Apigenin Provides Structural Protection to Human Fibrinogen against Nitrosative Stress: Biochemical and Molecular Insights.

BACKGROUND: Peroxynitrite (ONOO-) is an oxidant linked with several human pathologies. Apigenin, a natural flavonoid known for its health benefits, remains unexplored in relation to ONOO- effects. This study investigated the potential of apigenin to structurally protect fibrinogen, an essential blood clotting factor, from ONOO--induced damage. METHODS: Multi-approach analyses were carried out where fibrinogen was exposed to ONOO- generation while testing the efficacy of apigenin. The role of apigenin against ONOO--induced modifications in fibrinogen was investigated using UV spectroscopy, tryptophan or tyrosine fluorescence, protein hydrophobicity, carbonylation, and electrophoretic analyses. RESULTS: The findings demonstrate that apigenin significantly inhibits ONOO--induced oxidative damage in fibrinogen. ONOO- caused reduced UV absorption, which was reversed by apigenin treatment. Moreover, ONOO- diminished tryptophan and tyrosine fluorescence, which was effectively restored by apigenin treatment. Apigenin also reduced the hydrophobicity of ONOO--damaged fibrinogen. Moreover, apigenin exhibited protective effects against ONOO--induced protein carbonylation. SDS-PAGE analyses revealed that ONOO-treatment eliminated bands corresponding to fibrinogen polypeptide chains Aα and γ, while apigenin preserved these changes. CONCLUSIONS: This study highlights, for the first time, the role of apigenin in structural protection of human fibrinogen against peroxynitrite-induced nitrosative damage. Our data indicate that apigenin offers structural protection to all three polypeptide chains (Aα, Bß, and γ) of human fibrinogen. Specifically, apigenin prevents the dislocation or breakdown of the amino acids tryptophan, tyrosine, lysine, arginine, proline, and threonine and also prevents the exposure of hydrophobic sites in fibrinogen induced by ONOO-.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

A role of methionines in the functioning of oxidatively modified fibrinogen.

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/-OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/-OCl, 10 µM HOCl/-OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BßMet367, γMet264, and γMet94, identified in 10 µM HOCl/-OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.

Controlling nanoparticle-induced endothelial leakiness with the protein corona.

Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Selective binding of cationic fibrinogen-mimicking chitosan nanoparticles to activated platelets and efficient drug release for antithrombotic therapy.

Thrombosis is the main cause of catastrophic events including ischemic stroke, myocardial infarction and pulmonary embolism. Acetylsalicylic acid (ASA) therapy offers a desirable approach to antithrombosis through a reduction of platelet reactivity. However, major bleeding complications, severe off-target side effects, and resistance or nonresponse to ASA greatly attenuate its clinical outcomes. Herein, we report a cationic fibrinogen-mimicking nanoparticle, denoted as ASA-RGD-CS@TPP, to achieve activated-platelet-targeted delivery and efficient release of ASA for safer and more effective antithrombotic therapy. This biomimetic antithrombotic system was prepared by one-pot ionic gelation between cationic arginine-glycine-aspartic acid (RGD)-grafted chitosan (RGD-CS) and anionic tripolyphosphate (TPP). The platform exhibited selective binding to activated platelets, leading to efficient release of ASA and subsequent attenuation of platelet functions, including the remarkable inhibition of platelet aggregation through a potent blockage of cyclooxygenase-1 (COX-1). After intravenous administration, ASA-RGD-CS@TPP displayed significantly prolonged circulation time and successful prevention of thrombosis in a mouse model. ASA-RGD-CS@TPP was demonstrated to significantly enhance antithrombotic therapy while showing minimal coagulation and hemorrhagic risks and excellent biocompatibility in vivo as compared to free ASA. This platform provides a simple, safe, effective and targeted strategy for the development of antithrombotic nanomedicines.

Influence of Proteins and Building Direction on the Corrosion and Tribocorrosion of CoCrMo Fabricated by Laser Powder Bed Fusion.

