bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Lipid Rafts in the Assembly and Down-Regulation of the Factor X Activating Complex on the Activated Platelet Membrane.
Authors: Ahmad Syed S. , Su Ya-Chi , Walsh Peter N. 
Published: April 2020

Abstract
In a recent study of the role of detergent-insoluble platelet membrane microdomains (lipid rafts) in the assembly of the factor X (FX) activating complex, we have shown, contrary to expectations, that the formation of lipid rafts after incubation of platelets with the thrombin receptor activation peptide (SFLLRN, 25 μM, for 30 min) results in the down-regulation, rather than assembly, of the enzyme-cofactor-substrate complex by sequestering FVIIIa and FX in rafts and separating them from FIXa, which is excluded from raft fractions isolated by sucrose density gradient centrifugation of triton X-100 (0.25%) solubilized platelets. Since the FX-activating complex is assembled rapidly on platelets after incubation with low concentrations of either thrombin (&gt;1 nM) or SFLLRN (25 μM), we have now examined the kinetics of FX-activation complex assembly at early time points after exposure of platelets to agonists. Washed and gel-filtered human platelets, activated with SFLLRN (25 μM) in the presence of FVIIIa (5 U/ml, 1.5 nM) and FX (125 nM) rapidly (within 2 min) developed the capacity to support maximal rates of FIXa (1 nM) catalyzed FX activation that was transient and decayed to baseline within 5 min after exposure to agonist. At these early time points (0.5, 1 and 2 min), platelets activated with SFLLRN (25 μM) in the presence of 125I-labled FVIIIa (nM), FIXa (nM) or FX (nM) and analyzed by sucrose density gradient centrifugation after solubilization in triton X-100 (0.25%) were shown to sequester within the raft fractions ~15% of FIXa, ~15% of FVIIIa and ~15% of FX, whereas at later time points (5–30 min) only FVIIIa (~25%) and FX (~45%) were localized in rafts, from which FIXa was completely excluded. These results strongly suggest that platelet membrane microdomains (lipid rafts) form rapidly after exposure of platelets to PAR-1 agonists to colocalize the enzyme-cofactor-substrate complex, which is transient since at later time points FIXa dissociates from rafts that sequester the FVIIIa-FX complex to down-regulate FX activation.
Heparin Chain Length Dependence of Factor Xa Inhibition by Antithrombin in Plasma.
Authors: Rezaie Alireza R. 
Published: April 2020

Abstract
Heparin anticoagulants function by enhancing the inhibition of coagulation proteases by the serpin antithrombin (AT). Evaluation of the specific anti-factor Xa (fXa) activity of therapeutic heparins in the physiologically relevant plasma-based clotting assays has not been feasible since thrombin, the final protease of the cascade, is the primary target for inhibition by AT in the presence of heparin. To circumvent this problem, we developed an assay in which the native AT in plasma was replaced with an AT mutant which exhibits identical affinity for heparin and near normal reactivity for fXa, but does not react with thrombin and other coagulation proteases in either the absence or presence of heparin. This assay was used to distinguish the anti-fXa activity of different molecular weight heparins from their anti-thrombin activity in clotting assays which were initiated by triggers of either extrinsic or intrinsic pathways of coagulation. The results suggest that the acceleration of fXa inhibition by AT exhibits a marked heparin chain-length dependence, with fondaparinux (a pentasaccharide) having the lowest and unfractionated heparin having the highest effect. Interestingly, comparative studies revealed that the fondaparinux-catalyzed acceleration of thrombin inhibition by AT also makes a significant contribution to prolongation of the clotting time, possibly suggesting that the anticoagulant function of the therapeutic pentasaccharide is mediated though the inhibition of both fXa and thrombin.
B Domain of Coagulation Factor VIII Regulates Exposure of Its Heparin-Binding Site.
Authors: Makogonenko Evgeny M. , Sarafanov Andrey G. , Ananyeva Natalya M. , Radtke Klaus-Peter , Strickland Dudley K. , Saenko Evgueni L. 
Published: April 2020

