bluuebunny/crossref_metadata_2025_split huggingface dataset:
A3 Domain Residue Glu1829 Contributes to A2 Subunit Retention in Factor VIIIa.
Authors: Wakabayashi Hironao , Zhou Qian , Varfaj Fatbardha , Fay Philip J. 
Published: October 2019

Abstract
Factor VIII circulates as a heterodimer composed of a heavy chain (HC) and light chain (LC). Thrombin converts factor VIII into the active cofactor, factor VIIIa, by cleaving HC into A1 and A2 subunits. While the A1 subunit maintains a stable interaction with the LC-derived A3C1C2 subunit, the A2 subunit is weakly associated in the trimer and this low affinity interaction accounts for the instability of factor VIIIa activity. Although the majority of binding energy for A2 retention is thought to be derived from its interaction with the A1 subunit, the factor VIII structure model based on homology with ceruloplasmin suggests a binding interface between A2 and A3 domains. While exploring potential calcium binding sites in LC by Ala scanning mutagenesis of acidic amino acids, we encountered one mutant (factor VIII:E1829A) that exhibited an ~10-fold increase in the apparent decay rate of factor VIIIa activity compared with the wild type control, as monitored by a factor Xa generation assay (see figure). This decay rate was significantly reduced by the addition of exogenous A2 subunit. In addition, the activity of factor VIII:E1829A measured by one-stage clotting and two-stage factor Xa generation assays were ~20% and ~10% of wild type factor VIII, respectively, consistent with an assay discrepancy due to increased lability of the mutant factor VIIIa. Affinity values for A1 and A2 subunits for wild type and E1829 A3C1C2 subunits were assessed using subunits purified from the factor VIII proteins. To measure A1 subunit affinity for A3C1C2, wild type or E1829A A3C1C2 subunit (10 nM) was reacted with A1 subunit (0–200 nM), and subsequently factor VIIIa activity from the reconstituted A1/A3C1C2 plus excess A2 subunit (400 nM) was measured by factor Xa generation assay. To measure A2 subunit affinity for A3C1C2, wild type or E1829A A3C1C2 subunit (1 μM) and A1 subunit (100 nM) were reacted and then mixed with A2 (0–1 μM) and regenerated cofactor activity was determined. A1 binding affinity for E1829A-A3C1C2 was comparable to the wild type A3C1C2 (Kd = 20.1 ± 3.4 nM and 15.3 ± 3.7 nM, respectively) while A2 binding affinity was reduced (Kd = 385 ± 54 nM for E1829A, 153 ± 34 nM for wild type). Furthermore, examination of the hemophilia A database shows the mutation of Glu1829 to Gly as a mild phenotype consistent with a functional role for this acidic residue. These results suggest that residue E1829 in the A3 domain contributes to A2 subunit retention following activation of the procofactor, possibly by mediating a binding interaction between the two subunits.
Factor VIIIa Activity Decay Factor VIIIa Activity Decay
Identification and Characterization of a Sodium Ion Binding Site on the Staphylocoagulase-Prothrombin Complex.
Authors: Panizzi Peter R. , Bock Paul E. 
