Inhibiting thromboinflammation with a designed peptide that blocks the IL-1β-FXIa axis uncouples antithrombosis from bleeding risk

Abstract

The complex interplay between inflammation and coagulation drives thromboinflammatory disorders. While previous studies have shown that interleukin-1β (IL-1β) can induce a hypercoagulable state in response to tissue hypoxia and inflammation, the specific mechanisms of its action on the coagulation cascade remain unclear. Here, we demonstrate that IL-1β directly potentiates key coagulation factors (FXIa, FXa, thrombin, and kallikrein) to accelerate thrombosis. Mechanistically, IL-1β enhances FXIa enzymatic activity through specific binding to its exosite domain. Additionally, we established an IL-1β-FXIa interaction model, based on which the inhibitory peptide QK10 was derived to specifically disrupt their interaction interface and effectively block IL-1β’s potentiation of FXIa. Critically, in multiple thrombosis models, QK10 exhibited significant antithrombotic efficacy at doses equal to low molecular weight heparin (LMWH). Notably, bleeding assays revealed a significantly lower bleeding risk with QK10 compared to LMWH. Notably, QK10 also demonstrated promising anti-stroke efficacy in ischemic stroke models. Taken together, these findings establish QK10 as a viable therapeutic candidate that targets the IL-1β-FXIa prothrombotic axis for disruption, features enhanced safety, and offers a new approach to treating thrombo-inflammatory conditions, including ischemic stroke.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-026-06205-1.

Introduction

There is abundant evidence that the activation of coagulation is mediated by inflammatory activity [1–3]. While fibrin deposition secondary to inflammation serves a vital physiological role by spatially confining inflammatory responses to sites of injury or infection [4], it can also become profoundly pathological. This is exemplified by severe infection-associated coagulopathies like sepsis, and by the inflammation-rich microenvironments of ruptured atherosclerotic plaques, which drive thrombus formation and underpin acute arterial events [5]. Inflammation-induced coagulation activation is mediated by proinflammatory cytokines [6]. Multiple studies have shown that interleukin-6 (IL-6) significantly enhances thrombin generation by increasing tissue factor expression and inhibiting the anticoagulant system, such as reducing thrombomodulin [7, 8]. In addition, tumor necrosis factor (TNF-α) has been shown to activate platelets, promoting their aggregation and subsequent thrombus formation, particularly in the context of vascular injury [9, 10]. Despite the recognized influence of these proinflammatory cytokines, the precise regulatory mechanisms linking inflammation and thrombosis remain inadequately defined [11]. Elucidating these mechanisms is crucial for developing safer and more efficacious antithrombotic therapies. Current anticoagulants are limited by their poor specificity and consequent high bleeding risk [12–14]. Thus, there is an urgent need to explore novel antithrombotic targets with more precision and safer profiles.

IL-1β, a potent pro-inflammatory cytokine with central immunomodulatory functions, has emerged as a pivotal therapeutic target for inflammatory diseases [15]. A series of targeted therapeutics have been developed, including monoclonal antibodies and receptor antagonists [16]. Previous research has shown that administering 150 mg of canakinumab at three-month intervals significantly reduces the recurrence of cardiovascular events [17]. Concurrently, preclinical studies demonstrated that gevokizumab improves cardiac remodeling in heart failure rats [18]. These findings support IL-1β targeting as a promising therapeutic strategy. Beyond these anti-inflammatory effects, emerging evidence suggests that IL-1β plays a role in directly regulating coagulation. For instance, during neutrophil extracellular trap (NET) formation, IL-1β enhances tissue factor (TF) expression on activated neutrophils, thereby accelerating arterial thrombus development [19, 20]. Mechanistic investigations further reveal that IL-1β increases whole blood viscoelasticity, induces hypercoagulable phenotypes in erythrocytes [21]. In sepsis models, NLRP3 inflammasome-mediated IL-1β release upregulates TF expression, exacerbating endotoxin-induced coagulopathy [22]. Collectively, these findings underscore IL-1β as a critical mediator linking inflammation and hemostasis [23]. However, it remains unclear whether IL-1β affects coagulation through direct regulation of the coagulation cascade. Concurrently, existing IL-1β-targeted therapies (antibodies and receptor antagonists) face significant clinical limitations [24]. For instance, canakinumab treatment is associated with an increased risk of fatal infections and sepsis due to systemic immunosuppression, compromising long-term therapeutic safety [17, 25]. Further, IL-1β exerts regulatory effects on fibrinogen synthesis and thrombin generation, which are two core components of hemostasis [26]. Therefore, inhibiting IL-1β may disrupt the normal hemostatic balance and potentially elevate the risk of bleeding [27]. Hence, clarifying the direct mechanisms underlying IL-1β-mediated coagulation regulation is critical for overcoming the limitations of current therapies.

