Sheng-Yu Jin

Amino acid residues Arg659, Arg660 and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor

Previous studies have shown that ADAMTS13 spacer domain is required for cleavage of von Willebrand factor (VWF). However, the exact amino acid residues within this domain critical for substrate recognition are not known. Epitope mapping of anti-ADAMTS13 immunoglobulin G from patients with thrombotic thrombocytopenic purpura and sequence alignment of the ADAMTS13 spacer domains of human, mouse, and zebrafish with these of human and murine ADAMTS1, a closely related member of ADAMTS family, have provided hints to investigate the role of the amino acid residues between Arg(659) and Glu(664) of the ADAMTS13 spacer domain in substrate recognition. A deletion of all these 6 amino acid residues (ie, Arg(659)-Glu(664)) from the ADAMTS13 spacer domain resulted in dramatically reduced proteolytic activity toward VWF73 peptides, guanidine-HCl denatured VWF, and native VWF under fluid shear stress, as well as ultralarge VWF on endothelial cells. Site-directed mutagenesis, kinetic analyses, and peptide inhibition assays have further identified a role for amino acid residues Arg(659), Arg(660), and Tyr(661) in proteolytic cleavage of various substrates under static and fluid shear stress conditions. These findings may provide novel insight into the structural-function relationship of ADAMTS13 and help us to understand pathogenesis of thrombotic thrombocytopenic purpura and other arterial thromboses associated with compromised VWF proteolysis.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this 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 (approximately 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 (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (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.

Genetic Ablation of Adamts13 Gene Dramatically Accelerates the Formation of Early Atherosclerosis in a Murine Model

Objective—ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves von Willebrand factor, thereby modulating thrombosis and inflammation. Low plasma ADAMTS13 activity is associated with cardiovascular events, including myocardial and cerebral infarction. Here, we investigated the role of ADAMTS13 in the development of early atherosclerosis in a murine model. Methods and Results—Apolipoprotein E–null (ApoE−/−) and Adamts13-null (Adamts13−/−) ApoE−/− mice were fed with a high-fat Western diet for 12 weeks. Atherosclerotic lesions in the aorta and aortic roots were quantified after staining. Leukocyte rolling and adhesion onto cremaster venules after oxidative injury were determined by intravital microscopy. Although plasma cholesterol levels were largely similar in both groups, the extent of atherosclerotic lesions in the aorta en face and in the aortic roots in the Adamts13−/−ApoE−/− mice increased ≈5.5-fold (P=0.0017) and ≈6.1-fold (P=0.0037), respectively. In addition, the ratio of plasma high- to low-molecular-weight von Willebrand factor multimers increased ≈3-fold. The leukocyte rolling velocities were significantly reduced (P<0.001), with an increased number of leukocyte rolling (P=0.0026) and macrophage infiltration into the atherosclerotic lesions in the Adamts13−/−ApoE−/− mice. Conclusion—Our results suggest that ADAMTS13 plays a critical role in modulating the development of early atherosclerosis, likely through the proteolytic cleavage of ultra-large von Willebrand factor multimers, thereby inhibiting platelet deposition and inflammation.

Essential Domains of A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13 Metalloprotease Required for Modulation of Arterial Thrombosis

Objective—A disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) inhibits platelet aggregation and arterial thrombosis by cleavage of von Willebrand factor. However, the structural components of ADAMTS13 required for inhibition of arterial thrombosis are not fully defined. Methods and Results—Using recombinant proteins and a murine model, we demonstrated that an ADAMTS13 variant truncated after either the eighth thrombospondin type 1 repeat or the spacer domain inhibits ferric chloride–induced arterial thrombosis in ADAMTS13−/− mice with efficacy similar to that of full-length ADAMTS13. The results obtained from monitoring thrombus formation in carotid and mesenteric arteries were highly concordant. Further analyses by site-directed mutagenesis and human monoclonal antibody inhibition assay revealed that the Cys-rich and spacer domains of ADAMTS13, particularly the amino acid residues between Arg559 and Glu664 in the spacer domain, may be critical for modulation of arterial thrombosis in vivo. Finally, the thrombosis-modulating function of ADAMTS13 and variants/mutants was highly correlated with the von Willebrand factor–cleavage activity under fluid shear stress. Conclusion—Our results suggest that the amino terminus of ADAMTS13, specifically the variable region of the spacer domain, is crucial for modulation of arterial thromboses under (patho)physiological conditions. These findings shed more light on the structure-function relationship of ADAMTS13 in vivo and may be applicable for rational design of protein- or gene-based therapy of arterial thromboses.

Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice

Development of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 domain of FVIII. We created a C1 domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4(+) T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.

von Willebrand factor cleaved from endothelial cells by ADAMTS13 remains ultralarge in size

Von Willebrand factor (vWF) is synthesized in all vascular endothelial cells and megakaryocytes 1. The mature vWF is stored in the Weibel-Palade bodies of endothelial cells and the α-granules of platelets. A small fraction of vWF is constitutively secreted from the cells. Upon stimulation by thrombin, hormones, and inflammatory cytokines, vWF is released from the Weibel-Palade bodies of the endothelial cells as ultra large (UL) vWF, which forms polymers and remains attached to the endothelial cell surface via poorly characterized mechanisms, although recent reports have suggested that UL-vWF might be anchored on endothelial cells via interactions with P-selectin 2 and αvβ3 integrin 3. Proteolytic cleavage of the newly synthesized UL-vWF on endothelial cells by ADAMTS13 may be critical to maintain normal hemostasis and inflammatory response. For instance, certain mutations in the vwf gene may result in increased proteolysis of vWF by ADAMTS13 and significant reduction of vWF multimer sizes, leading to compromised hemostatic function as seen in patients with von Willebrand disease 4. Conversely, an inability to cleave the newly released UL-vWF strings 5;6 due to hereditary or acquired deficiency of ADAMTS13 may result in an accumulation of UL-vWF that results in formation of disseminated thrombi in the microvasculature as in patients with thrombotic thrombocytopenic purpura (TTP) 7. Moreover, ADAMTS13 deficiency appears to result in increased leukocyte rolling on unstimulated veins, leukocyte adhesion and extravasation of neutrophils in the inflamed veins 8. All these processes seem to depend on the presence of UL-vWF multimers 8. Therefore, the removal of cell bound UL-vWF by ADAMTS13 may help attenuate systemic inflammatory responses in addition to arterial thrombosis. In contrast to proteolytic cleavage of vWF in solution, which requires denaturant (urea 9 or guanidine 10) in a low ionic and alkaline buffer or high shear stress 11;12, the removal of UL-vWF polymers on cultured endothelial cells by ADAMTS13 occurs very efficiently under various shear stresses 5;6. The rapid removal of the UL-vWF strings by infused recombinant ADAMTS13 also occurs in both arteries (high shear) and venules (low shear) in Adamts13-/- mice 13. A recent study has shown that UL-vWF strings on histamine stimulated endothelial cells can be removed by ADAMTS13 in the absence of flow shear stress 14. It remains poorly understood what ADAMTS13 domains are required for cleavage of cell bound UL-vWF, and whether proteolytic cleavage of UL-vWF by ADAMTS13 on endothelial cell membranes is sufficient to reduce the multimer sizes of UL-vWF in the presence or absence of flow shear stress. In this study, we demonstrated by immunofluorescent microscopy that UL-vWF polymers were readily formed on the membrane of human umbilical vein endothelial cells (HUVECs) after being stimulated for 2 min with histamine (100 μM) in phosphate buffered saline (Fig. 1A-i). Recombinant ADAMTS13 (10 nM) (Fig. 1A-ii) or normal human plasma (NHP) (1:2 dilution) (not shown) in an assay buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 5 mM CaCl2) within 5 min could efficiently remove all surface bound UL-vWF polymers in the absence of flow shear stress. Plasma (1:2 dilution) from a patient with acquired idiopathic TTP with known high titer of anti-ADAMTS13 IgG autoantibodies, however, did not result in the removal of the cell bound UL-vWF polymers under the same conditions (not shown). These results are consistent with what has been reported by Turner et al 14, and are the premises for our further study of the structural and functional relationship of ADAMTS13 in cleavage of UL-vWF at the cellular levels. Figure 1 Cleavage of cell bound UL-vWF by recombinant ADAMTS13 and variants To better quantify the amount of UL-vWF released from endothelial cells, we employed an enzyme linked immunoassay (ELISA) using two polyclonal anti-vWF antibodies as described previously 15. In these experiments, HUVECs cultured on 6-well plate were stimulated with histamine (100 μM) for 2 min, washed with PBS and then incubated for 0~60 minutes without or with a fixed concentration (10 nM) of purified recombinant ADAMTS13 in 20 mM HEPES, 150 mM NaCl and 5 mM CaCl2, pH 7.4 (Fig. 1B-i) or for 5 min with various concentrations of recombinant ADAMTS13 (0~ 40 nM) (Fig. 1B-ii). The amount of vWF antigen in the conditioned media was increased in an incubation time and ADAMTS13 concentration dependent manner (Fig. 1B). After 60 min of incubation with purified recombinant ADAMTS13 (10 nM), the vWF antigen in the conditioned media (~24 ng/ml) was approximately 4-fold higher than in the buffer-treated control (~6 ng/ml) (Fig. 1B-i). The concentration of ADAMTS13 that achieved the half maximal levels of vWF released from endothelial cells was approximately 3.0 nM (Fig. 1B-ii), which is within the physiological range of ADAMTS13 in plasma. To determine whether or not the C-terminal domains of ADAMTS13 were required for cleavage of cell bound UL-vWF, histamine-stimulated and washed HUVECs