Browsing by Subject "COMPLEX"

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  • Silva, Sofia Marques; Townsend Peterson, A.; Carneiro, Lincoln; Tortola Burlamaqui, Tiberio Cesar; Ribas, Camila C.; Sousa-Neves, Tiago; Miranda, Leonardo S.; Fernandes, Alexandre M.; d'Horta, Fernando M.; Araujo-Silva, Lucas Eduardo; Batista, Romina; Bandeira, Cinthia H. M. M.; Dantas, Sidnei M.; Ferreira, Mateus; Martins, Denise M.; Oliveira, Joiciane; Rocha, Taina C.; Sardelli, Carla H.; Thom, Gregory; Rego, Pericles Sena; Santos, Marcos Persio; Sequeira, Fernando; Vallinoto, Marcelo; Aleixo, Alexandre (2019)
    The Amazon is the primary source of Neotropical diversity and a nexus for discussions on processes that drive biotic diversification. Biogeographers have focused on the roles of rivers and Pleistocene climate change in explaining high rates of speciation. We combine phylogeographic and niche-based paleodistributional projections for 23 upland terra firme forest bird lineages from across the Amazon to derive a new model of regional biological diversification. We found that climate-driven refugial dynamics interact with dynamic riverine barriers to produce a dominant pattern: Older lineages in the wetter western and northern parts of the Amazon gave rise to lineages in the drier southern and eastern parts. This climate/drainage basin evolution interaction links landscape dynamics with biotic diversification and explains the east-west diversity gradients across the Amazon.
  • Colombo, Jessica; Antkowiak, Adrien; Kogan, Konstantin; Kotila, Tommi; Elliott, Jenna; Guillotin, Audrey; Lappalainen, Pekka; Michelot, Alphée (2021)
    Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.
  • Lechuga, Susana; Cartagena-Rivera, Alexander X.; Khan, Afshin; Crawford, Bert; Narayanan, Vani; Conway, Daniel E.; Lehtimäki, Jaakko; Lappalainen, Pekka; Rieder, Florian; Longworth, Michelle S.; Ivanov, Andrei I. (2022)
    The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.
  • El-Khoury, Riyad; Dufour, Eric; Rak, Malgorzata; Ramanantsoa, Nelina; Grandchamp, Nicolas; Csaba, Zsolt; Duvillie, Bertrand; Benit, Paule; Gallego, Jorge; Gressens, Pierre; Sarkis, Chamsy; Jacobs, Howard T.; Rustin, Pierre (2013)
  • Hematy, Kian; De Bellis, Damien; Wang, Xin; Mähönen, Ari Pekka; Geldner, Niko (2022)
    The exocyst is the main plasma membrane vesicle-tethering complex in eukaryotes and is composed of eight different subunits. Yet, in plant genomes, many subunits display multiple copies, thought to reflect evolution of complex subtypes with divergent functions. In Arabidopsis thaliana root endodermal cells, the isoform EXO70A1 is required for positioning of CASP1 at the Casparian Strip Domain, but not for its non-targeted secretion to the plasma membrane. Here, we show that exo84b resembles exo70a1 mutants regarding CASP1 mistargeting and secretion of apoplastic proteins, but exo84b additionally affects secretion of other integral plasma membrane proteins. Moreover, conditional, cell-type-specific gene editing of the single-copy core component SEC6 allows visualization of secretion defects in plant cells with a complete lack of exocyst complex function. Our approach opens avenues for deciphering the complexity/diversity of exocyst functions in plant cells and enables analysis of central trafficking components with lethal phenotypes. Genetic analysis of exocyst isoforms reveals their distinct roles in cargo secretion.
