15 research outputs found
TFPIα Interacts with FVa and FXa to Inhibit Prothrombinase During the Initiation of Coagulation
Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase, the thrombin-generating complex of factor Xa (FXa) and factor Va (FVa), during the initiation of coagulation. This inhibition requires binding of a conserved basic region within TFPIα to a conserved acidic region in FXa-activated and platelet-released FVa. In this study, the contribution of interactions between TFPIα and the FXa active site and FVa heavy chain to prothrombinase inhibition were examined to further define the inhibitory biochemistry. Removal of FXa active site binding by mutation or by deletion of the second Kunitz domain (K2) of TFPIα produced 17- or 34-fold weaker prothrombinase inhibition, respectively, establishing that K2 binding to the FXa active site is required for efficient inhibition. Substitution of the TFPIα basic region uncharged residues (Leu252, Ile253, Thr255) with Ala (TFPI-AAKA) produced 5.8-fold decreased inhibition. This finding was confirmed using a basic region peptide (Leu252-Lys261) and Ala substitution peptides, which established that the uncharged residues are required for prothrombinase inhibitory activity but not for binding the FVa acidic region. This suggests that the uncharged residues mediate a secondary interaction with FVa subsequent to acidic region binding. This secondary interaction seems to be with the FVa heavy chain, because the FV Leiden mutation weakened prothrombinase inhibition by TFPIα but did not alter TFPI-AAKA inhibitory activity. Thus, efficient inhibition of prothrombinase by TFPIα requires at least 3 intermolecular interactions: (1) the TFPIα basic region binds the FVa acidic region, (2) K2 binds the FXa active site, and (3) Leu252-Thr255 binds the FVa heavy chain
Tricistronic operon expression of the genes <em>gcaD</em> (<em>tms</em>), which encodes <em>N-</em>acetylglucosamine 1-phosphate uridyltransferase, <em>prs</em>, which encodes phosphoribosyl diphosphate synthetase, and <em>ctc</em> in vegetative cells of <em>Bacillus</em> subtilis
Inactivation of <em>Escherichia</em> <em>coli</em> phosphoribosylpyrophosphate synthetase by the 2',3'-dialdehyde derivative of ATP. Identification of active site lysines
Characterisation and Crystallisation of an Active N-terminally Truncated Form of the <em>Escherichia coli</em> Glycogen Branching Enzyme
Thrombin Generation Assay in Plasma from Hemophilia a Patients with or without Inhibitors on Concizumab Prophylaxis: Spiking with rFVIIa or aPCC
Effect of Unfractionated Heparin on TFPI Elimination
Background: Tissue factor pathway inhibitor (TFPI) plays an important role for the
anticoagulant effect of heparin. Depletion of intravascular TFPI by treatment with
unfractionated heparin (UFH), and not by low molecular weight heparin (LMWH), has been
suggested to explain the superiority of LMWH in treatment of both arterial and venous
thrombosis. The present study was undertaken to investigate the impact of UFH on clearance
kinetics, and organs and cells responsible for the clearance of recombinant human full length TFPI purified from baby hamster kidney cells (TFPIBHK) and from E.Coli (TFPIE.Coli).
Methods: Male Sprague-Dawley rats were used as research animals. TFPIBHK and TFPIE.Coli were labelled with 125I, and used to study clearance in vivo.
Results: Surface Plasmon Resonance (SPR) analysis revealed that both types of TFPI bound to UFH in vitro, but TFPIE.Coli exhibited a faster association rate and a much slow dissociation
rate. Intravenous administration of 100 IU/kg UFH immediately prior to TFPI decreased the circulatory survival (t1/2α) of TFPIBHK from 1.99 ± 0.10 min to 1.17 ± 0.13 min (pE.Coli. Presence of UFH significantly increased the
circulatory survival during the slow t1/2β phase of TFPIE.Coli from 27.44 ± 1.91 min to 36.88 ±
1.87 min (p1/2β of TFPIBHK. Hepatocellular distribution of radiolabeled ligands showed that both forms of TFPI were mainly taken up by PCs in the absence of UFH (≥ 90%). UFH administration switched the hepatocellular distribution of
TFPIE.Coli from PCs towards LSECs, without affecting the distribution of TFPIBHK.
Conclusions: Our findings revealed a specific increase in the elimination of TFPIBHK during UFH treatment. This observation may represent the underlying mechanism for depletion of
endogenous TFPI in humans during UFH treatment
Rat liver sinusoidal endothelial cells (LSECs) express functional low density lipoprotein receptor-related protein-1 (LRP-1)
The low density lipoprotein receptor-related protein-1 (LRP-1) is a large, multifunctional endocytic receptor from the LDL receptor family, highly expressed in liver parenchymal cells (PCs), neurons, activated astrocytes, and fibroblasts. The aim of the study was to investigate if liver sinusoidal endothelial cells (LSECs), highly specialized scavenger cells, express LRP-1. To address this question, experiments were performed in vivo and in vitro to determine if receptor associated protein (RAP) and trypsin-activated α2-macroglobulin (α2M∗) were endocytosed in LSECs. Both ligands were cleared from the circulation mainly by the liver. Hepatocellular distribution of intravenously administered ligands, assessed after magnetic bead cell separation using LSEC- and KC-specific antibodies, showed that PCs contained 93% and 82% of liver-associated 125I-RAP and 125I-α2M∗, whereas 5% and 11% were associated with LSECs. Uptake of RAP and α2M∗ in the different liver cell population in vitro was specific and followed by degradation. The uptake of 125I-RAP was not inhibited by ligands to known endocytosis receptors in LSECs, while uptake of 125I-α2M∗ was significantly inhibited by RAP, suggesting the involvement of LRP-1. Immunofluorescence using LRP-1 antibody showed positive staining in LSECs. Ligand blot analyses using total cell proteins and 125I-RAP followed by mass spectrometry further confirmed and identified LRP-1 in LSECs. LSECs express functional LRP-1. An important implication of our findings is that LSECs contribute to the rapid removal of blood borne ligands for LRP-1 and may thus play a role in lipid homeostasis