Abstract
B domain of coagulation factor VIII (fVIII) was previously considered to be dispensable for fVIII function. Recently, it was found that the B domain is important for fVIII intracellular interaction with its chaperon and likely involved in fVIII clearance via asialoglycoprotein receptor. At the same time, the major clearance mechanism of fVIII involves initial interaction with heparan sulfate proteoglycans (HSPGs) followed by internalization via low-density lipoprotein receptor-related protein (LRP), member of low-density lipoprotein receptor (LDLR) family (Saenko et al, 1999; Sarafanov et al, 2001). It is possible that recently discovered clearance of fVIII via LDLR (Bovenschen et al, 2005) occurs in the same way. Since it was previously shown that fVIII binding sites for LRP are not located within B domain, we investigated if the latter regulates fVIII interaction with HSPGs. To explore this role of B domain, we compared the binding of plasma-derived fVIII (pd-fVIII) and recombinant B domain-deleted fVIII (BDD-fVIII) to immobilized LRP and heparin (a model of HSPGs) in surface plasmon resonance-based assay. The corresponding affinities were assessed by processing the binding signals obtained for five different concentrations of each analyte. Both pd-fVIII and BDD-fVIII showed similar affinities for LRP (KD 42–60 nM). The LRP-binding site of BDD-fVIII was partially blocked by pre-incubation with its carrier protein von Willebrand factor (vWf) indicating that it is only partially accessible within fVIII/vWf complex. This was further confirmed by the finding that monoclonal antibody 413, which recognizes a high-affinity LRP-binding site within the fVIII A2 domain, interacted with ~25% of BDD-fVIII molecules bound to immobilized vWf. The affinities of pd-fVIII and recombinant BDD-fVIII for immobilized heparin were similar (KD ~20 nM) and 2-fold higher than that for purified A2 domain (KD ~46 nM). Noteworthy, the maximal binding level (Rmax) proved to be 10-fold lower for pd-fVIII in comparison with BDD-fVIII indicating that in the circulation intact fVIII may have limited interaction with HSPGs. Importantly, pre-incubation with vWf did not interfere with the interaction between BDD-fVIII and heparin (KD ~ 19.5 and 21.8 nM, Rmax ~ 194 and 354 RU for BDD-fVIII and BDD-fVIII/vWf, respectively) thus revealing that heparin-binding site of fVIII is completely exposed in BDD-fVIII/vWf complex. These findings suggest that the presence of B domain in circulating fVIII/vWf complex may regulate fVIII clearance by preventing its interaction with HSPGs. The absence of B domain leads to exposure of heparin-binding site within fVIII and binding of fVIII/vWf complex to HSPGs. This binding may be a driving force in fVIII clearance which involves subsequent exposure of LRP (LDLR)-binding site(s) and internalization of fVIII from its complex with vWf via these receptors.
pH-Dependent Association of Factor VIII Chains: Enhancement of Affinity at Physiological pH by Cu2+.
Authors: Wakabayashi Hironao , Zhou Qian , Nogami Keiji , Ansong Charles , Varfaj Fatbardha , Miles Stephen , Fay Philip J. 
Published: April 2020

Abstract
Factor VIII circulates as a heterodimer composed of a heavy chain (HC) and light chain (LC). The structural integrity of the heterodimer is crucial for factor VIII function. In this study we found that the reconstitution of factor VIII from isolated HC and LC was pH-dependent. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH values. The affinity of HC and LC was maximal at pH 6.5-6.75 (Kd ~ 4 nM), whereas a Kd of ~20 nM was observed at physiological pH (7.25) (Figure). The effect of Cu2+ (0.5 μM total Cu2+) on maximal activity regenerated was negligible at pH 6.25–8.0. However, this level of Cu2+ increased the inter-chain affinity by ~5 fold at pH 7.25 (Figure). This effect resulted from an ~1.5-fold increased association rate constant (kon) and an ~3-fold reduced dissociation rate constant (koff). Addition of Cu2+ to a mixture of HC and LC at equilibrium resulted in an increase in cofactor activity that followed a single exponential, suggesting that association was defined by a simple reaction model. High affinity (Kd = 5.3 fM) of the factor VIII chains for Cu2+, as estimated by increases in cofactor activity, suggested a rapid association rate for the metal ion. Together, these results suggest that the protonation state of specific residues, His in particular, modulate inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.
Figure Figure
Mapping Factor VIIIa Inter-Subunit Interactions Using Chemical Modification and Zero-Length Cross-Linking.
Authors: Miles Stephen M. , Freas Jan , Fay Philip J. 
Published: April 2020

