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1.
Methods Mol Biol ; 2043: 63-73, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463903

RESUMO

ADAMTS7 is a secreted protease that is predominantly expressed in tissues of the cardiovascular system and tendon. Although recent evidence suggests that it may play a role in the etiology of coronary artery disease, its physiological function and substrates are unknown. The enzyme undergoes extensive posttranslational modifications, including chondroitin sulfate attachment, N and O-linked glycosylation, and a two-step activation process. For the benefit of scientists who study the function of ADAMTS7 and its role in disease, this chapter provides an introduction to the chemical and functional properties of the various ADAMTS7 domains, as well as a protocol for the recombinant expression and purification of ADAMTS7.

2.
Nat Commun ; 10(1): 3781, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439947

RESUMO

Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.

3.
Sci Rep ; 9(1): 10914, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358852

RESUMO

ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M-1 s-1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M-1 sec-1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739-744 and 837-844) and ADAMTS-4 (residues 717-724 and 788-795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.

4.
J Biol Chem ; 294(20): 8037-8045, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-30926607

RESUMO

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729-Val-730 and Glu-732-Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-ß-binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1-binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.

5.
Br J Pharmacol ; 176(1): 52-66, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29488211

RESUMO

The metzincin clan of metalloproteinases includes the MMP, disintegrin and metalloproteinase (ADAM) and ADAM with thrombospondin motifs families, which cleave extracellular targets in a wide range of (patho)physiological processes. Antibodies constitute a powerful tool to modulate the activity of these enzymes for both therapeutic and research purposes. In this review, we give an overview of monoclonal antibodies (mAbs) that have been tested in preclinical disease models, human trials and important studies of metzincin structure and function. Initial attempts to develop therapeutic small molecule inhibitors against MMPs were hampered by structural similarities between metzincin active sites and, consequently, off-target effects. Therefore, more recently, mAbs have been developed that do not bind to the active site but bind to surface-exposed loops that are poorly conserved in closely related family members. Inhibition of protease activity by these mAbs occurs through a variety of mechanisms, including (i) barring access to the active site, (ii) disruption of exosite binding, and (iii) prevention of protease activation. These different modes of inhibition are discussed in the context of the antibodies' potency, selectivity and, importantly, the effects in models of disease and clinical trials. In addition, various innovative strategies that were used to generate anti-metzincin mAbs are discussed. LINKED ARTICLES: This article is part of a themed section on Translating the Matrix. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.1/issuetoc.

6.
EBioMedicine ; 2(8): 942-52, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26425702

RESUMO

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease in which anti-ADAMTS13 autoantibodies cause severe enzyme deficiency. ADAMTS13 deficiency causes the loss of regulation of von Willebrand factor multimeric size and platelet-tethering function, which results in the formation of disseminated microvascular platelet microthrombi. Precisely how anti-ADAMTS13 autoantibodies, or antibody subsets, cause ADAMTS13 deficiency (ADAMTS13 activity generally < 10%) has not been formally investigated. METHODS: We analysed 92 acquired TTP episodes at presentation, through treatment and remission/relapse using epitope mapping and functional analyses to understand the pathogenic mechanisms of anti-ADAMTS13 IgG. RESULTS: 89/92 of TTP episodes had IgG recognising the ADAMTS13 N-terminal domains. The central spacer domain was the only N-terminal antigenic target detected. 38/92 TTP episodes had autoantibodies recognising the N-terminal domains alone; 54/92 TTP episodes also had antibodies against the ADAMTS13 C-terminal domains (TSP2-8 and/or CUB domains). Changes in autoantibody specificity were detected in 9/16 patients at relapse, suggesting a continued development of the disease. Functional analyses on IgG from 43 patients revealed inhibitory IgG were limited to anti-spacer domain antibodies. However, 15/43 patients had autoantibodies with no detectable inhibitory action and as many as 32/43 patients had autoantibodies with inhibitory function that was insufficient to account for the severe deficiency state, suggesting that in many patients there is an alternative pathogenic mechanism. We therefore analysed plasma ADAMTS13 antigen levels in 91 acquired TTP presentation samples. We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen. ADAMTS13 antigen in the lowest quartile at first presentation was associated with increased mortality (odds ratio 5.7). CONCLUSIONS: Anti-spacer domain autoantibodies are the major inhibitory antibodies in acquired TTP. However, depletion of ADAMTS13 antigen (rather than enzyme inhibition) is a dominant pathogenic mechanism. ADAMTS13 antigen levels at presentation have prognostic significance. Taken together, our results provide new insights into the pathophysiology of acquired TTP.


Assuntos
Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/imunologia , Especificidade de Anticorpos , Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Púrpura Trombocitopênica Trombótica/imunologia , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Adolescente , Adulto , Idoso , Autoanticorpos/sangue , Mapeamento de Epitopos , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Trombótica/sangue
7.
Blood ; 125(12): 1968-75, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25564400

RESUMO

ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.


Assuntos
Proteínas ADAM/fisiologia , Fator de von Willebrand/química , Proteínas ADAM/química , Proteína ADAMTS13 , Sequência de Aminoácidos , Sítios de Ligação , Cisteína/química , Humanos , Dados de Sequência Molecular , Mutagênese , Mutação Puntual , Polissacarídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato
9.
Blood ; 118(12): 3212-21, 2011 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-21715306

RESUMO

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.


