The Carboxyl-Terminal Domains of ADAMTS13 Cause Substrate-Dependent Divergence of ADAMTS13 Activity.

ADAMTS13, a circulating metalloprotease that cleaves the Y1605-M1606 bond of VWF, is critical for preventing intravascular platelet thrombosis of thrombotic thrombocytopenic purpura. Unlike genetic deficiency of ADAMTS13 in patients, inactivation of ADAMTS13 created by homologous recombination causes either no or delayed-onset phenotypic abnormalities in mice, depending on the strains examined. In order to further understand the role of ADAMTS13 in VWF homeostasis, we investigated the structure and function of the enzyme in various mouse strains. As in human subjects, RT PCR analysis revealed that ADAMTS13 was expressed primarily in the mouse liver. However, the enzyme activity levels of mouse plasma ADAMTS13 segregated in two groups: a low group (C56BL/6J strain, 24%±10% of normal human plasma, N=21; and DBA/2J strain, 24%±7%, N=6), and a high group (FVB/NJ strain, 288%±60%, N=23; and 129×1/SvJ strain, 313%±39%, N=6). No such difference was detected when GST-His fusion or FRET form of VWF73 (D1596-R1668) peptide was used for activity measurement. Real-time analysis of RNA extracted from mouse livers showed that the ADAMTS13 transcript levels were similar between the two groups. Cloning of the mouse liver ADAMTS13 yielded a predominant 3.5kb instead of the expected 4.3kb species from the low-activity group. This truncated cDNA contained the first 23 exons of the full-length (FL) ADAMTS13 and an extraneous sequence derived from the long terminal repeat sequence of a previously described IAP-type retrotransposone located in intron 23 of affected mouse genome. Protein expression analysis of the cloned cDNA in HEK 293 and MDCK cells showed that both full-length and IAP-type ADAMTS13 proteins were secreted efficiently without evidence of polarity. Nevertheless, the IAP-type ADAMTS13 was only 10% as active as FL ADAMTS13 in cleaving VWF multimers, but was 50% – 70% as effective in cleaving human or mouse VWF73 peptides, suggesting that truncation and/or presence of IAP sequence at the C-terminus sequence of ADAMTS13 markedly impeded cleavage of VWF multimers. Crossbreeding between C57BL/6J and FVB/NJ strains of mice confirmed that the low-activity phenotype was linked to homozygous IAP genotype. In SDS agarose gel analysis, both C57BL/6J and FVB/NJ strains contained ultra large multimers similar to those observed in ADAMTS13-null mice (kindly provided by D. Motto and D. Ginsburg). Analysis of recombinant human ADAMTS13 proteins also showed that proteins truncated at the disintegrin or spacer domain were relatively more active in cleaving VWF peptide substrates than cleaving VWF multimers. In conclusion, the presence of an IAP-type retrotransposone in the ADAMTS13 gene enhances alternative splicing, resulting in predominant expression of IAP types of ADAMTS13 in the affected mouse strains. Nevertheless, ADAMTS13 does not regulate VWF multimer size, and deficiency of ADAMTS13 is phenotypically silent in the mice examined. The carboxyl-terminus sequence of ADAMTS13 may enhance interaction with VWF multimers and its subsequent cleavage. Clinically, ADAMTS13 assay results should be interpreted with caution when VWF peptide-based proteins are used as the substrate, because they yield higher activity values for ADAMTS13 proteins lacking the carboxyl terminus sequence.