X. Long Zheng

Pediatrics, Pathology, Hematology

M.D. and Ph.D.
38.49

Publications

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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.
    Blood 03/2015; 125(21). DOI:10.1182/blood-2014-07-587139 · 10.43 Impact Factor
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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.
    Journal of Thrombosis and Haemostasis 03/2015; 13(6). DOI:10.1111/jth.12901 · 5.55 Impact Factor
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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.
    Circulation 02/2015; 131(13). DOI:10.1161/CIRCULATIONAHA.114.012669 · 14.95 Impact Factor
  • 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.
    Annual Review of Medicine 01/2015; 66(1):211-225. DOI:10.1146/annurev-med-061813-013241 · 15.48 Impact Factor
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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.
    Blood 10/2014; 124(25). DOI:10.1182/blood-2014-04-569392 · 10.43 Impact Factor
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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.
    Cellular and Molecular Life Sciences CMLS 06/2014; 72(2). DOI:10.1007/s00018-014-1667-3 · 5.86 Impact Factor
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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.
    Blood 01/2014; 123(1):10-11. DOI:10.1182/blood-2013-11-538322 · 10.43 Impact Factor
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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.
    Arteriosclerosis Thrombosis and Vascular Biology 12/2013; 34(2). DOI:10.1161/ATVBAHA.113.302547 · 5.53 Impact Factor
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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.
    Leukemia research 09/2013; 37(12). DOI:10.1016/j.leukres.2013.09.019 · 2.69 Impact Factor
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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.
    08/2013; 1(3). DOI:10.11648/j.ab.20130103.11
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    Zheng X.L
    Journal of Thrombosis and Haemostasis 06/2013; In press. · 5.55 Impact Factor
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    X. L. Zheng
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    ABSTRACT: ADAMTS-13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS-13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS-13 activity are emerging risk factors for developing myocardial and cerebral infarction, pre-eclampsia, and malignant malaria. Moreover, Adamts13−/− mice develop more severe inflammatory responses, leading to increased ischemia/perfusion injury and formation of atherosclerosis. Structure–function studies demonstrate that the N-terminal portion of ADAMTS-13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS-13 (TSP1 2–8 repeats and CUB domains) may function as a disulfide bond reductase to prevent an elongation of ultra-large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS-13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS-13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure–function and autoantibody mapping studies allow us to identify an ADAMTS-13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.
    Journal of Thrombosis and Haemostasis 06/2013; 11(s1). DOI:10.1111/jth.12221 · 5.55 Impact Factor
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    Thrombosis and Haemostasis 04/2013; DOI:10.1160/TH13-01-0053 · 5.76 Impact Factor
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    British Journal of Haematology 03/2013; 161(6). DOI:10.1111/bjh.12303 · 4.96 Impact Factor
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    ABSTRACT: 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 x 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.
    Blood 03/2013; 121(19). DOI:10.1182/blood-2013-02-486779 · 10.43 Impact Factor
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    ABSTRACT: Thienopyridine-derivatives (ticlopidine, clopidogrel, and prasugrel) are the primary antiplatelet agents. Thrombotic thrombocytopenic purpura (TTP) is a rare drug-associated syndrome, with the thienopyridines being the most common drugs implicated in this syndrome. We reviewed 20 years of information on clinical, epidemiologic, and laboratory findings for thienopyridine-associated TTP. Four, 11, and 11 cases of thienopyridine-associated TTP were reported in the first year of marketing of ticlopidine (1989), clopidogrel (1998), and prasugrel (2010), respectively. As of 2011, the FDA received reports of 97 ticlopidine-, 197 clopidogrel-, and 14 prasugrel-associated TTP cases. Severe deficiency of ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) was present in 80% and antibodies to 100% of these TTP patients on ticlopidine, 0% of the patients with clopidogrel-associated TTP (p < 0.05), and an unknown percentage of patients with prasugrel-associated TTP. TTP is associated with use of each of the three thienopyridines, although the mechanistic pathways may differ.
    Seminars in Thrombosis and Hemostasis 10/2012; 38(8). DOI:10.1055/s-0032-1328894 · 3.69 Impact Factor
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    Zheng XL
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    Zheng XL
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    Zheng XL
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    ABSTRACT: Prevailing approaches to manage autoimmune thrombotic disorders, such as heparin-induced thrombocytopenia (HIT), antiphospholipid syndrome (APS) and thrombotic thrombocytopenic purpura (TTP) include immunosuppression and systemic anticoagulation, though neither provides optimal outcome for many patients. A different approach is suggested by the concurrence of autoantibodies and their antigenic targets in the absence of clinical disease, such as platelet factor 4 (PF4) in HIT and β(2)-glycoprotein-I (β(2)GPI) in APS. The presence of autoantibodies in the absence of disease suggests that conformational changes or other alterations in endogenous protein autoantigens are required for recognition by pathogenic autoantibodies. In TTP, the clinical impact of ADAMTS13 deficiency caused by autoantibodies likely depends on the balance between residual antigen, i.e. enzyme activity, and demand imposed by local genesis of ultralarge multimers of von Willebrand factor (ULvWF). A corollary of these concepts is that disrupting PF4 and β(2)GPI conformation (or ULvWF oligomerization or function) might provide a disease-targeted approach to prevent thrombosis without systemic anticoagulation or immunosuppression. Validation of this approach requires a deeper understanding of how seemingly normal host proteins become antigenic or undergo changes that increase antibody avidity, and how they can be altered to retain adaptive functions while shedding epitopes prone to elicit harmful autoimmunity.
    Blood 09/2012; 120(20). DOI:10.1182/blood-2012-06-389445 · 10.43 Impact Factor

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