  • Sobral-Leite, Marcelo; Wesseling, Jelle; Smit, Vincent T. H. B. M.; Nevanlinna, Heli; van Miltenburg, Martine H.; Sanders, Joyce; Hofland, Ingrid; Blows, Fiona M.; Coulson, Penny; Patrycja, Gazinska; Schellens, Jan H. M.; Fagerholm, Rainer; Heikkila, Paivi; Aittomaki, Kristiina; Blomqvist, Carl; Provenzano, Elena; Ali, Hamid Raza; Figueroa, Jonine; Sherman, Mark; Lissowska, Jolanta; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Phillips, Kelly-Anne; Couch, Fergus J.; Olson, Janet E.; Vachon, Celine; Visscher, Daniel; Brenner, Hermann; Butterbach, Katja; Arndt, Volker; Holleczek, Bernd; Hooning, Maartje J.; Hollestelle, Antoinette; Martens, John W. M.; van Deurzen, Carolien H. M.; van de Water, Bob; Broeks, Annegien; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Easton, Douglas F.; Pharoah, Paul D. P.; Garcia-Closas, Montserrat; de Graauw, Marjo; Schmidt, Marjanka K.; kConFab-AOCS Investigators (2015)
    Background: Annexin A1 (ANXA1) is a protein related with the carcinogenesis process and metastasis formation in many tumors. However, little is known about the prognostic value of ANXA1 in breast cancer. The purpose of this study is to evaluate the association between ANXA1 expression, BRCA1/2 germline carriership, specific tumor subtypes and survival in breast cancer patients. Methods: Clinical-pathological information and follow-up data were collected from nine breast cancer studies from the Breast Cancer Association Consortium (BCAC) (n = 5,752) and from one study of familial breast cancer patients with BRCA1/2 mutations (n = 107). ANXA1 expression was scored based on the percentage of immunohistochemical staining in tumor cells. Survival analyses were performed using a multivariable Cox model. Results: The frequency of ANXA1 positive tumors was higher in familial breast cancer patients with BRCA1/2 mutations than in BCAC patients, with 48.6 % versus 12.4 %, respectively; P <0.0001. ANXA1 was also highly expressed in BCAC tumors that were poorly differentiated, triple negative, EGFR-CK5/6 positive or had developed in patients at a young age. In the first 5 years of follow-up, patients with ANXA1 positive tumors had a worse breast cancer-specific survival (BCSS) than ANXA1 negative (HRadj = 1.35; 95 % CI = 1.05-1.73), but the association weakened after 10 years (HRadj = 1.13; 95 % CI = 0.91-1.40). ANXA1 was a significant independent predictor of survival in HER2+ patients (10-years BCSS: HRadj = 1.70; 95 % CI = 1.17-2.45). Conclusions: ANXA1 is overexpressed in familial breast cancer patients with BRCA1/2 mutations and correlated with poor prognosis features: triple negative and poorly differentiated tumors. ANXA1 might be a biomarker candidate for breast cancer survival prediction in high risk groups such as HER2+ cases.
  • Nohynek, Hanna; Jokinen, Jukka; Partinen, Markku; Vaarala, Outi; Kirjavainen, Turkka; Sundman, Jonas; Himanen, Sari-Leena; Hublin, Christer; Julkunen, Ilkka; Olsen, Paivi; Saarenpaa-Heikkila, Outi; Kilpi, Terhi (2012)
  • Myllymaki, Satu-Marja; Kämäräinen, Ulla-Reetta; Liu, Xiaonan; Cruz, Sara Pereira; Miettinen, Sini; Vuorela, Mikko; Varjosalo, Markku; Manninen, Aki (2019)
    Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific beta 4-integrin that, as alpha 6 beta 4-heterodimer, forms the core of HDs. The analysis identified similar to 150 proteins that were specifically labeled by BirA-tagged integrin-beta 4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-beta 4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate beta 4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of alpha 6 beta 4-heterodimers, the assembly of beta 4-interactome was not strictly dependent on alpha 6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the beta 4-integrin.
