycerol, 2 mM EDTA and 0.02% bromphenal blue. The denatured sample (30 μl) was fractionated in 1 % (wt/vol) SeaKem HGT agarose (Cambrex, East Rutherford, NJ) gel by electrophoresis at 15 mA for 2.5 hours on ice. After being transferred onto nitrocellulose membrane (Bio-Rad) at 100 mA for 60 min on a Hoefer TE77 apparatus, the membrane was blocked by 1% (wt/vol) casein in TBST for 30 minutes and incubated with rabbit anti-vWF IgG (A0082 DAKO) (1:1,500) in 1% (wt/vol) casein TBST overnight, followed by IRDye800 fluorescent -conjugated goat anti-rabbit IgG (1:12,500), 25 °C for 1 hour. The membrane was washed 3 times with TBST, once with PBS and scanned in an Odyssey Imaging System at intensity of 5.0.
The mice at the age of 24 weeks (at the end of observation for long-term expression) were anesthetized with intraperitoneal injection of 10 mg/ml Nembutal (0.1 ml/10 g) and the right carotid artery was exposed by blunt dissection. A Doppler flow probe (Model 0.5VB, Transonic Systems, Ithaca, NY) was placed around the artery. [46, 47] Thrombosis was induced in the exposed carotid artery by applying a piece of filter paper (1 × 2 mm) saturated with 10% (vol/vol) ferric chloride to the adventitia for 1 minute. The field was flushed with PBS, and the blood flow was monitored for 30 minutes. The time until initial complete occlusion and the presence or absence of arterial occlusion at 30 minutes was recorded in the experimental and control mice.
Student t-test was used for continuous variables and ANOVA and Chi-square tests were used to determine the differences among various groups. P values less than 0.05 and 0.01 were considered to have statistically significant and very significant differences among the groups, respectively.
This study was supported by grants from National Institute of Health (R01-HL079027 to X.L.Z and P50-HL081012 to Dr. Joel Bennett at The University of Pennsylvania with X.L.Z. as a co-investigator) and R01-HL64715 and HL/DK63434 to A.W.F.). Authors thank Dr. David Ginsburg, Department of Human Genetics and Internal Medicine, and Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan for providing us with Adamts13−/− mice and Dr. Mortimer Poncz for providing us with the Doppler ultrasound arterial flow detection system. Authors also thank Dr. Fritz Scheiflinger for rabbit anti-ADAMTS13 IgG and Nicole DiRogatis for assaying murine vWF multimers.
M Niiya, M Endo and XL Zheng designed, performed the experiments, analyzed the data and wrote the manuscript; D Shang, W Cao, SY Jin, CG. Skipwith and NE Muvarak performed experiments; D Motto generated the Adamts13-null mice, helped with genotyping and revised the manuscript; PW Zoltick and AW Flake helped design the experiments, constructed lentiviral vectors and revised the manuscript.