Diagnosis of thrombotic thrombocytopenic purpura based on modulation by patient plasma of normal platelet adhesion under flow condition

Шенкман, Борис; Inbal, Aida; Tamarin, Ilia; Lubetsky, Aharon; Savion, Naphtali; Varon, David

doi:10.1046/j.1365-2141.2003.04119.x

Cited by 25 publications

(20 citation statements)

References 41 publications

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“…Plasma of patients with other thrombocytopenic entities including hemolytic uremic syndrome did not change the platelet deposition [27]. In another yet not published study we found a reverse correlation between the increased normal platelet adhesion under shear condition (2050 s -1 ) caused by acute TTP plasma and ADAMTS-13 activity measured by collagen-binding assay.…”

Section: Testing Ulvwf Multimers and Adamts-13 Activitysupporting

confidence: 52%

The Role of ADAMT-13 in Platelet Adhesion in Flow: methods for Diagnosis of Thrombotic Thrombocytopenic Purpura

Шенкман

2006

Pathophysiol Haemos Thromb

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Section: Testing Ulvwf Multimers and Adamts-13 Activitysupporting

confidence: 52%

The Role of ADAMT-13 in Platelet Adhesion in Flow: methods for Diagnosis of Thrombotic Thrombocytopenic Purpura

Шенкман

2006

Pathophysiol Haemos Thromb

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“…In the FRETS‐VWF73 assay, cleavage is enabled by the use of a short peptide substrate, whereas in the VWF multimer and CB assays, a denaturing agent is used to expose the cleavage site in full‐length VWF. To circumvent the potential pitfalls of such assays, various shear‐based systems for determining ADAMTS‐13 activity have been developed . In one such study, the presence of inhibitors was qualitatively assessed with a shear assay, and the results obtained were largely consistent with those of the FRETS‐VWF73 assay .…”

Section: Introductionmentioning

confidence: 99%

A novel flow‐based assay reveals discrepancies in ADAMTS‐13 inhibitor assessment as compared with a conventional clinical static assay

Grillberger¹,

Gruber²,

Skalicky³

et al. 2014

Journal of Thrombosis and Haemostasis

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To cite this article: Grillberger R, Gruber B, Skalicky S, Schrenk G, Kn€ obl P, Plaimauer B, Turecek PL, Scheiflinger F, Rottensteiner H. A novel flow-based assay reveals discrepancies in ADAMTS-13 inhibitor assessment as compared with a conventional clinical static assay. J Thromb Haemost 2014; 12: 1523-32.Summary. Background: Several static Bethesda-type assays are routinely used to determine ADAMTS-13-neutralizing autoantibodies in acquired thrombotic thrombocytopenic purpura (TTP), but the inhibitory activity of these antibodies has not been thoroughly evaluated under the more physiologic condition of flow. Objectives: We investigated whether ADAMTS-13 inhibitor assessment with the FRETS-VWF73 assay is predictive for evaluation under flow. Methods: Anti-ADAMTS-13 autoantibodies were purified from patients with acquired TTP by chromatography involving an ADAMTS-13 affinity matrix and/or protein G. ADAMTS-13 activity was measured with the FRETS-VWF73 assay and a novel flow assay determining the ADAMTS-13-mediated decrease in platelet aggregate surface coverage, caused by perfusion of a suspension containing platelets, erythrocytes and von Willebrand factor (VWF) over a surface coated with extracellular matrix components. The neutralizing activities of ADAMTS-13 inhibitors were compared under static conditions and under flow by use of the two assays. Results: The suitability of the flow-based ADAMTS-13 activity assay for quantification of ADAMTS-13 inhibitors could be demonstrated by reversibility of the ADAMTS-13-dependent decrease in surface coverage upon addition of goat AD-AMTS-13 antiserum. Testing the neutralizing activity of purified autoantibodies from six patients in the flow assay according to their FRETS-VWF73-based inhibitor titers gave rise to vastly different inhibitory effects, indicating a discrepancy in inhibitor assessment between static and flow conditions. Conclusions: Anti-ADAMTS-13 autoantibodies may show inhibitory properties in vivo that are not consistent with the ADAMTS-13 inhibitor levels determined in routine static assays, possibly because certain epitopes are selectively exposed under shear. Consequently, the course of disease and treatment efficacy may vary among TTP patients, despite common inhibitor titers.

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“…First, Shenkman et al [18] evaluated the ability of TTP plasma to increase the platelet deposition on a polystyrene surface from normal plasma under flow conditions using a cone and plate(let) analyzer (CPA) method. Second, we recently described that ULVWF multimers, upon release, form extremely long and platelet-decorated string-like structures on the surface of endothelial cells under fluid shear stress [19].…”

Section: Introductionmentioning

confidence: 99%

ADAMTS‐13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF‐A2 domain as substrate

Whitelock

Nolasco

Bernardo

et al. 2004

Journal of Thrombosis and Haemostasis

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To cite this article: Whitelock JL, Nolasco L, Bernardo A, Moake J, Dong J-F, Cruz MA. ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrate. J Thromb Haemost 2004; 2: 485-91.Summary. The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS-13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are nonphysiological and time consuming. We have expressed in bacteria a recombinant VWF-A2 peptide (aa 718-905) that contains both a 6xHis tag at the N-terminal end and a Tag-100 epitope at the C-terminal end. Diluted plasma was mixed with the VWF-A2 peptide and digestion was allowed to proceed in a Ni 2+ -coated microtiter well plate for 2 h. The immobilized Ni 2+ captures the VWF-A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C-terminus fragment of the A2 peptide that contains the Tag-100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag-100 epitope by the antiTag-100 monoclonal antibody. The assay was completed in <5 h. We then used the assay to analyze ADAMTS-13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura. The average of enzyme activity ± SEM for normal plasmas diluted 1 : 50 was 40 ± 4.2% while the value obtained for the patients was 2.4 ± 0.7%. These results were validated by a traditional long incubation assay (24 h). Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS-13 activity under physiological and non-denaturing conditions. This assay is clinically useful and significant in measuring ADAMTS-13 activity in plasma.

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Diagnosis of thrombotic thrombocytopenic purpura based on modulation by patient plasma of normal platelet adhesion under flow condition

Cited by 25 publications

References 41 publications

The Role of ADAMT-13 in Platelet Adhesion in Flow: methods for Diagnosis of Thrombotic Thrombocytopenic Purpura

The Role of ADAMT-13 in Platelet Adhesion in Flow: methods for Diagnosis of Thrombotic Thrombocytopenic Purpura

A novel flow‐based assay reveals discrepancies in ADAMTS‐13 inhibitor assessment as compared with a conventional clinical static assay

ADAMTS‐13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF‐A2 domain as substrate

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