Collagen-binding Activity Assay Research Articles

Diagnostic relevance of ADAMTS13 activity: Evaluation of 28 patients with thrombotic thrombocytopenic purpura - hemolytic uremic syndrome clinical diagnosis

The significance of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motif-13) activity for diagnosis and therapy of thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) is still a controversial issue. The aim of this report was to analyze the value of ADAMTS13 measurements in the diagnosis of TTP and HUS. At presentation, we analyzed patients with idiopathic TTP (n = 18), secondary TTP (n = 4), diarrhea positive HUS (n = 3) and diarrhea negative HUS (n = 3) treated in Belgrade, Serbia from 2004 to 2010. ADAMTS13 activity from acute phase samples was measured using the residual collagen binding activity assay at the Haemophilia and Thrombosis Centre, Milan, Italy. There was a significant correlation between reduced ADAMTS13 activity and idiopathic TTP diagnosis (p = 0.000) as well as between lower ADAMTS13 activities and higher reticulocytes (p = 0.017) and lactate dehydrogenase levels (p = 0.027). Significant correlation was also found between higher protease activity and diagnosis of HUS (p = 0.000). There was a statistically significant correlation between higher ADAMTS13 activities and higher platelets count (p = 0.002), blood urea nitrogen (p = 0.000), and creatinine level (p = 0.000). Severe ADAMTS13 deficiency points at the diagnosis of idiopathic TTP and it is present in the secondary TTP but not in HUS.

Detection of Continuous Flow Left Ventricular Assist Device -Associated Acquired Von Willebrand Factor (VWF) Abnormality by An Automated Immunoturbidimetric VWF Activity Assay

Abstract▪2273▪This icon denotes a clinically relevant abstract Background:Continuous flow left ventricular assist device (CF-LVAD) recipients have high tendency of post-surgical gastrointestinal (GI) bleeding. We previously described the loss of high molecular weight VWF multimers (HMWM), due to either an accelerated clearance or enhanced cleavage of the HMWM, in most CF-LVAD recipients (Ann Thorac Surg. 90:1263–9). However, besides the tedious plasma VWF multimer analysis, neither VWF ristocetin cofactor nor collagen binding activity assay provides sufficient sensitivity for detecting such an acquired VWF abnormality (AVWA). In this study, we examined a new automated latex particle-enhanced immunoturbidimetric VWF activity assay (VWF:Lx) for its ability of detecting CF-LVAD-AVWA. We also analyzed the VWF pro-peptide (VWF:pp) to explored the potential mechanism of CF-LVAD-AVWA. Design:As part of an on-going prospective multicenter study, pre- and post-CF-LVAD implantation (7, 30 days and 5–7 months) blood samples were collected from 15 LVAD recipients (median age 52 years; 10 male and 5 female; 2008∼2009). Plasma VWF antigen (VWF:Ag), VWF:Lx activity, VWF:pp/Ag ratios were measured; and plasma VWF multimer analyses were performed on all available plasma samples. Standard statistical analyses were employed. Result:Loss of VWF HMWM was observed in 3 patients prior to CF-LVAD implantation and virtually all patients after surgery. VWF:Ag, VWF:Lx or VWF:pp/Ag ratios of the pre- and post-implantation samples were not significantly different (P> 0.1). However, VWF:Lx/Ag ratios of the post-implantation samples were significantly decreased (P<0.05). At a cut off of 0.8, the VWF:Lx/Ag ratio has a 90% sensitivity and specificity for detecting AVWA. Conclusions:VWF:Lx/Ag ratio has excellent sensitivity and specificity in detecting CF-LVAD-AVWA, and its impact on predicting post-surgical bleeding tendency in CF-LVAD recipients is being investigated. The distinct CF-LVAD-AVWA is most likely caused by enhanced cleavage, rather than clearance, of the HMWM.Table 1:Laboratory Characteristics of VWF in Pre- and Post-CF-LVAD Implantation Patient Plasma Samples.Laboratory Results (Mean±Standard Deviation)Pre-implantation (n=15)Day 7 Post (n=13)Day 30 Post (n=13)5–7 month Post (n=4)VWF:Ag (IU/dL)318 ± 178340 ± 136263.8 ± 122.7224 ± 78VWF:Lx (U/dL)258 ± 145246 ± 87102.5 ± 64.6165 ± 44VWF:Lx/Ag Ratio0.82 ± 0.120.74 ± 0.080.75 ± 0.060.76 ± 0.12VWF:pp/Ag Ratio1.04 ± 0.361.00 ± 0.300.99 ± 0.231.02 ± 0.10Samples with HMWM Loss (Present/Total)3/1510/1312/134/4Agreement between abnormal VWF:Lx/Ag ratio (<0.8) and VWF Multimer Analysis13/1511/1312/134/4 Disclosures:No relevant conflicts of interest to declare.