were incubated with 10 nM of purified ADAMTS13 and various C-terminal truncated variants that were well characterized previously 12. The remaining UL-vWF on endothelial cells was determined by immunofluorescent microscopy using polyclonal anti-vWF IgG (Dako). We showed that the variants lacking the CUB domains (delCUB) (Fig. 1D-i) and truncated after the spacer domain (MDTCS) (Fig. 1D-ii) also efficiently removed UL-vWF polymers from histamine-stimulated endothelial cells, similar to full-length ADAMTS13 (Fig. 1A-ii). However, the variant truncated after the first thrombospondin type 1 (TSP1) repeat (MDT) (Fig. 1D-iii) or the metalloprotease domain (M) (Fig. 1D-iv) exhibited markedly impaired activity in removing the cell bound UL-vWF under the same conditions. The removal of cell bound UL-vWF polymers was consistent with the about 3~4 fold of increase in vWF antigen in the conditioned medium after incubation of the histamine-stimulated endothelial cells with full-length ADAMTS13, delCUB and MDTCS, but not with MDT and M or buffer alone (Fig. 1C-i). The proteolytic cleavage products (176kDa and 140 kDa) were also detectable in the conditioned media of HUVECs treated with FL-A13, delCUB and MDTCS, but not with MDT and M or buffer alone (Fig. 1C-ii). The amount of vWF antigen detected in the MDTCS-treated medium appeared to be slightly low compared with the FL-A13 and delCUB-treated media (Fig. 1C-ii), but such a difference was not statistically significant (p>0.05). The reduced cleavage products in the MDTCS-treated medium compared with full-length ADAMTS13 and delCUB may be a result of imperfect protein loading, rather than the lower proteolytic activity. Thus, our results suggest that the Cys-rich and spacer domains are required for recognition of the cell bound UL-vWF, but the TSP1 2-8 repeats and the CUB domains are dispensable. These results, however, do not fully agree with those reported 14. The reason for the discrepancy is not completely understood, but may be related to the assay methodologies being used. The domain requirement for proteolytic cleavage of cell bound UL-vWF is reminiscent of that seen in cleavage of soluble vWF by ADAMTS13 under denaturing conditions 16;17, suggesting that membrane association of UL-vWF results in a conformational change that permits ADAMTS13 binding in the absence of fluid shear stress. The mechanism underlying such a conformational change remains to be determined. To determine whether proteolytic cleavage of cell bound UL-vWF by ADAMTS13 was sufficient to reduce UL-vWF multimer size, the conditioned media were assessed by SDS-agarose (1%) gel electrophoresis and Western blot as previously described 15. Unexpectedly, whether in the absence of flow (Fig. 1E-i) or in the presence of flow (2.5 dynes/cm2) (Fig. 1E-ii), vWF multimers released into the conditioned media after being incubated with ADAMTS13 (10 nM) were indistinguishable from those in the conditioned media after being incubated with buffer alone or immediately after histamine stimulation (Fig. 1E-i & ii). When compared with normal human plasma, the conditioned media were highly enriched with UL-vWF as indicated by UL (Fig. 1E-i & ii). Incubation of the histamine-stimulated endothelial cells with recombinant ADAMTS13 (10 nM) for up to 60 min in the absence of flow (Fig. 1E-i, lanes 2-8) or for 5 min under various flow shear stresses up to ~25 dynes/cm2 (Fig. 1E-ii, lanes 3-7) did not appear to alter the vWF multimer distribution in the conditioned media, as compared with the UL-vWF released from the Weibel-Palade bodies after histamine stimulation (Fig. 1E-i, lane 9 & Fig. 1E-ii, lane 8), although some of the low molecular weight vWF (L) may be resulted from proteolytic degradation of the UL-vWF in the conditioned medium during storage. Addition of 0.1% protease inhibitor cocktail (Sigma) reduced the intensity of the smaller vWF bands. Nevertheless, the vWF cleaved off from endothelial cells by ADAMTS13 remains ultra large in size. This suggests further proteolytic processing downstream by ADAMTS13 or other leukocyte proteases 18, likely to occur in the small arteries and capillaries, where high fluid shear stress and perhaps physiological cofactors such as factor VIII 19 and platelets 20 are present, may be necessary to eliminate the ultra large vWF polymers.

AAV-mediated expression of an ADAMTS13 variant prevents shigatoxin-induced thrombotic thrombocytopenic purpura

Severe deficiency of plasma ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome for which plasma is the only effective therapy currently available. As much as 5% of TTP cases are hereditary, resulting from mutations of the ADAMTS13 gene. Here, we report the efficacy and safety of recombinant adeno-associated virus serotype 8 (AAV8)-mediated expression of a murine ADAMTS13 variant (MDTCS), truncated after the spacer domain, in a murine model of TTP. Administration of AAV8-hAAT-mdtcs at doses greater than 2.6 × 10(11) vg/kg body weight resulted in sustained expression of plasma ADAMTS13 activity at therapeutic levels. Expression of the truncated ADAMTS13 variant eliminated circulating ultralarge von Willebrand factor multimers, prevented severe thrombocytopenia, and reduced mortality in Adamts13(-/-) disease-prone mice triggered by shigatoxin-2. These data support AAV vector-mediated expression of a comparable truncated ADAMTS13 variant as a novel therapeutic approach for hereditary TTP in humans.