Quantifying crystallinity of amlodipine maleate in amorphous solid dispersions produced by fluidized bed granulation using PAT tools.

One of the main concerns with formulations containing amorphous solid dispersions (ASDs) is their physical stability. Stability can be compromised if a formulation contains any residual crystallinity of an active pharmaceutical ingredient (API) that could act as seeds for further crystallisation. This study presents four methods for crystalline amlodipine maleate quantification in ASD, which were developed using one Raman and three NIR process analysers.

Quantification of Amlodipine Maleate Content in Amorphous Solid Dispersions Produced by Fluidized Bed Granulation Using Process Analytical Technology Tools.

Active pharmaceutical ingredient (API) content is a critical quality attribute (CQA) of amorphous solid dispersions (ASDs) prepared by spraying a solution of APIs and polymers onto the excipients in fluid bed granulator. This study presents four methods for quantifying API content during ASD preparation. Raman and three near-infrared (NIR) process analysers were utilized to develop methods for API quantification.

Identification of Amino Acid Residues Critical for the Interaction of Fibrin with N-Cadherin.

We recently identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within the fibrin βN-domains and the third and fifth extracellular domains (EC3 and EC5) of N-cadherin. We also hypothesized that the His16 and Arg17 residues of the βN-domains and the (Asp/Glu)-X-(Asp/Glu) motifs present in the EC3 and EC5 domains may play roles in the interaction between fibrin and N-cadherin. The primary objectives of this study were to test these hypotheses and to further clarify the structural basis for this interaction.

Identification of Neural (N)-Cadherin as a Novel Endothelial Cell Receptor for Fibrin and Localization of the Complementary Binding Sites.

Based on the high structural homology between vascular endothelial (VE)-cadherin and neural (N)-cadherin, we hypothesized that fibrin, which is known to interact with VE-cadherin and promote angiogenesis through this interaction, may also interact with N-cadherin. To test this hypothesis, we prepared fibrin and its plasmin-produced and recombinant fragments covering practically all parts of the fibrin molecule. We also prepared the soluble extracellular portion of N-cadherin (sN-cadherin), which includes all five extracellular N-cadherin domains, and studied its interaction with fibrinogen, fibrin, and the aforementioned fibrin fragments using two independent methods, ELISA and SPR.

Current View on the Molecular Mechanisms Underlying Fibrin(ogen)-Dependent Inflammation.

Numerous studies have revealed the involvement of fibrinogen in the inflammatory response. To explain the molecular mechanisms underlying fibrinogen-dependent inflammation, two bridging mechanisms have been proposed in which fibrin(ogen) bridges leukocytes to endothelial cells. The first mechanism suggests that bridging occurs via the interaction of fibrinogen with the leukocyte receptor Mac-1 and the endothelial receptor ICAM-1 (intercellular adhesion molecule-1), which promotes leukocyte transmigration and enhances inflammation.

Dual functions of the fibrin βN-domains in the VLDL receptor-dependent pathway of transendothelial migration of leukocytes.

Our previous studies revealed that fibrin interacts with the VLDL receptor (VLDLR) through a pair of its βN-domains and this interaction promotes transendothelial migration of leukocytes and, thereby, inflammation. In agreement, the NDSK-II fragment representing the central part of the fibrin molecule and containing these domains stimulates leukocyte transmigration. However, the recombinant (β15-66) fragment corresponding to a pair of the βN-domains inhibits NDSK-II-stimulated leukocyte transmigration.

The Story of the Fibrin(ogen) αC-Domains: Evolution of Our View on Their Structure and Interactions.

Although much has been established concerning the overall structure and function of fibrinogen, much less has been known about its two αC regions, each consisting of an αC-connector and an αC-domain, but new information has been accumulating. This review summarizes the state of our current knowledge of the structure and interactions of fibrinogen's αC regions. A series of studies with isolated αC regions and their fragments demonstrated that the αC-domain forms compact ordered structures consisting of N- and C-terminal subdomains including β sheets and suggested that the αC-connector has a poly(L-proline) type II structure.

