Analysis on the Molecular Species and Concentration of Circulating ADAMTS13 in Blood

Abstract

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.

Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13

“…21 HUVECs used either the Targefect-HUVEC transfection reagent (Targeting Systems, Santee, CA) or lentivirus. 21 ADAMTS13 was produced using a vector kindly provided by Kenji Soejima (Kaketsuken, Japan 22 ). ADAMTS13 unit activity was calculated based on the World Health Organization 1st International Standard.…”

von Willebrand factor–cleaving protease ADAMTS13 reduces ischemic brain injury in experimental stroke

“…ADAMTS13 is a metalloprotease of approximately 190 kDa 10 present in plasma at a concentration of 0.5 to1 g/mL in humans. 11 Ischemia is a potent inducer of Weibel-Palade body secretion, 12 thus making the infarct area highly thrombogenic even after thrombolysis.…”

“…ADAMTS13 is a metalloprotease of approximately 190 kDa 10 present in plasma at a concentration of 0.5 to1 g/mL in humans. 11 Ischemia is a potent inducer of Weibel-Palade body secretion, 12 thus making the infarct area highly thrombogenic even after thrombolysis.VWF deficiency is associated with the most common bleeding disorder in humans, von Willebrand disease. 13 In contrast, deficiency of ADAMTS13 is seen in patients with thrombotic thrombocytopenic purpura, which is often characterized by neurologic symptoms because of cerebral ischemia caused by microthrombi in the cerebral microvasculature.…”

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

“…[15][16][17][18][19] Anti-ADAMTS13 antibodies present in the plasma of patients with acquired TTP target an antigenic surface including residues Arg660, Tyr661 and Tyr665. 14 However in three out of six patients' analyzed it was seen that there was residual binding to an MDTCS variant in which Arg660, Tyr661 and Tyr665 were replaced by an alanine.…”