Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF

Gerritsen, Helena E.; Turecek, Peter L.; Schwarz, Hans; Lämmle, Bernhard; Furlan, Miha

doi:10.1055/s-0037-1614780

Cited by 291 publications

(280 citation statements)

References 20 publications

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“…The VWF cleaving protease ADAMTS13 activity was measured using a rapid functional assay that determines the digestion of VWF by ADAMTS13, based on the method described by Gerritsen et al 14 The activity of ADAMTS13 in pooled normal plasma was arbitrarily defined as 1 U/mL. The values of ADAMTS13 in tested plasma samples are read from a calibration curve achieved by incubating the VWF substrate with dilutions of a normal plasma pool.…”

Section: Methodsmentioning

confidence: 99%

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity

et al. 2006

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Cirrhosis of the liver is frequently accompanied by complex alterations in the hemostatic system, resulting in a bleeding tendency. Although many hemostatic changes in liver disease promote bleeding, compensatory mechanisms also are found, including high levels of the platelet adhesive protein von Willebrand Factor (VWF). However, conflicting reports on the functional properties of VWF in cirrhosis have appeared in literature. We have measured a panel of VWF parameters in plasma from patients with cirrhosis of varying severity and causes. Furthermore, we assessed the contribution of VWF to platelet adhesion, by measuring the ability of plasma from patients with cirrhosis to support adhesion of normal or patient platelets under flow conditions. VWF antigen levels were strongly increased in patients with cirrhosis. In contrast, the relative collagen binding activity, as well as the relative ristocetin cofactor activity, was significantly lower in patients as compared with controls, indicating loss of function. Accordingly, patients had a reduced fraction of high-molecular-weight VWF multimers. Both strongly elevated and reduced activity and antigen levels of the VWF cleaving protease ADAMTS13 were found in individual patients. Adhesion of either normal or patient platelets to a collagen surface was substantially increased when these platelets were resuspended in plasma of patients with cirrhosis, as compared with control plasma. In conclusion, highly elevated levels of VWF in patients with cirrhosis contribute to the induction of primary hemostasis despite reduced functional properties of the molecule. This phenomenon might compensate for defects in platelet number and function in patients with cirrhosis. (HEPATOLOGY 2006;44:53-61.)

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Section: Methodsmentioning

confidence: 99%

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity

et al. 2006

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“…10,11 ADAMTS13 activity was expressed as a percentage relative to the activity in pooled normal plasma (normal range 55%-126%; lower limit of detection 5%).…”

Section: Assaysmentioning

confidence: 99%

A phase 2 study of the safety and efficacy of rituximab with plasma exchange in acute acquired thrombotic thrombocytopenic purpura

Scully

McDonald

Cavenagh

et al. 2011

Blood

378

388

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The safety and efficacy of weekly rituximab 375 mg/m 2 (؋4), given within 3 days of acute TTP admission, with standard therapy (PEX and steroids) was evaluated. Clinical outcomes were compared to historical controls (n ‫؍‬ 40) who had not received rituximab. Within the trial group, 15 of 40 required ICU admission and 15% of all cases with the highest troponin T levels on admission were ventilated. Before the second rituximab infusion, 68% of cases had a platelet count > 50 ؋ 10 9 /L and 38% > 150 ؋ 10 9 /L. Fewer PEX were required in whites compared to nonwhite in the rituximab group (mean 14 vs 21, P ‫؍‬ .0095). Inpatient stay was reduced by 7 days in the non-ICU trial cases compared to historical controls (P ‫؍‬ .04), especially in whites, with a mean reduction of 7 days (P ‫؍‬ .05). Ten percent of trial cases relapsed, median, 27 months (17-31 months), compared to 57% in historical controls, median 18 months (3-60 months; P ‫؍‬ .0011).There were no excess infections or serious adverse events with rituximab. In conclusion, rituximab appears a safe and effective therapy. Inpatient stay and relapse are significantly reduced in the rituximab cohort. Rituximab should be considered in conjunction with standard therapy on acute presentation of TTP. This study was registered at www.clinicaltrials.gov as NCT009-3713. (Blood. 2011;118(7): 1746-1753)

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“…We chose to use Western blotting so that persistence of high molecular weight multimers and appearance of digested multimers could be visualized in a manner similar to the original assay of Gerritsen et al [10]. Our data indicate that high-resolution gel electrophoresis was unnecessary to demonstrate VWF cleavage.…”

Section: Discussionmentioning

confidence: 90%

“…*Correspondence to: Mary Ann Knovich, Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, NC 27157. E-mail: mknovich@wfubmc.edu Despite the publication of several assays [10][11][12][13] designed to measure ADAMTS13 activity in plasma, the test is not yet widely available to the practicing clinician. It is offered by very few reference laboratories, with a turnaround time that precludes timely incorporation of results into diagnosis and management decisions.…”

Section: Introductionmentioning

confidence: 99%

Simplified assay for VWF cleaving protease (ADAMTS13) activity and inhibitor in plasma

et al. 2004

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The absence of VWF cleaving protease activity (VWFcp, ADAMTS13) has recently been identified as a central component in the pathogenesis of TTP. Several assays for the measurement of ADAMTS13 activity have been described, but most are cumbersome and employ techniques not easily adapted to routine laboratories. Thus, ADAMTS13 assays are available at only a few reference or research laboratories. Prompt identification of absent or impaired ADAMTS13 activity could prove invaluable to clinical decision-making regarding diagnosis and treatment of suspected TTP patients. We describe an assay for ADAMTS13 that uses a commercial source of VWF substrate and obviates dialysis for sample and substrate preparation. Patient and normal plasma are prepared by desalting over Micro-Spin columns, and barium is added to activate the cleaving protease. Humate-Pâ is prepared over a desalting column and reconstituted in urea prior to use as the exogenous VWF substrate. Desalted plasma is mixed with prepared substrate, and digestion is carried out at 37 C. Maximum sensitivity to very low levels of enzyme activity is achieved by using undiluted plasma and allowing digestion to proceed for 14 hr. The extent of VWF multimer cleavage is then demonstrated by Western blot. We used this assay to analyze VWF cleaving protease activity in plasma samples from 30 patients treated at our institution for TTP. Plasma from patients lacking ADAMTS13 was incubated with PNP and then analyzed by the same methods to determine the presence of an inhibitor. Our results indicate that the assay is suitable for providing timely information regarding ADAMTS13 activity for the diagnosis and treatment of patients with suspected TTP. Am.

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Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF

Cited by 291 publications

References 20 publications

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity

A phase 2 study of the safety and efficacy of rituximab with plasma exchange in acute acquired thrombotic thrombocytopenic purpura

Simplified assay for VWF cleaving protease (ADAMTS13) activity and inhibitor in plasma

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