Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation
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Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation. / Letzer, Anton; Lehmann, Katja; Mess, Christian; König, Gesa; Obser, Tobias; Peine, Sven; Schneppenheim, Sonja; Budde, Ulrich; Schneider, Stefan W; Schneppenheim, Reinhard; Brehm, Maria A.
in: PLOS ONE, Jahrgang 15, Nr. 5, 2020, S. e0232637.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation
AU - Letzer, Anton
AU - Lehmann, Katja
AU - Mess, Christian
AU - König, Gesa
AU - Obser, Tobias
AU - Peine, Sven
AU - Schneppenheim, Sonja
AU - Budde, Ulrich
AU - Schneider, Stefan W
AU - Schneppenheim, Reinhard
AU - Brehm, Maria A
PY - 2020
Y1 - 2020
N2 - 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 VWF-platelet 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.
AB - 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 VWF-platelet 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.
KW - ADAMTS13 Protein/genetics
KW - Blood Platelets/metabolism
KW - Catalytic Domain
KW - Codon, Nonsense
KW - Endothelial Cells/metabolism
KW - Genetic Variation
KW - HEK293 Cells
KW - Hemostasis
KW - Humans
KW - Mutation, Missense
KW - Platelet Aggregation
KW - Purpura, Thrombotic Thrombocytopenic/congenital
KW - Recombinant Proteins/genetics
KW - Shear Strength
KW - Time Factors
KW - von Willebrand Factor
U2 - 10.1371/journal.pone.0232637
DO - 10.1371/journal.pone.0232637
M3 - SCORING: Journal article
C2 - 32365113
VL - 15
SP - e0232637
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 5
ER -