Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants

Abstract

The hirudin‐like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech‐derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF‐Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin‐inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF‐Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin‐inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.

Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

“…rHV1 was additionally tested at a concentration of 31.0 nmol/l. Data for the activity of rHV1 at a concentration of 310 nmol/l were taken from Müller et al 71 . For determination of clotting times double determination was used as "proof of principle" (n = 2).…”

“…To verify the anti-coagulatory activity of the different HV1 preparations, two blood coagulation assays were performed: the activated partial thromboplastin time test (aPTT; reference range 22.7-28.9 s) and the thrombin time test (TT; reference range 16.8-21.4 s) using a BFT II analyzer (Siemens Healthcare, Erlangen, Germany). All steps followed the instructions outlined by the manufacturer and were described in detail in our previous works with small deviations 48,71 . 10 µl of the respective SN2 fraction of the cell-free expressed protein were directly transferred into the cuvette immediately before the plasma was added.…”

The hirudin-like factors HLF3 and HLF4—hidden hirudins of European medicinal leeches

“…1; Table 2). According to previous investigations, the presence of the central globular domain of HLF2 renders HLF hybrids unable to inhibit thrombin (Müller et al 2020). We hence constructed three different hybrids of the highly active thrombin inhibitor HLF1V and HLF3l: HLF-Hyb3a (N-and C-termini of HLF1V fused to the central globular domain of HLF3), HLF-Hyb3b (N-and Ctermini of HLF3l fused to the central globular domain of HLF1V), and HLF-Hyb3c (N-terminus and central globular domain of HLF1V fused to the C-terminal tail of HLF3l) (Fig.…”

“…However, the construction and functional Section Editor: Boris R. Krasnov characterization of hybrid variants of HLF1 and HLF2 bypassed this limitation and subsequently revealed strong evidence for the crucial importance of the central globular domain of HLFs on the anti-thrombin activity of the whole molecule as well. The central globular domain of HLF1 enabled thrombin-inhibitory potency, whereas the central globular domain of HLF2 did not (Müller et al 2020). In the current work, we wanted to apply the same strategy and technical approach to analyze and functionally characterize the hirudin-like factors HLF3 and HLF4.…”

“…Strikingly, HLF1 variants with the N-terminal five amino acid residues IVYGP (HLF1V and HLF1long) exhibited very high anti-coagulatory activities comparable to hirudins. In contrast, variants with either four (IYGP, HLF1) or different five N-terminal amino acid residues, specifically IDYEP (HLF1D), had only very low or no detectable activities (Müller et al 2020). These results were in very good accordance with examinations of the N-termini of hirudins like HV1 of Hirudo medicinalis (Betz et al 1992;Wallace et al 1989;Lazar et al 1991) or in HM1 of Hirudinaria manillensis (De Filippis et al 1995, 1998.…”