X. Long Zheng

Pediatrics, Pathology, Hematology

M.D. and Ph.D.
38.98

Publications

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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via FcγRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase (Syk) and leads to the generation of tissue factor and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases annexin and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.
    No preview · Article · Oct 2015 · Blood
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    ABSTRACT: Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.
    Full-text · Article · Jul 2015 · Proceedings of the National Academy of Sciences
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    Catherine B Zander · Wenjing Cao · X Long Zheng
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    ABSTRACT: ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13-VWF interaction is essential for developing novel treatments to this disorder. Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2-8 repeats and the CUB domains have proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. Significant progress has been made in our understandings of the structure-function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13-VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.
    Full-text · Article · Jul 2015 · Current opinion in hematology
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    ABSTRACT: ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultra large VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer (FRETS) assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild type (WT) and Adamts13(-/-) (KO) platelets. The expressed rADAMTS13 is released upon stimulation with thrombin and collagen, but less with 2MesADP. Platelet-delivered rADAMTS13 is able to inhibit arterial thrombosis after vascular injury and prevent the onset and progression of Shigatoxin-2 or recombinant murine VWF (rmVWF)-induced TTP syndrome in mice despite lack of plasma ADAMTS13 activity resulting from the ADAMTS13 gene deletion or the antibody-mediated inhibition of plasma ADAMTS13 activity. These findings provide a proof-of-concept that platelet-delivered ADAMTS13 may be explored as a novel treatment for arterial thrombotic disorders including hereditary and acquired TTP in the presence of anti-ADAMTS13 autoantibodies. Copyright © 2015 American Society of Hematology.
    No preview · Article · Mar 2015 · Blood
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    X Long Zheng
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    ABSTRACT: In this issue of Blood, de Groot et al identify a hydrophobic pocket in the Cys-rich domain of ADAMTS13 that appears to interact with the hydrophobic pocket in the central A2 domain of von Willebrand factor (VWF) for its proteolysis.
    Full-text · Article · Mar 2015 · Blood
  • Rui-Nan Lu · Shangbin Yang · Haifeng M Wu · X Long Zheng
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    ABSTRACT: Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. unconjugated bilirubin directly inhibits ADAMTS13's ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Mar 2015 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: -Genome-wide association studies (GWAS) have established ADAMTS7 as a locus for coronary artery disease (CAD) in humans. Yet, these studies fail to provide directionality for the association between ADAMTS7 and CAD. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell (VSMC) migration, but a role and direction of impact for this gene in atherogenesis has not been shown in relevant model systems. -We bred an Adamts7 whole body knockout (KO) mouse onto both the Ldlr and Apoe KO hyperlipidemic mouse models. Adamts7(-/-)/Ldlr(-/-) and Adamts7(-/-)/Apoe(-/-) mice displayed significant reductions in lesion formation in aortas and aortic roots as compared to controls. Adamts7 KO mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later timepoints, and primary Adamts7 KO VSMCs showed reduced migration in the setting of TNFα stimulation. ADAMTS7 localized to cells positive for SMC markers in human CAD lesions, and sub-cellular localization studies in cultured VSMCs placed ADAMTS7 at the cytoplasm and cell membrane, where it co-localized with markers of podosomes. -These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.
    Preview · Article · Feb 2015 · Circulation
  • X. Long Zheng
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    ABSTRACT: Pathogenesis of thrombotic thrombocytopenic purpura (TTP) was a mys- tery for over half a century until the discovery of ADAMTS13. ADAMTS13 is primarily synthesized in the liver, and its main function is to cleave von Willebrand factor (VWF) anchored on the endothelial surface, in circula- tion, and at the sites of vascular injury. Deficiency of plasma ADAMTS13 activity (<10%) resulting from mutations of the ADAMTS13 gene or autoantibodies against ADAMTS13 causes hereditary or acquired (idiopathic) TTP. ADAMTS13 activity is usually normal or modestly reduced (>20%) in other forms of thrombotic microangiopathy secondary to hematopoietic progenitor cell transplantation, infection, and disseminated malignancy or in hemolytic uremic syndrome. Plasma infusion or exchange remains the initial treatment of choice to date, but novel therapeutics such as recombinant ADAMTS13 and gene therapy are under development. Moreover, ADAMTS13 deficiency has been shown to be a risk factor for the development of myocardial infarction, stroke, cerebral malaria, and preeclampsia.
    No preview · Article · Jan 2015 · Annual Review of Medicine
  • X. Long Zheng
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    ABSTRACT: ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF), has been shown to have both antithrombotic and anti-inflammatory activities. Severe deficiency of plasma ADAMTS13 activity is a primary cause of thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome, but mild to moderate deficiency of plasma ADAMTS13 activity may be associated with increased risk for many other pathologies, including myocardial/cerebral infarction, malignant malaria, preeclampsia, and acute and chronic inflammation. Structure function analyses demonstrate that N-terminal fragment containing a metalloprotease domain, a disintegrin domain, the first thrombospondin type 1 repeat (TSP1 1), a cysteine-rich domain, and a spacer domain (i.e., MDTCS) appears to be sufficient for recognition and proteolytic cleavage of VWF in vitro and in vivo. The role of more distal C-terminal domains including TSP1 2-8 repeats and CUB (the complement C1r/C1s, Uegf, and Bmp1) domains remains to be determined. Recent studies suggest that TSP12-8 and CUB domains may negatively regulate ADAMTS13 activity. Further investigations of the binding sites of anti-ADAMTS13 autoantibodies from acquired TTP patients may help redesign ADAMTS13 protease for therapeutic use.
    No preview · Chapter · Jan 2015
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    Hayley A Hanby · X Long Zheng

