Desmolaris is a major anticoagulant from vampire bat saliva. (A) Clustal alignment. Desmolaris (gi527498655) and TFPI-1 from Homo sapiens (gi5454114), Macaca mullatta (gi297264502), Equis cabalus (gi338715615), Bos taurus (gi346644729), and TFPI-2 from Homo sapiens (gi5730091) were aligned. (B) Predicted secondary structure of Desmolaris based on human TFPI.12 Amino acids are identified by a single letter code. Basic (positively charged) residues are colored in blue and acidic (negatively charged) residues in red. Arrows indicate P1 sites. N glycosylation sites were found for Asn77, Asn 78, Asn 122, and Asn 149. Putative phosphorylation sites were identified at Ser 42, Thr 133, Thr 141, Thr 151, Ser 152, and Ser 155. The Kunitz domains are labeled. Dotted lines correspond to Kunitz-1 of TFPI. The putative sites of introns in the Desmolaris gene (based in human TFPI)12 are labeled with lines and the capital letters A, B, C, and D. (C) Recombinant expression. Desmolaris was expressed in HEK293 cells and purified with an Ni2+ column followed by reverse-phase chromatography. (Inset) Samples were loaded in a NuPAGE gel under reducing conditions. Gels were stained with Coomassie Blue. Molecular mass markers are shown (right). The arrow indicates Desmolaris. The Desmolaris sequences depicted in red and underlined are the peptides identified by mass spectrometry, with more than 70% coverage. (D) Screening for enzyme inhibition by Desmolaris. Enzymes at indicated concentrations were incubated with Desmolaris (250 nM), and catalytic activity was estimated by fluorogenic substrate hydrolysis. FVIIa/TF complex activity was determined with chromogenic substrate (S2366). Results are the mean ± standard error of triplicates. (E) SPR experiments. Desmolaris was immobilized in a CM5 chip, and several coagulation enzymes and zymogens (all at 64 nM) were injected as analytes. Association phase of 60 seconds was followed by a 60-second dissociation. Resonance values are based on binding at stability. A typical result is shown. (F) PT and aPTT. Desmolaris was added to plasma at the indicated concentrations, followed by addition of a PT or aPTT reagent as described in the “Methods” section. Clotting was estimated using a coagulometer. Control human plasma: PT, 14.6 ± 0.20 seconds; aPTT, 35.9 ± 0.15 seconds (each data point is the average of duplicate or triplicate determinations).
