Structural, Biochemical Characterization and Molecular Mechanism of Cerastokunin: A New Kunitz-Type Peptide with Potential Inhibition of Thrombin, Factor Xa and Platelets.

The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75 kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% ß-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6 mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration.

Conformation of factor Xa in solution revealed by single-molecule spectroscopy.

BACKGROUND: All current X-ray structures of factor (F)Xa are devoid of the γ-carboxyglutamate (Gla) domain and fail to reveal the overall conformation of the free protein. The recent cryogenic electron microscopy (cryo-EM) structure of FXa in the prothrombinase complex is the only structure of full-length FXa and shows that the Gla domain is positioned at an angle relative to the epidermal growth factor 1 domain. OBJECTIVES: Establish if the curved conformation of FXa revealed by cryo-EM is also present in solution. METHODS: The conformation of FXa in solution was studied by single-molecule Förster resonance energy transfer. RESULTS: The conformation of full-length FXa in solution is resolved for the first time. The conformation is curved and extremely sensitive to Ca2+. It does not differ significantly from its zymogen form or from that present in the prothrombinase complex free or bound to the physiologic substrates prothrombin and meizothrombin. CONCLUSION: Measurements by single-molecule Förster resonance energy transfer reveal that FXa has a curved conformation in solution, free or bound to physiologic ligands, and validate the recent cryo-EM structures of prothrombinase. The drastic conformational changes observed in the absence of Ca2+ suggest that the structural architecture of FXa changes upon administration of vitamin K antagonists that perturb the interaction of the Gla domain with divalent cations.

Bait and Cleave: Exosite-Binding Peptides on Quantum Dots Selectively Accelerate Protease Activity for Sensing with Enhanced Sensitivity.

The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological roles─substrate, receptor, and inhibitor─and were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.

Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors.

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

Changes in Internal Structure and Dynamics upon Binding Stabilise the Nematode Anticoagulant NAPc2.

Abnormal blood coagulation is a major health problem and natural anticoagulants from blood-feeding organisms have been investigated as novel therapeutics. NAPc2, a potent nematode-derived inhibitor of coagulation, has an unusual mode of action that requires coagulation factor Xa but does not inhibit it. Molecular dynamics simulations of NAPc2 and factor Xa were generated to better understand NAPc2. The simulations suggest that parts of NAPc2 become more rigid upon binding factor Xa and reveal that two highly conserved residues form an internal salt bridge that stabilises the bound conformation. Clotting time assays with mutants confirmed the utility of the salt bridge and suggested that it is a conserved mechanism for stabilising the bound conformation of secondary structure-poor protease inhibitors.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Molecular Dynamics Simulation Study of the Selective Inhibition of Coagulation Factor IXa over Factor Xa.

Thromboembolic disorders, arising from abnormal coagulation, pose a significant risk to human life in the modern world. The FDA has recently approved several anticoagulant drugs targeting factor Xa (FXa) to manage these disorders. However, these drugs have potential side effects, leading to bleeding complications in patients. To mitigate these risks, coagulation factor IXa (FIXa) has emerged as a promising target due to its selective regulation of the intrinsic pathway. Due to the high structural and functional similarities of these coagulation factors and their inhibitor binding modes, designing a selective inhibitor specifically targeting FIXa remains a challenging task. The dynamic behavior of protein-ligand interactions and their impact on selectivity were analyzed using molecular dynamics simulation, considering the availability of potent and selective compounds for both coagulation factors and the co-crystal structures of protein-ligand complexes. Throughout the simulations, we examined ligand movements in the binding site, as well as the contact frequencies and interaction fingerprints, to gain insights into selectivity. Interaction fingerprint (IFP) analysis clearly highlights the crucial role of strong H-bond formation between the ligand and D189 and A190 in the S1 subsite for FIXa selectivity, consistent with our previous study. This dynamic analysis also reveals additional FIXa-specific interactions. Additionally, the absence of polar interactions contributes to the selectivity for FXa, as observed from the dynamic profile of interactions. A contact frequency analysis of the protein-ligand complexes provides further confirmation of the selectivity criteria for FIXa and FXa, as well as criteria for binding and activity. Moreover, a ligand movement analysis reveals key interaction dynamics that highlight the tighter binding of selective ligands to the proteins compared to non-selective and inactive ligands.

The Interaction of Factor Xa and IXa with Non-Activated Antithrombin in Michaelis Complex: Insights from Enhanced-Sampling Molecular Dynamics Simulations.