Published: October 2019

Abstract
Sodium ion binding regulates allosterically the pro- and anti-coagulant functions of the central coagulation serine proteinase, thrombin (T). The conversion of the prothrombin (ProT) zymogen into T results in expression of the fibrinogen (Fbg) recognition site (exosite I) and may facilitate binding of a single Na+. Na+ binding stabilizes the “fast” form of T that mediates the efficient conversion of Fbg into fibrin. The “slow” form of T, lacking Na+, shows decreased Fbg substrate activity and slightly enhanced activity towards activation of protein C, which results in a net increase in the anticoagulant activity of T (Di Cera et al., 1995). Conversion of protein C to activated protein C results in cleavage and inactivation of factor Va and VIIIa, which inhibits reactions central to hemostasis. Staphylocoagulase (SC), a conformational activator of ProT, binds ProT with high affinity through proexosite I and imparts T-like activity to the zymogen-activator complex. To determine whether the Na+ binding site is present on the SC-(1-325)·ProT complex, kinetic studies in the presence and absence of Na+ were performed, using the non-interacting cation, choline to maintain constant ionic strength. Results indicate that indeed Na+ binds to the SC-(1-325)·ProT complex with a KD of 17 ± 2 mM and causes an ~7-fold increase in the activity of SC-(1-325)·ProT complex toward a T-specific tripeptide chromogenic substrate. The chromogenic substrate activity of mouse ProT·SC-(1-325), unlike the human zymogen, was independent of Na+, consistent with the recent finding that mouse T lacks a functional Na+ binding site (Bush et al., 2006). Fbg clotting studies indicated that human T has 24-fold increased clotting activity in the presence of Na+, and the SC-(1-325)·T complex has 13-fold enhanced activity. Surprisingly, much unlike the results obtained from the chromogenic substrate studies, the Fbg clotting activity of the SC-(1-325)·ProThuman complex was less dependent on the presence of Na+, as indicated by a 4.1-fold increase in clotting activity for the complex in the presence of Na+. Further studies will be necessary to elucidate the functional significance of the Na+ binding site on the SC·ProT complex and its effect on Fbg recognition and cleavage.
Characterization of an Anti-Factor VIII Monoclonal Antibody with A1 and A3 Epitopes Which Increases Factor VIII Activity.
Authors: Takeyama Masahiro , Nogami Keiji , Soeda Tetsuhiro , Suzuki Tsukasa , Hattori Kunihiro , Tatsumi Kohei , Fujita Yuri , Tanaka Ichiro , Yoshioka Akira , Shima Midori 
Published: October 2019

Abstract
Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.
Tyrosine Kinase 2 (Tyk2) Interacts with and Phosphorylates Siva-1, and Auguments the Apoptotic Effect Induced by Siva-1.
Authors: Kakumitsu Haruko , Shimoda Kazuya , Haro Takashi , Shide Kotaro , Kumano Takashi , Oku Seido , Kamezaki Kenjirou , Numata Akihiko , Takenaka Katsuto , Harada Mine 
Published: October 2019

Abstract
Jak-Stat pathway is essential to transduce cytokine signalings. We previously reported that tyrosine kinase 2 (Tyk2), one of the Jak kinases, was essential but Stat1 was dispensable in IFN-alpha-induced B lymphocyte apoptosis. This indicates the existence of other signaling molecules besides Stat1 downstream of activated Tyk2. Therefore, in order to find Tyk2-activated signaling molecules which transduce IFN-alpha signal and induce B lymphocyte apoptosis, we performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Siva-1, which had been originally cloned as a CD27 binding protein. Siva-1 has a death domain (DD) homology region and induces apoptosis in various cells through binding CD27, which belongs to the tumor necrosis factor receptor (TNFR) family. We found that Tyk2 interacts with and phosphorylates Siva-1. Two regions of Siva-1, the N-terminus and the middle portion of the protein containing a death domain homology region, contributed to binding to Tyk2, and two tyrosines of Siva-1, Tyr53 and Tyr162, were phosphorylated by Tyk2. Because Siva-1 is so far thought to be a proapoptotic protein, we assessed whether Tyk2 had any effects on Siva-1-mediated apoptosis through the Tyk2-Siva-1 interaction. First, we established BaF3 cell (IL-3 dependent murine proB cell) lines stably expressing either wild-type Tyk2 (BaF3/WT Tyk2) or constitutively activated Tyk2 which Valine678 was replaced by Phenylalanine (BaF3/V678F Tyk2). In BaF3/V678F Tyk2 cells, Tyk2 and Stats are constitutively activated, and they grow in the absence of IL-3. We transiently transfected either GFP-Siva-1 or control GFP vector into BaF3/parent cells, BaF3/WT Tyk2 cells or BaF3/V678F Tyk2 cells. Thirty-six hours after transfection, we measured annexin V positive cells by flow cytometry on GFP-positive cells. Approximately 24% of both BaF3/parent cells and BaF3/WT Tyk2 cells transfected with GFP-Siva-1 fell into apoptosis, whereas only less than 7% of the cells transfected control vector showed apoptosis. The frequency of this Siva-1-induced apoptosis was increased in BaF3/V678F Tyk2 cells (over 43% vs <10% of expressing control vector), suggesting that activated Tyk2 augmented the apoptotic function of Siva-1. In conclusion, we show that Tyk2 associates with and phosphorylates Siva-1 and that this Tyk2-Siva-1 interaction enhances the apoptotic effect induced by Siva-1. This indicates that Siva-1 might be a probable key molecule downstream of Tyk2 which is activated in response to IFN-alpha.
