A new Factor Xa inhibitor from Amblyomma cajennense with a unique domain composition

Abstract

Bioactive compounds of great interest are found in the saliva of hematophagous organisms. While exploring a cDNA library derived from the salivary glands of the tick Amblyomma cajennense, a transcript that codes for a protein with unique structure (containing an N-terminal Kunitz-type domain and a C-terminus with no homology to any annotated sequences) was found. The recombinant mature form of this protein ( approximately 13.5kDa) was produced in Escherichia coli BL21 (DE3), and it was able to inhibit Factor Xa (FXa) and extend global blood clotting times in vitro and ex vivo. Static and dynamic predictions of its tertiary structure indicate regions that may be related to its FXa inhibitor function.

The Essential Role of Tick Salivary Glands and Saliva in Tick Feeding and Pathogen Transmission

“…TAP, the Kunitz-type tick anticoagulant peptide from O. moubata (Waxman et al, 1990), the TAP-like protein from O. savignyi (Joubert et al, 1998), amblyomin-X from Amblyomma cajennense (Batista et al, 2010) and Salp14 (a basic tail-secreted protein) from I. scapularis (Narasimhan et al, 2002) are all inhibitors of coagulation factor Xa. Kunitz-type inhibitors that display similarity to the tissue factor (TF) pathway inhibitor have been identified, e.g., in I. scapularis (Ixolaris and penthalaris) (Francischetti et al, 2002, 2004), and Rhipicephalus hemaphysaloides (Rhipilin-1 and -2) (Gao et al, 2011; Cao et al, 2013).…”

Serine Protease Inhibitors in Ticks: An Overview of Their Role in Tick Biology and Tick-Borne Pathogen Transmission

“…Amblyomin-X was initially discovered following SG transcriptome sequencing of the Amblyomma cajennense Cayenne tick (Batista et al, 2010). This protein harbors one Kunitz-type domain, and is observed in monomeric, dimeric, trimeric, and tetrameric conformations.…”

“…This protein harbors one Kunitz-type domain, and is observed in monomeric, dimeric, trimeric, and tetrameric conformations. Amblyomin-X inhibits FXa in a non-competitive manner but also increases PT and aPTT, suggesting an effect on prothrombin conversion (Batista et al, 2010; Branco et al, 2016). Four hypotheses have been proposed to explain the FXa inhibitory mechanism of Amblyomin-X.…”

“…Thirdly, an additional binding of the Amblyomin-X NH 2 -terminus to the FXa active site could exist. Lastly, Lys30 from the characteristic Kunitz domain loop could bind to the FXa active site (Batista et al, 2010). …”

A Kunitz-type FXa inhibitor affects tumor progression, hypercoagulable state and triggers apoptosis

“…It is a recombinant protein form (GenBank accession No. AAT68575) identified in the transcriptome of the salivary glands of the Amblyomma cajennense tick [14,15]. The studies performed with Amblyomin-X have shown that this is also able to induce cell cycle arrest and apoptosis in different tumor cell lines, and in vivo experiments, it promotes regression and reduction of metastases of some tested tumors [16,17].…”