The interaction between coagulation factors Xa and IXa and the activated state of their inhibitor, antithrombin (AT),have been investigated using X-ray diffraction studies. However, only mutagenesis data are available for non-activated AT. Our aim was to propose a model based on docking and advanced-sampling molecular dynamics simulations that can reveal the conformational behavior of the systems when AT is not binding a pentasaccharide. We built the initial structure for non-activated AT-FXa and AT-FIXa complexes using HADDOCK 2.4. The conformational behavior was studied using Gaussian accelerated molecular dynamics simulations. In addition to the docked complexes, two systems based on the X-ray structures were also simulated, with and without the ligand. The simulations revealed large variability in conformation for both factors. In the docking-based complex of AT-FIXa, conformations with stable Arg150-AT interactions can exist for longer time periods but the system also has a higher tendency for reaching states with very limited interaction with the "exosite" of AT. By comparing simulations with or without the pentasaccharide, we were able to gain insights into the effects of conformational activation on the Michaelis complexes. RMSF analysis and correlation calculations for the alpha-carbon atoms revealed important details of the allosteric mechanisms. Our simulations provide atomistic models for better understanding the conformational activation mechanism of AT against its target factors.

A molecular dynamics simulation study to understand the effect of cholesterol and tissue factor palmitoylation on tissue factor-factor VIIa-factor Xa ternary complex in different lipid environments.

BACKGROUND: Tissue factor (TF), a transmembrane glycoprotein, plays a profound role in the formation of the tissue factor-factor VIIa (TF-FVIIa) complex that initiates factor Xa (FXa) generation followed by thrombin activation and clot formation. Previous reports suggest that TF-FVIIa coagulant activity at the cell surface may be affected by various processes, including changes in cholesterol content and posttranslational modifications of TF. Numerous studies were conducted but yielded inconclusive results about the effect of cholesterol on TF expression. OBJECTIVE: The present study aimed to understand how cholesterol affects structural modulations on the tissue factor-factor VIIa-factor Xa ternary complex (TF-FVIIa-FXa). Additionally, we aimed to illustrate the effect of palmitoylation on the Cys245 residue of TF and understand its structural implications on the TF-FVIIa-FXa. METHODS: We set up the following 4 systems in different lipid environments: TF-FVIIa-FXa in POPC:POPS (CS), TF-FVIIa-FXa in POPC:POPS:CHOL (CSL), Palmitoylated TF-FVIIa-FXa in POPC:POPS:CHOL (CSLP), and Palmitoylated TF-FVIIa-FXa in POPC:CHOL (CLP), respectively, and subjected them to molecular dynamics simulation. RESULTS: Hydrogen-bond and contact probability analysis were performed between various important domains of TF-FVIIa-FXa and notable novel interactions: Asn93FVIIa:L-Lys48TF, Arg178FVIIa:H-Asp95FXa:B, Lys20FVIIa:H-Glu193FXa:A, Arg178FVIIa:H-Asp97FXa:B, and Arg153FVIIa:H-Gln135FXa:B have been reported. The protein stability study implies that the CS and CLP systems are thermodynamically less stable than CSL and CSLP systems. CONCLUSION: Analysis of molecular dynamic simulation data suggests that the presence of cholesterol and palmitoylation may contribute to structural rigidity, stability, and compactness of key domains of TF-FVIIa-FXa by augmenting protein-protein and protein-lipid interactions.

Investigation of the tight binding mechanism of a new anticoagulant DD217 to factor Xa by means of molecular docking and molecular dynamics.

A new promising drug candidate DD217 has been proposed recently as a potent anticoagulant acting on factor Xa (fXa) target. It exhibits the lowest concentration of doubling the prothrombin time among the known anticoagulants. In order to explain the efficacy of DD217 in terms of molecular interactions with its target we studied the hypothesis of the tight binding mechanism by means of molecular dynamics simulations and statistical analysis of the trajectory. The conducted analysis confirms the significant contributions to the MM/GBSA estimated binding free energy of the S4 pocket residues as well the crucial role of establishing the hydrogen bonds between the ligand and the backbone amides of Gly216 and Gly218 of the target. The simulation results support the hypothesis of the tight binding mechanism of DD217 to fXa.Communicated by Ramaswamy H. Sarma.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.

Combinatorial assembly, traceless generation and in situ evaluation of inhibitors for therapeutically relevant serine proteases.

A combinatorial method was devised and applied for the design and identification of substrate-analogue inhibitors of therapeutically relevant serine proteases, such as thrombin and factor Xa. We conceptualized imino acid derived diketomorpholines as generally applicable key intermediates prepared through solid-phase synthesis and prone to be cleaved with primary amines in a traceless fashion. The approach led to a compound library whose members were prepared under bioassay-compatible conditions and directly subjected to the in situ evaluation, allowing a fast prediction of hit compounds. Highly active inhibitors for serine proteases of the coagulation cascade have been identified. The most potent dual inhibitor, 16K, has a binding affinity of 23.9 nM to thrombin and 32.8 nM to factor Xa.

Effect of variations in the conserved residues E371 and S359 on the structural dynamics of protein Z dependent protease inhibitor (ZPI): a molecular dynamic simulation study.

Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin superfamily, it possesses unique structural features such as activation by PZ, regulating its inhibitory function. In order to understand the Reactive Centre Loop (RCL) dynamics of ZPI, which is absolutely critical for its activity, we performed Molecular Dynamics (MD) simulation on ZPI and its E371 and S359 variants located at important conserved functional sites. Unexpectedly, the RCL of E371 variants, (E371K, E371R, and E371Q), were shown to be very stable due to compensatory interactions at the proximal end of RCL. Interestingly, RCL flexibility was shown to be enhanced in the double mutant K318E-E371K due to the repulsive effect of increased negative charge on top of the breach region. Principal component analysis (PCA) coupled with residue wise interaction network analysis(RIN) revealed correlated motion between the RCL and the PZ binding regions in the WT. However, a loss of regulation in correlated motion between RCL and PZ binding hotspot Tyr240 in the double mutant was also observed. Additionally, the S359F and S359I mutations resulted in increased RCL flexibility owing to the disruption of stabilizing hydrogen bonding interaction at the distal end of strand S5A. Thus, the current study proposes that the overall stabilizing interactions of S5A is a major regulator of proper loop movement of ZPI for its activity. The results would be beneficial to engineer activity compromised ZPI as a prophylactic agent for the treatment of hemophilia.Communicated by Ramaswamy H. Sarma.

Induced forms of α2-macroglobulin neutralize heparin and direct oral anticoagulant effects.

Alpha2-macroglobulin (α2M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α2M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α2M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α2M were depicted by computer-aided energy minimization modeling. GDFXa-induced α2M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α2M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α2M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α2M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α2M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Enoxaparin Dose Requirements to Achieve Therapeutic Low-molecular-weight Heparin Anti-factor Xa Levels in Infants and Young Children.

INTRODUCTION: Enoxaparin is commonly used to treat pediatric thrombosis. Several small retrospective studies have suggested that infants and young children require higher enoxaparin doses to achieve therapeutic anti-factor Xa levels compared with adults. MATERIALS AND METHODS: This is a retrospective study of hospitalized children who received enoxaparin for the treatment of thrombosis at a free-standing children's hospital. The primary objective was to ascertain the enoxaparin dose required to achieve an anti-factor Xa level of 0.5 to 1.0 U/mL among 4 age groups in a large cohort of infants and young children between 60 days and 5 years of age. RESULTS: A total of 176 infants and children were evaluated. The majority of patients were less than 1 year of age (n=104). An inverse relationship between enoxaparin dose needed to achieve therapeutic anti-factor Xa levels and patient age was noted, particularly in the first year of life. Patients who were 60 days to less than 7 months at the time of enoxaparin initiation (n=73) required the highest mean dose among the age groups at 1.73 mg/kg subcutaneously every 12 hours (P<0.0001). CONCLUSION: Infants and young children require higher doses of enoxaparin to achieve therapeutic anti-factor Xa levels compared with adults.

Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.

Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic ß-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1-4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

Immobilisation of an anti-platelet adhesion and anti-thrombotic drug (EP224283) on polydopamine coated vascular stent promoting anti-thrombogenic properties.

Current vascular drug-eluting stents based on immuno-proliferative drugs would reduce the rate of in-stent restenosis (ISR) but may be associated with a higher risk of acute stent thrombosis due to non-selective activity. In this paper, we aimed to develop a polydopamine (PDA) coated chromium­cobalt (CoCr) stent functionalised with EP224283 (Endotis Pharma SA), which combines both a GPIIbIIIa antagonist (tirofiban moiety) and a factor Xa inhibitor (idraparinux moiety) to reduce acute stent thrombosis. PDA-coated chromium­cobalt (CoCr) samples were first immersed in a polyethylenimine (PEI, pH 8.5) solution to increase amine function density (36.0 ± 0.1 nmol/cm2) on the CoCr surface. In a second step, avidin was grafted onto CoCr-PDA-PEI through the biotin linkage (strategy 1) or directly by coupling reactions (strategy 2). The HABA titration proved the fixation of biotin onto CoCr-PDA-PEI surface with a density of 0.74 nmol/cm2. The fixation of avidin was demonstrated by water contact angle (WCA) and surface plasmon resonance (SPR). SEM micrograph shows the flexibility of the thin layer coated onto the stent after balloon inflation. Independently of the strategy, a qualitative SEM analysis showed a reduction in platelet activation when the molecule EP224283 was immobilised on avidin. In parallel, the measurement of anticoagulant activity (anti-Xa) revealed a higher anti-factor Xa activity (2.24 IU/mL vs. 0.09 IU/mL in control) when EP224283 was immobilised on avidin. Interestingly, after seven days of degradation, the anticoagulant activity was persistent in both strategies and looked more important with the strategy 2 than in strategy 1. Throughout this work, we developed an innovative vascular stent through the immobilisation of EP224283 onto CoCr-PDA-PEI-(avidin) system, which provides a promising solution to reduce ISR and thrombosis after stent implantation.