In the current study, we demonstrate that IL-1β directly potentiates key coagulation factors (thrombin, kallikrein, FXa, FXIa) to accelerate thrombosis. There is increasing evidence that a novel category of FXI-targeting agents can exert antithrombotic effects while maintaining normal hemostatic function [28]. Notably, we developed QK10, a peptide that disrupts IL-1β/FXIa interaction, and showed that it reduced thrombus formation and mitigated cerebral ischemic stroke in vivo with a low bleeding risk.

Methods

Ethics statement

Human plasma samples were collected from healthy volunteers with written informed consent. The study was approved by the Ethics Committee of The Affiliated Hospital of Qingdao University (No. QYFYWZLL28251) and complied with the Declaration of Helsinki.

The experimental protocols involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Qingdao University (Approval No. QDU-AEC-2025065). All procedures were conducted in strict accordance with internationally recognized guidelines for laboratory animal welfare and experimentation. BALB/c mice (8 weeks old) were obtained from HFK Bioscience Co., Ltd. (Beijing, China). The animal study was conducted following the ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines). We strictly applied ethical standards and the 3R principles (Replacement, Reduction, and Refinement) to minimize the number of animals used and optimize experimental protocols. Mice were randomly assigned to experimental groups by an independent researcher. All surgical and outcome assessments were performed by blinded investigators.

Recalcification time

Citrated plasma (from healthy donors) was obtained by centrifugation (3000 rpm, 30 min, 4 °C). IL-1β (HY-P73149, MCE) or QK10 in 10 µL H₂O were added to 40 µL plasma and incubated at 37 °C for 10 min. Subsequently, 50 µL of preheated 25 mM CaCl₂ (37 °C) was added. Absorbance at 650 nm was recorded kinetically using a microplate reader (Readmax 1500, Shanpu, Shanghai), and the time to reach 50% of maximal absorbance was defined as the coagulation time [29, 30].

Thromboelastography (TEG) analysis

A thromboelastograph analyzer (CWPS-8800, PMDT, China) was used to dynamically monitor blood coagulation, platelet aggregation, fibrinolysis, and other parameters during the coagulation process. After mixing 340 µL of whole blood with 20 µL of 0.2 M CaCl₂ in the experimental cup, we measured the clot reaction time (R time), clot kinetic time (K time), thrombus generation angle (α angle), and thrombus maximum amplitude (MA).

Effects of IL-1β on activity of coagulation factors

As we previously described [29–31], the effect of IL-1β on coagulation factors, including kallikrein, FXIIa, FXIa, FXa, FIXa, FVIIa, thrombin, plasmin, and antithrombin, was evaluated using the corresponding chromogenic substrates. Human α-thrombin (20 nM, HT 1002a, Enzyme Research Laboratories, USA) interacts with 0.2 mM chromogenic substrate S-2238 (Chromogenix AB, Sweden); kallikrein (20 nM, HPKa 1303, Enzyme Research Laboratories, USA) with 0.2 mM chromogenic substrate S-2302 (Chromogenix AB, Sweden); FXa (HFXa 1011, Enzyme Research Laboratories, USA) with 0.2 mM chromogenic substrate S-2222 (Chromogenix AB, Sweden); and FXIa (HFXIa, Enzyme Research Laboratories, USA) with 0.2 mM chromogenic substrate S-2366 (Chromogenix AB, Sweden). IL-1β and coagulation factors were combined in 60 µL of Tris-HCl buffer (1 M, pH 7.4) and incubated for 5 min at 37 °C, followed by the addition of 40 µL of the corresponding chromogenic substrates (0.15 mM). QK10’s effects on IL-1β’s potentiation of FXIa/FXa/thrombin/kallikrein was also investigated as the same method described above. The relative enzyme activity was quantitatively determined by measuring the substrate hydrolysis rate.

To determine whether IL-1β modulates the interaction between FXIa and its natural substrate FIX, we assessed FIX cleavage by FXIa in the presence of IL-1β. Briefly, FXIa and FIX (HFIX1009, Enzyme Research Laboratory, USA) were mixed and incubated in Tris-HCl buffer (1 M, pH = 7) for 3 min at 37 °C. IL-1β (20, 40, 80 ng/mL) was then added, and the incubation continued for an additional 30 min at 37 °C. Samples were boiled for 3 min and the effect of IL-1β on the enzymatic activity of FXIa against FIX was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue.

