Affinity For Fibrinogen Research Articles

A Small-Molecule NIR-II Probe for the Diagnosis of Hemorrhagic Diseases.

Numerous hemorrhagic disorders, particularly those presenting deep hemorrhage, pose diagnostic challenges, often leading to delayed treatment and severe outcomes. Near-infrared (NIR)-II fluorescence imaging offers advantages such as deep tissue penetration, real-time visualization, and a high signal-to-background ratio, making it highly suitable for diagnosing hemorrhagic diseases. In this study, an NIR-II fluorescent probe LJ-2P carrying carboxylic and phosphoric acid groups is successfully applied for imaging hemorrhagic diseases. LJ-2P demonstrates a strong affinity for fibrinogen and fibrin clots both computationally and experimentally, thus exhibiting increased brightness upon coagulation. As compared to Indocyanine Green, LJ-2P provides a longer imaging window, higher imaging specificity, and signal-to-background ratio, as well as superior photobleaching resistance in three disease models: gastric, pulmonary, and cerebral hemorrhages. These results reveal that LJ-2P demonstrates enhanced imaging capabilities, enabling precise identification of hemorrhagic sites.

Investigation of the affinity and interaction of fibrinogen with trehalose as a protein stabilizer

Trehalose, a remarkable substance, holds great importance in biomedicine for its anti-inflammatory properties and its capacity to hinder scar formation at wounds. This study aims to explore how trehalose interacts with fibrinogen, a crucial protein in wound healing, paying particular attention to their structural characteristics. An array of analytical methods, such as steady-state fluorescence, UV–Vis analysis, circular dichroism (CD), Zeta potential measurements, Dynamic light scattering (DLS), and Fourier-transform infrared spectroscopy (FT-IR), are employed to examine the influence of trehalose on fibrinogen. The findings demonstrate that trehalose induces structural alterations on the surface of fibrinogen, resulting in its compaction. Moreover, computational modeling approaches like docking and molecular dynamics simulations are utilized to enhance the understanding of the interactions between trehalose and fibrinogen. The findings indicate the establishment of a stable complex between the two entities, accompanied by slight modifications in the protein's structure due to their interaction. The main forces facilitating the bonding of trehalose (the ligand) and the protein are hydrogen bonds and van der Waals forces, resulting in an entropy-driven spontaneous bonding process. FT-IR data reveals the emergence of fresh bonds between fibrinogen and trehalose post their interaction, whereas Zeta potential investigations suggest that the engagement with trehalose enhances the stability of the protein structure.Tests assessing cytotoxicity on a typical fibroblast cell line demonstrated that the fibrinogen-trehalose compound displays lower toxicity levels compared to trehalose alone. This suggests a milder impact on cells from the compound. Moreover, molecular docking analysis supports these experimental results, indicating that trehalose molecules primarily bind to the C-terminal end of the coiled-coil segment and the globular area of the Bβ chain within the Dβ fragment. These outcomes highlight the promise of trehalose as a valuable material for wound dressing purposes.

Frequency and risk factors of thromboembolic complications in patients with inflammatory bowel diseases

