ERISTOSTATIN-ALKALINE PHOSPHATASE FUSION PROTEIN: AN ENZYMATIC MARKER FOR a_IIbb_{3 INTEGRIN}

Butera, D.(1,2), McLane, M. A.(3), Paquette-Straub, C.(3), Ducancel, F.(4), Moura-da-Silva, A. M.(1,2)

(1)Laboratório de Imunopatologia, InstitutoButantan, Brazil, (2)Departamento de Bioquímica, IQ/USP, Brazil, (3)University of Delaware, USA, (4)CEA Saclay, France.

The a_IIbb_{3 integrin is the platelet fibrinogen receptor, essential for aggregation. Its low expression is related to severe coagulation disorders such as Glanzmann disease. In addition,} a_IIbb_{3  is over-expressed during metastasis in tumor cells of different origins. Thus, quantitation of} a_IIbb_{3 integrin using selective markers as Eristostatin, a snake venom disintegrin, is an important task for evaluating these pathologies. In this work, an enzymatic marker selective for} a_IIbb₃ was produced using alkaline phosphatase(APv) tagged eristostatin (Er). Fusion protein was obtained by cloning and expressing Eristostatin DNA into the pLIP6-GN vector. The Er/APv fusion protein was identified by SDS-PAGE and by western blotting using both anti-Er and anti-AP antibodies. The fusion protein showed enzymatic AP activity similar to wild APv. The potential use of the hybrid protein for a_IIbb_{3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and} a_IIbb_{3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. The dot-blot test was then standardized with respect to platelet concentration dotted onto nitrocellulose membranes, concentration of Er/APv, time and temperature of incubation. The optimized assay uses physiological concentrations of platelets, incubation times of 30 minutes, room temperature, with minimal concentrations of 10 pMEr/APv. Our data present a novel tool, Er/APv, with potential use in diagnosis of disorders where the} a_IIbb_{3 integrin is involved.}

Support: FAPESP 00-11732/2, FAPESP 00-13651/0 and FAPESP-CEPID/CAT

CORRESPONDENCE TO:

Diego Butera, Rua Rocha 318, Apto. 21, São Paulo, SP, CEP: 01330-000, Brasil, Email: dbutera@hotmail.com