A mathematical framework for group analysis of von Willebrand factor multimeric composition following luminography

Lopes, Antônio Augusto; Soares, R.P.S.; Maeda, Nair Yukie

doi:10.1590/s0100-879x2002001100002

Cited by 9 publications

(10 citation statements)

References 21 publications

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“…[18][19][20][21][22][23] A comparison of the cost of reagents between the previous method that used chemiluminescence and the current method (ECL Plex CyDye) shows major differences; the reduced time to ❚Image 4❚ Densitometric von Willebrand factor (vWF) quantification (low-resolution gel) of platelet-rich plasma (PRP; black line), plasma (blue line), and platelet (red line) visualized by high-sensitivity fluorescence detection. The arrow demonstrates an additional rapidly migrating band, present in platelets in vWF.…”

Section: Discussionmentioning

confidence: 99%

Analysis of von Willebrand Factor Multimers by Simultaneous High- and Low-Resolution Vertical SDS-Agarose Gel Electrophoresis and Cy5-Labeled Antibody High-Sensitivity Fluorescence Detection

et al. 2010

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Analysis of von Willebrand factor (vWF) multimers allows classification of the subtypes of von Willebrand disease (vWD) in human serum and platelet lysates. A novel method for multimer analysis of vWF by 2-chamber, vertical (sodium dodecyl sulfate), agarose gel electrophoresis, designed for comparing discontinuous high- and low-resolving gels for plasma and platelets, followed by Western blotting and high-sensitivity fluorescence detection (HSFD) of cyanine (Cy)5-labeled vWF multimers is presented. HSFD shows that this method has high discriminatory power for visualization and densitometric analysis of platelets and plasma vWF multimers in various types of vWD and allows rapid classification of vWD types, to separate types 2A and 2B. The described procedures of vWF multimer analysis with high-sensitivity Cy5 fluorescence detection and direct comparison of high- and low-resolving gels for screening and detection of the complete range of high- and low-molecular vWF multimers is efficient and useful for screening, detecting, and classifying vWD subtypes and makes this method diagnostically and clinically relevant.

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Section: Discussionmentioning

confidence: 99%

Analysis of von Willebrand Factor Multimers by Simultaneous High- and Low-Resolution Vertical SDS-Agarose Gel Electrophoresis and Cy5-Labeled Antibody High-Sensitivity Fluorescence Detection

et al. 2010

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“…Blood was collected [1:10 (v/v) into 3.2% sodium citrate] for analysis of plasma VWF:Ag and VWF biological activity (VWF:CB), ADAMTS-13 antigen (ADAMTS-13:Ag) and ADAMTS-13 activity. Protease inhibitors were added for the analysis of VWF multimeric structure 19.…”

Section: Methodsmentioning

confidence: 99%

“…The multimeric structure of VWF was detected and analyzed by Western blotting as previously described 19. Luminographs were subjected to densitometric analysis, and the relative density of low molecular weight VWF multimers was calculated.…”

Section: Methodsmentioning

confidence: 99%

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

Soares

Bydlowski

Nascimento

et al. 2013

Braz J Med Biol Res

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Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

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“…Protease inhibitors were added to analyze the VWF multimeric composition (11). Plasma VWF:Ag and ADAMTS-13:Ag were determined using enzyme-linked immunosorbent assays (Diagnostica Stago, Asnières, France, and Technoclone, Vienna, Austria, respectively).…”

Section: Methodsmentioning

confidence: 99%

“…The results were expressed as percent activity. Proteolysis of VWF multimers was analyzed using Western immunoblotting as previously described (11). The results were expressed as the density of VWF low-molecular-weight fractions relative to the density of all the fractions (laser densitometry).…”

Section: Methodsmentioning

confidence: 99%

Decreased plasma ADAMTS-13 activity as a predictor of postoperative bleeding in cyanotic congenital heart disease

Soares¹,

Bydlowski²,

Jatene³

et al. 2013

Clinics

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OBJECTIVE:To analyze the preoperative plasma antigenic concentration and activity of von Willebrand factor and its main cleaving protease ADAMTS-13 in pediatric patients with cyanotic congenital heart disease undergoing surgical treatment and investigate possible correlations with postoperative bleeding.METHODS:Plasma antigenic concentrations (von Willebrand factor:Ag and ADAMTS-13:Ag) were measured using enzyme-linked immunoassays. Collagen-binding assays were developed to measure biological activities (von Willebrand factor:collagen binding and ADAMTS-13 activity). The multimeric structure of von Willebrand factor was analyzed using Western immunoblotting. Demographic, diagnostic, and general and specific laboratory data and surgery-related variables were subjected to univariate, bivariate, and multivariate analysis for the prediction of postoperative bleeding.RESULTS:Forty-eight patients were enrolled, with ages ranging from 9 months to 7.6 years (median 2.5 years). The plasma concentrations of von Willebrand factor:Ag and ADAMTS-13:Ag were decreased by 65 and 82%, respectively, in the patients compared with the controls (p<0.001). An increased density of low-molecular-weight fractions of von Willebrand factor, which are suggestive of proteolytic degradation (p = 0.0081), was associated with decreased ADAMTS-13 activity, which was likely due to ADAMTS-13 consumption (71% of controls, p = 0.0029) and decreased von Willebrand factor:collagen binding (76% of controls, p = 0.0004). Significant postoperative bleeding occurred in 13 patients. The preoperative ADAMTS-13 activity of <64.6% (mean level for the group), preoperative activated partial thromboplastin time, and the need for cardiopulmonary bypass were characterized as independent risk factors for postoperative bleeding, with respective hazard ratios of 22.35 (95% CI 1.69 to 294.79), 1.096 (95% CI 1.016 to 1.183), and 37.43 (95% CI 1.79 to 782.73).CONCLUSION:Low plasma ADAMTS-13 activity is a risk factor for postoperative bleeding in children with cyanotic congenital heart disease, particularly in children undergoing cardiopulmonary bypass.

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A mathematical framework for group analysis of von Willebrand factor multimeric composition following luminography

Cited by 9 publications

References 21 publications

Analysis of von Willebrand Factor Multimers by Simultaneous High- and Low-Resolution Vertical SDS-Agarose Gel Electrophoresis and Cy5-Labeled Antibody High-Sensitivity Fluorescence Detection

Analysis of von Willebrand Factor Multimers by Simultaneous High- and Low-Resolution Vertical SDS-Agarose Gel Electrophoresis and Cy5-Labeled Antibody High-Sensitivity Fluorescence Detection

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

Decreased plasma ADAMTS-13 activity as a predictor of postoperative bleeding in cyanotic congenital heart disease

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