Abstract Inflammatory bowel diseases (IBD) are characterised by chronic immune inflammation of the mucous membrane and/or the thickness of the intestinal wall, and are also accompanied by disorders of the blood clotting system and the development of a hypercoagulation state. The aim of this study was to identify the frequency of thromboembolic complications (TEC) in IBD patients and to determine the influence of acquired and inherited hypercoagulation factors that contribute to the development of TEC. The clinical status of 1238 IBD patients who were treated in 2019 was evaluated. Of these, 748 patients suffered from ulcerative colitis (UC) and 490 had Crohn's disease (CD). Among the UC patients, there were 369 men (49.3%) and 379 women (50.7%). In 10.2% of patients with UC, there were clinically significant TEC. There were 227 men (46.3%) and 263 women (53.7%) among patients with CD. 7.3% of patients with CD had clinically significant TEC. 112 (9.0 %) of 1238 IBD patients had clinically significant TEC. Among patients with UC (n = 748), 76 (10.2%) showed clinically significant TEC. Among patients with CD (n = 490), 36 (7.3%) had TEC. Of 112 IBD patients with clinically significant TEC, 45 (40.2%) had genetic polymorphisms that increased affinity for fibrinogen, increased platelet aggregation, and contributed to a decrease in the activity of folate cycle enzymes, including methylenetetrahydrofolate reductase, which may be manifested by a moderate increase in homocysteine levels. Of the 45 IBD patients with clinically significant TEC due to inherited factors, 30 (66.6%) patients had UC and 15 (33.7%) patients had CD (HR – 1.038, 95% CI 0.746–1.444; x2 – 0.049; p = 0.83921). 67 patients with IBD (59.8%) who had clinically significant TEC did not have genetic polymorphisms leading to hypercoagulation. Based on the analysis, we can conclude that risk factors for the development of TEC like the status of a smoker, long bed rest, taking hormonal contraceptives, varicose veins of the lower extremities, high activity of the disease, glucocorticosteroids therapy, the extent of intestinal damage in patients with IBD, and genetic factors, should be taken into account by gastroenterologists in the treatment of patients with UC and CD. The hereditary factor of hypercoagulation equally affects the development of TEC, both in patients with UC and CD.

Frequency and risk factors for thromboembolic complications in patients with inflammatory bowel diseases

Inflammatory bowel diseases (IBD) are characterized by chronic immune inflammation of the mucous membrane and/or the thickness of the intestinal wall, and are also accompanied by disorders of the blood clotting system and the development of a hypercoagulation state. To identify the frequency of thromboembolic complications (TEC) in IBD patients and to determine the influence of acquired and inherited hypercoagulation factors that contribute to the development of TEС. The clinical status of 1,238 IBD patients who were treated in 2019 was evaluated. Of these, 748 patients with ulcerative colitis (UC) and 490 patients with Crohn's disease (CD). Among UC patients, there were 369 (49.3%) men and 379 (50.7%) women. In 10.1% of patients with UC, there were clinically significant feasibility studies. There were 227 (46.3%) men and 263 (53.7%) women among patients with CD; 7.3% of patients with CD had clinically significant feasibility studies. In general 112 (9.0%) of 1,238 IBD patients had clinically significant feasibility studies. Among patients with UC (n=748), 76 (10.2%) showed clinically significant feasibility studies. Among patients with CD (n=490), 36 (7.3%) had a feasibility study. Of 112 IBD patients with clinically significant TEC, 45 (40.2%) had genetic polymorphisms that increase affinity for fibrinogen, increase platelet aggregation, and contribute to a decrease in the activity of folate cycle enzymes, including methylenetetrahydrofolate reductase, which may be manifested by a moderate increase in homocysteine levels. Of the 45 IBD patients with clinically significant TEC due to inherited factors, 30 (66.6%) patients had UC, 15 (33.7%) patients had CD (hazard ratio 1.038, 95% confidence interval 0.7461.444; 2=0.049; p=0.83921); 67 (59.8%) patients with IBD who had clinically significant TEC did not have genetic polymorphisms leading to hypercoagulation. Based on the analysis, we can conclude that such risk factors for the development of TEC as the status of a smoker, long bed rest, taking hormonal contraceptives, varicose veins of the lower extremities, high activity of the disease, glucocorticoids therapy, the extent of intestinal damage in patients with IBD, genetic factors, should be taken into account by gastroenterologists in the treatment of patients with UC and CD. The hereditary factor of hypercoagulation equally affects the development of TEC, both in patients with UC and CD.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

AbstractThe αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Hypoxic Exercise Training Elevates Erythrocyte Aggregation

