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Título:
VWF-CLEAVING SERINE PROTEASES ISOLATION DIFFERENT FROM ADAMTS13
Autor/es:
KEMPFER A C; FARIAS CE; POWAZNIAK Y; WOODS AI; CALDERAZZO JC; KELLER L; LAZZARI MA
Lugar:
Ginebra
Reunión:
Congreso; XXIst International Society on Thrombosis and Haemostasis Congress; 2007
Institución organizadora:
International Society on Thrombosis and Haemostasis
Resumen:
Introduction: In the assay of VWF-cleaving activity, 4-2-aminoethyl-benzenesulfonyl fluoride hydrochloride (AEBSF) is added to citrate plasma as inhibitor of serine proteases (SP). AEBSF does not prevent VWF cleavage by ADAMTS13. The VWF-cleaving activity had a significant decrease when AEBSF was added to plasma in our detection assay. So, we decided to separate ADAMTS13 from this SP (which have not been eliminated by the double plasma centrifugation) in order to discard interferences between them or another protease(s). Methods: ACD plasmas were double centrifuged and applied on Sephadex G-100 column. The void volume was applied on Gelatin-Sepharose 4B column. Urea-eluted fractions containing fibronectin (FN) and VWF-cleaving activity substances were applied to Butyl-Sepharose 4B column (buffer Tris with 0.35M ammonium sulfate). We collected two peaks separated on the basis of their hydrophobic interaction, P1 low and P2 high (n=3). P1 and P2 were assayed for VWF-cleaving activity adding 1mM AEBSF or plasma with circulating inhibitory ADAMTS13 antibody (ADAMTS13ab). FN was determined by ELISA. Results: P1 showed: 220% of VWF-cleaving activity not affected by AEBSF; in presence of ADAMTS13ab decreased 76% of VWF-cleaving activity; contained 30% of FN. P2 showed: 162% of VWF-cleaving activity not affected by ADAMTS13ab; in presence of AEBSF decreased 64%; contained 200% of FN. Normal citrate plasma (control), in presence of ADAMTS13ab decreased 48% and in presence of AEBSF decreased 52% (n=6). Conclusions: We had made the separation of the two VWF-cleaving activities: ADAMTS13 and SP, without interferences between them. The origin of these SP was uncertain. As we avoid the use of protease inhibitors, we can not assure that they were not derived of contamination from white cells and platelets. ADAMTS13 and SP had high activity; however, due to the residual activity detected in P1 and P2, we can not discard the presence of other VWF-cleaving activity substance