Production and functional characteristics of a novel hirudin variant with better anticoagulant activities than bivalirudin

Abstract

Current antithrombotic therapies face dual constraints of bleeding complications and monitoring requirements. Although natural hirudin provides targeted thrombin inhibition, its clinical adoption is hindered by sourcing limitations. This study developed a recombinant hirudin variant HMg (rHMg) with enhanced anticoagulant activity through genetic engineering and established cost-effective large-scale production methods. The synthesised HMg gene was expressed in E. coli BL21 via a pET vector plasmid, followed by nickel-affinity purification. Systematic evaluations demonstrated rHMg’s antithrombin activity of 9573 ATU/mg, dose-dependent prolongation of APTT/PT/TT. It has superior thrombin inhibition with the IC₅₀ and K_i values were 2.8 and 0.323 nM respectively compared to FDA approved drug bivalirudin (p < 0.001). The high-yield prokaryotic expression of rHMg with enhanced anticoagulant efficacy provides a novel strategy for developing affordable antithrombotic drugs, showing significant potential for cardiovascular disease management.

Introduction

Thromboembolism is a key pathological factor in the development and worsening of major cardiovascular diseases, such as ischaemic heart disease, ischaemic stroke, and venous thromboembolism¹. In the clotting cascade, thrombin fulfils a key catalytic function by linking intrinsic and extrinsic pathways. This positions thrombin as the primary enzymatic factor driving thrombus formation and a central target for anticoagulant therapies².

Although traditional anticoagulants continue to see broad clinical use, they exhibit notable limitations. These include heightened bleeding risks, the emergence of drug resistance, and the necessity for regular therapeutic monitoring^3,4. Furthermore, heparin-based indirect thrombin inhibitors require interaction with antithrombin to achieve effective anticoagulation. This indicates that the anticoagulant activity of these inhibitors depends not only on their concentration but also on the presence of antithrombin. Consequently, this therapeutic approach is not applicable to all patients and has several limitations^5,6. In contrast, hirudin directly and effectively inhibits the catalytic function of thrombin through specific binding. Unlike indirect thrombin inhibitors, it does not require dependence on antithrombin levels or supplemental antithrombin injections. This mechanism provides superior anticoagulant efficacy while reducing bleeding risk, solidifying hirudin’s position as an ideal candidate for development as a next-generation anticoagulant⁷.

Hirudin, a polypeptide constituted by 64–66 amino acid residues with a molecular weight of approximately 7000 Da, was first isolated from Hirudo medicinalis, the European medicinal leech^8,9. To date, over ten distinct hirudin variants (HVs) with high stability and bioactivity have been identified across various leech species¹⁰. As a potent natural thrombin inhibitor, hirudin exhibits multiple beneficial biological activities including anticoagulant and antithrombotic¹¹, anti-atherosclerotic¹², anti-angiogenic, and antitumor effects¹³, antifibrotic¹⁴, demonstrating broad clinical application prospects.

Hirudin’s anticoagulant advantage originates from its distinct three-dimensional structure. The N-terminal domain creates a rigid globular configuration stabilised by three disulphide linkages (Cys6-Cys14, Cys16-Cys28, Cys32-Cys39), which accurately occupy the catalytic triad (His57-Asp102-Ser195) of thrombin through its first three N-terminal amino acid residues^15,16. The acidic domain at the C-terminal binds to thrombin’s Exosite I, inducing competitive inhibition of fibrinogen recognition¹⁷. This dual-target inhibition mechanism efficiently suppresses the conversion of fibrinogen to fibrin. It also disrupts thrombin-mediated platelet activation through positive feedback loops. Clinical validation across multiple trials has confirmed the therapeutic effectiveness of this bimodal mechanism in preventing and managing cerebrovascular and cardiovascular diseases¹⁴.