Pathological erythrocyte aggregation reduces capillary perfusion and oxygen transfer to tissue, which is determined by the negative surface charge on the erythrocyte membrane (intrinsic aggregability) and fibrinogen–erythrocyte interaction (extrinsic factor). Exercise-induced oxidative stress is important for rheological adaptation to training but may also cause erythrocyte senescence. This study clarifies the effects of hypoxic exercise training on intrinsic/extrinsic factors of aggregation. In total, 60 healthy sedentary males were randomly assigned to either hypoxic (HE; FIO2 = 0.15) or normoxic exercise training (NE; FIO2 = 0.21) groups for 30 min·d−1, 5 d·wk−1 for 6 weeks at 60 % of the maximum work rate or to a control group (CTL). A hypoxia exercise test (HET, FIO2 = 0.12) was performed before and after the intervention. Erythrocyte aggregation was assessed by ektacytometry, and fibrinogen binding affinity and senescence biomarkers were assessed by flow cytometry. An acute 12% oxygen HET significantly enhanced erythrocyte global aggregation through intrinsic aggregability. Resting aggregation is promoted by both intrinsic aggregability and fibrinogen binding probability and force after HE, whereas NE is mainly associated with ameliorated fibrinogen–erythrocyte interactions. The HET still facilitated global aggregation after HE because of the augmented fibrinogen-related factors, even though the intrinsic factor was suppressed. Additionally, HE further increased reticulocyte counts while reducing the expression of CD47 and CD147. Resting aggregability is promoted by both intrinsic and extrinsic factors after HE, whereas NE is mainly associated with an ameliorated affinity for fibrinogen. Although an accelerated turnover rate was observed, HE further led to erythrocyte senescence.

Staphylococcus epidermidis Has Growth Phase Dependent Affinity for Fibrinogen and Resulting Fibrin Clot Elasticity.

Bacterial infection and thrombosis are highly correlated, especially in patients with indwelling medical devices. Coagulase-negative staphylococci, typified by Staphylococcus epidermidis, are a common cause of medical device infections owing to their biofilm forming capacity which provides protection from antibiotics and host immune response. Attention has been drawn to the interaction between S. epidermidis and host proteins, specifically fibrinogen. However, little is known regarding the impact of the transition from planktonic to biofilm forming phenotype on this interaction. Here we investigate the growth phase dependence of bacteria-fibrinogen interaction and the resulting effect on fibrin clot formation, structure, and mechanics. Flow cytometry demonstrated growth phase dependent affinity for fibrinogen. To mimic intravascular device seeding, we quantified the adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow conditions in vitro. The bacterial deposition rate onto fibrinogen was significantly greater for stationary (5,360 ± 1,776 cells/cm2s) versus exponential phase (2,212 ± 264, cells/cm2 s). Furthermore, the expression of sdrG–a cell surface adhesion protein with specificity for fibrinogen–was upregulated ∼twofold in the stationary versus the exponential phase. Rheometry and confocal microscopy demonstrated that stationary phase S. epidermidis slows clot formation and generates a more heterogeneous fibrin network structure with greater elasticity (G′ = 5.7 ± 1.0 Pa) compared to sterile fibrinogen (G′ = l.5 ± 0.2 Pa), while exponential phase cells had little effect. This work contributes to the current understanding of the growth phase dependent regulation of bacterial virulence factors and the correlation between bacterial infection and thrombosis.

