Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p. Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p. Cys2771Arg and p. Cys2773Arg, we found the majority of monomers (87 +/- 5% and 73 +/- 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 +/- 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p. Cys2771Arg, p. Cys2773Arg, and p. Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini;a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis

The importance of the intramembrane protease SPPL2a for the development of murine B lymphocytes

In dieser Arbeit wurde die physiologische Bedeutung der Intramembranproteasen Signal peptide peptidase like 2a und 2b (SPPL2a und SPPL2b) mit Hilfe SPPL2a- und SPPL2b-defizienter Mäuse untersucht. Die invariante Kette (CD74), ein Chaperon von MHCII Molekülen, konnte als erstes in vivo validiertes Substrat von SPPL2a identifiziert werden. Aufgrund fehlender Intramembranproteolyse von CD74 in SPPL2a-defizienten B-Lymphozyten kommt es zur Akkumulation eines N-terminalen Fragmentes von CD74. Die Auswirkungen dieser Akkumulation auf die Entwicklung und Funktionalität der B-Zellen und die humorale Immunantwort werden beschrieben

Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS)

Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS).BackgroundThrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS) are now considered to be variants of one single syndrome called thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS). Key features are thrombocytopenia, hemolytic anemia, and subsequently impaired function of different organs, especially the kidneys and the central nervous system (CNS). One possible reason is the deficiency of von Willebrand factor-cleaving protease (vWF-CP) resulting in persistence of uncleaved, ultralarge von Willebrand factor multimers (ULvWFM).MethodsWe report a patient who was initially diagnosed with Evans syndrome (hemolytic anemia and autoimmune thrombocytopenia) as infant. At 10 years of age he developed HUS-like disease with gastrointestinal tract infection, hemolytic anemia, thrombocytopenia,and acute renal failure. However, enteropathogenic Escherichia coli–like or Shiga-like toxins were not detected.ResultsFurther investigations revealed severe deficiency (<3%; normal >40%) of vWF-CP activity caused by compound heterozygosity of two novel ADAMTS13 gene mutations (1170 G>C [W390C] and 3735 G>A [W1245X]. vWF-CP autoantibodies were not detected. Periodic (every 2 weeks) treatment with fresh frozen plasma (FFP) maintained both platelet level and kidney function within normal range and prevented new episodes of TTP/HUS.ConclusionEnteropathogenic E. coli– and Shiga-like toxin-negative patients who present with hemolytic or thrombocytopenic episodes and HUS like symptoms should be tested for vWF-CP deficiency and other noninfectious reasons for TTP/HUS since plasma substitution possibly provides an efficient therapeutic option for this subgroup of patients

Exponential Size Distribution of von Willebrand Factor

AbstractVon Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications

Shear-Induced Unfolding Activates von Willebrand Factor A2 Domain for Proteolysis

To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13-mediated proteolysis in a shear flow dependent manner. We examined if tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. We find this mutant to feature ADAMTS13-resistant behavior in vitro. Our results yield molecular-detail evidence for the force-sensoring function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional knotted Rossman fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we can explain the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage-activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases

Mortalidade entre Idosos no Estado do Paraná e no município de Foz do Iguaçu

Trabalho de Conclusão da Residência apresentado ao Programa de Residência Multiprofissional em Saúde da Família da Universidade Federal da Integração Latino- Americana, como requisito parcial para obtenção do título de Especialista em Saúde da Família na modalidade de residência. Orientador: Walfrido Svoboda. (Prof. Dr. do Curso de Saúde Coletiva - UNILA) e Coorientadora: Carmen Justina Gamarra. (Profa. Dra. do Curso de Saúde Coletiva - UNILA)Objetivo: Analisar a tendência da mortalidade entre idosos residentes no estado do Paraná e no município de Foz do Iguaçu, no período de 2001 a 2012, e mortalidade proporcional por capítulos CID-10. Método: Trata-se de um estudo descritivo, ecológico de série temporal. Os dados dos óbitos e da população idosa de 60 anos ou mais foram obtidos por meio do Sistema de Informação de mortalidade SIM/DATASUS. Foram calculadas as taxas anuais de mortalidade bruta e padronizadas por sexo e faixa etária. Para os dados de mortalidade proporcional por capítulos CID-10 foram calculados os percentuais para os anos selecionados de 2001; 2006; 2011; 2016. As informações foram tabuladas em planilhas do Excel, sendo construídas tabelas e gráficos e analisados por meio de regressão linear simples. Resultados: Observou-se tendência declinante da mortalidade dos idosos no estado do Paraná e no município de Foz do Iguaçu. Ao longo do estudo verificou-se que as doenças do aparelho circulatório constituem-se como a primeira e principal causa de óbito, seguido das neoplasias e doenças do aparelho respiratório. Conclusão: No período analisado, a tendência da mortalidade padronizadas dos idosos obteve decréscimo tanto no Estado como no município, sendo observados maiores coeficientes de mortalidade entre os homens quando comparado às mulheres.Objective: To analyze the mortality trend among elderly people living in the state of Para- ná and in the municipality of Foz do Iguaçu, from 2001 to 2012, and proportional mortality by ICD-10 chapters. Method: This is a descriptive, ecological time series study. Data on deaths and the elderly population aged 60 years and over were obtained using the SIM / Datasus Mortality Infor- mation System. Gross annual mortality rates were standardized and standardized by gen- der and age group. The information was tabulated in Excel spreadsheets, and tables and graphs were constructed and analyzed using simple linear regression. Results: In general terms, a declining trend in mortality among the elderly was observed in the state of Paraná and in the city of Foz do Iguaçu. Throughout the study it was verified that diseases of the circulatory system constitute the first and main cause of death, fol- lowed by neoplasias and diseases of the respiratory system. Conclusion: In the period analyzed, the general trend of standardized mortality among the elderly decreased both in the state and in the municipality, with higher mortality rates among men when compared to womenObjetivo: Analizar la tendencia de la mortalidad entre ancianos residentes en el estado de Paraná y en el municipio de Foz do Iguaçu, en el período de 2001 a 2012, y mortali- dad proporcional por capítulos CID-10. Método: Se trata de un estudio descriptivo, eco- lógico de serie temporal. Los datos de mortalidad y de la población de la tercera edad (mayor de 60años) fueron obtenidos por medio del sistema de información de mortalidad SIM/DATASUS. Fueron calculadas las tasas anuales de mortalidad bruta y estandariza- das por sexo y edad. Las informaciones fueron tabuladas en planillas de cálculo Micro- soft Excel, siendo construidas tablas, gráficos e analizados por medio de regresión linear simple. Resultados: En términos generales fue observada la tendencia declinante de la mortali- dad en personas de la tercera edad en el Estado de Paraná y en el Municipio de Foz de Iguazú. A lo largo de este estudio, se verifico que las enfermedades del aparato circula- torio se consideran como la primera causa de muerte, seguido por Cáncer e enfermeda- des del Aparato Respiratorio. Conclusión: En el periodo analizado, la tendencia de la mortalidad estandarizada en personas de la tercera edad obtuvo decrecimos tanto en el Estado de Paraná como en el Municipio, siendo observados mayores coeficientes de mortalidad en hombres compara- do con mujere

Force-Sensitive Autoinhibition of the von Willebrand Factor ls Mediated by Interdomain Interactions

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis

Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86976/1/j.1538-7836.2011.04268.x.pd