Although natural hirudin exhibits notable pharmacological benefits, its large-scale industrial production encounters substantial challenges. Traditional extraction methods face limitations from scarce leech resources, complicated purification procedures, and inadequate yields. Modern separation technologies enhance product quality but significantly increase production costs¹⁸. Genetic engineering advancements have facilitated recombinant hirudin synthesis through sequence optimisation, preserving bioactivity and improving production efficiency alongside batch consistency¹⁹. Multiple engineered hirudin derivatives, such as lepirudin²⁰, bivalirudin²¹, and desirudin^22,23, have secured FDA approval for anticoagulant therapeutic use. The development of genetic engineering techniques has provided new strategies for the production and optimisation of hirudin. Through rational design or directed evolution of amino acid sequences, the expression efficiency of recombinant hirudin, thermal stability, and anticoagulant activity can be enhanced^24–26.

A novel hirudin variant named HMg derived from Hirudinaria manillensis, an Asian endemic leech species, demonstrates superior antithrombotic properties compared to European medicinal leech variants due to its unique molecular structure. Lin’s team identified 21 antithrombotic gene families in Hirudinaria manillensis through transcriptomic analysis²⁷. This study selected one novel hirudin gene from these candidates, achieving high-efficiency prokaryotic expression with elevated yield and activity. Through integrated bioinformatics analysis and multimodal activity assays, including thrombin titration, thrombin kinetic analysis, and coagulation parameter detection, we comprehensively characterised rHMg’s structural properties and anticoagulant potency. Parallel comparisons with the clinical first-line drug bivalirudin provided theoretical foundations for developing novel anticoagulants.

Materials and methods

Bioinformatics analysis

Homologous hirudin sequences were retrieved from the NCBI BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), followed by multiple sequence alignment of representative Hirudo species sequences using MEGA 7.0 software. A phylogenetic tree was constructed via the Neighbour-Joining (NJ) method.

Construction of pET-HMg prokaryotic expression vector

Primers containing Nde I and Xho I restriction enzyme digestion site were designed using SnapGene software based on the HMg gene sequence and pET vector multiple cloning sites:

• FP: 5′-GGAATTCCATATGGTTTCTTACACTGGTTGTACTGA-3′

• RP: 5′-CCGCTCGAGCTTTTGTTCAATATCATCCAAAGAAAAT-3′

The HMg gene was amplified by PCR, digested with restriction enzymes, gel-purified, and ligated into similarly digested pET vectors. The recombinant plasmids were transformed into E. coli Top10 competent cells. Positive clones were screened by colony PCR and confirmed by sequencing, yielding the recombinant plasmid pET-HMg (containing a C-terminal 6 × His tag).

Restriction endonucleases Nde I and Xho I, T4 DNA ligase, Gel Extraction Kit, PCR Purification Kit, and Plasmid Extraction Kit were obtained from Sangon Biotech (Shanghai, China).

Expression, purification, and detection of rHMg

The pET-HMg plasmid was transformed into E. coli BL21. Single colonies were cultured in LB medium at 37 °C until OD₆₀₀ reached 1.0–1.2, followed by induction with 1 mM IPTG at 18 °C for 24 h. Cells were harvested by centrifugation at 4,000 ×g for 30 min, resuspended in PBS, and lysed by sonication at 90% amplitude with 3 s pulse/7 s pause cycles for 120 min. Supernatants were collected after centrifugation at 12,000 ×g for 10 min.

Recombinant HMg was purified using Ni-NTA affinity chromatography with PBS buffers containing 20 mM, 200 mM, and 500 mM imidazole for gradient elution. Target fractions were dialysed to remove imidazole.

Protein samples from each stage were analysed by SDS-PAGE. Purified protein was subjected to denaturing mass spectrometry for molecular weight verification and purity assessment.

Thrombin titration assay for rHMg activity

Hirudin samples were diluted to 0.0039 mg/mL with PBS. Thrombin was diluted to 40 U/mL and fibrinogen dissolved to 5 mg/mL in physiological saline. Bovine fibrinogen and bovine thrombin were purchased from Solarbio (Beijing, China).

A 200 μL fibrinogen solution was mixed with 100 μL sample solution in porcelain plate wells, pre-incubated at 37 °C for 5 min. Thrombin solution (40 U/mL) was titrated at 5 μL/min under continuous stirring. The coagulation endpoint was determined by visual observation of fibrin strand formation within 1 min, with total thrombin volume recorded at clot formation. PBS served as negative control.

