Fibrinogen Conformational Changes Research Articles

Anti-Platelet Effect Induced by Iron Oxide Nanoparticles: Correlation with Conformational Change in Fibrinogen.

Iron oxide nanoparticles are developed for various biomedical applications, however, there is limited understanding regarding their effects and toxicity on blood components. The particles traveling in circulation inevitably interact with blood cells and plasma proteins and may interfere with hemostasis. Specifically, this study focuses on the influence of superparamagnetic iron oxide nanoparticles (SPIONs) coated with a biocompatible polymer, polyvinyl alcohol (PVA), on platelet function. Here, engineered SPIONs that are functionalized with various PVA coatings to provide these particles with different surface charges and polymer packing are described. These formulations are assessed for any interference with human platelet functions and coagulation, ex vivo. Positively charged SPIONs induce a significant change in platelet GPIIb-IIIa conformation, indicative of platelet activation at the dose of 500µg mL-1 . Remarkably, engineered PVA(polyvinyl alcohol)-SPIONs all display a robust dose-dependent anti-platelet effect on platelet aggregation, regardless of the PVA charge and molecular weight. After assessing hypotheses involving SPION-induced steric hindrance in platelet-platelet bridging, as well as protein corona involvement in the antiplatelet effect, the study concludes that the presence of PVA-SPIONs induces fibrinogen conformational change, which correlates with the observed dose-dependent anti-platelet effect.

Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S-nitroso-N-acetylpenicillamine, but not S-nitrosoglutathione.

Previous work in a 4 h rabbit thrombogenicity model has shown that a nitric oxide- (NO) generating polymer extracorporeal circuits (ECC) with infusion of S-nitroso-N-acetyl-penicillamine (SNAP) preserved platelets eventhough platelets were activated as shown by an increase in the glycoprotein, p-selectin. The platelet preservation mechanism was shown to be due to a changing fibrinogen structure leading to attenuation of platelet aggregation. Understanding the effects that SNAP, another RSNO, S-nitroso-glutathione (GSNO) as well as the non-RSNO, sodium nitroprusside (SNP), may have on human fibrinogen polymerization, this in vitro study evaluated the released NO effects on the thrombin-mediated fibrin formation and fibrinogen structure. Thrombin-induced fibrin formation at 300 μM SNAP (50 + 11% of baseline) was significantly reduced compared to SNAP's parent, N-acetyl-penicillamine (NAP) (95 + 13%) after 1 h of RSNO exposure. GSNO, its parent, glutathione (GSH) and 1000 ppm NO gas did not attenuate the thrombin-mediated fibrin formation. SNAP, NAP and SNP exposure for 1 h, however, did not decrease thrombin activity by directly inhibiting thrombin itself. Changes in fibrinogen conformation as measured by intrinsic tryptophan fluorescence significantly decreased in the 300 μM SNAP (38057 + 1196 mean fluorescence intensity (MFI) and SNP (368617 + 541 MFI) groups versus the NAP control (47937 + 1196 MFI). However, infused 1000 ppm NO gas had no direct effect on the ITF after 1 h incubation at 37°C. High performance liquid chromatography (HPLC) showed that fibrinogen degradation by 0.03 U/ml thrombin was concentration-dependently reduced after 1 h with SNAP but not with NAP or SNP. Western blotting showed RSNOs, SNAP, NAP and the non-RSNO, SNP-incubated fibrinogen solutions showed that the percent level of the Aγ dimer to total Aγ dimer + γ monomer was significantly reduced in the case of the SNAP group when compared to SNP group. These results suggest that NO donors such as SNAP and SNP induce fibrinogen conformational changes by potentially nitrosating fibrinogen tyrosine residues. These NO-mediated fibrinogen changes induced via NO donors may provide another mechanism of NO for improving thromboresistance in ECC.