Cobalt-chromium-molybdenum (CoCrMo) alloys are common wear-exposed biomedical alloys and are manufactured in multiple ways, increasingly using additive manufacturing processes such as laser powder bed fusion (LPBF). Here, we investigate the effect of proteins and the manufacturing process (wrought vs LPBF) and building orientation (LPBF-XY and XZ) on the corrosion, metal release, tribocorrosion, and surface oxide composition by means of electrochemical, mechanical, microscopic, diffractive, and spectroscopic methods. The study was conducted at pH 7.3 in 5 g/L NaCl and 5 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer, which was found to be necessary to avoid metal phosphate and metal-protein aggregate precipitation. The effect of 10 g/L bovine serum albumin (BSA) and 2.5 g/L fibrinogen (Fbn) was studied. BSA and Fbn strongly enhanced the release of Co, Cr, and Mo and slightly enhanced the corrosion (still in the passive domain) for all CoCrMo alloys and most for LPBF-XZ, followed by LPBF-XY and the wrought CoCrMo. BSA and Fbn, most pronounced when combined, significantly decreased the coefficient of friction due to lubrication, the wear track width and severity of the wear mechanism, and the tribocorrosion for all alloys, with no clear effect of the manufacturing type. The wear track area was significantly more oxidized than the area outside of the wear track. In the reference solution without proteins, a strong Mo oxidation in the wear track surface oxide was indicative of a pH decrease and cell separation of the anodic and cathodic areas. This effect was absent in the presence of the proteins.

A novel mutation in the FGG gene causes hypofibrinogenemia in a Chinese family.

Congenital fibrinogen disorders are a group of coagulation deficiencies caused by fibrinogen defects and are divided into four types, including afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. In this study, we collected a family with hypofibrinogenemia, and genetics analysis identify a novel pathogenic variants (c.668G > C, p.Arg223Thr) in the FGG gene. And electron microscope observation revealed significant changes in the ultrastructure of fibrin of the proband. Our research expands the phenotypic and genetic spectrum associated with the FGG gene, which would facilitate in genetic counselling and prenatal genetic diagnosis.

Degradation protection and enhanced biocompatibility of Mg alloys pretreated with plasma proteins.

After implantation of the Mg alloy in the human body, the adsorption of plasma protein on surface will cause a series of cell reactions and affect the degradation of Mg alloys. Herein, in vitro biological reactions of the ZK60 and AZ31 Mg alloys are analyzed in plasma protein environment. Combined with mass spectrometry analysis of the type of adsorbed proteins, it is shown that proteins such as fibrinogen, vitronectin, fibronectin, and prothrombin are prone to get adsorbed on the surface of the alloys than other proteins, leading to the promotion of MG63 cell adhesion and proliferation. The effect of selected proteins (fibrinogen, fibronectin, and prothrombin) on degradation of ZK60 and AZ31 Mg alloys is investigated using immersion tests. The degradation of AZ31 Mg alloy is significantly restrained with the presence of proteins. This is due to the protein adsorption effect on the sample surface. The molecular dynamics simulation results indicate that both fibrinogen and fibronectin tend to adsorb onto the AZ31 rather than ZK60, forming a stable protein layer on the AZ31 Mg alloy retarding the degradation of the samples. As to ZK60 alloy, the addition of protein inhibits the degradation in the short term, however, the degradation increases after a long time of immersion. This phenomenon is particularly pronounced in fibronectin solution.

Mesoscopic Model for Reversible Adsorption Stage of Albumin and Fibrinogen on TiO2 Surface.

The competitive behavior of proteins in the reversible adsorption stage plays a crucial role in determining the composition of the protein layer and the subsequent biological responses to the biomaterial. However, such competitive adsorption is a mesoscopic process at physiological protein concentration, and neither a macroscopic experiment nor microscopic MD (molecular dynamics) simulation is suitable to clarify it. Here, we proposed a mesoscopic DPD (dissipative particle dynamics) model to illustrate the competitive process of albumin and fibrinogen on TiO2 surface with its parameters deduced from our previous MD simulation, and proved the model well retained the diffusion and adsorption properties of proteins in the competitive adsorption on the plane surface. We then applied the model to the competitive adsorption on the surfaces with different nanostructures and observed that when the nanostructure size is much larger than that of protein, the increase in surface area is the main influencing factor; when the nanostructure size is close to that of protein, the coordination between the nanostructure and the size and shape of protein significantly affects the competitive adsorption process. The model has revealed many mechanical phenomena observed in previous experimental studies and has the potential to contribute to the development of high-performance biomaterials.