Antithrombin units (ATU/mg) were calculated using: U = (C₁ × V₁)/(C₂ × V₂), Where C₁ (U/mL) and V₁ (mL) represent thrombin concentration/volume, C₂ (mg/mL) and V₂ (mL) denote sample concentration/volume.

Viscoelastic haemostatic assay

For viscoelastic haemostatic assay, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) coagulation experiments, we require fresh, healthy human blood samples. Three volunteers participated. Blood collection was performed by professional medical staff at the Jinggangshan University Medical Clinic using disposable sterile equipment via venipuncture from the arm. Each person donated 20 ml of blood. Blood collection was approved by the Jinggangshan University Ethics Committee on October 28, 2024, and volunteers signed an informed consent form.

The anticoagulant effect of hirudin on whole blood was evaluated in accordance with the instruction manual of the Century Yikang coagulation instrument. A 400 μL whole blood was incubated with 100 μL hirudin (five-step gradient dilution) at 37 °C for 5 min, and 20 μL 0.25 M CaCl₂ was added and inverted 10 times to activate coagulation. 360 μL treated blood was immediately loaded into test cups for analysis. Finally, read the values of ACT (activated clotting time), CR (coagulation rate), and PF (platelet function).

APTT/PT/TT assays for rHMg activity and EC₅₀ determination

Recombinant HMg was serially diluted (50–7500 nM) and mixed with plasma at 1:4 (v/v) ratio. PBS served as negative control. The APTT assay was conducted by mixing 100 μL sample with 100 μL APTT reagent, incubating at 37 °C for 5 min, and then recording the clotting time after adding 100 μL pre-warmed 0.025 M CaCl₂. For the PT assay, 100 μL sample was incubated at 37 °C for 5 min, and the clotting time was measured after the addition of 200 μL PT reagent. In the TT assay, 200 μL sample was incubated at 37 °C for 5 min, followed by determining the clotting time upon adding 200 μL TT reagent. The APTT, PT, and TT coagulation assay kits were obtained from Sun Biotech (Shanghai, China). Dose - response curves for APTT/PT/TT prolongation were analysed via nonlinear regression (GraphPad Prism 10.1.2) to calculate EC₅₀.

Chromogenic substrate assay for thrombin inhibition (K_i and IC₅₀)

Substrate S2238 was diluted (0–1000 μM) in Tris-HCl buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% PEG6000, 0.1% BSA, pH 8.0). Bivalirudin and rHMg and were diluted to final concentrations of 0.023–11.466 μM and 0.00003–4.9 μM respectively. Human thrombin was acquired from EKEAR Biotech (Shanghai, China). Chromogenic substrate S2238 was provided by Dulai Biotechnology (Nanjing, China).

The reaction mixture containing 10 μL of thrombin (40 U/mL) and 10 μL of inhibitor was pre-incubated at 37 °C for 10 min, followed by the addition of 180 μL S2238 to initiate the reaction. Absorbance at 405 nm was monitored every 20 s for 5 min to get the 4-nitroaniline release rate.

To calculate the inhibition constant (K_i) and half-maximal inhibitory concentration (IC₅₀), nonlinear regression analysis was performed using GraphPad Prism 10.1.2. This analysis systematically compared reaction rates across different substrate (S2238) and inhibitor concentrations to characterise the inhibition kinetics. The IC₅₀ value was specifically determined under a fixed S2238 concentration of 1000 μM.

Data analysis

EC₅₀, IC₅₀, and K_i values were calculated using GraphPad Prism 10.1.2 with curve fitting algorithms. Statistical significance was determined by independent-sample t-tests (SPSS 26.0).

Results

Sequence alignment and phylogenetic analysis of HMg

BLAST analysis of the HMg amino acid sequence against the NCBI database identified 20 homologous sequences from leech species including Hirudinaria manillensis, Hirudo medicinalis, Hirudo verbana, Hirudo troctina, Hirudo orientalis, and Whitmania pigra. Multiple sequence alignment performed using MEGA 7.0 and visualised via GeneDoc (Figure 1) revealed high sequence conservation, with black shading indicating strictly conserved residues and gray shading representing moderately conserved regions. This conservation pattern underscores the evolutionary stability and functional preservation of these hirudin variants.

Figure 1.

