The effect of water dynamics on conformation changes of albumin in pre-denaturation state:photon correlation spectroscopy and simulation

Water is essential for protein three-dimensional structure, conformational dynamics, and activity. Human serum albumin (HSA) is one of major blood plasma proteins, and its functioning is fundamentally determined by the dynamics of surrounding water. The goal of this study is to link the conformational dynamics of albumin to the thermal motions in water taking place in the physiological temperature range. We report the results of photon correlation spectroscopy and molecular dynamics simulations of HSA in aqueous solution. The experimental data processing produced the temperature dependence of the HSA hydrodynamic radius and its zeta potential. Molecular dynamics reproduced the results of experiments and revealed changes in the secondary structure caused by the destruction of hydrogen bonds in the macromolecule's globule

The fibrin Bβ125-135 site is involved in the lateral association of protofibrils

Earlier we reported that during the human fibrinogen to fibrin transition a neoantigenic determinant was exposed in the Bβ119-133 fragment, where a hinge locus is situated. The fibrin-specific mAb FnI-3c and its Fab-fragment with epitope in this fragment inhibited the lateral association of protofibrils. We suggested that the epitope coincided with a site involved in this process. In this work we investigated the epitope location more precisely and defined a functional role for its exposure in the hinge locus of the molecule. It was found that mAb FnI-3c bound to human, horse and rabbit fibrins, all of which have Lys in the position corresponding­ to human BβK130, but not to bovine and rat fibrins, which have other amino acid residues in this position, strongly suggesting that BβK130 provides the integral part of the epitope. This fact, homology data, and structural biological analysis of the amino acid sequences around BβK130 indicate that the site of interest is localized within Bβ125-135. The synthetic peptides Bβ121-138 and Bβ125-135, unlike their scrambled versions, bound to mAb FnI-3c in SPR analysis. Both peptides, but not their scrambled versions, inhibited the lateral association of protofibrils. The FnI-3c epitope is exposed after fibrinopeptide A cleavage and desA fibrin monomer formation. Structural biological analysis of the fibrinogen to fibrin transition showed a distinct increase of flexibility in the hinge locus. We propose that the structural transformation in the fibrin hinge regions leads to the conformation necessary for lateral association of protofibrils

Comparative analysis of the influence of chlorine and fluorine anions on the fibrin polymerization

The effect of NaCl and NaF salts in the range of 0.1-0.225 M concentrations on individual stages of fibrin polymerization was investigated, namely: the rate of fibrinogen activation by thrombin, the rate of protofibrils formation, the rate of lateral association of protofibrils, and the maximum clot absorbance value at 350 nm. It was found that the chlorine and fluorine anions equally inhibit the rate of formation of fibrin from fibrinogen and the formation of protofibrils activated with thrombin. Chlorine anions were shown to be significantly more effective than fluorine anions, inhibiting the rate of lateral association and maximal clot absorban­ce level from fibrin desA and desAB. A component of the inhibitory action of chlorine anions, not related to the ionic strength of the solution, was identified and its effect on the individual polymerization steps was shown. Chlorine anions were found to bind to a fibrin clot. Using the surface plasmon resonance method and fibrin-specific mAb FnI-3c, it was established that the rate of exposure of neoantigenic determinants of mAb in the hinge regions of the fibrinogen molecule during its transformation into fibrin under the action of thrombin is inhibited by chlorine anions in correlation with inhibition of the protofibril lateral association rate. It has been suggested that the inhibitory effect of chlorine anions consists of an ionic component and a component that blocks the conformational mobility of the molecule by the chlorine anions binding to its hinge regions and polymerization sites

Interaction of the fibronectin COOH-terminal Fib-2 regions with fibrin: Further characterization and localization of the Fib-2-binding sites

Incorporation of fibronectin into fibrin clots is important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. Previous studies revealed that this incorporation occurs through non-covalent interaction between two NH(2)-terminal Fib-1 regions of fibronectin (one on each chain) and the αC-regions of fibrin, and is further reinforced by factor XIIIa-mediated covalent cross-linking of fibronectin to the fibrin matrix. To clarify the role of another pair of fibrin-binding regions, Fib-2, located at the disulfide-linked COOH-terminal ends of fibronectin, we prepared by limited proteolysis a dimeric 140-kDa (Fib-2)(2) fragment containing both Fib-2 regions and tested its interaction with recombinant fragments corresponding to the αC regions of fibrin(ogen). In both ELISA and surface plasmon resonance (SPR) experiments 140-kDa (Fib-2)(2) bound to the immobilized Aα221-610 αC-fragment. However, the affinity of binding was substantially lower than that for Fib-1. Ligand blotting and ELISA established that the Fib-2 binding site is located in the connector part of the αC region including residues Aα221-391. Analysis of the SPR-detected binding of fibronectin to the immobilized Aα221-610 αC-fragment revealed two types of fibronectin-binding sites, one with high affinity and another one with much lower affinity. Competition experiments revealed about 30% inhibition of the Fib-2 mediated binding by increasing concentrations of Fib-1 fragment suggesting partial overlap of the two sets of binding sites. Based on these results and our previous studies we propose a mechanism of interaction of fibronectin with fibrin in which both Fib-1 and Fib-2 play a role

Level of overall hemostasis potential in donor and patient plasma in pathology

Coagulation potential (CP), overall hemostasis potential (OHP) and fibrinolysis potential (FP) in plasma of donors and patients with myocardial infarction (MI), stroke (St) and hip joint diseases (HJD) have been investigated using M. Blomback’s global hemostasis assay. Plasma samples of the patients were analyzed with APTT reagent in the presence or absence of t-PA. It was found that the ratio of values of СP, OHP and FP in plasma of patients to those of donors plasma were 78, 60 and 123% at MI; 157, 155 and 162% at St; 128, 131 and 124% at HJD. CP to FP ratio that indicated balance between coagulation and fibrinolytic systems activities were 4.13, 2.5, 4.0 and 4.26 in plasma of donors and MI, St and HJD patients, respectively. These results are evidence of increased fibrinolytic activity in plasma of MI patients. Lag-periods of plasma clotting of MI, St and HJD patients were prolonged by 2.3, 7.2 and 1.5-fold, respectively. Pearson’s correlation analysis between parameters, obtained in vitro studies using global hemostasis assay, and concentrations of the molecular markers (soluble fibrin and D-dimer), which formed in vivo in plasma of MI, St and HJD patients, did not reveal any relationship between them