Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone‐binding proteins (PBPs) could be identified in pheromone‐sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI‐TOF MS of sensillum lymph droplets from pheromone‐sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant‐binding proteins, immunoreactivity was found only with an anti‐(Apol PBP) serum. Free‐flow IEF, ion‐exchange chromatography and Western blot analyses revealed at least three anti‐(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N‐Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215–2218] and a new PBP having only 57% identity with this amino‐acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N‐terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin‐labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277–284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)‐6,11‐hexadecadienyl acetate (AC1) and the (E,Z)‐6,11‐hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H‐labelled acetate, whereas no binding of the 3H‐labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.

A cDNA coding for the thrombin inhibitor dipetalogastin has been isolated from a stomach library of Dipetalogaster maximus, a blood-sucking insect. The open reading frame of the cloned inhibitor cDNA codes for a protein of 344 amino-acid residues. Sequence analysis reveals the existence of three repeated homologous main regions, indicating that the inhibitor consists of three domains. Each domain shows a double-headed structure with an internal sequence homology like rhodniin, the thrombin inhibitor from the blood-sucking insect Rhodnius prolixus. Peptide sequence comparisons of the deduced amino-acid sequence exhibit a high homology of the domains I and II to the natural inhibitor dipetalogastin from the stomach content of D. maximus and to rhodniin, respectively. Significant sequence similarities to Kazal-type inhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine residues, indicate that the thrombin inhibitor from D. maximus is a further blood-sucking insect which belongs to the Kazal-type family (besides rhodniin). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated recombinant dipetalogastin with a molecular mass of 12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The K i value of the recombinant dipetalogastin was determined to be 49.3^22.28 fm.Keywords: thrombin inhibitor; Kazal-type; blood-sucking insect; bug; cDNA cloning.Hematophagous animals are a particularly rich source of anticoagulant substances. In order to prevent the clotting during sucking of host blood and during digestion of fed blood, they have developed various mechanisms to interfere with blood coagulation. Among the highly specific enzymes and inhibitors involved in the coagulation [1], the thrombin inhibitors are the most prominent anticoagulants, having been described and characterized from leeches [2±4], snake venoms [9] and also from insects [5±8,10,11].The trypsin-like serine protease thrombin is a multifunctional key enzyme in the blood coagulation cascade. During coagulation thrombin converts fibrinogen into fibrin, activates other coagulation factors such as factor V, VIII, XIII and protein C [12,13], interacts with different cells, induces platelet aggregation and stimulates platelet secretion [14].While the anticoagulants from the blood-sucking insects Rhodnius prolixus and Triatoma pallidipennis have been investigated intensively [10,11,15±22], only sparse knowledge about anticoagulant activities from a further blood-sucking insect Dipetalogaster maximus [23,24] is available. Recently, we have isolated and characterized biochemically the specific natural thrombin inhibitor dipetalogastin from the stomach content of D. maximus [24]. This inhibitor consists of four isoforms each with a molecular mass of approximately 12 kDa. Natural dipetalogastin forms only 1 : 1 molar complexes with thrombin and acts as a slow, tight-binding inhibitor of throm...

A new sensitive and precise method for quantitative determination of direct thrombin inhibitors is described, the ecarin chromogenic assay (ECA). Ecarin is used as the specific prothrombin-activating principle. The cleavage of a chromogenic substrate by meizothrombin is inhibited in a concentration-dependent fashion by direct thrombin inhibitors. For the ECA, the linear measuring range is about 0.1–3.0 µg hirudin/ml plasma. Coefficients of variations between 2.3 and 4% over the whole concentration range were achieved. The ECA has proved to be more sensitive than the compared tests (ecarin clotting time and a thrombin-based chromogenic assay); a detection limit of 0.011 µg hirudin/ml and a quantitation limit of 0.032 µg hirudin/ml were calculated. The ECA is independent of the variations of the coagulation variables fibrinogen and prothrombin. Neither heparin nor oral anticoagulants interfere with the ECA.

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a Km value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the Km for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.

From the bloodsucking bug Dipetalogaster maximus, a protein with anticoagulant activity was isolated and biochemically characterized. The isolated protein, named dipetalogastin, possesses an average molecular mass of 11.8 kD. Its N-terminal sequence shows homology to rhodniin, a thrombin inhibitor isolated from the bug Rhodnius prolixus. The in vitro anticoagulant activity of dipetalogastin occurs via the inhibition of thrombin. The anticoagulant and thrombin inhibitory potency of dipetalogastin is comparable to that of recombinant hirudin. Its specific thrombin inhibitory activity is 9,300 antithrombin units/mg protein. Dipetalogastin forms only 1:1 molar complexes with thrombin. It is a tight-binding inhibitor of thrombin possessing a dissociation constant of 125 fM. It does not inhibit factor Xa or α-chymotrypsin and only weakly inhibits trypsin.