    Full-text · Article · Oct 2014
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    ABSTRACT: Platelet-VWF interactions must be tightly regulated in order to promote effective hemostasis and prevent occlusive thrombus formation. However, it is unclear what role the inherent properties of the bond formed between the platelet receptor GPIbα and the A1 domain of VWF play in these processes. Using VWF-A1 knock-in mice with mutations that enhance (I1309V) or disrupt (R1326H) GPIbα binding, we now demonstrate that the kinetic interplay between two distinct contact surfaces influences the site and extent to which platelets bind VWF. Incorporation of R1326H mutation into the major site shortened bond lifetime, yielding defects in hemostasis and thrombosis comparable to VWF deficient animals. Similarly, disrupting this region of contact with an allosteric inhibitor impaired human platelet accrual in damaged arterioles. In contrast, the I1309V mutation near the minor site prolonged bond lifetime, which was essential for the development of a type 2B-like VWD phenotype. However, combining the R1326H and I1309V mutations normalized both bond kinetics and the hemostatic and thrombotic properties of VWF. These findings broaden our understanding of mechanisms governing platelet-VWF interactions in health and disease, and underscore the importance of combined biophysical and genetic approaches in identifying potential therapeutic avenues for treating bleeding and thrombotic disorders.
    No preview · Article · Oct 2014 · Blood
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    ABSTRACT: Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. The goals of this study were to understand the angiogenic pathways ADAMTS13 activates and to identify the important structural components of ADAMTS13 that stimulate angiogenesis. Incubation of human umbilical vein endothelial cells (HUVEC) with 150 ng/mL (1 nM) of recombinant human ADAMTS13 induced VEGF expression by 53 % and increased VEGF mRNA by over sixfold, both within 10 min; the measured VEGF levels steadily decreased over 2 h, as shown by Western blot and ELISA. Phosphorylation of VEGFR2 was significantly enhanced in HUVEC after incubation with ADAMTS13 (1 nM). Structure-function analysis showed that an ADAMTS13 variant containing thrombospondin type 1 (TSP1) 2-8 repeats (TSP1 2-8), TSP1 2-8 plus CUB domains (TSP1 2-8 plus CUB), or TSP1 5-8 repeats plus CUB domains (TSP1 5-8 plus CUB) increased HUVEC proliferation by 41-54 % as compared to the EBM-2 controls. Chemotaxis assays further demonstrated that the TSP1 domains of ADAMTS13 increased HUVEC migration by 2.65-fold. Incubation of HUVEC with both ADAMTS13 variants containing TSP1 repeats and anti-VEGF IgG abrogated the enhanced effect of ADAMTS13 on proliferation, migration, and VEGFR2 phosphorylation. In conclusion, ADAMTS13-induced endothelial cell angiogenesis occurs via the upregulation of VEGF and phosphorylation of VEGFR2. This angiogenic activity depends on the C-terminal TSP1 repeats of ADAMTS13.
    Full-text · Article · Jun 2014 · Cellular and Molecular Life Sciences CMLS
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    Full-text · Article · Feb 2014 · Haematologica
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    X. Long Zheng
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    ABSTRACT: In this issue of Blood, Savchenko et al demonstrate that removal of neutrophil extracellular traps (NETs) and inhibition of neutrophil recruitment by DNase I with or without ADAMTS13 or inactivation of peptidyl arginine deiminase 4 (iPAD4) protects from cardiac damage after myocardial infarction in mice.
    Full-text · Article · Jan 2014 · Blood
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    Jialing Bao · Juan Xiao · Yingying Mao · X Long Zheng
