Lopap, a prothrombin activator from Lonomia obliqua belonging to the lipocalin family: recombinant production, biochemical characterization and structure-function insights.

Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.

A prothrombin activator (Lopap) modulating inflammation, coagulation and cell survival mechanisms.

A severe hemorrhagic syndrome produced by contact with Lonomia obliqua caterpillars has become epidemic in southern Brazil. A significant thrombin production with intense consumption of fibrinogen and high D-dimer production indicates a consumption coagulopathy and secondary fibrinolysis in patients. Lopap is a single-chain 69kDa serine protease isolated from the crude extract of L. obliqua bristles. Experiments in mice showed that the purified protein, similar to the crude extract, causes uncoagulable blood by fibrinogen depletion. In order to characterize the effects of Lopap on cells involved with hemostatic system, we performed experiments using human umbilical vein endothelial cells (HUVECs). Our results show that Lopap exerts a direct effect on endothelial cells by increasing the liberation of molecules involved in the regulation of vascular tone, inhibiting platelet activation and chemotaxis, apart from inducing the expression of cell adhesion molecules which participate in inflammatory responses. The release or new synthesis of mediators involved in coagulation as von Willebrand factor and tissue factor, or in fibrinolysis as tissue plasminogen activator, was not affected by Lopap. Also our results demonstrated that Lopap acts on cell survival of HUVECs, regulating the expression of molecules as NO and avoiding cell death.

Clonagem, sequenciamento, expressäo e caracterizaçäo parcial da estrutura do Lopap, um ativador de protrombina da lagarta Lonomia obliqua / Cloning, sequencing, expression and partial structure characterization of Lopap, a prothrombin activator from Lonomia obliqua caterpillar

O contato da pele humana com cerdas de lagartas Lonomia obliqua, espécie encontrada principalmente na região Sul do Brasil, causa, entre outros sintomas, urna severa sindroine hemorrágica devido a uma coagulopatia de consumo ocasionada por agentes procoagulantes (ativadores de protrombina e fator X da coagulação), presentes no veneno. O Lopap (Lonomia obliqua prothrornbin activator protease) é urna proteína de 69 kDa, ativadora de protrotnbina, purificada do extrato bruto das cerdas da taturana L. obliqua através de cromatografia de gel filtração (Sephadex G 75) e fase reversa etn HPLC (coluna C4). A proteína purificada é capaz de ativar a protrombina de forma dose-dependente. Através de análise por SDS-PAGE, observou-se que o Lopap pode ser uma molécula composta por 4 cadeias idênticas de 18 kD.a A atividade do Lopap é aumentada pela adição de íons cálcio, entretanto outros íons bivalentes, como magnésio ou zinco, não produzem o mesmo efeito. O Lopap é inibido por imbidores de serino proteases como o PMSF e Trasylol. O peptídeo fluorogênico, derivado da protrombina na região hidrolisada pela trombina (Abz-YQTFFNPRTGSQ-EDDnp), teve sua clivagem no sítio principal entre a ligação Arg-Thr. Os parâmetros cinéticos obtidos para este substrato foram: Kmapp de 4,5 pM, kcat de 5,32 seg 1 e wn kcatlKrnaPP1,2 x 106 M-1 s 1. O soro anti-lonômico reconhece o Lopap quando analisado através de reação cruzada por imunodifusão radial O Lopap é capaz de provocar a formação de trombos, induzir a incoagulabilidade sanguínea, a depleção do fibrinogênio e causar urna diminuição na contagem de plaquetas quando injetada na corrente sangüínea de ratos. Também foi observada congestão e hemorragia nos...(au)