Assuntos
Proteínas ADAM , Coagulação Sanguínea/fisiologia , Trombose/metabolismo , Fator de von Willebrand , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Animais , Sítios de Ligação , Plaquetas/citologia , Plaquetas/metabolismo , Ativação Enzimática , Hemorragia/metabolismo , Hemorragia/patologia , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Reologia , Especificidade por Substrato , Trombose/patologia , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo
10.
Proc Natl Acad Sci U S A ; 108(28): 11602-7, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21705658

RESUMO

The platelet-tethering function of von Willebrand factor (VWF) is proteolytically regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the Tyr1605-Met1606 (P1-P1') bond in the VWF A2 domain. To date, most of the functional interactions between ADAMTS13 and VWF that have been characterized involve VWF residues that are C terminal to the scissile bond. We now demonstrate that the substrate P3 position in VWF, Leu1603, is a critical determinant of VWF proteolysis. When VWF Leu1603 was substituted with Ser, Ala, Asn, or Lys in a short VWF substrate, VWF115, proteolysis was either greatly reduced or ablated (up to 400-fold reduction in k(cat)/K(m)). As Leu1603 must interact with residues proximate to the Zn(2+) ion coordinated in the active center of ADAMTS13, we sought the corresponding S3 interacting residues. Substitution of 10 candidate residues in the metalloprotease domain of ADAMTS13 identified two spatially separated clusters centered on Leu198 or Val195 (acting with Leu232 and Leu274, or with Leu151, respectively), as possible subsites interacting with VWF. These experimental findings using the short VWF115 substrate were replicated using full-length VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported remote exosites C terminal of the cleavage site, suggests a mechanism whereby the VWF P1-P1' scissile bond is brought into position over the active site for cleavage. Together with recently characterized remote exosite interactions, these findings provide a general framework for understanding the ADAMTS family substrate interactions.


Assuntos
Proteínas ADAM/metabolismo , Fator de von Willebrand/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAMTS13 , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico/genética , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fator de von Willebrand/química , Fator de von Willebrand/genética
11.
Blood ; 116(16): 3064-72, 2010 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-20647566

RESUMO

ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (k(cat)/K(m) reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1' M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1' amino acid specificity, we hypothesize, by shaping the S1' pocket. It is concluded that 2 subsites, D187-R193 and D252-P256, in the metalloprotease domain play an important role in cleavage efficiency and site specificity.


Assuntos
Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Mutação , Fator de von Willebrand/metabolismo , Proteínas ADAM/química , Proteína ADAMTS13 , Sequência de Aminoácidos , Linhagem Celular , Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Fator de von Willebrand/genética
12.
Anal Bioanal Chem ; 396(7): 2547-58, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20195581

RESUMO

A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatin-hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein-hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin-cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody-HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose-response curves for enzyme activities were measured within ranges of 0-550 microunits mL(-1) for chymotrypsin, 0-12 microunits mL(-1) for type IX, 0-35 microunits mL(-1) for type XIV and 0-100 microunits mL(-1) for type XXIV. The detection limits of the proteases studied were 89 microunits mL(-1) for chymotrypsin, 0.26 microunits mL(-1) for type IX, 5.8 microunits mL(-1) for type XIV and 6.5 microunits mL(-1) for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format.


Assuntos
Proteínas de Bactérias/análise , Ensaio de Imunoadsorção Enzimática/métodos , Gelatina/química , Haptenos/química , Peptídeo Hidrolases/análise , Extração em Fase Sólida/métodos , Proteínas de Bactérias/imunologia , Haptenos/imunologia , Peptídeo Hidrolases/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
Blood ; 113(22): 5609-16, 2009 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-19234142

RESUMO

ADAMTS13 is a highly specific multidomain plasma metalloprotease that regulates the multimeric size and function of von Willebrand factor (VWF) through cleavage at a single site in the VWF A2 domain. The precise role that the ADAMTS13 disintegrin-like domain plays in its function remains uncertain. Truncated ADAMTS13 variants suggested the importance of the disintegrin-like domain for both enzyme activity and specificity. Targeted mutagenesis of nonconserved regions (among ADAMTS family members) in the disintegrin-like domain identified 3 of 8 ADAMTS13 mutants (R349A, L350G, V352G) with reduced proteolytic activity. Kinetic analyses revealed a 5- to 20-fold reduction in catalytic efficiency of VWF115 (VWF residues 1554-1668) proteolysis by these mutants. These residues form a predicted exposed exosite on the surface of the disintegrin-like domain that lies approximately 26 A from the active site. Kinetic analysis of VWF115 carrying the D1614A mutation suggested that Arg349 in the ADAMTS13 disintegrin-like domain interacts directly with Asp1614 in VWF A2. We hypothesize that this interaction assists in positioning the scissile bond within the active site of ADAMTS13 and therefore plays a major role in determining cleavage parameters (K(m) and k(cat)), as opposed to binding affinity (K(d)) of ADAMTS13 for VWF, the latter being primarily determined by the spacer domain.


Assuntos
Proteínas ADAM/química , Proteínas ADAM/fisiologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Sequência de Aminoácidos , Substituição de Aminoácidos/fisiologia , Domínio Catalítico/genética , Células Cultivadas , Desintegrinas/química , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/fisiologia , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
14.
Blood ; 113(5): 1149-57, 2009 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-19047683

RESUMO

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca(2+) ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca(2+) concentrations, a high-affinity functional ADAMTS13 Ca(2+)-binding site was suggested with K(D(app)) of 80 muM (+/- 15 muM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca(2+)-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca(2+) dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca(2+)-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K(D(app)) for Ca(2+) of ADAMTS13, and for D187A and E212A also reduced the V(max) to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca(2+) revealed an approximately 13-fold reduction in specificity constant, k(cat)/K(m), contributed by changes in both K(m) and k(cat). These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca(2+) upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.


Assuntos
Proteínas ADAM/química , Cálcio/química , Modelos Moleculares , Fator de von Willebrand/química , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Substituição de Aminoácidos , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Linhagem Celular , Humanos , Cinética , Mutagênese , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína/fisiologia , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
15.
Blood ; 112(5): 1548-9, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18725567
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