Abstract
Activated factor VIII (FVIIIa) is a complex of three subunits, designated A1, A2, and A3C1C2, that serves as a cofactor for factor IXa in the proteolytic activation of factor X. The structure and surface interactions between factor VIIIa subunits have yet to be fully defined, but a homology-based model is available as a predictive tool (Stoylova-McPhie et al, Blood, 2002). To investigate FVIIIa inter-subunit interactions we used a chemical modification approach to determine surface-exposed and potentially interactive residues. The protein modification reagents acetic anhydride and diethylpyrocarbonate (DEPC), that modify lysine and histidine residues, respectively, were used to identify residues that are modified in the free subunits but protected in the bound complex. The modified samples were digested with various proteases and the resulting digests were analyzed by MALDI-TOF mass spectrometry to determine sites of peptide modification (Figure 1). The majority of data observed was in agreement with the A1/A3C1C2 interface in the predicted model. Protection was observed at Lys-89, Lys-142, and Lys-230 in the A1 subunit, all of which are near the A3C1C2 interface. While most His residues were completely modified by DEPC, partial protection was seen at His-1954, His-1957, and His-1961 in A3C1C2, which we have previously shown to be an A1-interactive region (Ansong and Fay, Biochemistry, 2005). In addition to this data, we attempted to define inter-subunit contacts by covalently cross-linking the FVIIIa complex. Using the zero-length cross-linker 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), which cross-links Glu/Asp residues to Lys, four distinct cross-linked bands were identified by SDS-PAGE (Figure 2). The composition of the cross-linked products was determined using proteolytic digestion and MALDI analysis. The four products correspond to each possible combination of the three subunits. Interestingly, there are no candidate cross-link sites between A1 and A2 in the model, yet this cross-link is the most predominant of the four. One alternative is that the A1–A2 cross-link may involve the A1 337–372 region that is not represented in the model. Taken together, the data are directed toward physically defining interactive regions between FVIIIa subunits and serve to test and supplement current structure models.
FIGURE 1, FIGURE 2 FIGURE 1, FIGURE 2.
Identification of a Plasmin-Interactive Site within the A2 Domain of the Factor VIII Heavy Chain.
Authors: Nogami Keiji , Shima Midori , Nishiya Katsumi , Saenko Evgueni L. , Takeyama Masahiro , Tatsumi Kohei , Tanaka Ichiro , Yoshioka Akira 
Published: April 2020

Abstract
Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991, BLOOD102, 2002). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by &lt;20%). To investigate the responsible residues in A2 domain for plasmin binding, the mutant forms of the A2 domain were expressed in baculovirus system and purified. Compared with wild type (23 nM), the affinity of R484A mutant was dramatically decreased by ~250-fold, and the affinities of K377A, K466A, R471A, and K523A mutants also were decreased by 10~40-folds, respectively. Especially, the addition of R484A mutant was reduced inactivation rate constant of plasmin-catalyzed FVIIIa by only ~40% of that of wild type. These findings demonstrate that Arg484 in the A2 domain contains plasmin-binding site responsible for plasmin-catalyzed FVIII(a) inactivation.
Fragments of Activated Coagulation Factor VIII Bind to Multiple Sites within Low-Density Lipoprotein Receptor-Related Protein.
Authors: Sarafanov Andrey G. , Makogonenko Evgeny M. , Andersen Olav M. , Khrenov Alexey V. , Mikhailenko Irina A. , Ananyeva Natalya M. , Strickland Dudley K. , Saenko Evgueni L. 
Published: April 2020

Abstract
Catabolism of coagulation factor VIII (fVIII) is mediated by the hepatic multiligand receptor low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CRs) organized in four clusters, among which clusters II and IV bind most of LRP ligands. In turn, fVIII contains two major LRP-binding sites, located in A2 and A3 domains (Saenko et al, JBC 1999; Bovenschen et al, JBC 2003). In present work, we characterized binding sites in LRP for A2 domain (A2) and heterodimer A1/A3-C1-C2 (HD), the products of dissociation of activated fVIII. Using a baculovirus expression system, we generated CR clusters II, III and IV, along with eight overlapping CR triplets encompassing clusters II and IV. Surface plasmon resonance-based assays demonstrated that both A2 and HD bind to clusters II and IV, and to the same sets of their CR triplets with similar affinities (KDs 25–50 nM). The same kinetic parameters of interaction of both A2 and HD were observed for several CR doublets from cluster II, shown previously to be minimal binding sites for a classical ligand of LRP, receptor associated protein (RAP) (Andersen et al, JBC 2000). The specificity of A2 and HD interactions with all tested fragments of LRP was confirmed by the ability of RAP to inhibit these interactions, and by the ability of these fragments to inhibit binding of 125I-A2 and 125I-HD to immobilized LRP in a solid-phase assay, and LRP-mediated catabolism of 125I-A2 and 125I-HD in cell culture. Notably, some mutations of the LRP-binding site in A2 resulted in significant reduction or abolishment of its binding to certain fragments of LRP, while the binding to other LRP fragments was less affected. In summary, we demonstrated that i) A2 and HD interact with LRP via its multiple binding sites spanning CRs 3–8 in cluster II and CRs 24–29 in cluster IV, and ii) the elementary binding unit of LRP is formed by at least two adjacent CRs, similar to that shown for RAP. The above data also suggest that besides regulating fVIII levels, LRP also plays a role in clearance of the products of dissociation of activated fVIII.
Loss of TM-Dependent PC-Activation Predisposes to Diabetic Nephropathy: Potential Role of Endothelial Apoptosis.
Authors: Isermann Berend , Thati Madhusudhan , Vinnikov Ilya , Herzog Stefanie , Huntscha Sina , Bünder Robert , Weiler Hartmut , Bierhaus Angelika , Nawroth Peter P. 
Published: April 2020