Facile conjugation of heparin onto titanium surfaces via dopamine inspired coatings for improving blood compatibility

Immobilization of heparin on biomaterials surface has been proven to be an effective strategy for preventing thrombus formation. However, the procedures of most immobilization methods (physical adsorption, covalent linkage, electrostatic interaction) are complicated and time-consuming. In the present study, heparin with various concentrations immobilized on a titanium (Ti) substrate via polydopamine layer for improving blood compatibility was investigated. Water contact angle measurement showed that the immobilization of heparin resulted in an increase of the hydrophilicity. X-ray photoelectron spectroscopy (XPS) and Toluidine Blue O (TBO) tests displayed that the heparin molecules were successfully immobilized on Ti surface. The evaluations of blood compatibility (hemolysis rate, APTT, platelet adhesion and activation, fibrinogen conformational change) showed that the immobilization of heparin decreased hemolysis rate, prolonged blood coagulation time, reduced platelets adhesion and activation, and induced less fibrinogen conformational change. Moreover, a significant inhibition of blood coagulation and platelet adhesion was obtained when the heparin concentration was higher than 5 mg/mL, indicating that only with a certain surface densities could heparin perform its anticoagulant properties well. The results suggest that the immobilization of heparin via polydopamine layer can confer excellent antithrombotic properties, and the heparin immobilization method via polydopamine layer provides an alternative approach for other biomolecules immobilization on biomaterials surface. Thus it is envisaged that this method will be potentially useful for the surface modification of blood-contacting biomaterials.

In vitro blood compatibility of polyethylene terephthalate with covalently bounded hirudin on surface

Polyethylene terephthalate (PET, Dacron) was modified by surface immobilization of hirudin with glutaraldehyde(GA) as coupling reagent to improve the blood compatibility. Hirudin-immobilized PETs were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The blood compatibility of the PETs was evaluated by platelet adhesion evaluation and fibrinogen conformational change measurements in vitro. The results showed the decrease of platelet adhesion and activation on hirudin-immobilized PET with increasing of glutaraldehyde concentration. Fibrinogen experiment showed that fibrinogen adherence and conformational changes of PET-HRD were less than those of untreated PET, which made the materials difficult to form thrombus. The proper reason of blood compatibility improvement was low interface tension between hirudin-immobilized PETs and blood, as well as blood proteins, and low ratio of dispersive/polar component of the surface energy(γsd/γsp) and high hydrophilicity.

The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization

Currently available cardiovascular implants, such as heart valves and stents, exhibit suboptimal biocompatibility because of the incomplete endothelialization and sequential thrombosis formation especially after a long-term implantation. To improve the blood compatibility and endothelialization simultaneously and ensure the long-term effect of the cardiovascular implants, a technique of combining electrostatic interaction and coimmobilization was developed to form heparin and fibronectin (Hep/Fn) films on aminosilanized titanium (Ti) surfaces. The Hep/Fn coimmobilized films were stable after immersion in PBS for five days, probed by wettability studies and by the release kinetics of heparin and fibronectin. Blood compatibility tests showed that the coimmobilized Hep/Fn films displayed lower hemolysis rate, prolonged blood coagulation time, higher AT III binding density, less platelets activation and aggregation, and less fibrinogen conformational change compared with Ti surface. Endothelial cells (ECs) seeding and fibronectin bioactivity results showed more attached and proliferated ECs and exposed cell-binding sites on the Hep/Fn immobilized samples than that on Ti surfaces. Thus, the Hep/Fn coimmobilized films kept excellent bioactivity even after immersion in PBS for five days. Systemic evaluation suggests that the coimmobilization of Hep/Fn complex improves the blood compatibility and promotes the endothelialization simultaneously. We envisage that this method will provide a potential and effective selection for biomaterials surface modification of cardiovascular implants.

Tailoring of the Titanium Surface by Immobilization of Heparin/Fibronectin Complexes for Improving Blood Compatibility and Endothelialization: An in Vitro Study

To improve the blood compatibility and endothelialization simultaneously and to ensure the long-term effectiveness of the cardiovascular implants, we developed a surface modification method, enabling the coimmobilization of biomolecules to metal surfaces. In the present study, a heparin and fibronectin mixture (Hep/Fn) covalently immobilized on a titanium (Ti) substrate for biocompatibility was investigated. Different systems [N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide, electrostatic] were used for the formation of Hep/Fn layers. Atomic force microscopy (AFM) showed that the roughness of the silanized Ti surface decreased after the immobilization of Hep/Fn. Fourier transform infrared spectroscopy (FTIR), Toluidine Blue O (TBO) test, and immunochemistry assay showed that Hep/Fn mixture was successfully immobilized on Ti surface. Blood compatibility tests (hemolysis rate, APTT, platelet adhesion, fibrinogen conformational change) showed that the coimmobilized films of Hep/Fn mixture reduced blood hemolysis rate, prolonged blood coagulation time, reduced platelets activation and aggregation, and induced less fibrinogen conformational change compared with a bare Ti surface. Endothelial cell (EC) seeding showed more EC with better morphology on pH 4 samples than on pH 7 and EDC/NHS samples, which showed rounded and aggregated cells. Systematic evaluation showed that the pH 4 samples also had much better blood compatibility. All results suggest that the coimmobilized films of Hep/Fn can confer excellent antithrombotic properties and with good endothelialization. We envisage that this method will provide a potential and effective solution for the surface modification of cardiovascular implant materials.