Development of a collagen-binding activity assay as a screening test for type II von Willebrand disease in dogs

Development of a collagen-binding activity assay as a screening test for type II von Willebrand disease in dogs

Development of a collagen-binding activity assay as a screening test for type II von Willebrand disease in dogs

To develop an assay to measure canine von Willebrand factor (vWF):collagen-binding activity (CBA) to screen for type 2 von Willebrand disease (vWD) in dogs. 293 plasma samples submitted for analysis of canine vWF antigen (vWF:Ag) and 12 control plasma samples from dogs with inherited type 2 or 3 vWD. Bovine collagens were evaluated for suitability as binding substrate for vWF. Assay sensitivity to depletion, proteolytic degradation, or a genetic deficiency of high-molecular-weight vWF were determined. Amounts of vWF:Ag and vWF:CBA were measured. The ratio of vWF:Ag to vWF:CBA was used to discriminate between type 1 and type 2 vWD. An assay for canine vWF activity was developed by use of mixed collagen (types I and III). When vWF:Ag was used to subtype vWD, 48% of the dogs were classified as clinically normal, 9% as indeterminate, and 43% as type 1 vWD. Inclusion of vWF activity resulted in reclassification of 5% of those identified as type 1 to type 2 vWD. However, vWF:CBA of the reclassified dogs was not persistently abnormal, a finding compatible with acquired type 2 vWD. Some Doberman Pinschers had lower antigen-to-activity ratios than other breeds with type 1 vWD, suggesting that Doberman Pinschers have more functional circulating vWF. Analysis of canine vWF activity should be included among the vWF-specific assays used to confirm type 2 vWD. The prevalence of inherited forms of type 2 vWD in screened dogs is lower than acquired forms that can result secondary to underlying disease.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

Laboratory Diagnosis of von Willebrand Disorder: Use of Multiple Functional Assays Reduces Diagnostic Error Rates

Regular multilaboratory surveys of laboratories primarily in Australia, New Zealand, and Southeast Asia have been conducted over the past 8 years to evaluate testing proficiency in the diagnosis of von Willebrand disorder (VWD). We have reassessed the findings of these surveys with a particular emphasis on the diagnostic errors and error rates associated with particular tests or test panel limitations. The 37 plasma samples dispatched to survey participants include 9 normal samples, 4 type 1 VWD samples, 8 type 2 VWD samples (2A x 3, 2B x 3, 2M x 1, and 2N x 1), and 4 type 3 VWD samples. In addition to providing numerical test results, participant laboratories (average, n = 35) were asked to provide diagnostic interpretations of their test results regarding whether VWD was evident and, if so, the probable subtype. Although laboratories usually provided correct interpretative responses, diagnostic errors occurred in a substantial number of cases. On average, type 1 VWD plasma was misidentified as type 2 VWD plasma in 11% of cases, and laboratories that performed the ristocetin cofactor assay for von Willebrand factor (VWF:RCo) without performing the collagen-binding activity assay for VWF (VWF:CB) were 6 times more likely to make such an error than those that did perform the VWF:CB. Similarly, type 2 VWD plasma samples were misidentified as type 1 or type 3 VWD in an average of 20% of cases, and laboratories that performed the VWF:RCo without the VWF:CB were 3 times more likely to make such an error than those that performed the VWF:CB. Finally, normal plasma was misidentified as VWD plasma in an average of 5% of cases, and laboratories that performed the VWF:RCo without the VWF:CB were 10 times more likely to make such an error than those that performed the VWF:CB. We conclude that laboratories are generally proficient in their testing for VWD and that diagnostic error rates are substantially reduced when test panels are more comprehensive and include the VWF:CB.

