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

TY - JOUR

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 -