Structural Basis for the Interaction of Fibrin with the Very Low-Density Lipoprotein Receptor Revealed by NMR and Site-Directed Mutagenesis.

Interaction of fibrin with the very low-density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation. To establish the structural basis for this interaction, we have previously localized the VLDLR-binding site to fibrin βN-domains including fibrin β chain sequence 15-64 and determined the NMR solution structure of the VLDLR(2-4) fragment containing fibrin-binding CR domains 2-4 of VLDLR. In this study, we identified amino acid residues in VLDLR and the βN-domains that are involved in the interaction using NMR and site-directed mutagenesis.

Illustrated State-of-the-Art Capsules of the ISTH 2020 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person.

Structure and function of fibrinogen BβN-domains.

Two BβN-domains of fibrinogen are formed by the N-terminal portions of its two Bβ chains including amino acid residues Bβ1-65. Although their folding status is not well understood and the recombinant disulfide-linked (Bβ1-66) fragment corresponding to a pair of these domains was found to be unfolded, some data suggest that these domains may be folded in the parent molecule. In contrast, their major functional properties are well established.

Fibrin-VLDL Receptor-Dependent Pathway Promotes Leukocyte Transmigration by Inhibiting Src Kinase Fyn and is a Target for Fibrin β15-42 Peptide.

According to the current view, binding of fibrin degradation product E fragment to endothelial VE-cadherin promotes transendothelial migration of leukocytes and thereby inflammation, and fibrin-derived β15-42 peptide reduces leukocyte transmigration by competing with E for binding to VE-cadherin and, in addition, by signaling through Src kinase Fyn. However, the very low affinity of β15-42 to VE-cadherin raised a question about its ability to inhibit E-VE-cadherin interaction. Further, our previous study revealed that fibrin promotes leukocyte transmigration through the very-low-density lipoprotein (VLDL) receptor (VLDLR)-dependent pathway and suggested a possible link between the inhibitory properties of β15-42 and this pathway.

Nuclear Magnetic Resonance Solution Structure of the Recombinant Fragment Containing Three Fibrin-Binding Cysteine-Rich Domains of the Very Low Density Lipoprotein Receptor.

Our previous studies revealed that interaction of fibrin with the very low density lipoprotein (VLDL) receptor plays a prominent role in transendothelial migration of leukocytes and thereby inflammation. The major goal of our subsequent studies is to establish the structural basis for this interaction. As the first step toward this goal, we localized the fibrin-binding sites within cysteine-rich (CR) domains 2-4 of the VLDL receptor.

Effect of fibrinogen, fibrin, and fibrin degradation products on transendothelial migration of leukocytes.

In spite of numerous studies on the involvement of fibrinogen in transendothelial migration of leukocytes and thereby inflammation, there is still no clear understanding of which fibrin(ogen) species can stimulate leukocyte transmigration. Although we have previously proposed that interaction of fibrin with the VLDL receptor (VLDLR) promotes leukocyte transmigration, there is no direct experimental evidence for the involvement of fibrin in this process. To address these questions, we performed systematic studies of interaction of VLDLR with fibrinogen, fibrin, and their isolated recombinant BβN- and βN-domains, respectively, and the effect of various fibrin(ogen) species on transendothelial migration of leukocytes.

Interaction of Fibrin with the Very Low-Density Lipoprotein (VLDL) Receptor: Further Characterization and Localization of the VLDL Receptor-Binding Site in Fibrin βN-Domains.

Our recent study revealed that fibrin and the very low-density lipoprotein receptor (VLDLR) interact with each other through a pair of fibrin βN-domains and CR domains of the receptor and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major objectives of this study were to further clarify the molecular mechanism of fibrin-VLDLR interaction and to identify amino acid residues in the βN-domains involved in this interaction. Our binding experiments with the (β15-66) fragment, which corresponds to a pair of fibrin βN-domains, and the VLDLR(1-8) fragment, consisting of eight CR domains of VLDLR, revealed that interaction between them strongly depends on ionic strength and chemical modification of all Lys or Arg residues in (β15-66) results in abrogation of this interaction.