The alignment of hirudin HMg with 20 hirudin amino acid sequences is shown, where black indicates highly conserved regions and gray denotes moderately conserved regions.

Neighbor-Joining phylogenetic analysis (Figure 2) demonstrated that HMg clusters most closely with Hirullin-P18 from H. manillensis, sharing 90.62% sequence similarity.

Figure 2.

Phylogenetic tree of hirudin HMg and 20 hirudin variants, demonstrating the closest evolutionary relationship with Hirullin-P18.

Expression and purification of rHMg protein

Sequencing confirmed successful construction of the recombinant plasmid pET-HMg, encoding a 74-amino acid polypeptide with a theoretical molecular weight of ∼8 kDa, including restriction site sequences and a 6 × histidine tag. SDS-PAGE analysis revealed a predominant protein band (8–13 kDa) post-induction and purification (Figure 3). While gel migration anomalies precluded precise molecular weight determination due to differential denaturation effects and molecular marker resolution limits, denaturing mass spectrometry precisely identified the fusion protein at 8042.31 Da (Figure 4), aligning with theoretical predictions and confirming successful production of the His-HMg fusion protein.

Figure 3.

Electrophoresis analysis of the recombinant protein rHMg. Lane 1: Protein molecular weight marker; Lanes 2–7: Purified rHMg with loading amounts of 0.5, 0.75, 1, 1.25, 1.5, and 1.75 μg.

Figure 4.

Mass spectrometric analysis of rHMg, revealing a molecular weight of approximately 8042.31 Da.

Preliminary identification of rHMg anticoagulant activity

The anticoagulant activity of rHMg was preliminarily assessed using the porcelain plate thrombin titration method. Calculation via the specified formula revealed an antithrombin activity of 9573 ± 296.1 ATU/mg for rHMg.

Viscoelastic coagulation analysis, using the designated reagent kit and instrument, demonstrated that rHMg significantly prolonged whole blood ACT, reduced CR, and inhibited PF in a dose-dependent manner (Table 1).

Table 1.

Effects of rHMg on whole blood coagulation: prolonged ACT, reduced CR, and impaired PF. All experimental groups showed statistically significant differences compared to the control group (all p < 0.05), n = 3.

rHMg (nM)	ACT (s)	CR (coagulation signals/min)	PF
0	135	22.2	5.6
100	179	19.8	4.2
500	257	13.8	3.5
2500	781	10.8	0.3

Recombinant HMg prolongation of APTT, PT, and TT

The anticoagulant effects of rHMg on intrinsic and extrinsic coagulation pathways were evaluated using APTT, PT, and TT assay kits. Recombinant HMg significantly prolonged plasma APTT, PT, and TT (Figure 5), with half-maximal effective concentrations (EC₅₀) of 79.25 ± 7.00 nM, 1048 ± 176.70 nM, and 0.09 ± 0.12 nM, respectively. TT exhibited the highest sensitivity to rHMg inhibition (lowest EC₅₀), followed by APTT and PT. These results indicate that rHMg achieves anticoagulation through simultaneous modulation of thrombin activity and both intrinsic/extrinsic coagulation pathways.

Figure 5.

rHMg dose-dependently prolonged APTT, PT, and TT, with the most pronounced effect on TT, followed by APTT and PT. For the TT assay, the two concentrations of rHMg (740 and 1490 nM) were considered excessive, resulting in plasma failing to coagulate over an extended period. The error bars in all plots represent the standard deviation (SD) of the means, n = 3. All experimental groups showed statistically significant differences compared to the control group (all p < 0.05).

Inhibition constants (K_i) and half-maximal inhibitory concentration (IC₅₀)

Bivalirudin, a direct thrombin inhibitor clinically employed for cardiovascular therapeutics, served as the positive control. The chromogenic substrate S2238 (H-D-Phe-Pip-Arg-pNA-2HCl) specifically binds thrombin, releasing 4-nitroaniline upon hydrolysis. Comparative analysis via chromogenic substrate assay revealed rHMg’s superior thrombin-binding capacity and inhibitory efficacy relative to bivalirudin.

