Platelet ADAMTS13 and in vitro expression study of a patient affected by congenital TTP

Introduction : proteolysis of VWF is significantly reduced in congenital TTP patients due to ADAMTS13 deficiency. As 15-25% of circulating VWF is stored in platelets, the presence and function of platelet ADAMTS13 could play an important role in TTP pathogenesis. We studied a congenital TTP patient homozygous for a 6bp deletion in the exon 23 of ADAMTS13 gene. The effect of this mutation was verified either in plasma or in platelets and characterized by in vitro expression studies Methods : Furlan assay determined ADAMTS13 activity. The rADAMTS13 WT and mutant proteins were analyzed by Western Blot (WB), Furlan assay, immunofluorescence and pulse-chase labeling experiments. To analyze platelet ADAMTS13, the patient’s washed platelets (1.2x109/ml) were lysed with 1% Triton X-100 and evaluated by WB and immunofluorescence studies using two monoclonal antibodies against CUB-1 and TSP1-1 repeat domains of ADAMTS13 Results : the patient’s plasma showed no ADAMTS13 activity (<6%). WB and pulse-chase labeling experiments demonstrated a secretion pathway defect of the mutant protein, observed only in the conditioned media after 3 hours of chase. These data were also confirmed by immunofluorescence studies showing the presence of mutant protein diffused throughout the cytoplasm with only a minimal amount conserved at ER and Cis-Golgi. In WB both monoclonal antibodies detected a band of ~200 kDa either in the patient’s platelet lysate or normal subject, confirming the presence of platelet ADAMTS13 in a similar amount between patient and normal subject, independently of the conserved plasmatic ADAMTS13. Immunofluorescence also confirmed these data by showing ADAMTS13 localization at the platelet membrane region with no apparent colocalization with αIIbβIII. Conclusions : these data show that 6bp deletion leads to a secretion defect without changing the expression of platelet ADAMTS13, confirming that ADAMTS13 in platelets is not from plasma by endocytosis. The role of platelet ADAMTS13 needs to be further analysed