A Novel Mechanism of ADAMTS13 Deficiency in Mice.

Background. ADAMTS13, a circulating metalloprotease that cleaves conformationally altered von Willebrand factor (VWF), is critical for preventing microvascular thrombosis. Deficiency of the protease, due to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura. In the course of investigating the regulation of ADAMTS13, we noted that mice differed widely in their plasma ADAMTS13 activity levels. In order to understand the factors affecting plasma ADAMTS13 levels, we examined the molecular basis of ADAMTS13 variation in different strains of mice. Methods. Plasma ADAMTS13 activity level was determined by previously described SDS PAGE and immunoblotting. ADAMTS13 transcripts were analyzed by RT PCR, RACE, nucleotide sequencing, and real-time RT PCR. Plasmids containing the cDNA of mouse ADAMTS13 were constructed for transfection of mammalian cell lines. Results. The mouse strains FVB/NJ and 129X1/SvJ differed from C57BL/6J and DBA/2J by more than 10 folds in their plasma ADAMTS13 activity levels (3.09 +/− 0.45 vs 0.24 +/− 0.11 U/mL for FVB/NJ and C57BL/6J respectively, P < 0.0001). To determine the causes of the difference, we analyzed the ADAMTS13 transcripts by using RT PCR and RACE, which showed that the FVB/NJ mice contained the predicted full-length ADAMTS13 transcript with a domain structure similar to human ADAMTS13, while the C57BL/6J mice contained at least 4 isoforms: the full-length transcript, one internal splicing isoform, and two truncated forms that ended with an extraneous sequence homologous to the long-terminal repeat (LTR) of an retrotransposone of the IAP type. Comparison of the genomic sequences showed that the ADAMTS13 gene of C57BL/6J mice contained in its intron #23 an IAP-type retrotransposone sequence whose LTR sequence with a stop codon was included in the mouse transcripts. The IAP retrotransposone sequence, which contained one base substitution at the 5′-end 4-base repeat (tgtt>g) and was flanked at both ends by a 6-base repeat (cactag), was also present in the DBA/2J but not the 129X1/SvJ strains of mice. Real-time RT PCR showed that the FVB/NJ and C57BL/6J mice had similar levels of ADAMTS13 transcripts in the liver. Nevertheless in the C57BL/6J mice the IAP-truncated forms accounted for >90% of the ADAMTS13 transcripts. Expression of mouse ADAMTS13 cDNA in mammalian cell lines revealed that the both the full-length and the IAP-truncated forms of the ADAMTS13 protease were similar in VWF-cleaving activity. Conclusion. This study shows the presence of intragenic retrotransposone in the ADAMTS13 gene of some mouse strains. The presence of an IAP-retrotransposone within the ADAMTS13 gene of C57BL/6J mice affects the splicing of the ADAMTS13 transcripts, creating truncated forms of the protease that lack the last two TSP-1 and the CUB domains but remain proteolytically active in cleaving VWF. The lower plasma ADAMTS13 activity level of C57BL/6J may result from abnormal intravascular clearance of the protease or other post-secretory events.