Human Mesenchymal Cells Regulates Naive and Memory T Regulatory Cells.
Authors: Di Ianni Mauro , Del Papa Beatrice , Moretti Lorenzo , Sportoletti Paolo , Bonifacio Elisabetta , Cecchini Debora , De Ioanni Mariangela , Falzetti Franca , Tabilio Antonio 
Published: October 2019

Abstract
CD4(+)CD25(+)FoxP3(+) T regulatory (T reg) cells constitute 1–2% of peripheral blood cells in adults and function prevalently as immunosuppressors. 70% of T reg are memory/effector cells with a CD45RO+ phenotype. The other 30% have a naive T cell phenotype (CD45RA+). Both sub-populations exert a regulatory function and when sorted and/or purified each inhibits a mixed lymphocyte culture. In the present study, we investigated the effects of human recombinant IL-7 (rIL-7), human mesenchymal cells (hMSC) and hMSC engineered with the IL-7 gene (hMSC/IL-7) on regulatory T cells expressing a memory (CD4/CD25/CD45RO) or naive (CD4/CD25/CD45RA) phenotype. T cells from healthy donors were enriched by immnuselection to provide populations of CD45RA+ cells (95 % ± 2.9) and CD45RO+ cells (97 % ± 0.25). Enriched naive and memory cells were cultured in presence rIL-7 (100 ng/ml), hMSC (ratio 5:1) or hMSC/IL-7 (ratio 5:1). After 7 days’ culture we examined the CD4/CD25+ cells within the naive and memory populations. In the naive T cell population the T reg starting fraction of 0.05 % ± 0.01 of CD4/CD25 positive cells, did not change in the presence of rIL-7 while it rose to 0.2 % ± 0.14 in presence of human MSC and more interestingly reached 1.67 % ± 0.6 in presence of IL-7 engineered human MSC. In the memory T cell population the T reg starting fraction of 0.3 % ± 0.05 of CD4/CD25 positive cells, did not change in presence of rIL-7 while it rose 1.5 % ± 0.9 in the presence of human MSC and more interestingly reached 11.55 % ± 7.5 in the presence of human IL-7 engineered-MSC. We analyzed CD127 expression within different groups. In the naive T reg starting fraction 3 % ± 1.2 expressed CD127 which was down-regulated to 0.96 % ± 0.5 in the presence of rIL-7, to 0.29 % ± 0.2 with human MSC and to 0.37 % ± 0.02 with human IL-7engineered-MSC. Memory T reg cells expressed CD127 in 15% ± 1.2 of the starting fraction which was down-regulated to 1.2 % ± 0.45 in the presence of rIL-7, to 1.32 % ± 0.34 with hMSC and to 4.01% ± 0.74 in presence of hMSC/IL-7. FoxP3 expression was measured by real time quantitative PCR in sort-purified subsets of peripheral blood, identified by staining with a combination of CD4, CD25, CD45RA or CD45RO. In naive T reg FoxP3 expression was increased 1.15 fold in the presence of hMSC and 2.7 fold in presence of hMSC/IL-7. In memory T reg FoxP3 expression was increased 1.14 fold in the presence of hMSC and 2.67 fold in presence of hMSC/IL-7. In conclusion naive T reg cells are IL-7 independent and up-regulated by human MSC. Engineering human MSC with the IL-7 gene enhanced up-regulation. Memory T reg cells are also IL-7 independent and are up-regulated by human MSC. Engineering human MSC with the IL-7 gene markedly increased up-regulation. The different regulatory systems may underlie different functions within the T reg sub-populations.
