Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

Abstract

A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.

Rapid assembly and profiling of an anticoagulant sulfoprotein library

“…1). While unmodified variants of the hyalomin family have been shown to exhibit thrombin inhibitory activity (24,25), based on our prior work on madanin-1 we predict that each is likely to be sulfated based on the presence of conserved tyrosine residues flanked by a highly acidic amino acid sequence--a quintessential motif for posttranslational sulfation (26). To date MadL1, MadL2 (14,27), And82, and And310 (23) have only been characterized as putative thrombin inhibitors; however, based on the sequence alignment of these proteins ( Fig.…”

Accelerated Protein Synthesis via One-Pot Ligation-Deselenization Chemistry

“…The hyalomins are a family of four cysteine-free proteins produced within the salivary glands of the tick Hyalomma marginatum rufipes that support the bloodfeeding behavior of the organism. 39 The absence of Ala residues at a site that would permit assembly through other ligation approaches, together with the wealth of acidic residues within the sequences (pI < 4), made these amenable to assembly with our ligation technology. Disconnection of hyalomins 2-4 (38-40) was made at Asp residues close to the middle of the sequences.…”

“…(38)(39)(40), we next assessed the activity of the synthetic proteins as inhibitors of human thrombin (see Supplemental Information for details). Both hyalomin 2(38) and hyalomin 3 (39) proved to be extremely potent thrombin inhibitors with inhibition constants (K i ) of 1.24 G 0.05 nM and 14.73 G 0.64 nM, respectively (Figures S99…”

Peptidase inhibitors in tick physiology

“…In particular, the huge number of thrombin inhibitors found in the saliva of both hard and soft ticks demonstrates the importance of interfering with coagulation to keep the blood flowing. It is interesting to note that most of the small peptide inhibitors found in ticks are thrombin inhibitors (Zhu et al, 1997;Ibrahim et al, 2001;Iwanaga et al, 2003;Motoyashiki et al, 2003;Ciprandi et al, 2006;Nakajima et al, 2006;Koh et al, 2007;Jablonka et al, 2015).…”