Immunoprecipitation

Normal plasma (0.5 µL) was immunoprecipitated with 2 µg of IL-1β antibody (50101-R001, Sino Biological, China) and 20 µL of protein A agarose (P2051, Beyotime, China) in 200 µL of phosphate-buffered saline (PBS; pH 7.4, 0.01 M) at 4 °C for 16 h. Following incubation, the mixture was centrifuged at 2,500 rpm for 5 min at 4 °C and washed seven times with PBST (PBS + 0.05% Tween 20). Subsequently, 10 µL of 1 × protein loading buffer (P1040, Solarbio, China) was added, and the sample was boiled for 3 min. SDS-PAGE was performed using a 12% polyacrylamide gel. The primary antibodies used for detection of the target proteins were as follows: anti-FXIa (dilution 1:1000, MabHFXI, Enzyme Research Laboratory, USA), anti-human kallikrein (dilution 1: 1000, ab1006, Abcam, USA), anti-FXa (dilution 1: 1000, MabHFX, Enzyme Research Laboratory, USA), anti-thrombin (dilution 1: 1000, P00734, Abmart, China) antibodies.

Pull-down assays

Protein A agarose (20 µL; P2051, Beyotime) was first incubated with an anti-IL-1β antibody (50101-R001, Sino Biological) and IL-1β in 200 µL of PBS (0.01 M, pH 7.4) at 4 °C for 2 h. Subsequently, the beads were washed with washing buffer. The beads were then incubated with the potential interacting proteins (thrombin, kallikrein, FXIa, and FXa) in 200 µL of PBS at 4 °C for 16 h. Finally, following separation by SDS-PAGE on a 12% polyacrylamide gel, proteins were immunoblotted with antibodies against IL-1β, FXIa, kallikrein, FXa, and thrombin to assess their interactions.

Protein-protein docking and peptide synthesis

For the simulation of the IL-1β-FXIa/FXa/thrombin/kallikrein complex, we employed the structures of IL-1β (PDB ID: 8C3U), FXIa (PDB ID: 7MBO), FXa (PDB ID: 4BTI), thrombin (PDB ID: 1TB6), and kallikrein (PDB ID: 5TJX) to conduct protein docking through ZDOCK. As a tool for predicting protein-protein complexes, ZDOCK takes shape complementarity, electrostatics, and desolvation free energy into consideration. The docking process was directed by activity and surface plasmon resonance experimental data. It involved the inclusion of interface-disrupting residues and the exclusion of those residues that had no impact on binding. Eventually, the most favorable ZDOCK pose representing the interaction between IL-1β and FXIa was chosen as the final result. The peptides, including QK10 (QEIIIHDQYK), YE5 (YKMAE), TS16 (TTVNYTDSQRPISLPS), AK11 (AHCFYGVESPK), and HG12 (HTTSPTQRHLCG), were synthesized by GL Biochem (Shanghai, China) with a purity exceeding 98%.

Activated partial thromboplastin time (APTT) and prothrombin time (PT) assays The APTT was measured by incubating 100 µL of plasma with 100 µL of APTT reagent (Product No. R41056-50T, Yuanye, China) for 5 min at 37 °C, followed by the addition of 100 µL of preheated 37 °C CaCl₂ (25 mM) to initiate clotting. The PT assay was performed using a PT assay kit (Product No. GMS10176, Genmed Scientifics, USA) according to the manufacturer’s instructions. Briefly, 100 µL of plasma was incubated at 37 °C for 3 min, and clotting was then initiated by adding 200 µL of pre-warmed PT reagent.

FeCl₃-induced carotid artery thrombosis

Following our previous studies [29, 30], mice were anesthetized with isoflurane via an anesthetic respirator (R540IP, RWD Life Science, China), and the carotid artery was exposed through a longitudinal incision in the carotid region. Thrombosis was induced by placing filter paper discs soaked in 10% FeCl₃ onto the carotid arteries. Blood flow in the carotid arteries was monitored for 60 min or until complete occlusion using a laser speckle perfusion imager (RFLSI III, RWD Life Science, China). Blood flow was quantified in perfusion units (PU) throughout this period.

Mouse deep vein thrombosis (DVT) model

We established the DVT model using the standard methods from our previous publications [29, 30]. The anesthetized mice were placed supine on the operating table, and the abdomen was incised for 2.5 cm in the middle of the abdomen, and the abdominal wall muscles were separated and the intestinal tubes were pushed away to expose the posterior peritoneum. The inferior vena cava (IVC) was freed under a stereomicroscope, and the dorsal lumbar vein and bilateral branches of the genitourinary vein were ligated. The trunk of the IVC was doubly ligated with a 7 − 0 suture 5 mm below the renal vein until the lumen was occluded. Subsequently, the muscular layer and peritoneal closure were closed with 4 − 0 and 5 − 0 sutures, respectively. Mice were killed 24 h after surgery, and the ligated sections of the IVC were rinsed with pre-cooled PBS. The thrombus tissue was fixed in 10% neutral formalin for 24 h, embedded in paraffin wax, and then cut into 5-µm slices and stained with HE staining to visualize the pathologic pattern. Thrombus weight was measured after drying.