Inhibition of γδ T Cell Proliferation by CD4+CD25+FOXP3+ Regulatory T Cells (Treg).
Authors: Kunzmann Volker , Kimmel Brigitte , Engert Judith , Wilhelm Martin , Einsele Hermann 
Published: October 2019

Abstract
CD4+CD25+FOXP3+ regulatory T cells (Treg) are a unique population of T cells that maintain immune tolerance by suppressing self-reactive cells. Treg also contribute to the establishment of a dominant tolerance during infections and after allogeneic transplantation and suppress immune response to tumors. Increasing evidence supports the existence of elevated numbers of Treg in both solid tumors and hematological malignancies. In this study we show that Treg may also suppress other arms of an effective immune response. In vitro, purified human CD4+CD25+FOXP3+ Treg (by immunomagnetic selection) directly inhibit phosphoantigen (BrHPP)-mediated proliferation of Vγ9Vδ2 T cells, the major γδ T lymphocyte subset in humans. Importantly, suppression of γδ T cell proliferation by Treg was maintained when Treg where separated from γδ T cells by Transwells, suggesting that the inhibitory function of Treg on γδ T cell proliferation is not cell-contact independent and rather soluble factors produced by Treg contribute to this suppressive effect. However, Treg do neither influence the expression of activation markers (CD69, CD25) nor the production of IFN-γ by Vγ9Vδ2 T cells stimulated with phosphoantigen indicating that not all effector functions of Vγ9Vδ2 T cells are suppressed by Treg. As we have recently reported, phosphoantigen-mediated γδ T cell proliferation is frequently suppressed in cancer patients. This observation prompted us to address the role of Treg in controlling γδ T cell proliferation in cancer patients. An inverse correlation between Treg frequencies (i.e. the ratio between Treg and Vγ9Vδ2 T cells) in peripheral blood and phosphoantigen-mediated γδ T cell proliferation was found (mean Treg/Vγ9Vδ2 T cell ratio in cancer patients with maintained phosphoantigen-mediated γδ T cell proliferation (n=14): 4.06; mean Treg/Vγ9Vδ2 T cell ratio in cancer patients without phosphoantigen-mediated γδ T cell proliferation (n=55): 33.36)). Therefore, the Treg/Vγ9Vδ2 T cell ratio in peripheral blood can predict the capacity of γδ T cells to proliferate in response to phosphoantigens. In conclusion, these findings support a role for Treg in blunting the γδ T cell arm of the innate immune response in cancer patients and highlight the potential of Treg depletion (e.g. by anti-CD25 antibodies, cyclophosphamide or fludarabine) to promote γδ T cell mediated antitumor activity.
A Novel Use of Rehydrated, Lyophilized Platelets Increases Recombinant FVIIa Procoagulant Activity in a Model of Hemophilia.
Authors: Campbell Robert A. , Fischer Thomas H. , Wolberg Alisa S. 
