Characterization of interaction between blood coagulation factor VIII and LRP1 suggests dynamic binding by alternating complex contacts.

Background: Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes.

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors.

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling.

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products.

Background: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIII ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIII can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated.

Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes.

Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.

Low-density lipoprotein receptor-related protein 1 (LRP) mediates clearance of blood coagulation factor VIII (FVIII). In LRP, FVIII binds the complement-type repeats (CRs) of clusters II and IV, which also bind a majority of other LRP ligands. No ligand is known for LRP cluster I, and only three ligands, including the LRP chaperone alpha-2 macroglobulin receptor-associated protein (RAP), bind cluster III.

Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode.

Insect cell-based expression and characterization of a single-chain variable antibody fragment directed against blood coagulation factor VIII.

A recombinant single-chain variable antibody fragment (scFv) KM33 was previously described as a ligand that can inhibit the function of blood coagulation factor VIII (FVIII). This scFv was previously derived from an individual with anti-FVIII antibodies manifested in FVIII functional deficiency (Hemophilia A) and expressed in bacteria. In the present work, we describe an alternative approach for fast and easy production of KM33 in insect cells (Spodoptera frugiperda).

Prostate cancer outcome and tissue levels of metal ions.

Background: There are several studies examining prostate cancer and exposure to cadmium, iron, selenium, and zinc. Less data are available on the possible influence of these metal ions on prostate cancer outcome. This study measured levels of these ions in prostatectomy samples in order to examine possible associations between metal concentrations and disease outcome.

Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects.

Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484-509.

Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays.

Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.

Catabolism of coagulation factor VIII (FVIII) is mediated by low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains.

Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509.