Mouse-tail thrombosis model induced by Carrageenan

To induce thrombosis, as previously described in our study [29–31], mice were administered an intraperitoneal injection of 1% Type I Carrageenan (60 mg/kg, 9062-07-1, Coolaber, China) and then housed at 16 °C. After this, the lengths of the thrombotic regions in the tails were measured and photographed at the 24-hour time point. Finally, pathological changes in the mouse tails were examined through H&E staining.

Bleeding time measurement

Bleeding duration was quantified through a standardized tail transection protocol adapted from previous methodologies [29, 30]. In brief, a 4.5 mm segment was excised from the distal tail tip using aseptic technique, and the wound was immediately immersed in 20 mL saline solution preheated to 37 °C. Hemostasis duration was tracked for up to 40 min to avoid lethal blood loss, with continuous observation until complete cessation of bleeding. This temporal threshold was implemented in accordance with institutional animal care guidelines to ensure ethical compliance.

Saphenous vein bleeding model

The saphenous vein bleeding model was performed as previously described in our established protocols [29, 30]. Briefly, the saphenous vein was surgically exposed and subjected to partial transection, with the time to initial hemostasis recorded. Following thrombus removal to induce rebleeding, the procedure was iterated over a 30-min observational period.

Mice stroke model

A mouse model of transient middle cerebral artery occlusion (tMCAO) was established, as previously described [13]. Mice were anesthetized with isoflurane using an anesthetic ventilator. A longitudinal incision was made in the neck to expose the common, internal, and external carotid arteries. The external carotid artery was ligated, and the common carotid artery was clamped. An incision was made proximal to the ligature of the external carotid artery, and a monofilament (6023910PK10, Doccol, Sharon, MA) was inserted through the bifurcation of the common carotid artery into the internal carotid artery. The monofilament was advanced until slight resistance was felt, then fixed in place and removed after 1 h. The brains were harvested after 24 h, and 2 mm thick coronal sections were prepared using a Rodent Brain Matrice (Harvard Apparatus, Holliston, MA). These sections were stained with 2% 2,3,5-triphenyl tetrazolium chloride (TTC, Sigma, St. Louis, MO) to assess the ischemic area.

Statistical analysis

Data from independent experiments are presented as mean ± standard deviation (SD). All statistical analyses were performed using a two-tailed test with a 95% confidence interval (CI). The normality of the data was assessed using the Kolmogorov-Smirnov test (K-S test). A one-way analysis of variance (ANOVA) was performed, followed by post hoc Dunnett’s test. Unpaired t-tests were used for between-group comparisons, and the Mann-Whitney U test was employed for non-parametric data. Data analysis was conducted using Prism 9.5 (GraphPad Software, USA) and SPSS 26.0 (SPSS Inc, USA). Differences were considered statistically significant at p < 0.05.

Results

IL-1β promotes coagulation in a concentration-dependent manner

As a key inflammatory mediator, IL-1β is hypothesized to be a critical link between inflammation and hypercoagulability [32], but its direct effects on the coagulation cascade remain incompletely characterized. Our investigation demonstrates that IL-1β significantly shortens human plasma recalcification time in a concentration-dependent manner (from 13.22 to 10.50 and 9.30 min; Fig. 1a, b). Furthermore, TEG analysis revealed that treatment with IL-1β led to a decrease in clot reaction time (8.02 min vs. 6.9 min vs. 5.9 min) and clot kinetic time (4.22 min vs. 2.86 min vs. 2.74 min), concurrent with increased α angle (49.26° vs. 57.04° vs. 60.6°), while maximum amplitude showed no significant alterations (Fig. 1c, g). Collectively, these findings indicate that IL-1β promotes a hypercoagulable state by accelerating the initiation of coagulation and the rate of fibrin clot formation.

Fig. 1.

IL-1β accelerates the coagulation process. a, b IL-1β (20 ng/mL, 40 ng/mL) shortens the recalcification time of human plasma in a dose-dependent manner. c-g Effects of IL-1β (20 and 40 ng/mL) on thromboelastography parameters, including reaction time (R), kinetics time (K), angle (α), and maximum amplitude (MA). Data represent mean ± SD of 5 independent experiments, **P < 0.01 by one-way ANOVA with Dunnett’s post hoc test. NC, negative control, ns.: no significance