As shown in Figure 6 (p < 0.001), the inhibition constant (K_i) of rHMg (0.323 ± 0.144 nM) is 542-fold lower than that of bivalirudin (175.1 ± 65.4 nM), indicating that recombinant hirudin HMg has a stronger binding affinity for thrombin.

Figure 6.

Determination of inhibition constants (K_i) of bivalirudin and rHMg against thrombin. rHMg exhibited stronger binding affinity to thrombin than bivalirudin. (A) K_i for bivalirudin: 175.1 ± 65.4 nM; (B) K_i for rHMg: 0.323 ± 0.144 nM. Compared with the bivalirudin group, p < 0.001. Data are expressed as mean ± SD, n = 3.

Figure 7 shows rHMg exhibits a significantly lower IC₅₀ (2.8 ± 0.03 nM) compared to bivalirudin (376.0 ± 23.64 nM) (P < 0.001), reflecting a 135-fold increase in thrombin inhibition potency. This enhanced pharmacological profile highlights rHMg as a promising candidate for antithrombotic drug development.

Figure 7.

Determination of the half-maximal inhibitory concentration (IC₅₀) of bivalirudin and rHMg against thrombin. The IC₅₀ of rHMg (2.8 ± 0.03 nM) was 135-fold lower than that of bivalirudin (376.0 ± 23.64 nM), p < 0.001, indicating superior inhibitory efficacy of rHMg on thrombin activity. Data are expressed as mean ± SD. The error bars in the plot represent the standard deviation (SD) of the means, n = 3.

Discussion

The limited natural sources, complex extraction protocols, and low yields of native hirudin severely restrict its large-scale clinical application. These limitations have positioned recombinant genetic engineering as the predominant strategy for hirudin production. Although expression systems such as E. coli and yeast have been successfully utilised for recombinant hirudin preparation²⁸, prokaryotic expression—despite its cost-effectiveness and operational simplicity-faces critical challenges including inclusion body formation and the absence of essential post-translational modifications (e.g. tyrosine sulphation). Studies demonstrate that Tyr63 sulphation at the C-terminus of native hirudin is structurally critical for anchoring thrombin’s Exosite I via electrostatic interactions with basic residues (e.g. Lys81), forming stable salt bridges. Proper Tyr sulphation is indispensable for hirudin’s anticoagulant activity^29–31; however, this modification cannot be achieved in prokaryotic systems like E. coli, significantly compromising recombinant hirudin potency.

Multiple sequence alignment and phylogenetic analysis in this study revealed that the hirudin HMg shares 93.55% sequence identity with Hirullin-P18 from Hirudinaria manillensis, with critical residues in the active centre showing strict conservation. Notably, although recombinant HMg lacks Tyr63 sulphation, a post-translational modification critical for native hirudin activity, its C-terminal anionic cluster effectively anchors the basic pocket of thrombin’s Exosite I through charge compensation, thereby mitigating functional deficits caused by the absence of sulphation³².

In this study, rHMg was successfully expressed in E. coli BL21 using a prokaryotic expression system. Soluble expression was increased through low-temperature (18 °C) induction and extended induction time to 24 h. The purification method is simple, yielding about 36 mg of high-purity rHMg from 1 L of culture medium. Thrombin titration assay showed rHMg’s activity at 9573 ATU/mg, equivalent to 345 ATU per millilitre of culture medium, indicating its activity ranks relatively high among reported prokaryotically expressed recombinant hirudins. For example, Wang et al. reported rHV3 with 3.187 ATU/mL in C. glutamicum³³, and Kongwei et al. obtained recombinant hirudin with only 114 ATU/mg³⁴. Notably, rHMg achieved high efficacy without Tyr63 sulphation.

