Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A.

doi:10.1016/j.thromres.2014.08.013

Cited by 8 publications

(8 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have used two methods that allow determining the ability of ADAMTS13 variants to proteolize VWF strings [31] and VWF-platelet complexes [32,33], importantly, under shear flow conditions. In the used assays, cleaved VWF remains bound to GPIbα-beads or platelets, rendering multimer analysis after cleavage impossible.…”

Section: Discussionmentioning

confidence: 99%

“…However, the specificity of our assays for Fig 2B) Mean ± SEM of 3-4 experiments. � For variant p.Gly702Arg, this value was determined in a previously published study [31]. Activity in simulated circulation was quantified by analyzing turbidity before and after addition of ADAMTS13 in LTA measurements (Fig 3).…”

Section: Discussionmentioning

confidence: 99%

“…Then 1.45 � 10 6 single donor Human Umbilical Vein Endothelial Cells (HUVEC, Promocell) were seeded in the μ-slides in Endothelial Cell Growth Medium (Promocell) supplemented with 1 mM MgSO 4 and fetal bovine serum (Thermo Fisher Scientific, final concentration 5% (v/v)) and cultured overnight at 37˚C and 5% CO 2 in a humidified chamber (OLAF, ibidi). Then, the shear-flow assays were performed as previously described by us [31]. In brief, the cells were washed with 1 ml pre-warmed (37˚C) HBRS buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, pH 7.4) and the channel slide was connected to a perfusion set air bubble-free and mounted in an incubation unit (Tokai-Hit, 5% CO 2 , 37˚C) within a fluorescence microscope (BZ-9000, Keyence).…”

Section: Surface Shear Flow Assaymentioning

confidence: 99%

“…The overall string length at 0 min was defined as 0% cleavage and % cleavage for each frame was determined by the decrease of the overall string length in %. Details on assay performance and validation as well as detailed description of data analysis was previously described [31].…”

Section: Surface Shear Flow Assaymentioning

confidence: 99%

“…To obtain comparable data, independent of the secretion efficiency, we always measured equal concentrations of all recombinant proteins. We have previously described that 10% of the normal ADAMTS13 plasma concentration (100 ng/ml) is the most feasible to use in our shear flow assay [31]. We thus also tested the activity of this protein concentration under static conditions employing the TECHNOZYM 1 ADAMTS-13 Activity ELISA Kit (…”

Section: Proteolytic Activity Of Adamts13 Variants Under Static Condimentioning

confidence: 99%

See 4 more Smart Citations

Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation

et al. 2020

Self Cite

View full text Add to dashboard Cite

ADAMTS13 regulates the hemostatic activity of von Willebrand factor (VWF). Determined by static assays, proteolytic activity <10IU/dL in patient plasma, in absence of ADAMTS13 autoantibodies, indicates Upshaw-Schulman syndrome (USS); the congenital form of Thrombotic Thrombocytopenic Purpura (TTP). We have recently functionally characterized sixteen USS-associated ADAMTS13 missense variants under static conditions. Here, we used two assays under shear flow conditions to analyze the activity of those seven mutants with sufficiently high residual secretion plus two newly identified variants. One assay determines cleavage of VWF strings bound to the surface of endothelial cells. The other, light transmission aggregometry-based assay, mimics degradation of VWF-platelet complexes, which are likely to be present in the circulation during TTP bouts. We found that 100 ng/ml of all variants were able to cleave about 80-90% of VWF strings even though 5 out of 9 exhibited activity �1% in the state-of-the-art static assay at the same concentration. These data indicate underestimation of ADAMTS13 activity by the used static assay. In simulated circulation, two variants, with missense mutations in the vicinity of the catalytic domain, exhibited only minor residual activity while all other variants were able to effectively break down VWFplatelet complexes. In both assays, significant proteolytic activity could be observed down to 100 ng/ml ADAMTS13. It is thus intriguing to postulate that most variants would have ample activity if secretion of 10% of normal plasma levels could be achieved.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Surface Shear Flow Assaymentioning

confidence: 99%

Section: Surface Shear Flow Assaymentioning

confidence: 99%

Section: Proteolytic Activity Of Adamts13 Variants Under Static Condimentioning

confidence: 99%

See 3 more Smart Citations

Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

Assessing thrombogenesis and treatment response in congenital thrombotic thrombocytopenic purpura

Alwan

Vendramin

Budde

et al. 2021

eJHaem

View full text Add to dashboard Cite

Despite clinical remission and normal platelet counts, congenital TTP (cTTP) is associated with non-overt symptoms. Prophylactic ADAMTS13 replacement therapy such as plasma infusion (PI) prevents acute episodes and improves symptomatology. There is no current method to investigate disease severity or monitor the impact of treatment.We utilize a dynamic high shear flow assay to further understand disease pathophysiology and determine the impact of cTTP on symptomatology and therapy, despite normal platelet counts. Whole blood, under high shear, was run over collagen-coated channels, causing platelet adhesion to von Willebrand factor (VWF) multimers. The resulting surface coverage by platelet-VWF thrombus was assessed. The normal range was 6-39% in 50 controls. Twenty-two cTTP patients with normal platelet counts were evaluated.Median pre-treatment surface coverage was 89%, and PI reduced coverage to a median of 44% (p = 0.0005). Patients taking antiplatelets had further reduced coverage when combined with PI and improved non-overt symptoms such as headache, lethargy, and abdominal pain in 100% of patients compared to 74% with PI alone (p = 0.046). We use a dynamic assay to report increased in vitro platelet adhesion and aggregation and additionally demonstrate significantly decreased thrombi following PI, with levels in the normal range levels achieved in patients taking additional antiplatelet therapy. K E Y W O R D Scongenital TTP, prophylaxis, shear flow, upshaw schulman syndrome INTRODUCTIONCongenital TTP (cTTP) is an ultra-rare disorder defined by an inherited deficiency of the von Willebrand factor (VWF) cleaving enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) leading to the presence of highly adhesive ultra large VWF multimers. Acute events present similarly to immune-mediated TTP with thrombocytopenia and microangiopathic hemolytic anemia, and there is broad consensus that such events should be treated withThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

show abstract

von Willebrand Disease: Differential Diagnosis and Diagnostic Approach to Specific Subtypes

Ragni

2016

Nonmalignant Hematology

View full text Add to dashboard Cite

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

Cited by 8 publications

References 17 publications

Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation

Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation

Assessing thrombogenesis and treatment response in congenital thrombotic thrombocytopenic purpura

von Willebrand Disease: Differential Diagnosis and Diagnostic Approach to Specific Subtypes

Contact Info

Product

Resources

About