IL-1β potentiates FXIa, kallikrein, FXa, and thrombin

We next investigated the mechanism of IL-1β-induced hypercoagulability by assessing its direct effects on the activity of multiple coagulation factors. As shown in Fig. 2a, b, treatment with IL-1β at 5, 10, and 20 ng/mL increased the activity of FXIa by ~ 2.6-, ~ 4.9-, and ~ 12.5-fold, kallikrein by ~ 2.9-, ~ 6.5-, and ~ 8.7-fold, FXa by ~ 1.1-, ~ 2.9-, and ~ 4.5-fold, and thrombin by ~ 1.2-, ~ 1.3-, and ~ 1.7-fold, respectively. However, it did not affect the activity of FVIIa, FIXa, FXIIa, APC, and plasmin (Fig. 2c). Using immunoprecipitation analysis, we revealed a physiological interaction between IL-1β and FXI/prekallikrein/prothrombin/FX in plasma (Fig. 2d). Pull-down experiments further demonstrated that IL-1β directly bound to kallikrein, FXa, FXIa, and thrombin (Fig. 2e). Furthermore, IL-1β increased the enzymatic activity of FXIa toward its natural substrate, FIX. The results demonstrated that at concentrations of 20, 40, and 80 ng/mL, IL-1β enhanced FXIa’s ability to hydrolyze FIX by approximately ~ 0.6-, ~ 1.1-, and ~ 1.5-fold, respectively (Fig. 2f, g). We employed Lineweaver-Burk plots to assess kinetic activity of FXIa, with and without IL-1β, revealing a reduced Michaelis constant (Km) and increased maximum velocity of reaction (Vmax) value in the IL-1β-treated group (Fig. S1).

Fig. 2.

IL-1β enhances the activity of FXIa, kallikrein, FXa, and thrombin. a, b Potentiating effect of IL-1β (5 ng/mL, 10 ng/mL, 20 ng/mL) on FXIa, kallikrein, FXa, and thrombin and statistics of their relative activities. c IL-1β (5 ng/mL, 10 ng/mL, 20 ng/mL) shows no significant effect on the activity of FVIIa, FIXa, FXIIa, APC, or plasmin. d Co-immunoprecipitation of IL-1β with Thrombin, Kallikrein, FXIa, or FXa in human normal plasma. IB, immunoblotting; IP, immunoprecipitation. e Pull-down assay analysis of the interaction between thrombin, kallikrein, FXIa, or FXa and IL-1β. f Representative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of FIX hydrolysis by FXIa (lane 1: 500 ng FIX; lane 2: 500 ng FIX + 500 ng FXIa; lane 3, 4, 5: 500 ng FIX + 500 ng FXIa + 80, 40, or 20 ng/mL IL-1β, respectively) and experimental combinations indicated by “+” or “-“. g Quantification of panel f is shown below. Data represent mean ± SD of 3 to 5 independent experiments, *P < 0.05, **P < 0.01 by one-way ANOVA with Dunnett’s post hoc test. NC, negative control

Inhibitory peptide QK10 inhibits IL-1β’s potentiation on FXIa

Growing evidence indicates that a novel class of drugs targeting FXI is capable of preventing thrombosis while preserving hemostasis [28, 33], and coupled with the fact that IL-1β exerts its most potent effect on FXIa, we therefore focused our mechanistic investigation on this specific interaction. We established an IL-1β-FXIa protein interaction docking model (Fig. 3a), as well as potential IL-1β-FXa/thrombin/kallikrein interaction models (Fig. S2a-c). To accurately identify the interaction sites between IL-1β and FXIa, we designed five peptides derived from the FXIa sequence (QK10: QEIIIHDQYK; YE5: YKMAE; TS16: TTVNYTDSQRPISLPS; AK11: AHCFYGVESPK; HG12: HTTSPTQRHLCG) aimed at disrupting the IL-1β-FXIa protein interaction based on IL-1β-FXIa interaction model. Plasma recalcification assays revealed that QK10 inhibited plasma coagulation, while the other inhibitory peptides had no significant effect (Fig. 3b, c). Enzymatic kinetic assays confirmed that QK10 concentration-dependently counteracted IL-1β-induced potentiation of FXIa activity against chromogenic substrates (Fig. 3d, e). Furthermore, the plasma recalcification time was prolonged from the original 5.9 min to 7.8 min and 9.3 min at QK10 concentrations of 15 µΜ and 30 µΜ, respectively (Fig. 3f, g). Pull-down assay was performed to investigate the effect of QK10 on the binding interaction between IL-1β and FXIa. As shown in Fig. 3h, IL-1β can directly bind to FXIa, and QK10 can attenuate the interaction between them in a concentration-dependent manner. These findings indicate that the critical region mediating the interaction between IL-1β and FXIa is located within the exosite loop of the FXIa protease domain, which corresponds to the sequence of peptide QK10. Therefore, the QK10 peptide can block the IL-1β-FXIa interaction by competitively binding to IL-1β. Additionally, we assessed effects of QK10 on the IL-1β-FXa/thrombin/kallikrein interaction. As illustrated in Fig. S3a-c, pull-down assay demonstrated that QK10 did not influence IL-1β-FXa/thrombin/kallikrein interaction. Additionally, QK10 can not reverse IL-1β’s potentiation of FXa/thrombin/kallikrein (Fig. S3d-f).

