Lonomia obliqua: A BIOCHEMICAL AND MOLECULAR VIEW

CHUDZINSKI-TAVASSI, A.M.(1)

(1)Laboratório de Bioquímica e Biofísica, Instituto Butantan

Accidental contact with Lonomia obliqua induces an acquired hemorrhagic diathesis. Prolongation of coagulation times with significant reduction of fibrinogen, decrease of factors V, VIII and protein C, and an increase of fragments 1+2, TAT were found in 105 plasma patients.  A prominent increase in D-dimerlevels occured without alterations of t-PA or U-PA. A moderate reduction of plasminogen and AP were also observed. These data suggested a consumption coagulopathy and a secondary fibrinolysis. Lonomia obliqua bristles extract exerted procoagulant activity in human plasma but is not able to clot purified fibrinogen and shows FX and FII activation activity in purified systems. In addition, neither inhibition of FXIII nor degradation occured.Cross-linked fibrin were not hydrolysed by the extract, however fibrinogen a chain degradation was observed after long time pré-incubation. The generated products are not similar to fragments D and E, plasmin derived. Plasminogen activation was not observed in SOFIA system. Furthermore, bristles extract not affected platelet aggregation. From the extract, we purified Lopap,(Lo) a serine-protease of 69 kDa prothrombin activator. In vivo, Lo induces thrombin formation, neutrophil margination in pulmonary rat microvasculature, fibrinogen depletion and 30% platelet reduction, human platelet aggregation Lopap was able to activate prothrombin independently of  prothrombinase complex components but Ca++ , Va and  PL increase its activity. On endothelial cells (EC)  fromHUVECs, flow cytometric analysis showed that the incubation with Lo (5 ug/ml) during 1 h produced an increased expression of ICAM-1 (7±1 vs. 19±3 arbitrary units of fluorescence (UAF), p<.05, n=7) and E-Selectin(4±1 vs. 24±5 UAF, p<.05, n=3) without modifying VCAM-1 levels (3±1 vs. 4±1 UAF, n/s, n=6). The E-selectin-increased expression was specific since EC pretreatment with serum against L. obliqua almost completely inhibited this response. It was not associated with thrombin formation, once hirudin did not modify the ICAM-1 upregulation. An increase of IL-8 (1.4±0.2 x 5.3±1.7 ng/ml) and  PGI2, time dependent,  is also observed (0.46± 0.05 control x 4.9 ±1.1 induced by 30 min) and (2.8 ± 0.9 control x 15.6 ± 2.7 induced by 4h).  ELISA studies showed that Lo was not able to induce von Willebrand factor release nor its synthesis (n=5). Lo had no effect on washed platelets aggregation, which were induced by several agonists, suggesting that it does not contribute for platelet function in vivo. Also, AT was able to inhibit amidolytic activity of thrombin generated by incubation of Lo with prothrombin. Lo hydrolysed the fluorescent peptide, a prothrombin sequence derived, having the hydrolysis site recognized by thrombin (Arg284-Thr285) (Km 0,3269mM and Kcat 2,91 s^-1). Recombinat Lopap was obtained and DNA from aleatory clones are being sequenced. In conclusion, the direct action of Lo on EC could have an important role in the pathogenesis of inflammatory and coagulation disorders observed during the Lo infusion in rats or in human accidental contact with L.obliqua . Probably, during prothrombin activation, Lo induces prethrombin 2 formation and possible meizothrombin.

Supported by FAPESP and CNPq

CORRESPONDENCE TO:

CHUDZINSKI-TAVASSI, A.M., Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av Vital Brazil, 1500 - Lab Bioquímica, São Paulo, SP, CEP: 05503-900, Brasil, amchudzinski@hotmail.com