Localization and Function of Platelet ADAMTS-13.

The haemostatic activity of von Willebrand factor (VWF) is strongly dependent on its multimeric size, with the highest activity in ‘unusually large’ multimers (ULVWF) secreted from endothelial cells. The multimeric size is regulated by a plasma metalloprotease, ADAMTS-13. Since 15–25% of circulating VWF is stored in platelets, the presence and function of ADAMTS-13 in platelets could be an important issue to be investigated. In our study, we showed the presence of ADAMTS-13 in human platelets consistently with observations reported by Suzuki M. et al (Biochem Biophys Res Commun, 2004). Total mRNA isolated from platelet lysates was reverse-transcribed and amplified with specific oligonucleotide primers spanning from exon 2 to 5 of ADAMTS-13 cDNA. The nucleotide sequence of amplified fragment corresponded to ADAMTS-13 cDNA reported in the NCBI database. The immunoblot analysis performed on platelet lysates, using a monoclonal antibody against ADAMTS-13 CUB domains (13E2/75), revealed a band with an apparent molecular weight of ~200 kDa, which was similar to the band corresponding to recombinant ADAMTS-13 protein. To investigate ADAMTS-13 activity, washed platelets in free calcium Tyrode buffer in absence of EDTA were lysed and used as a source of ADAMTS-13. We demonstrated by measuring the extent of VWF multimer degradation, using a quantitative immunoblotting assay, that platelet ADAMTS-13 was enzymatically active, being able to cleave recombinant VWF by roughly 10% compared to plasmatic ADAMTS-13. The activity was concentration dependent and inhibited by EDTA. No VWF cleaving activity was observed in the supernatant in which platelets were resupended before lysis, meaning that plasmatic ADAMTS-13 was completely removed. Immunofluorescence microscopy analysis was performed to investigate the localization of ADAMTS-13 in platelets. Washed platelets were let adhere to glass coverslip coated with collagen (10 μg/ml) and then fixed with paraformaldehyde. Permeabilized platelets were then incubated with a primary murine antibody anti-ADAMTS-13 (13E2/75) and labelled with a secondary antibody rhodamine-conjugated. After washing, the different slides were restained with FITC conjugated anti αIIbβ3 (10E5), anti-p-selectin (CD62) or antiGpIb (CD42) antibodies. Immunofluorescence analysis revealed that ADAMTS-13 was mainly localized at the platelet membrane surface with no colocalization with αIIbβ3, GpIb or p-selectin. Hence the results of this study confirmed that ADAMTS-13 mRNA was present in the human platelets. Immunoblotting and immunofluorescence microscopy showed the presence of ADAMTS-13 protein in platelets detectable specifically at the level of membrane surface. We further demonstrated that platelet ADAMTS-13 is able to cleave recombinant VWF under static conditions. The presence of ADAMTS-13 in platelets may imply the functional role of this enzyme in the local regulation of platelet function at the site of vascular injury or thrombus formation.