Fig. 3.

QK10 inhibits the potentiation of FXIa by IL-1β. a A structural model from molecular docking provides the predicted three-dimensional model demonstrating the binding of human IL-1β (green) to the exosite loop of FXIa (red). b, c To interfere with the IL-1β-FXIa protein interaction, five peptides (QK10: QEIIIHDQYK; YE5: YKMAE; TS16: TTVNYTDSQRPISLPS; AK11: AHCFYGVESPK; HG12: HTTSPTQRHLCG) were designed based on the FXIa sequence and used to perform plasma recalcification assays. d, e The peptide QK10 (15 µM, 30 µM) was tested for its ability to inhibit the potentiation of FXIa activity by IL-1β. f, g Dose-dependent inhibition of plasma recalcification by QK10 (15 µM, 30 µM). h Pull - down assays were conducted to explore the interaction between FXIa and IL-1β, as well as the impact of peptide QK10 (lane 1: IgG + FXIa, lane 2: IL-1β mAb + FXIa, lane 3, 4: IL-1β mAb + FXIa with peptide QK10 at concentrations of 15 µM and 30 µM). Data represent mean ± SD of 5 independent experiments, **P < 0.01 by one-way ANOVA with Dunnett’s post hoc test. NC, negative control, ns.: no significance

QK10 shows potent antithrombotic efficacy and low bleeding risk

We firstly evaluated preliminary plasma stability, immune activation, and toxicity of QK10. PK/PD modeling using non-compartmental analysis and a sigmoidal Emax model was included, and key parameters are shown in Table S1. The time-course correlation revealed that although QK10 is rapidly cleared, its antithrombotic effect significantly outlasts plasma presence, indicating that brief exposure suffices for a durable response (Fig. S4a-d, Table S1). QK10 treatment (20 µM) showed no significant changes in the plasma levels of pro-inflammatory cytokines, e.g., TNF-α and IL-6 (Fig. S4e, f). In addition, peptide QK10 had no significant inhibitory effect on cell viability, even at the concentration of 640 µM (Fig. S4g). To investigate the anticoagulant function of QK10 in vivo, a remarkably low single dose of only 0.2 mg/kg was administered intravenously to mice. As illustrated in Fig. 4a, b, treatment with QK10 at 0.2 mg/kg resulted in a significant prolongation of APTT (from 29.8 s to 52.6 s) and PT (from 15 s to 29.8 s), demonstrating potent systemic anticoagulant activity even at a low dose. The role of QK10 in hypercoagulability and thrombosis was further investigated using a mouse carotid artery thrombosis model induced by 10% FeCl₃, with low molecular weight heparin (LMWH) (S26247, Yuanye Bio-Technology, China) used as a positive control. As depicted in Fig. 4c, d, the carotid artery occlusion time was 10.8 and 16.8 min in mice injected with 0.1 and 0.2 mg/kg QK10, respectively, whereas those in the LMWH and negative control groups were 9.7 and 7.3 min, respectively. In addition, we demonstrated that QK10 also significantly suppressed thrombus formation in a deep vein thrombosis model under the same experimental groupings (Fig. 4e-h).

Fig. 4.

QK10 exerts anti-thrombotic effects with a low risk of bleeding. a, b The mice were injected with QK10 (0.2 mg/kg) via intravenous injection. Effects of QK10 on APTT and PT. Saline, LMWH (0.2 mg/kg), and QK10 (0.1 mg/kg, 0.2 mg/kg) were injected intravenously into the mice. c, d Carotid blood flow in FeCl₃-treated mice was observed using laser speckle perfusion imaging to quantify changes in blood flow and calculate the time to vascular occlusion in mice. e, f Representative images of IVC thrombus specimens and statistical analysis of thrombus length. g, h Histological staining sections of IVC thrombus and statistical analysis of thrombus weight. i The mice were given saline, and QK10 (0.1 mg/kg, 0.2 mg/kg) intravenously. j At 6 h after carrageenan injection, thrombus formation was quantified by measuring the length of the tail vein thrombus and photographed. k H&E staining of the tail sections. l Statistical analysis of thrombus area. m, n After QK10 (0.2 mg/kg) or LMWH (0.2 mg/kg) was injected intravenously into the mice, the effect of QK10 and LMWH on the time of bleeding in the tail and saphenous vein were recorded. Data represent mean ± SD (n = 5), *P < 0.05, **P < 0.01 by one-way ANOVA with Dunnett’s post hoc test or t-test. ns.: no significance

