Poster 236 - Isolation of angiostatin from normal human plasma using affinity chromatography

Poster 236. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.595.

Isolation of Angiostatin from Normal Human Plasma Using Affinity Chromatography with a Myotoxic PLA2 from Bothrops asper Venom

Chermont, S.A^1., Jurgilas, P.B¹., Neves-Ferreira, A.G.C¹., Chapeaurouge, A¹., Domont, G.B². and Perales, J¹.

1 Departamento de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro; 2 Departamento de Bioquímica, IQ, UFRJ, Rio de Janeiro.

DM64 is a glycoprotein isolated from opossum serum through an affinity chromatography with immobilized snake venom myotoxin. This myotoxin inhibitor was cloned and its deduced protein sequence showed homology to a1B-glycoprotein, a human plasma protein of unknown function (Rocha et al., 2002). In this study, we have applied crude human plasma to the affinity column coupled with myotoxin I from Bothrops asper in an attempt to isolate and investigate a1B-glycoprotein. The fraction bound to the column was eluted with glycine/HCl and analysed by SDS-PAGE. Protein bands of 94, 65 and 38 kDa were detected. The 94 kDa band corresponds to a precursor of angiostatin (plasminogen), as identified by MALDI-TOF peptide mass fingerprint. The smallest protein band (38 kDa) is the active form of angiostatin, as confirmed by positive staining with a monoclonal antibody against human angiostatin. The 65 kDa band is under investigation and may represent an intermediate form of angiostatin, as already described in the literature. Angiostatin is a potent inhibitor of angiogenesis that selectively inhibits endothelial cell proliferation through an unknown mechanism. Breast adenocarcinoma (MDA) supernatant, which is the main source of commercial angiostatin, was also submitted to this same affinity chromatography. SDS-PAGE revealed the presence of either angiostatin and its precursor interacting to the myotoxin column. The identity of the precursor was confirmed by mass spectrometry. It is well known that myotoxins from Bothrops venoms have fosfolipase (PLA2) structure. Using synthetic inhibitors, Chen et al (2002) had already proposed that PLA2 inhibition could be controling angiogenesis. Our results demonstrate the non-covalent interaction between human angiostatin and a molecule with PLA2 structure. Therefore, we are now working with the hypothesis that angiostatin down regulates the angiogenesis process though PLA2 inhibition. It is also important to note that, for the first time, we have reported the isolation of angiostatin from normal human plasma.