ADAMTS13 P475S polymorphism causes a lowered enzymatic activity and urea labilityin vitro

Abstract

To cite this article: Akiyama M, Kokame K, Miyata T. ADAMTS13 P475S polymorphism causes a lowered enzymatic activity and urea lability in vitro. J Thromb Haemost 2008; 6: 1830-2.von Willebrand factor (VWF) is a plasma glycoprotein synthesized primarily in vascular endothelial cells and megakaryocytes [1]. VWF is released into plasma as ultra-large multimeric forms that are highly active in platelet aggregation. A plasma metalloprotease ADAMTS13 specifically cleaves the Tyr 1605 -Met 1606 peptidyl bond within the A2 domain of VWF [2]. Deficiency of the ADAMTS13 enzymatic activity, caused by genetic mutations or acquired autoantibodies against ADAMTS13, results in the accumulation of ultra-large VWF multimers in plasma that lead to the hyper-aggregation of platelets. This prothrombotic condition can cause thrombotic thrombocytopenic purpura (TTP) [3]. A number of nonsynonymous mutations and polymorphisms of ADAMTS13 have been identified [4,5]. Among them, the P475S (c.1423C > T) polymorphism is noteworthy.The allele frequency of ADAMTS13 P475S was 5.1% in Japanese subjects [6], 4.0% in Koreans [7], and 1.5% in Chinese [8], but absent in Caucasians [9]. The recombinant P475S mutant was normally secreted from cultured cells but showed greatly reduced enzymatic activity (10%) in the VWFmultimer assay [6]. Recently, we have developed a quantitative ADAMTS13 activity assay using a synthetic fluorogenic substrate FRETS-VWF73 [10] and it is now widely utilized [11][12][13][14]. The assay can be performed in the absence of urea that is required for the VWF-multimer assay. In this study, we evaluated the activity of the P475S mutant using FRETS-VWF73 and found that the mutant exhibited the more profound loss of activity than the wild-type in the presence of urea.We have prepared four forms of recombinant ADAMTS13 using a transient expression system of HeLa cells: the fulllength form (Met ) with the wild-type sequence (MDTCS) or with the P475S polymorphism (MDTCS-P475S). FL and FL-P475S were tagged by the C-terminal FLAG sequence. MDTCS and MDTCS-P475S were tagged by the C-terminal 6xHis sequence. The culture media of cells expressing FL and FL-P475S were collected and concentrated 5-fold by centrifugal filtration (Ultrafree-MC; Millippore, Billerica, MA, USA). The media of cells expressing MDTCS and MDTCS-P475S were collected, and the recombinant proteins were purified by the Ni-NTA column chromatography. The relative amounts of the recombinant proteins were adjusted by their band intensities of Western blotting analysis using anti-FLAG or anti-6xHis antibodies, within the linear detection range. Enzymatic activity of each protein was measured by the FRETS-VWF73 assay [10] in the absence or presence of urea.First, we compared the enzymatic activities between FL and FL-P475S in the absence of urea, and found that FL-P475S showed 71 ± 3.6% (n = 3) activity of FL. We previously reported that FL-P475S showed only approximately 10% activity of FL in the VWF-multimer assay [6]. This paradoxical observation led us to further stud...

Survival and relapse in patients with thrombotic thrombocytopenic purpura

“…28,39 Some genetic variants of ADAMTS13 may be sensitive to urea, resulting in lower ADAMTS13 activity measured by the immunoblotting assay. 40,41 Circulating immune complexes of ADAMTS13 with low affinity autoantibodies may dissociate during in vitro assays, with different ADAMTS13 activities Clinical course of patients who had ADAMTS13 Ͻ 10% at the time of their initial diagnosis with TTP and who had ADAMTS13 activity measured during remission. Data are presented for the 37 patients who had ADAMTS13 activity Ͻ 10% at the time of their initial diagnosis of TTP and who also had ADAMTS13 activity measured during clinical remission, documented by clinical evaluation, platelet count, and hematocrit.…”

Crystal structures of the noncatalytic domains of ADAMTS13 reveal multiple discontinuous exosites for von Willebrand factor

“…Five polymorphisms have been identified within the DTCS region (Table S2). Approximately 10% of the Japanese population are heterozygous for P475S substitution, located in the V-loop (C A ), which reduces VWF-cleaving activity (40,42). The P618A substitution reduces secretion efficiency in cultured cells (43).…”

Association of ADAMTS13 polymorphism with cerebral malaria

“…The SNP rs11575933 (P475S) has been reported to influence the proteolytic activity of ADAMTS13 [11-13]. In 128 Thai malaria patients, rs11575933-T (475S) allele was not observed.…”