EFFECTS OF LOSAC (A FACTOR X ACTIVATOR FROM LONOMIA OBLIQUA CATERPILLAR) ON HUVECs AND SEQUENCING. 

¹FLORES, M.P.A.; ^{2KONNO, K.;} 1FRITZEN, M. AND ¹CHUDZINSKI-TAVASSI, A.M.

1 LABORATÓRIO DE BIOQUÍMICA E BIOFÍSICA, 2 LABORATÓRIO DE ESPECTROMETRIA DE MASSA, CAT-CEPID, INSTITUTO BUTANTAN, SÃO PAULO, SP.

Losac is the first factor X activator purified from lepidopter. It is a 43 kDa protein that cleaves the Arg⁵²-Ile⁵³ bond in the heavy chain of factor X, producing the activated factor Xaa. Apoptosis of vascular endothelial cells is related to angiogenesis, vascular regression, atherosclerosis and restenosis. Effect of Losac on proliferation, apoptosis, metabolites production and adhesion molecules expression were here studied using human umbilical vein endothelial cells (HUVECs). Nitric oxide concentration in the supernatans was determined by chemiluminescence in gaseous phase, elicited by the reaction of NO with ozone, after nitrate and nitrite reduction. PGI2 concentration in the supernatans was determined by ELISA. Inhibition and induction of apoptosis and viability were determined by immunouorescence cell stained by acridine orange and ethidium bromide in cell incubated during 48h in RPMI containing 1% and 10% FBS with or without Losac. Expression of ICAM-1 and DAF were determined by flow cytometry. Losac (0.35 mM) was able to reduce the percentage of apoptotic cells (79 ± 4,3) compared to control samples (more of than 90%). On the other hand, Losac increased the cell proliferation of HUVECs treated with 10% FBS and incubated during 72h with Losac 0.12 mM ( 26833 control cells ± 1421 vs. 31456 ± 3970) and 1.16 mM (26833 ± 1421 vs. 42350 ± 5696). Enhanced concentration of NO was found in HUVECs supernatans treated with Losac 0.12 mM (7.12 mM ± 2.3 vs. 10.47 ± 3.13) and 0.58 mM (6.2 mM ± 0.25 vs. 24.5 ± 5.4). However, the concentration of PGI2 was not altered with Losac 0.58 mM after 1 and 24h incubation. Also, Losac (0.12 mM) did not have effect on the expression of ICAM-1 and DAF. A small increase of ICAM-1 and DAF was observed only with 1.16 mM of protein. Purified Losac was digested with trypsin and sequencing using a Q-TOF MS. Ten peptide fragments were obtained, apparently Losac do not presents similarity with other well-know factor X activators. A cDNA library was constructed and recombinant Losac is being obtained. Thus, we concluded that Losac is a new factor X activator protein and probably posses an important role in the human envenoming by L. obliqua. Also we suggest that Losac can modulates cell survival.

Supported by FAPESP.

Correspondence to: mpalvarez@butantan.gov.br