Published: October 2019

Abstract
The anti-bleeding therapy recombinant factor VIIa (rFVIIa) is used to abrogate bleeding in hemophiliacs with inhibitors, bypassing the need for replacement factors. RFVIIa is hypothesized to work by increasing Xa generation on the platelet’s surface. However, high plasma levels of rFVIIa are required, in part due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. One platelet preparative that may be used in this application is rehydrated, lyophilized (RL) platelets. RL platelets are fixed with paraformaldehyde, which allows them to be frozen and lyophilized while retaining their hemostatic capabilities. Previously, we have shown RL platelets are capable of supporting rFVIIa-mediated thrombin generation and that thrombin generation is increased in a rFVIIa dose-dependent manner (Blood, 106:4057, 2005). In this current study, we have characterized the ability of RL platelets to modulate rFVIIa-mediated thrombin generation and fibrin clot formation in a cell-based complete model of hemophilia. The addition of RL platelets with 50 nM rFVIIa increased the thrombin generation rate in hemophilia 2.8-fold more than 50 nM rFVIIa, alone. Further, the addition of RL platelets with 50 nM rFVIIa normalized clot formation and stability in a fibrinolytic environment, which did not occur in the presence of rFVIIa, alone. In contrast, the addition of RL platelets, alone, to hemophilic conditions had minimal to no effect on thrombin generation rate or the onset of clot formation, suggesting that the effects were due to a specific interaction between rFVIIa and RL platelets. When rFVIIa plus RL platelets were added to platelet-rich plasma from patients with hemophilia A in the presence of tissue-type plasminogen activator, clot formation and stability were improved more than the addition of either agent alone. To examine the mechanism of RL platelets’ augmentation of rFVIIa activity, we titrated the phosphatidylserine (PS) binding protein, annexin V, into reactions with RL platelets in the presence of factors Xa, Va, and prothrombin and measured thrombin generation. The addition of annexin V reduced thrombin generation equally in reactions that contained RL platelets stimulated with or without SFLLRN. Further, thrombin generation was similar on RL platelets simulated with or without SFLLRN in the absence of annexin V. These data suggest that RL platelets already have PS exposed on their surface. We conclude that RL platelets can support rFVIIa-mediated thrombin generation in the absence of factor IX and may enhance rFVIIa activity in part due to PS exposure on the RL platelet surface. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa, at lower doses of rFVIIa than are currently required to achieve hemostasis.
Characterization of TFPI and TFPI-beta in Mouse Tissues.
Authors: Maroney Susan A. , Ferrel Josephine P. , Mast Alan E. 
Published: October 2019

Abstract
Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor. The first two Kunitz domains efficiently inhibit factor VIIa and factor Xa and thereby abrogate tissue factor initiated blood coagulation. Two isoforms of TFPI have been identified. Full-length TFPI is thought to indirectly associate with endothelium via binding to a GPI-anchored co-receptor. TFPI-beta is produced by alternative splicing of full-length TFPI. It consists of the first two Kunitz domains followed by a unique C-terminal region with a direct GPI-anchor attachment site. The physiological significance of TFPI-beta is not known. We performed 3′RACE of mouse placental cDNA to determine if additional isoforms of TFPI exist. These studies identified only TFPI and TFPI-beta. Of importance, a full-length form of TFPI containing a direct GPI-anchor attachment site was not identified. This finding supports the hypothesis that full-length TFPI binds to endothelium indirectly through association with a GPI-anchored co-receptor. In order to identify potential physiological roles for TFPI and TFPI-beta, we analyzed mRNA expression and protein production in mouse tissues. Message for both TFPI and TFPI-beta was present in all tissues examined. Real time PCR showed TFPI message 2.5- to 65-fold higher than TFPI-beta message in the different tissues (heart (2.5)>lung (5.1)>testicle (5.8)>spleen (9.3)>thymus (10)>uterus (11)>kidney (26)> liver (35)> placenta (37)>brain (65)). The high relative amount of TFPI-beta in heart, lung and testicle suggest that it may have an important physiological role in these tissues. In situ hybridization studies did not identify vascular bed specific expression of TFPI or TFPI-beta. These studies demonstrated that TFPI and TFPI-beta localize predominantly within endothelial cells with identical staining patterns in all tissues studied. Western blot analysis for TFPI and TFPI-beta in detergent lysates of the different tissues demonstrated the presence of full-length TFPI in all tissues in amounts proportional to the vascularization of the tissue (placenta>>heart>lung>spleen>kidney>liver>>>brain).
TFPI-beta was not observed by western blot analysis in any of the tissues indicating that TFPI-beta message may be transcriptionally repressed. Brain contains much less TFPI than other tissues, yet has high amounts of tissue factor. The low TFPI in the brain could be due to either low numbers of endothelial cells within the brain or to low expression of TFPI by brain endothelial cells. We used real time PCR to compare the level of TFPI and TFPI-beta mRNA expression to that of the endothelial specific proteins VE-cadherin and PECAM. When compared to these markers, the relative amount of TFPI mRNA brain is similar to that present in the heart and lung, indicating that TFPI likely has an important role in prevention of thrombosis within the cerebral vasculature.