P192 Frequency of hereditary and acquired thromboembolic complications in patients with inflammatory bowel diseases in Moscow Clinical Scientific Center named after A.S. Loginov

Abstract Background Thromboembolic complications (TC), which are one of the characteristic manifestations of inflammatory bowel diseases (IBD). Objective to identify the frequency of acquired and inherited hypercoagulation factors that contribute to the development of TC in patients with IBD. Methods The clinical status of 1238 IBD patients undergoing treatment in 2019 was evaluated in the Department of IBD. 748 patients with ulcerative colitis (UC) and 490 patients with Crohn’s disease (CD). In 112 patients with IBD (9.0%), clinically significant TC (venous thrombosis of the lower extremities, upper extremities and others) was detected. In patients with clinically significant feasibility studies, DNA isolated from peripheral blood lymphocytes was examined to identify molecular genetic mutations that lead to hypercoagulation. Results Of the 112 patients with TC, 76 (67.8%) patients had UC, and 36 (32.2 %) patients had Crohn’s disease. Of 112 IBD patients with clinically significant TC, 45 (40.2 %) had genetic mutations that increase affinity for fibrinogen, increase platelet aggregation, disrupt folic acid metabolism, and reduce the activity of the methylentetrahydrofolate reductase enzyme, which may be manifested by a moderate increase in homocysteine levels. 67 patients with IBD (59.8%) did not have genetic mutations that lead to hypercoagulation. Of the 45 IBD patients with clinically significant feasibility studies due to hereditary factors, 30 (66.6%) patients had UC, 15 (33.7%) patients had CD (RR-1.038, 95% CI 0.746–1.444; x2-0.049; p=0.83921). Conclusion Clinically significant feasibility studies were found in 9.0% of IBD patients. More than 40% of patients with clinically significant feasibility studies (n=112) have inherited factors that contribute to the development of feasibility studies. About 60 % of IBD patients with clinically significant feasibility studies do not have hereditary factors that lead to the development of feasibility studies.

P003 Frequency of Hereditary and Acquired Thromboembolic Complications in Patients With Inflammatory Bowel Diseases in Moscow.

BACKGROUND: Thromboembolic complications (TC), which are one of the characteristic manifestations of inflammatory bowel diseases (IBD). OBJECTIVE: To identify the frequency of acquired and inherited hypercoagulation factors that contribute to the development of TC in patients with IBD. METHODS: The clinical status of 1238 IBD patients undergoing treatment in 2019 was evaluated in the Department of IBD. 748 patients with ulcerative colitis (UC) and 490 patients with Crohn's disease (CD). In 112 patients with IBD (9.0%), clinically significant TC (venous thrombosis of the lower extremities, upper extremities and others) was detected. In patients with clinically significant feasibility studies, DNA isolated from peripheral blood lymphocytes was examined to identify molecular genetic mutations that lead to hypercoagulation. RESULTS: Of the 112 patients with TC, 76 (67.8%) patients had UC, and 36 (32.2%) patients had Crohn's disease. Of 112 IBD patients with clinically significant TC, 45 (40.2%) had genetic mutations that increase affinity for fibrinogen, increase platelet aggregation, disrupt folic acid metabolism, and reduce the activity of the methylentetrahydrofolate reductase enzyme, which may be manifested by a moderate increase in homocysteine levels. 67 patients with IBD (59.8%) did not have genetic mutations that lead to hypercoagulation. Of the 45 IBD patients with clinically significant feasibility studies due to hereditary factors, 30 (66.6%) patients had UC, 15 (33.7%) patients had CD (HR 1.038, 95% CI 0.746–1.444; x2-0.049; P = 0.83921). CONCLUSION: Clinically significant feasibility studies were found in 9.0% of IBD patients. More than 40% of patients with clinically significant feasibility studies (n = 112) have inherited factors that contribute to the development of feasibility studies. About 60% of IBD patients with clinically significant feasibility studies do not have hereditary factors that lead to the development of feasibility studies.

Biocompatibility enhancement of hemodialysis membranes using a novel zwitterionic copolymer: Experimental, in situ synchrotron imaging, molecular docking, and clinical inflammatory biomarkers investigations.