Surface Characterization and in Vitro Blood Compatibility of Poly (Ethylene Terephthalate) Immobilized with Hirudin

Poly (ethylene terephthalate)(dacron, PET) films were exposed under argon plasma glow discharge with different glows and induced polymerization of acrylic acid(AA) in order to introduce carboxylic acid group onto PET (PET-AA) assisted by ultraviolet radiation(UV). Hirudin-immobilized PET (PET-HRD) films were prepared by the grafting of PET-AA, followed by chemical reaction with hirudin. The surface structure of the treated PET was determined by X-ray photoelectron spectroscopy (XPS). The wettability, surface free energy, and interface free energy of the films were investigated by contact angle measurement. The blood compatibility of the films was assessed by platelet-adhesion test and fibrinogen conformational change measurements to evaluate the viability of the materials in biomedical engineering. Measurement by scanning electron microscopy (SEM) revealed that the amounts of adhered, aggregated and morphologically changed platelets were reduced on the hirudin-immobilized PET films. Enzyme-linked-immunoassay measurements that disclosed fibrinogen conformational changes showed results consistent with the platelets' behavior.

Measurement of interaction forces between fibrinogen coated probes and mica surface with the atomic force microscope: The pH and ionic strength effect

The study of protein-surface interactions is of great significance in the design of biomaterials and the evaluation of molecular processes in tissue engineering. The authors have used atomic force microscopy (AFM) to directly measure the force of attraction/adhesion of fibrinogen coated tips to mica surfaces and reveal the effect of the surrounding solution pH and ionic strength on this interaction. Silica colloid spheres were attached to the AFM cantilevers and, after plasma deposition of poly(acrylic acid), fibrinogen molecules were covalently bound on them with the help of the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of N-hydroxysulfosuccinimide (sulfo-NHS). The measurements suggest that fibrinogen adsorption is controlled by the screening of electrostatic repulsion as the salt concentration increases from 15 to 150 mM, whereas at higher ionic strength (500 mM) the hydration forces and the compact molecular conformation become crucial, restricting adsorption. The protein attraction to the surface increases at the isoelectric point of fibrinogen (pH 5.8), compared with the physiological pH. At pH 3.5, apart from fibrinogen attraction to the surface, evidence of fibrinogen conformational changes is observed, as the pH and the ionic strength are set back and forth, and these changes may account for fibrinogen aggregation in the protein solution at this pH.

Si–N–O Films Synthesized by Plasma Immersion Ion Implantation and Deposition (PIII&D) for Blood-Contacting Biomedical Applications

Silicon-Oxynitride (Si-N-O) films were fabricated on silicon wafers by silicon cathodic arc combined with plasma immersion ion implantation and deposition. The blood compatibility of the films was assessed by platelet-adhesion test and fibrinogen conformational change measurements to evaluate the viability of the materials in biomedical engineering. Significantly, a better platelet-adhesion behavior, as manifested by a smaller number and weaker aggregation as well as pseudopodium, was observed on the Si-N-O samples compared to the low-temperature isotropic pyrolytic carbon, which is the most common material used in blood-contacting biomedical devices such as artificial heart valves. Enzyme-linked-immunoassay measurements that disclose fibrinogen conformational changes show results that are consistent with the platelets' behavior, which is believed to be involved in the activation process. The good blood compatibility of the films can be attributed to the high hydrophilicity and surface free energy arising from the Si-N, Si-N-O, and Si-O bonding states. The interfacial reactions between fibrinogen, platelets, and material surface are discussed from the perspective of thermodynamics. The promising blood compatibility of the Si-N-O films is of both scientific and commercial interests in biomedical engineering