Recombinant hirudin HMg was used for whole blood anticoagulation. Thromboelastography evaluation showed its dose-dependent prolongation of ACT. With 2500 nM rHMg, ACT extended from a baseline of 135 s to 781 s. This value significantly exceeded clinical thresholds of 100–240 s. The compound also notably suppressed CR and PF. Coagulation regulation includes intrinsic, extrinsic, and common pathways³⁵. Coagulation tests showed rHMg most significantly prolonged TT, with an EC₅₀ of 0.09 nM. It also extended APTT with an EC₅₀ of 79.25 nM and PT with an EC₅₀ of 1048 nM, in descending order of efficacy. After comparison of the data, its anticoagulant efficacy was superior to most of the natural and recombinant hirudins reported to date^36,37. Mechanistically, rHMg exerts anticoagulant effects via two pathways. It directly inhibits thrombin activity, blocking the conversion of fibrinogen to fibrin. Also, it interferes with the thrombin - mediated positive feedback in the intrinsic pathway, which leads to the APTT effect. Recombinant HMg has a weaker impact on the extrinsic pathway (the PT effect), which is in line with the thrombin - independent activation mechanism of the VIIa - TF complex³⁸. Collectively, hirudin rHMg achieves potent anticoagulation by directly inhibiting thrombin activity and indirectly regulating the intrinsic and extrinsic coagulation pathways, highlighting its potential applications in extracorporeal circulation or blood preservation while establishing a foundation for further research into anticoagulant mechanisms.

Bivalirudin, a hirudin-derived synthetic peptide with molecular weight 2180 Da, approved by the FDA as a direct thrombin inhibitor (DTI) for percutaneous coronary intervention³⁹, faces clinical limitations including a short half-life (25 min) and requirement for continuous intravenous infusion⁴⁰. Our kinetic analysis revealed that under identical experimental conditions, rHMg demonstrated a 542-fold lower inhibition constant (K_i amounted to 0.323 nM) compared to bivalirudin (K_i was determined to be 175.1 nM). Its half - maximal inhibitory concentration was also remarkably superior, with an IC₅₀ of 2.8 nM, showing 135-fold better performance than bivalirudin’s IC₅₀ of 376 nM. The data demonstrate superior performance compared to previously reported recombinant hirudins. Boyle et al. detailed derivative peptides with IC₅₀ values of 140 nM and 2400 nM, along with K_i values of 290 nM and 54 nM⁴¹. It is worth noting that while Cho et al. determined the K_i for bivalirudin to be 1.78 ± 0.152 nM⁴², our study observed a significantly higher K_i of 175.1 nM for bivalirudin. This discrepancy may potentially be attributed to methodological differences in chromogenic substrate assays (S2238), which could be influenced by factors such as ionic strength or thermal fluctuations. Crucially, rHMg’s K_i remains lower than all comparators, underscoring its exceptional thrombin-binding capacity.

Conclusion

Although hirudin faces challenges including poor stability in vivo, susceptibility to proteases, low bioavailability, immunogenicity, and limited natural sources, these limitations can be addressed through protein engineering, drug delivery system development, and expression optimisation. This study established a prokaryotic expression system for high-efficiency production of recombinant hirudin rHMg without requiring post-translational modifications, achieving a specific activity of 9573 ATU/mg, showing a significant performance advantage over many reported recombinant variants and demonstrating up to 134-fold higher activity than the clinical drug bivalirudin. As the most potent direct thrombin inhibitor currently available, hirudin exhibits substantial potential in cardiovascular disease management, extracorporeal circulation anticoagulation, and blood preservation. The developed rHMg, with its high activity, low cost, and simplified production process, provides a novel candidate molecule for clinical translation of next-generation antithrombotic drugs, advancing the development of anticoagulant therapies.

Funding Statement

This research supported by grants from Natural Science Foundation of Jiangxi Province (20232BAB205022); Key Laboratory of Jiangxi Province for Biological Invasion and Biosecurity (2023SSY02111); Key Laboratory of Jiangxi Province for Functional Biology and Pollution Control in Red Soil Regions (2023SSY02051); Professor Zuhao Huang for the funding for gene synthesis: Jiangxi Double Thousand Plan (jxsq2023201063).

Ethics approval statement

This study involves the use of human blood samples from participants. The research protocol has been reviewed and approved by the Jinggangshan University Ethics Committee under Approval Number: (2024) lunpi (136). All human participants have signed the informed consent form prior to their involvement in the study. Research involving human participants was conducted in accordance with the Declaration of Helsinki.

Disclosure statement

The authors report no conflicts of interest.

Data availability statement

The data supporting this study are available from the corresponding author upon reasonable request. All figures in this study were prepared by the authors based on experimental data generated in this research.