The aim of the current research study is to conduct a comparative assessment of biocompatibility of zwitterionic-coated polyether sulfone (PES) clinical hemodialysis (HD) membranes using both theoretical and experimental methods. Fibrinogen plays a key role in assessing membrane hemocompatibility since its membrane-surface adsorption triggers several biological reactions, complete thrombosis and embolism. As a result, adsorption of fibrinogen on the untreated PES surface and novel synthesized PES coated with poly 3-((3-(3-carboxy-2,5-dimethyltridecanamido) propyl) dimethylammonio) propane-1-sulfonate as a zwitterion (ZW) was compared. Specifically, the comparison was conducted using in situ synchrotron based micro computed tomography imaging (SR-μCT), Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy, and Scanning Electron Microscopy (SEM). The in situ SR-μCT showed that fibrinogen adsorption and membrane fouling were intense on PES membrane surface. However, there was insignificant fouling in the middle layer of zwitterion coated PES membrane (PES-ZW). Moderate shifting of peaks was observed in ATR-FTIR spectra of the adsorbed fibrinogen when compared to the bulk protein spectra, which may be due to the conformational transformations occurring during the adsorption process. The spectral features indicate that PES-ZW surface has a lower adsorption affinity for fibrinogen than that for the PES surface. In this innovative study, the use of molecular modeling docking to evaluate the interaction of fibrinogen active pose with PES-ZW and PES models with the aim of gaining an in depth understanding of the functional group responsible for the interactions was explored. The PES and PES/zwitterion hemodialysis membrane models indicated minimum binding energies with fibrinogen by -6.00 and -6.70kcal/mol, respectively. Docking studies thus suggest that the membrane's sulfone functional groups play an essential key role during the fibrinogen interaction and adsorption. The HD patients' uremic samples were incubated in vitro with PES and PES-ZW membranes for the inflammatory biomarkers released of Serpin/Antithrombin-III, Properdin, C5a, IL-1α, IL-1β, TNF-α, and IL6. This study's results emphasize that even though a neutral charge of synthesized novel zwitterion PES, which enhances biocompatibility, the sulfone group still significantly affected the interactions with fibrinogen.

Cerebral amyloid angiopathy-linked β-amyloid mutations promote cerebral fibrin deposits via increased binding affinity for fibrinogen

Cerebral amyloid angiopathy (CAA), where beta-amyloid (Aβ) deposits around cerebral blood vessels, is a major contributor of vascular dysfunction in Alzheimer's disease (AD) patients. However, the molecular mechanism underlying CAA formation and CAA-induced cerebrovascular pathology is unclear. Hereditary cerebral amyloid angiopathy (HCAA) is a rare familial form of CAA in which mutations within the (Aβ) peptide cause an increase in vascular deposits. Since the interaction between Aβ and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, we investigated the role of the Aβ-fibrinogen interaction in HCAA pathology. Our work revealed the most common forms of HCAA-linked mutations, Dutch (E22Q) and Iowa (D23N), resulted in up to a 50-fold stronger binding affinity of Aβ for fibrinogen. In addition, the stronger interaction between fibrinogen and mutant Aβs led to a dramatic perturbation of clot structure and delayed fibrinolysis. Immunofluorescence analysis of the occipital cortex showed an increase of fibrin(ogen)/Aβ codeposition, as well as fibrin deposits in HCAA patients, compared to early-onset AD patients and nondemented individuals. Our results suggest the HCAA-type Dutch and Iowa mutations increase the interaction between fibrinogen and Aβ, which might be central to cerebrovascular pathologies observed in HCAA.

Affinity of Serum Albumin and Fibrinogen to Cellulose, Its Hydrophobic Derivatives and Blends.