Influence of titanium nanoscale surface roughness on fibrinogen and albumin protein adsorption kinetics and platelet responses.

Biomaterials with nanoscale topography have been increasingly investigated for medical device applications to improve tissue-material interactions. This study assessed the impact of nanoengineered titanium surface domain sizes on early biological responses that can significantly affect tissue interactions. Nanostructured titanium coatings with distinct nanoscale surface roughness were deposited on quartz crystal microbalance with dissipation (QCM-D) sensors by physical vapor deposition. Physico-chemical characterization was conducted to assess nanoscale surface roughness, nano-topographical morphology, wettability, and atomic composition. The results demonstrated increased projected surface area and hydrophilicity with increasing nanoscale surface roughness. The adsorption properties of albumin and fibrinogen, two major plasma proteins that readily encounter implanted surfaces, on the nanostructured surfaces were measured using QCM-D. Significant differences in the amounts and viscoelastic properties of adsorbed proteins were observed, dependent on the surface roughness, protein type, protein concentration, and protein binding affinity. The impact of protein adsorption on subsequent biological responses was also examined using qualitative and quantitative in vitro evaluation of human platelet adhesion, aggregation, and activation. Qualitative platelet morphology assessment indicated increased platelet activation/aggregation on titanium surfaces with increased roughness. These data suggest that nanoscale differences in titanium surface roughness influence biological responses that may affect implant integration.

Development of biosensors for detection of fibrinogen: a review.

Fibrinogen as a major inflammation marker and blood coagulation factor has a direct impact on the health of humanity. The variations in fibrinogen content lead to risky conditions such as bleeding and cardiovascular diseases. So, accurate methods for monitoring of this glycoprotein are of high importance. The conventional methods, such as the Clauss method, are time consuming and require highly specialized expert analysts. The development of fast, simple, easy to use, and inexpensive methods is highly desired. In this way, biosensors have gained outstanding attention since they offer means for performing analyses at the points-of-care using self-testing devices, which can be applied outside of clinical laboratories or hospital. This review indicates that different electrochemical and optical sensors have been successfully implemented for the detection of fibrinogen under normal levels of fibrinogen in plasma. The biosensors for the detection of fibrinogen have been designed based on the quartz crystal microbalance, field-effect transistor, electrochemical impedance spectroscopy, amperometry, surface plasmon resonance, localized surface plasmon resonance, and colorimetric techniques. Also, this review demonstrates the utility of the application of nanoparticles in different detection techniques.

Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

Biomechanics, Energetics, and Structural Basis of Rupture of Fibrin Networks.

Fibrin provides the main structural integrity and mechanical strength to blood clots. Failure of fibrin clots can result in life-threating complications, such as stroke or pulmonary embolism. The dependence of rupture resistance of fibrin networks (uncracked and cracked) on fibrin(ogen) concentrations in the (patho)physiological 1-5 g L-1 range is explored by performing the ultrastructural studies and theoretical analysis of the experimental stress-strain profiles available from mechanical tensile loading assays. Fibrin fibers in the uncracked network stretched evenly, whereas, in the cracked network, fibers around the crack tip showed greater deformation. Unlike fibrin fibers in cracked networks formed at the lower 1-2.7 g L-1 fibrinogen concentrations, fibers formed at the higher 2.7-5 g L-1 concentrations align and stretch simultaneously. Cracked fibrin networks formed in higher fibrinogen solutions are tougher yet less extensible. Statistical modeling revealed that the characteristic strain for fiber alignment, crack size, and fracture toughness of fibrin networks control their rupture resistance. The results obtained provide a structural and biomechanical basis to quantitatively understand the material properties of blood plasma clots and to illuminate the mechanisms of their rupture.

ROS-driven structural and functional fibrinogen modifications are reverted by interleukin-6 inhibition in Giant Cell Arteritis.

BACKGROUND: Cranial and extra-cranial vascular events are among the major determinants of morbidity and mortality in Giant Cell Arteritis (GCA). Vascular events seem mostly of inflammatory nature, although the precise pathogenetic mechanisms are still unclear. We investigated the role of oxidation-induced structural and functional fibrinogen modifications in GCA. The effects of the anti-IL6R tocilizumab in counteracting these mechanisms were also assessed. MATERIALS AND METHODS: A cross-sectional study was conducted on 65 GCA patients and 65 matched controls. Leucocyte reactive oxygen species (ROS) production, redox state, and fibrinogen structural and functional features were compared between patients and controls. In 19 patients receiving tocilizumab, pre vs post treatment variations were assessed. RESULTS: GCA patients displayed enhanced blood lymphocyte, monocyte and neutrophil ROS production compared to controls, with an increased plasma lipid peroxidation and a reduced total antioxidant capacity. This oxidative impairment resulted in a sustained fibrinogen oxidation (i.e. dityrosine content 320 (204-410) vs 136 (120-176) Relative Fluorescence Units (RFU), p < 0.0001), with marked alterations in fibrinogen secondary and tertiary structure [intrinsic fluorescence: 134 (101-227) vs 400 (366-433) RFU, p < 0.001]. Structural alterations paralleled a remarkable fibrinogen functional impairment, with a reduced ability to polymerize into fibrin and a lower fibrin susceptibility to plasmin-induced lysis. In patients receiving tocilizumab, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features (p < 0.001). CONCLUSIONS: An impaired redox status accounts for structural and functional fibrinogen modifications in GCA, suggesting a potential role of tocilizumab for cardiovascular prevention in GCA.

Antiadhesive Copolymers at Solid/Liquid Interfaces: Complementary Characterization of Polymer Adsorption and Protein Fouling by Sum Frequency Generation Vibrational Spectroscopy and Quartz-Crystal Microbalance Measurements with Dissipation Monitoring.

Amphiphilic copolymers comprising hydrophilic segments of poly(ethylene glycol) and hydrophobic domains that are able to adhere to solid/liquid interfaces have proven to be versatile ingredients in formulated products for various types of applications. Recently, we have reported the successful synthesis of a copolymer designed for modifying the surface properties of polyesters as mimics for synthetic textiles. Using sum frequency generation (SFG) spectroscopy, it was shown that the newly developed copolymer adsorbs effectively on the targeted substrates even in the presence of surfactants as supplied by common detergents. In the present work, these studies were extended to evaluate the ability of the formed copolymer adlayers to passivate polyester surfaces against undesired deposition of bio(macro)molecules, as represented by fibrinogen as model protein foulants. In addition, SFG spectroscopy was used to elucidate the structure of fibrinogen at the interface between polyester and water. To complement the obtained data with an independent technique, analogous experiments were performed using quartz-crystal microbalance with dissipation monitoring for the detection of the relevant interfacial processes. Both methods give consistent results and deliver a holistic picture of brush copolymer adsorption on polyester surfaces and subsequent antiadhesive effects against proteins under different conditions representing the targeted application in home care products.

Fbg αC 389-402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions.