Anti-VLDL receptor monoclonal antibodies inhibit fibrin-VLDL receptor interaction and reduce fibrin-dependent leukocyte transmigration.

Our previous studies revealed that the interaction of fibrin with the very low density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation, and localised the fibrin-binding site to CR-domains 2-4 of this receptor. In the present study, we tested interaction of three anti-VLDLR monoclonal antibodies, mAb 1H10, 1H5, and 5F3, with recombinant fragments of VLDLR containing various combinations of its CR-domains and found that the epitopes for mAb 1H10 and mAb 1H5 overlap with the fibrin-binding site of VLDLR. Based on these findings, we hypothesised that mAb 1H10 and mAb 1H5 should inhibit fibrin-VLDLR interaction and modulate leukocyte transmigration.

Interaction of Fibrin with the Very Low Density Lipoprotein Receptor: Further Characterization and Localization of the Fibrin-Binding Site.

Our recent study revealed that fibrin interacts with the very low density lipoprotein receptor (VLDLR) on endothelial cells through its βN domains, and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major aims of this study were to further characterize this interaction and localize the fibrin-binding site in the VLDLR. To localize the fibrin-binding site, we expressed a soluble extracellular portion of this receptor, sVLDLRHT, its N- and C-terminal regions, VLDLR(1-8)HT and des(1-8)VLDLRHT, respectively, and a number of VLDLR fragments containing various combinations of CR domains and confirmed their proper folding by fluorescence spectroscopy.

Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation.

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells.

[Family-school interaction in solving the problem of pupils' health preservation].

The paper covers the most methodological priority forms of a family-school interaction, which are necessary for effective collaboration in molding children's health. It gives the results of a study conducted at experimental schools, which showed the level of performance of heath-preserving, tutorial, and educational functions of a family.

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes.

While testing the effect of the (β15-66)(2) fragment, which mimics a pair of fibrin βN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (β15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance.

Structure, stability, and interaction of fibrin αC-domain polymers.

Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa.

Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrin-derived fragments.

Background: The interaction of the fibrin βN-domain with VE-cadherin on endothelial cells is implicated in transendothelial migration of leukocytes, and the β15-42 fragment representing part of this domain has been shown to inhibit this process. However, our previous study revealed that only a dimeric (β15-66)(2) fragment, corresponding to the full-length βN-domain and mimicking its dimeric arrangement in fibrin, bound to VE-cadherin.

Objective: To test our hypothesis that dimerization of β15-42-containing fragments increases their affinity for VE-cadherin and ability to inhibit transendothelial migration of leukocytes.

Fibrinopeptides A and B release in the process of surface fibrin formation.

Fibrinogen adsorption on a surface results in the modification of its functional characteristics. Our previous studies revealed that fibrinogen adsorbs onto surfaces essentially in 2 different orientations depending on its concentration in the solution: "side-on" at low concentrations and "end-on" at high concentrations. In the present study, we analyzed the thrombin-mediated release of fibrinopeptides A and B (FpA and FpB) from fibrinogen adsorbed in these orientations, as well as from surface-bound fibrinogen-fibrin complexes prepared by converting fibrinogen adsorbed in either orientation into fibrin and subsequently adding fibrinogen.

Evaluation of fibrinogen self-assembly: role of its αC region.

Background: Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood.

Objective: To investigate hydrophobic surface-induced fibrinogen aggregation.

Noncovalent interaction of alpha(2)-antiplasmin with fibrin(ogen): localization of alpha(2)-antiplasmin-binding sites.

Covalent incorporation (cross-linking) of plasmin inhibitor alpha(2)-antiplasmin (alpha(2)-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that alpha(2)-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of alpha(2)-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance.