Some studies on the salivary anticoagulant components of the black fly Simulium vittatum (Diptera: Simuliidae).

Abstract

Three major experiments on the anticoagulant components of the saliva of some black fly species were conducted. These included investigations on anticoagulant activities against factor Xa and thrombin in the salivary gland extracts (SGE) of Simulium argus Williston, S. vittatum Zettersted, S. metallicum Bellardi, and S. ochraceum Walker using in vitro, chromogenic substrate assays, biochemical and molecular characterization of the anti-thrombin from the saliva of S. vittatum and determination of the target enzyme in the vertebrate coagulation cascade of a novel anticoagulant from the saliva of S. vittatum. The study on anticoagulant activities in the four simuliid species revealed factor Xa inhibition to be common in all four species while thrombin inhibition was detected only in S. argus and S. vittatum. Both bovine and human α-thrombins were inhibited with the highest activity occurring with S. argus SGE. Factor Xa inhibition was highest in S. ochraceum which is an anthropophilic species and vector of Onchocerca volvulus, and lowest in S. vittatum, a primiparous autogenous species that is also zoophilic. Total soluble SGE protein also varied among the four species with the highest concentration measured in S. ochraceum and the lowest in S. vittatum. In the second experiment, the anti-thrombin component of the saliva of S. vittatum was purified using a two-step reverse phase (RP) high performance liquid chromatography (HPLC) involving a C-8 macrosphere column. The molecular weight of the HPLC purified inhibitor was determined by laser desorption ionization mass spectrometry (LDI-MS) and was found to be 11,333 daltons. Studies on the effect of the molecule on other serine proteinases such as α-chymotrypsin, human neutrophil elastase, and human neutrophil cathepsin G showed that they are inhibited by the salivary anticoagulant. The N-terminal sequence for the first 35 amino acids was determined. The molecule has been named Simulidin. In the third experiment, a novel anticoagulant from the saliva of S. vittatum, with activities on factor V, was demonstrated using the activated partial thromboplastin time (APTT) in HPLC partially purified salivary lysate. Factor Xa and thrombin were unaffected by the inhibitor.