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    ABSTRACT: ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, 13) cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface-exposed free sulfhydryls on ADAMTS13 that seemed to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow. A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2 to 8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5 to 8 Thrombospondin type 1 (TSP1) repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging analysis showed that the carboxyl-terminal fragment of ADAMTS13 (T2C or T5C) was capable of inhibiting the formation and elongation of platelet-decorated ultra large (UL) VWF strings and the adhesion of platelets/leukocytes on endothelium in mesenteric venules after oxidative injury. The inhibitory activity of T2C and T5C on platelet aggregation and ULVWF string formation were dependent on the presence of their surface free thiols; pretreatment of T2C and T5C or full-length ADAMTS13 with N-ethylmaleimide that reacts with free sulfhydryls abolished or significantly reduced its antithrombotic activity. Our results demonstrate for the first time that the carboxyl terminus of ADAMTS13 has direct antithrombotic activity in a free-thiol-dependent manner. The free thiols in the carboxyl-terminal domains of ADAMTS13 may also contribute to the overall antithrombotic function of ADAMTS13 under pathophysiological conditions.
    Full-text · Article · Dec 2013 · Arteriosclerosis Thrombosis and Vascular Biology
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    ABSTRACT: Arsenic trioxide (As2O3) can induce apoptosis in many tumors. However, the associated mechanisms are not clearly understood. We found that As2O3 significantly inhibited the proliferation of WSU-CLL cells and induced apoptosis in dose- and time-dependent manners. WSU-CLL cells treated with 2μM As2O3 showed survivin down-regulation and p53 up-regulation. Survivin siRNA combined with As2O3 further inhibited the proliferation of WSU-CLL cells. p53 inhibition by siRNA prevented the down-regulation of survivin by As2O3 and prevented the As2O3-induced cytotoxicity of WSU-CLL cells. These results suggest that As2O3 may be of therapeutic value for chronic lymphocytic leukemia.
    Full-text · Article · Sep 2013 · Leukemia research
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    Hayley A Hanby · X Long Zheng
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    ABSTRACT: Here, we provide a comprehensive review of current findings concerning the biochemistry and physiological functions of ADAMTS7, a metalloprotease that is known to interact with cartilage oligomeric matrix protein, progranulin, and alpha2-macroglobulin. Such broad substrate specificity and potentially diverse physiological functions make ADAMTS7 an interesting enzyme to study. ADAMTS7 has been shown to play a role in the pathogenesis of arthritis and disc disorders. More recently, the ADAMTS7 locus is identified to have a strong association with coronary atherosclerotic disease. However, the role of ADAMTS7 in the development of atherosclerosis is yet to be determined. The development of an easy and high throughput assay for ADAMTS7 activity and appropriate animal models will allow us to uncover the novel mechanisms of coronary arterial disease.
    Full-text · Article · Aug 2013
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    Zheng X.L

    Full-text · Article · Jun 2013 · Journal of Thrombosis and Haemostasis
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    Full-text · Article · Apr 2013 · Thrombosis and Haemostasis
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    Full-text · Article · Mar 2013 · British Journal of Haematology

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