This work describes the preparation of spin-coated thin polymer films composed of cellulose (CE), ethyl cellulose (EC), and cellulose acetate (CA) in the form of bi- or mono-component coatings on sensors of a quartz crystal microbalance with dissipation monitoring (QCM-D). Depending on the composition and derivative, hydrophilicity can be varied resulting in materials with different surface properties. The surfaces of mono- and bi-component films were also analyzed by atomic force microscopy (AFM) and large differences in the morphologies were found comprising nano- to micrometer sized pores. Extended protein adsorption studies were performed by a QCM-D with 0.1 and 10 mg mL−1 bovine serum albumin (BSA) and 0.1 and 1 mg mL−1 fibrinogen from bovine plasma in phosphate buffered saline. Analysis of the mass of bound proteins was conducted by applying the Voigt model and a comparison was made with the Sauerbrey wet mass of the proteins for all films. The amount of deposited proteins could be influenced by the composition of the films. It is proposed that the observed effects can be exploited in biomaterial science and that they can be used to extent the applicability of bio-based polymer thin films composed of commercial cellulose derivatives.

Aminopyrimidine Class Aggregation Inhibitor Effectively Blocks Aβ-Fibrinogen Interaction and Aβ-Induced Contact System Activation.

Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an important role in circulatory dysfunction in Alzheimer's disease (AD). Previous work has shown that the interaction between fibrinogen and β-amyloid (Aβ), a hallmark pathological protein in AD, induces plasmin-resistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates cognitive function in mouse models of AD. Since Aβ oligomers have a much stronger affinity for fibrinogen than Aβ monomers, we tested whether amyloid aggregation inhibitors could block the Aβ-fibrinogen interaction and found that some Aβ aggregation inhibitors showed moderate inhibitory efficacy against this interaction. We then modified a hit compound so that it not only showed a strong inhibitory efficacy toward the Aβ-fibrinogen interaction but also retained its potency toward the Aβ42 aggregation inhibition process. Furthermore, our best hit compound, TDI-2760, modulated Aβ42-induced contact system activation, a pathological condition observed in some AD patients, in addition to inhibiting the Aβ-fibrinogen interaction and Aβ aggregation. Thus, TDI-2760 has the potential to lessen vascular abnormalities as well as Aβ aggregation-driven pathology in AD.

Influence of glyco-oxidation on complexes between fibrin(ogen) and insulin-like growth factor-binding protein-1 in patients with diabetes mellitus type 2

Fibrinogen and insulin-like growth factor-binding protein-1 (IGFBP-1) are tightly connected to metabolic changes and complications in patients with diabetes mellitus (DM), and since they mutually interact to form complexes in plasma, we investigated whether and to what extent IGFBP-1/fibrinogen complexes change due to glyco-oxidative processes in DM and whether they participate in fibrin clot formation. These complexes were determined by immunoblotting in plasma samples from healthy adults and patients with DM type 2 (DM2). The influence of glyco-oxidation in vitro on the complexes was also investigated. Amounts of IGFBP-1/fibrinogen complexes in plasma from patients with DM2 were slightly but not significantly lower than in healthy persons. Such complexes in patients’ samples participated in fibrin clot formation to a significantly decreased extent. In vitro experiments with glucose or methylglyoxal (MGO) as reactive agents demonstrated that the complexes underwent glyco-oxidative modification leading to reduced formation and/or stability. Extensively oxidized fibrinogen almost completely lost its ability to bind IGFBP-1. The reduced affinity of fibrinogen for IGFBP-1 accompanying diabetes may potentially shift the equilibrium to liberate more IGFBP-1 (and possibly insulin-like growth factor (IGF)-I) able to activate platelets during coagulation, so contributing to the hypercoagulation state together with other factors. This hypothesis, however, needs further examination.

Regulation of integrins in platelets.