Coagulation Factor XIII (FXIII) stabilizes blood clots by cross-linking glutamines and lysines in fibrin and other proteins. FXIII activity in the fibrinogen αC region (Fbg αC 221-610) is critical for clot stability and growth. Fbg αC 389-402 is a binding site for thrombin-activated FXIII, (FXIII-A*), with αC E396 promoting FXIII-A* binding and activity in αC. The current study aimed to discover additional residues within Fbg αC 389-402 that accelerate transglutaminase activity toward αC. Electrostatic αC residues (E395, E396, and D390), hydrophobic αC residues (W391 and F394), and residues αC 328-425 were studied by mutations to recombinant Fbg αC 233-425. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) cross-linking and gel-based fluorescence monodansylcadaverine (MDC) cross-linking assays. Truncation mutations 403 Stop (Fbg αC 233-402), 389 Stop (Fbg αC 233-388), and 328 Stop (Fbg αC 233-327) reduced Q237-GEE and MDC cross-linking compared to wild-type (WT). Comparable cross-linking between 389 Stop and 328 Stop showed that FXIII is mainly affected by the loss of Fbg αC 389-402. Substitution mutations E396A, D390A, W391A, and F394A decreased cross-linking relative to WT, whereas E395A, E395S, E395K, and E396D had no effect. Similar FXIII-A* activities were observed for double mutants (D390A, E396A) and (W391A, E396A), relative to D390A and W391A, respectively. In contrast, cross-linking was reduced in (F394A, E396A), relative to F394A. In conclusion, Fbg αC 389-402 boosts FXIII activity in Fbg αC, with D390, W391, and F394 identified as key contributors in enhancing αC cross-linking.

Investigation of the effect of pre-analytical factors on particle concentration and size in cryoprecipitate using nanoparticle tracking analysis.

BACKGROUND: Cryoprecipitate is used primarily to replenish fibrinogen levels in patients. Little is known about the presence of micro- or nano-sized particles in cryoprecipitate. Therefore, we aimed to quantify these particles and investigate some pre-analytical considerations. MATERIALS AND METHODS: Particle concentration and size distribution were determined in 10 cryoprecipitate units by nanoparticle tracking analysis (NTA). The effects of freeze-thawing cryoprecipitate and 0.45 µm filtration with either regenerated cellulose (RC) or polytetrafluoroethylene (PTFE) filters before sample analysis were examined. RESULTS: Neither the size nor concentration of particles were affected by two freeze/thaw cycles. PTFE filtration, but not RC filtration, significantly reduced particle mean and mode size compared to RC filtration and mode size compared to unfiltered cryoprecipitate. The 10 cryoprecipitate units had an average particle concentration of 2.50 × 1011 ± 1.10 × 1011 particles/mL, a mean particle size of 133.8 ± 7.5 nm and a mode particle size of 107.9 ± 11.1 nm. CONCLUSION: This study demonstrated that preanalytical filtration of cryoprecipitate units using RC filters was suitable for NTA. An additional freeze/thaw cycle did not impact NTA parameters, suggesting that aliquoting cryoprecipitate units prior to laboratory investigations is suitable for downstream analyses.

Development of a bioactive tunable hyaluronic-protein bioconjugate hydrogel for tissue regenerative applications.

Every year, there are approximately 500 000 peripheral nerve injury (PNI) procedures due to trauma in the US alone. Autologous and acellular nerve grafts are among current clinical repair options; however, they are limited largely by the high costs associated with donor nerve tissue harvesting and implant processing, respectively. Therefore, there is a clinical need for an off-the-shelf nerve graft that can recapitulate the native microenvironment of the nerve. In our previous work, we created a hydrogel scaffold that incorporates mechanical and biological cues that mimic the peripheral nerve microenvironment using chemically modified hyaluronic acid (HA). However, with our previous work, the degradation profile and cell adhesivity was not ideal for tissue regeneration, in particular, peripheral nerve regeneration. To improve our previous hydrogel, HA was conjugated with fibrinogen using Michael-addition to assist in cell adhesion and hydrogel degradability. The addition of the fibrinogen linker was found to contribute to faster scaffold degradation via active enzymatic breakdown, compared to HA alone. Additionally, cell count and metabolic activity was significantly higher on HA conjugated fibrinogen compared previous hydrogel formulations. This manuscript discusses the various techniques deployed to characterize our new modified HA fibrinogen chemistry physically, mechanically, and biologically. This work addresses the aforementioned concerns by incorporating controllable degradability and increased cell adhesivity while maintaining incorporation of hyaluronic acid, paving the pathway for use in a variety of applications as a multi-purpose tissue engineering platform.