Abstract
Increased levels of soluble thrombomodulin (TM) in patients with diabetes mellitus are considered a marker of endothelial damage. It is unknown whether the loss of endothelial TM-function contributes to the progression of vascular complications in diabetes mellitus. To address whether the loss of TM-dependent protein C (PC) activation contributes to diabetic complications such as diabetic nephropathy two animal models were employed: (1) TMPro mice, which have been previously described and carry a point mutation in the TM-gene (E404P), resulting in a loss of TM-dependent PC-activation, and (2) hPC mice, which carry a transgene resulting in the expression of a mutant “hyperactivatable” PC, which can be activated by thrombin in the absence of TM. The mutant PC could be captured from plasma samples of hPC mice and activated ex vivo by thrombin in the absence of TM. hPC mice had a prolonged bleeding time. Following induction of diabetes by streptozotocin TAT levels were increased in diabetic control (wild type) mice and to a larger extent in TMPro mice, but not in diabetic hPC mice. In comparison to diabetic control mice the kidney weight was increased in diabetic TMPro mice, but not in diabetic hPC mice. Albuminuria was increased in diabetic TMPro mice and reduced in diabetic hPC mice in comparison to diabetic control mice, indicating increased glomerular damage in TMPro mice and partial protection from glomerular damage in hPC mice. Consistently, using a histological score glomerular damage was more severe in diabetic TMPro mice in comparison to diabetic control mice, while diabetic hPC mice were protected. Preliminary data suggest that the observed changes are associated with increased apoptosis in glomeruli of diabetic TMPro mice. Using HUVECs we were able to establish that high glucose concentrations (30 mM) reduce TM-dependent PC activation. The reduced TM-dependent PC activation is associated with increased apoptosis. Glucose induced apoptosis in HUVECs is associated with an increased Bax/Bcl-2 ratio, increased translocation of Bax into mitochondria, and increased caspase-3 activation. Activated PC normalizes the Bax/Bcl-2 ratio, prevents translocation of Bax, and reduces caspase-3 activity. Further studies using TRAPs and inhibitory antibodies established that the antiapoptotic effect of aPC in glucose stressed endothelial cells is mediated through a Par-1 and EPCR-dependent mechanism. The current data strongly suggest that the loss of the endothelial TM-PC system is not just a marker of endothelial damage in diabetic patients, but rather contributes to the progression of diabetic vascular complications.
Genome-Wide Identification of Prednisolone-Responsive Genes in Primary Acute Lymphoblastic Leukemia Cells.
Authors: Tissing Wim J.E. , den Boer Monique L. , Meijerink Jules P.P. , Menezes Renee X. , Swagemakers Sigrid , van der Spek Peter J. , Sallan Stephen E. , Armstrong Scott , Pieters Robrecht 
Published: April 2020

Abstract
Glucocorticoids (GC) are keystone drugs in the treatment of childhood ALL and therefore it is important to get more insight in signal transduction pathways involved in GC induced apoptosis. Affymetrix U133A GeneChips were used to identify genes that are transcriptionally regulated upon 3 and 8 hours of prednisolone exposure in leukemic cells of 13 children newly diagnosed with ALL. After 3 hours of exposure no changes in gene expression could yet be found. After 8 hours exposure, 57 probesets (51 unique genes) were differentially expressed (p&lt;0.0005 and false discovery rate &lt;0.1) of which 44 probesets (39 genes) were upregulated (median 2.4-fold), whereas 13 probesets (12 genes) were downregulated (median 1.7-fold). Twenty-one genes were not previously found to be transcriptionally regulated by GC. Two tumor suppressor genes, Thioredoxin interacting protein (TXNIP) and Zinc finger and BTB domain containing 16 (ZBTB16), were 3.7-fold and 8.8-fold upregulated. Genes were functionally categorized in three major routes: i.e. MAPK pathways (9 genes), NF-k B signaling (11 genes) and carbohydrate metabolism (6 genes).
Biological characterization of these genes and pathways might elucidate the action of GC in leukemic cells. This knowledge may point to causes of GC resistance, ways to circumvent GC resistance and new potential targets for therapy.
DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.
Authors: Ikeda Kazuhiko , Shichishima Tsutomu , Yamashita Yoshihiro , Maruyama Yukio , Mano Hiroyuki 
Published: April 2020

Abstract
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of &gt;22,000 probe sets (corresponding to &gt;14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P &lt;0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.