Blood platelets prevent bleeding after trauma by forming occlusive aggregates at sites of vascular injury. Platelet aggregation is mediated by the integrin heterodimer αIIbβ3 and occurs when platelet agonists generated at the injury site convert αIIbβ3 from its resting to its active conformation. Active αIIbβ3 is then able to bind macromolecular ligands such as fibrinogen that crosslink adjacent platelets into hemostatic aggregates. Platelets circulate in a plasma milieu containing high concentrations of the principal αIIbβ3 ligand fibrinogen. Thus, αIIbβ3 activity is tightly regulated to prevent the spontaneous formation of platelet aggregates. αIIbβ3 activity is regulated at least three levels. First, intramolecular interactions involving motifs located in the membrane-proximal stalk regions, transmembrane domains, and the membrane-proximal cytosolic tails of αIIb and β3 maintain αIIbβ3 in its inactive conformation. Transmembrane domain interactions appear particularly important because disrupting these interactions causes constitutive αIIbβ3 activation. Second, the agonist-stimulated binding of the cytosolic proteins talin and kindlin-3 to the β3 cytosolic tail rapidly causes αIIbβ3 activation by disrupting the intramolecular interactions constraining αIIbβ3 activity. Third, the strength of ligand binding to active αIIbβ3 seems to be allosterically regulated. Thus, αIIbβ3 exists in a minimum of three interconvertible states: an inactive (resting) state that does not interact with ligands and two active ligand binding states that differ in their affinity for fibrinogen and in the mechanical stability of fibrinogen complexes they form.

Newly Exposed Membrane Glycoprotein II β (CD41) on Activated Platelets has Higher Affinity for Plasma Fibrinogen that Blocks Anti-CD41 Binding

The density of glycoprotein receptors on platelet membrane is dynamic and increase upon platelet activation by various stimulants to facilitate more ligand binding and functional outcome. Since CD41/61 receptor complex is responsible for platelet to platelet cross-linking, infusion of antagonists/antibodies against these molecules is considered an effective method for inhibiting platelet aggregation in certain clinical conditions. However, binding of anti-CD41 to the platelets stored in bags for transfusion was found to be lower. Probably, upon stimulation the newly exposed receptors may immediately bind fibrinogen and prevent access to the antibody. To prove this hypothesis, binding intensity of FITC-conjugated human fibrinogen to resting and stimulated platelets were compared using flow cytometry. In the presence of plasma fibrinogen, monoclonal antibody binding to the CD41 receptor decreased significantly, both onto the stored and agonistactivated platelets as compared to the resting cells. As compared to ADP-activated platelets, Original Research Article Bhatt and Krishnan; IBRR, 3(1): 1-11, 2015; Article no.IBRR.2015.001 2 binding of anti-CD41 to thrombin-activated platelets was significantly lower. Variable binding intensity of anti-CD41 to platelets activated by different stimulants observed in this study allowed us to suggest that therapeutic antibodies also may not bind to platelets that are stimulated by strong agonists. Our results signify the importance of validating such antibodies under conditions of stronger activation of platelets. We observed that more platelet microparticles (PMP) were shredded upon in vitro platelet activation by thrombin as compared to induction by ADP. However, fibrinogen did not bind to the shredded microparticles Therefore; PMP in circulation may not take part in pathological thrombus formation.

Research Topic: A Study Of Fibrinolysis

According to present concepts, the enzyme responsible for the dissolution of fibrin both intra - and extra - vascularly is generated by the action of an “activator” upon the enzyme precursor plasminogen. Plasminogen is a B-globulin, of molecular weight of the order of 143,000, and a normal constituent of human plasma. It is known to possess a high affinity for both fibrin and fibrinogen, a property fundamental to the Sherry hypothesis of the physiological function of plasminogen where any fibrin clot is visualised as having an intrinsic plasminogen content sufficient to ensure its lysis when activated. The activation is achieved by activator either trapped within the clot at the moment of polymerisation, or diffusing subsequently into the clot from the surrounding liquid phase.

