DOI: 10.1161/circ.148.suppl_1.17158 ISSN: 0009-7322

Abstract 17158: Effects of Human Monoclonal Antibodies on ADAMTS13 in Immune Thrombotic Thrombocytopenic Purpura

Konstantine Halkidis, Chan Meng, Donald L Siegel, X. Long Zheng
  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Introduction: Immune thrombotic thrombocytopenic purpura (iTTP) is caused by antibody-mediated deficiency of the metalloprotease ADAMTS13, which cleaves VWF. Myocardial infarction and ischemic strokes are common and often fatal sequelae of iTTP. Inhibitory anti-ADAMTS13 antibodies appear to affect catalytic turnover more than apparent substrate binding affinity (PMID 37023370). Exploring how antibodies affect VWF cleavage by ADAMTS13 in physiologic conditions is needed to better understand the pathophysiology of iTTP.

Hypothesis: Anti-ADAMTS13 antibodies may influence ADAMTS13 function by affecting catalytic turnover and/or apparent substrate binding affinity differently in physiologic conditions than in standard assay conditions.

Aims: Characterize how single chain fragments of the variable loop (scFv’s) previously identified from a phage display library derived from several iTTP patients’ antibodies affect ADAMTS13-mediated VWF cleavage in conditions that more closely represent plasma and see if/how they differ from scFv-mediated effects in standard conditions.

Methods: ADAMTS13 in normal human plasma was titrated with increasing concentrations of the substrate FRETS-VWF73 to derive maximum velocity ( V max ) and apparent Michaelis constant ( K M,app ) in standard assay conditions (pH 6.0) and conditions closer to physiologic (pH 7.0). The same parameters were derived in the presence of saturating concentrations of scFv’s.

Results: Compared to VWF cleavage by ADAMTS13 in the absence of scFv, the inhibitory scFv4-20 decreased V max at both pH 6.0 and pH 7.0; K M,app decreased at pH 6.0 and increased at pH 7.0. The stimulatory scFv3-3 and scFv4-41 increased V max at each pH, while K M,app in the presence of each scFv decreased only at pH 7.0.

Conclusions: Anti-ADAMTS13 antibody-mediated effects on VWF cleavage differ when comparing standard and more physiologic pH conditions, guiding future investigative directions into iTTP pathophysiology.

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