The von Willebrand factor collagen-binding activity assay: clinical application.

A collagen type III based collagen-binding assay was developed for measuring the functional activity of the von Willebrand factor. The assay had a low coefficient of variance (4.8%) for normal values under optimized conditions. The results of the collagen-binding activity (CBA) assay correlated with ristocetin cofactor activity tested in normal plasma samples (n=29). We found that the CBA of blood group O is lower than that of other blood groups. The test was used for the diagnosis of von Willebrand's disease (VWD) and for estimating the response to treatment with DDAVP (1-deamino-D-arginine-8 vasopressin) and factor VIII concentrate. A mean ratio of VWF antigen (VWF:Ag) to CBA of 1.5 indicated type 1 and of 2.7 indicated type 2 VWD. The increase in the collagen-binding activity of VWF released in type 1 VWD patients (n=7) after treatment with DDAVP was higher than the increase in the VWF antigen; this is characteristic of very high multimers with greater functional activity. Factor VIII concentrate Koate-HP (Bayer) administered to a patient with VWD type 3 had a mean residence time of 12.6 h for VWF:Ag and 11.2 h for CBA. These findings suggest that the collagen-binding assay is a useful test for measuring the functional activity of VWF in plasma samples, factor VIII concentrates, as well as for estimating the outcome of treatment.

Laboratory assays for von Willebrand Factor: relative contribution to the diagnosis of von Willebrand’s disease

Appropriate investigation of a patient with suspected von Willebrand's disease (VWD) involves a clinical assessment of the patient followed by laboratory testing. A variety of laboratory assays may be performed, not necessarily restricted to an assessment of von Willebrand factor (VWF). Due to the limitations of each assay, and because of VWD heterogeneity, no single test procedure is sufficiently robust to permit detection of all VWD variants. Indeed, these factors often lead to considerable confusion in the process of laboratory interpretation regarding the likelihood of VWD, and the subtype of VWD. This paper attempts to clarify some of the issues that lead to this confusion. It analyses the relative contribution of four separate assays for VWF [VWF:Multimer analysis, VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and VWF:Collagen Binding Activity assay (VWF:CBA)] to the diagnosis and classification of VWD. Although each assay detects VWF, it is important to recognise that each assay provides different information on the VWF so detected. For example, while the VWF:Ag assay is a quantitative assay and provides a very good measure of the overall level of VWF present in a patient's plasma, it is not a functional assay and yields no information concerning the quality of the VWF present. Thus, the VWF:Ag on its own will not permit detection of many qualitative defects (consequently, use of this assay alone will lead to many Type 2 VWD patients being missed by the laboratory). In contrast, the VWF:CBA is a functional assay which provides very useful information on the quality of VWF present. Although the VWF:CBA is also a quantitative assay, it is less sensitive than the VWF:Ag assay in terms of its ability to measure the overall level of VWF (i.e.; the VWF:CBA detects only highly adhesive VWF, and therefore only a proportion of overall VWF). In essence, the VWF:Ag and VWF:CBA assays are complementary assays and should be used in combination. The VWF:Multimer assay is a qualitative procedure, but at best is only semi-quantitative. The VWF:Multimer assay essentially provides a snap-shot of the VWF present. Unfortunately considerable technical and interpretive problems limits its overall applicability and usefulness. The VWF:RCof assay is both a quantitative and qualitative assay that provides information about the presence of VWF that lies between that provided individually by the VWF:Ag and VWF:CBA assays. Unfortunately, the VWF:RCof suffers considerable technical problems, including considerable assay variability, that also limits its overall usefulness. Laboratories performing assays for VWF need to develop diagnostic strategies which include the use of appropriate multiple test combinations so as to ensure that VWD is properly detected.