Interaction of fibrinogen and albumin with titanium dioxide nanoparticles of different crystalline phases

TiO2 nanoparticles (NPs) are contained in different kinds of industrial products including paints, self-cleaning glasses, sunscreens. TiO2 is also employed in photocatalysis and it has been proposed for waste water treatment. Micrometric TiO2 is generally considered a safe material, while there is concern on the possible health effects of nanometric titania. Due to their small size NPs may migrate within the human body possibly entering in the blood stream. Therefore studies on the interaction of NPs with plasma proteins are needed. In fact, the interaction with proteins is believed to ultimately influences the NPs biological fate. Fibrinogen and albumin are two of the most abundant plasma proteins. They are involved in several important physiological functions. Furthermore, fibrinogen is known to trigger platelet adhesion and inflammation. For these reasons the study of the interaction between these protein and nanoparticles is an important step toward the understanding of the behavior of NPs in the body. In this study we investigated the interaction of albumin and fibrinogen with TiO2 nanoparticles of different crystal phases (rutile and anatase) using an integrated set of techniques. The amount of adsorbed fibrinogen and albumin for each TiO2 surface was investigated by using the bicinchoninic acid assay (BCA). The variation of the surface charge of the NP-protein conjugates respect to the naked NPs was used to indirectly estimate both surface coverage and reversibility of the adsorption upon dilution. Surface charge was monitored by measuring the ζ potential with a conventional electrophoretic light scattering (ELS) system. The extent of protein deformation was evaluated by Raman Spectroscopy. We found that both proteins adsorb irreversibly against electrostatic repulsion, likely undergoing conformational changes or selective orientation upon adsorption. The size of primary particles and the particles aggregation rather than the crystal phase modulate the affinity of fibrinogen for the TiO2 surfaces.

Effect of DOPE and cholesterol on the protein adsorption onto lipid nanoparticles

Upon administration, nanoparticles (NPs) are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a “protein corona”. NP–protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here, we have investigated the effect of neutral dioleoylphosphatidylethanolamine (DOPE) and cholesterol on the adsorption of human plasma proteins onto the surface of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based cationic liposomes of 100 nm in diameter. Quantitative analysis of the protein corona revealed that replacing cationic DOTAP lipids with neutral lipids, being indifferently DOPE or cholesterol, reduces the affinity of fibrinogen, prothrombin, vitamin K, and vitronectin for the lipid surface. On the other side, DOPE specifically promotes the adsorption of apolipoproteins and serum albumin, while cholesterol induces the preferential binding of immunoglobulins and complement proteins. The results of this study will help to explain why NPs of different lipid compositions have a dramatic difference in their in vivo transfection efficiency and will be useful for design of lipid NPs with optimal circulation profiles.

Corona protein composition and cytotoxicity evaluation of ultra-small zeolites synthesized from template free precursor suspensions

The toxicity of two types of ultra-small zeolites (8–18 nm) with LTL- and EMT-type structures is reported. Both the LTL- and EMT-type zeolites belong to the same group of molecular sieves; they have large pores (7.1–7.5 A) and low silica content (Si/Al = 1.2–2.3). The zeolites are prepared by an environmentally friendly synthetic approach from precursor suspensions without using any organic template. Cellular interactions with the two types of zeolite nanocrystals are evaluated by cell viability, reactive oxygen species and cell life cycle assays. It is found that various concentrations of zeolites have negligible effects on the cell life cycle. Moreover, the LTL- and EMT-types zeolites did not cause extensive oxidative stress on the cells. Although it is seen that the zeolites extensively entered in the cells, there is no sign of toxicity for all employed concentrations of ultra-small EMT and LTL zeolites. Additionally, no abnormality in DNA replication while exposed to the zeolites is observed. Very importantly, the zeolite corona shows a high affinity for fibrinogen, moderate affinity for apoA-II and complement factor 3, and trace affinity for albumin, which is the most abundant protein of human plasma. Thus the zeolite nanoparticles can be considered as very promising material for purification of fibrinogen and lipoproteins. Since fibrinogen is considered as acute phase protein and found to be the most associated biomolecule in the composition of corona at the surface of zeolites, we propose that these nanoparticles can be potentially pro-inflammatory for in vivo applications.