Thrombosis is intimately linked to inflammation [6]. Carrageenan triggers systemic inflammation in mice, leading to subsequent thrombus formation in the tail [29, 30]. In this model, QK10 significantly reduced tail thrombus formation in mice in a dose-dependent manner (Fig. 4i-l). These results suggest that IL-1β mediates the cross talk between inflammation and thrombus. To evaluate the bleeding risk associated with inhibitory peptide QK10, we employed the mouse tail hemorrhage model and the saphenous vein hemorrhage model (with LMWH as a positive control). The bleeding time in QK10-treated mice was significantly shorter than that in LMWH-treated controls (tail bleeding: 10.7 min vs. 15.1 min; saphenous bleeding: 46.6 s vs. 105.4 s), indicating bleeding risk of QK10 is lower than that of LMWH (Fig. 4m, n). The long-term safety of QK10 depends on whether a prothrombotic rebound occurs after its effect wears off. To assess this risk, we systematically monitored thromboelastography in mice at multiple time points (3, 12, 24, and 48 min) post-administration, spanning from peak efficacy to clearance of QK10. As illustrated in the Fig. S5a-e, no risk of thrombosis rebound after QK10 wore off.

QK10 attenuates ischemic stroke in mice

The potent pro-inflammatory cytokine IL-1β plays a crucial role in stroke pathophysiology, driving neuroinflammation and exacerbating neuronal damage, which leads to acute and long-term neurological deficits [34]. We next evaluated whether targeting the IL-1β/FXIa axis with QK10 confers protection in a murine tMCAO model. The cerebral infarct volume in mice treated with QK10 was significantly lower than that in control group. QK10 treatment dose-dependently attenuated cerebral infarct volume (0.1 mg/kg: 41.4%; 0.2 mg/kg: 28.2%) compared to the control group (56.1%) (Fig. 5a, b). Furthermore, QK10-treated mice exhibited significantly lower Bederson scores and higher Grip test scores than control mice, indicating improved neurological and motor function (Fig. 5c, d).

Fig. 5.

Treatment with the QK10 attenuates cerebral ischemic stroke in mice. a Representative images of TTC-stained brain slices demonstrating infarct areas. b Statistical graph of infarct volume percentage. c, d Bederson neurological severity scores and grip test scores. Data represent mean ± SD (n = 6), **P < 0.01 by one-way ANOVA with Dunnett’s post hoc test

Discussion

The intricate crosstalk between inflammation and coagulation underpins the pathogenesis of thromboinflammatory disorders [6], and the majority of critically ill patients with a systemic inflammatory response manifest coagulopathy [35]. Substantial evidence indicates that the activation of coagulation is mediated by inflammatory activity [4, 36]. The activation of coagulation and deposition of fibrin induced by inflammation represent a key mechanism that confines inflammatory activity to sites of injury or infection [37, 38]. However, inflammation-driven coagulation may also significantly contribute to disease progression, as seen in coagulation dysfunction associated with severe infections such as sepsis [39]. Furthermore, the pathological basis of acute arterial thrombotic events lies in the formation of thrombin following the rupture of atherosclerotic plaques rich in inflammatory cells [39]. Current knowledge regarding the modulation of coagulation, hemostasis, and thrombosis by the innate immune system, particularly through inflammatory cytokines, remains incomplete and necessitates more thorough investigation [2]. Consequently, an ideal therapeutic strategy for thromboembolism should effectively counteract aberrant inflammation and coagulation while preserving physiological hemostasis and immune competence [29, 30]. The present study demonstrates that IL-1β directly potentiates key coagulation factors (thrombin, kallikrein, FXa, FXIa) to accelerate thrombogenesis. The developed peptide QK10 targets IL-1β to disrupt the IL-1β/FXIa interaction, thereby reducing thrombus formation, alleviating cerebral ischemic stroke in vivo, and with a low bleeding risk.

