Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation

Abstract

Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose-and time-dependent manner but not ␥-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X.

Toxins from Lonomia obliqua — Recombinant Production and Molecular Approach

“…Some years later, from a cDNA library of L. obliqua bristle transcripts [19], the specific clone encoding for Losac was identified and the recombinant protein produced in bacteria system (for details, see section 3.3). Studies using the native or recombinant form of Losac (rLosac) revealed specificity toward factor X [20,22,28]. Moreover, Losac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation, and it was recognized by the antilonomic serum produced in Butantan Institute.…”

“…Although there are strong functional similarities, the major difference is in the structure of both activators. Apparently, Losac activates factor X through a serine protease-like activity, while RVV-X has a typical metalloproteinase structure [20,28,84].…”

“…Biochemical characterization of Losac has shown that, although its sequence did not show an equivalent among other factor X activators, Losac possess a similar mechanism of action than RVV-X, a factor X activator purified from Russell's viper venom Daboia russelli [28,82,83]. Like RVV-X, factor X activation by Losac can be accelerated in the presence of calcium and phospholipids, two important cofactor in the assembling of blood coagulation complexes [69].…”

“…These molecules can point out novel mechanisms of action, undiscovered molecular interactions and new classes of enzymes and inhibitors. Interesting, some venom toxins have shown multifunctional properties [19,22,28]. The best examples are Lopap (a prothrombin activator with high similarity with lipocalins) and Losac (a factor X activator highly similar to hemolins).…”

“…Besides activation of blood coagulation, Lopap and Losac can modulate cellular functions and promote cell survival [22,45]. Both molecules were cloned and produced in its recombinant form in yeast and/or bacteria [19,25,28].…”