PROTHROMBIN ACTIVATOR TYPE C. (LOPAP) AND BRISTLES EXTRACT OF Lonomia obliqua ACTIONS ON HUMAN COAGULATION AND FIBRINOLYTIC FACTORS

Reis, C.V.(1), Fritzen, M. (1), Flores, M.P.A. (1), Juliano, L. (2), Romero-Ramos, C.R.(3), Azevedo, I.L.M.J. (3), Ho, P.L. (3), Chudzinski-Tavassi, A.M. (3)

(1)Laboratóriode Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP; (2)Departamento de Biofísica, UNIFESP, São Paulo; (3)Centro de Biotecnologia, Instituto Butantan, São Paulo.

Lonomia obliqua venom produces a severe consumption coagulopathy that can lead to a hemorrhagic syndrome. Here we describe some activities of the Lonomia obliqua bristles’ crude extract (LOBCE) and LOPAP on blood coagulation and fibrinolytic factors. LOBCE is able to clot the plasma, but not purified fibrinogen and shows prothrombin (LOPAP) and Factor X activators. In addition neither FXIII inhibition nor degradation (SDS-PAGE) is observed. Fibrinolytic activity on cross-linked fibrin is not found, however a fibinogenolytic activity on a-chains of fibrinogen occurred and the generated products are not similar from those induced by plasmin. In Solid Fibrin Assay no plasminogen activation is observed. Lopap hydrolysed fluorescent peptides derived from prothrombin sequences binding sites recognized by the thrombin (Arg284-Thr285) (Km 0,3269mM and Kcat2,91 s^-1) or by FXa (Arg320-Ile321). Lopap hydrolyse prothrombin independently of the prothrombinase complex, but its activity is strongly increased by ions calcium and phospholipids. A cDNA library from mRNA of bristles was constructed in pGEM11zf(+) plasmid. The LopapcDNA(600bp) was amplified from this library by PCR using degenerated primers corresponding to N-terminal sequence of protein. This fragment was sequenced and sub-cloned in E. coli expression vector and the active recombinant Lopap with 6xHis fusion at the N-terminus was obtained. Aleatory clones showed a great variety of genes for several proteins of the insects. Of these, some proteins already present similarity with other known and presents in databases, among which we can highlight a BPP, a catepsin, and a hemolysin. Other DNAs didn't present homology with any present well-known sequence in databases, and they can also be studied. Lonomia obliqua extract contains procoagulant proteins that leading to a consumption coagulopathy. A fibrinogenolytic activity occurs also, although the component responsible for this activity is not able to degrades cross-linked fibrin. Also Lopap can be classified as a type C prothrombin activator.

Supported by FAPESP and CNPq

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