Proinflammatory cytokines serve as the primary mediators in inflammation-induced coagulation activation [40]. For instance, studies have demonstrated the pivotal role of IL-6 in initiating coagulation activation, as well as the involvement of TNF-α and IL-1 in regulating physiological anticoagulation [41, 42]. An increasing body of evidence suggests considerable bidirectional connections between the inflammatory and coagulation systems, with inflammation stimulating coagulation and coagulation considerably modulating inflammatory activity [4, 36, 43]. Coagulation modulates inflammatory activity, which is then stimulated by cellular receptors on the surfaces of inflammatory and endothelial cells [44]. Yet the precise mechanisms by which pro-inflammatory cytokines drive hypercoagulability remain incompletely defined. Previous studies have demonstrated that IL-1β, as a central proinflammatory cytokine, plays a pivotal role in linking inflammation and coagulation [6]. It promotes the formation of NETs [19], upregulates procoagulant mediators such as TF [19] and plasminogen activator inhibitor-1 (PAI-1) [45, 46], and impairs nitric oxide-dependent anticoagulant signaling pathways [47]. Circulating levels in both normal and inflammatory conditions typically remain in the pg/mL range (from low pg/mL up to hundreds of pg/mL). Most studies exploring IL-1β-mediated effects in monocytes/macrophages, endothelial cells, smooth muscle cells, and fibroblasts utilize IL-1β concentrations in the ~ ng/mL range [48–50]. In the current study, we used concentrations in the ng/ml range to approximate the elevated local levels likely present within the thrombotic microenvironment. In the current study, we identify IL-1β as a direct activator of key coagulation factors, FXIa, FXa, thrombin, and kallikrein, with FXIa emerging as its primary target. While IL-1β is established as a central driver of inflammation-induced TF upregulation, our study reveals that it can directly modulate coagulation factors independently of transcriptional regulation [19]. Therefore, targeting the specific interaction between IL-1β and FXIa presents a promising therapeutic strategy for attenuating thromboinflammation without compromising hemostasis.

Furthermore, targeting FXIa confers a safer therapeutic profile than antagonizing other coagulation factors, as it is associated with a lower risk of bleeding complications and consequently provides a broader therapeutic window [31, 34]. Unlike traditional anticoagulants that systemically inhibit coagulation factors essential for hemostasis (such as thrombin or FXa), FXIa inhibition specifically attenuates the amplification phase of coagulation while leaving the initiation phase intact [51–53]. As demonstrated in our study, IL-1β improves FXIa activity via binding to the 456–465 amino acid portion of the FXIa exosite loop. Critically, based on this binding site, we synthesized the inhibitory peptide QK10, which selectively disrupts the IL-1β-FXIa interface. QK10 exhibited robust antithrombotic effects with minimal hemorrhagic risk. Administered at equimolar doses, QK10 demonstrated dose-dependent prolongation of FeCl₃-induced carotid occlusion time relative to controls and exerted potent suppression of IVC thrombogenesis. Furthermore, QK10 significantly reduced cerebral infarct volume, highlighting its dual role in mitigating thromboinflammation and ischemia-reperfusion injury. QK10 is designed to mitigate the risk of bleeding complications associated with prevailing antithrombotic regimens. Additionally, while QK10 exhibits a relatively short plasma half-life, its rapid action may be sufficient for the intended therapeutic window. Future studies will focus on structural modifications to enhance its pharmacokinetic profile.

In summary, we identify IL-1β as a direct enhancer of coagulation factor activity and establish QK10 as a mechanistically novel antithrombotic agent that confers a low risk of bleeding. Our study provides a targeted intervention strategy for the treatment of ischemic stroke, while advancing our understanding of the crosstalk between innate immunity and coagulation.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IL-1β

Interleukin-1β

IL-6

Interleukin-6

TNF-α

Tumor Necrosis Factor-α

NET

Neutrophil Extracellular Trap

TF

Tissue Factor

TEG

Thromboelastography

R time

Reaction Time

K time

Kinetics Time

α angle

Alpha Angle

MA

Maximum Amplitude

SDS-PAGE

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

PBS

Phosphate-Buffered Saline

PBST

Phosphate-Buffered Saline with Tween-20

SPR

Surface Plasmon Resonance

APTT

Activated Partial Thromboplastin Time

PT

Prothrombin Time

PV

Plasma Viscosity

DVT

Deep Venous Thrombosis

IVC

Inferior Vena Cava

tMCAO

Transient middle cerebral occlusion

TTC

2,3,5-Triphenyltetrazolium Chloride

SD

Standard Deviation

CI

Confidence Interval

K-S test

Kolmogorov-Smirnov Test

ANOVA

Analysis of Variance

LMWH

Low-Molecular-Weight Heparin

PAI-1

Plasminogen Activator Inhibitor-1

HUVECs

Human umbilical vein endothelial cells

CCK8

Cell counting kit-8

ELISA

Enzyme-linked immunosorbent assay

Author contributions

X.T., M.X. and Y.W. designed and conceptualized the research; Y.W., Q.Y., X.J., W.L., M.Y. and C.L. performed the experiments and analyzed the data; S.W. and M.W. established the animal model and performed the behavioral tests; S.L. and X.J. collected the animal samples; X.T. wrote the manuscript; M.X. and Y.W. revised the manuscript. All authors have reviewed the final version of the manuscript and approved its submission.

Data availability

The data are available upon reasonable request from the corresponding author.

Declarations

Competing interests

The authors declare that they have no competing interests.