  • Ilyas, Maria; Mietzsch, Mario; Kailasan, Shweta; Väisänen, Elina; Luo, Mengxiao; Chipman, Paul; Smith, J. Kennon; Kurian, Justin; Sousa, Duncan; McKenna, Robert; Söderlund-Venermo, Maria; Agbandje-McKenna, Mavis (2018)
    Bufavirus strain 1 (BuV1), a member of the Protoparvovirus genus of the Parvoviridae, was first isolated from fecal samples of children with acute diarrhea in Burkina Faso. Since this initial discovery, BuVs have been isolated in several countries, including Finland, the Netherlands, and Bhutan, in pediatric patients exhibiting similar symptoms. Towards their characterization, the structures of virus-like particles of BuV1, BuV2, and BuV3, the current known genotypes, have been determined by cryo-electron microscopy and image reconstruction to 2.84, 3.79, and 3.25 angstrom, respectively. The BuVs, 65-73% identical in amino acid sequence, conserve the major viral protein, VP2, structure and general capsid surface features of parvoviruses. These include a core -barrel (B-I), -helix A, and large surface loops inserted between these elements in VP2. The capsid contains depressions at the icosahedral 2-fold and around the 5-fold axes, and has three separated protrusions surrounding the 3-fold axes. Structure comparison among the BuVs and to available parvovirus structures revealed capsid surface variations and capsid 3-fold protrusions that depart from the single pinwheel arrangement of the animal protoparvoviruses. These structures provide a platform to begin the molecular characterization of these potentially pathogenic viruses.
  • Moles, Laura; Gomez, Marta; Heilig, Hans; Bustos, Gerardo; Fuentes, Susana; de Vos, Willem; Fernandez, Leonides; Rodriguez, Juan M.; Jimenez, Esther (2013)
  • Lutfullahoglu-Bal, Guleycan; Keskin, Abdurrahman; Seferoglu, Ayse Bengisu; Dunn, Cory D. (2017)
    Background: During the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was re-fashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol. Results: Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM. Conclusions: Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.
  • Manjur, A. B. M. Kaiser; Lempiainen, Joanna K.; Malinen, Marjo; Varjosalo, Markku; Palvimo, Jorma J.; Niskanen, Einari A. (2021)
    Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and antiinflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.
  • Bankina, Biruta; Stoddard, Frederick L.; Kaneps, Janis; Brauna-Morzevska, Elina; Bimsteine, Gunita; Neusa-Luca, Ingrida; Roga, Ance; Fridmanis, Davids (2021)
    Faba bean (Vicia faba L.) is gaining importance as a crop in northern Europe. In this region, the most important disease of faba bean is chocolate spot disease, attributed to the pathogen Botrytis fabae. However, other Botrytis species have been found to contribute to the disease. Hence, it was decided to isolate fungi from faba bean plants showing symptoms of chocolate spot disease in Latvia, identify the Botrytis species using the DNA sequences of three definitive genes, evaluate the morphological diversity of the isolates in vitro and, finally, to determine the pathogenicity of the isolates in a detached-leaf test. In addition to B. fabae, B. cinerea, B. pseudocinerea and B. fabiopsis were all identified. Phylogenetic analysis of the DNA sequences put all the obtained 44 isolates unequivocally into clusters with known examples of each species. Every species showed wide diversity in its in vitro colour, texture and growing pattern of mycelium, production of sclerotia and pigmentation of the growing medium with much overlap between species showing that this method is not adequate for species discrimination. B. fabae produced the largest lesions on infected leaves, followed closely by B. pseudocinerea and B. cinerea, while B. fabiopsis produced much smaller lesions. The results show that chocolate spot disease of faba bean is attributable to Botrytis four species in northern Europe. This knowledge needs to be considered when controlling the disease by genetic or agronomic means.
  • Patwardhan, Ardan; Brandt, Robert; Butcher, Sarah J.; Collinson, Lucy; Gault, David; Grunewald, Kay; Hecksel, Corey; Huiskonen, Juha T.; Iudin, Andrii; Jones, Martin L.; Korir, Paul K.; Koster, Abraham J.; Lagerstedt, Ingvar; Lawson, Catherine L.; Mastronarde, David; McCormick, Matthew; Parkinson, Helen; Rosenthal, Peter B.; Saalfeld, Stephan; Saibil, Helen R.; Sarntivijai, Sirarat; Valero, Irene Solanes; Subramaniam, Sriram; Swedlow, Jason R.; Tudose, Ilinca; Winn, Martyn; Kleywegt, Gerard J. (2017)
    The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.
  • Kallijärvi, Jukka; Stratoulias, Vassilis; Virtanen, Kristel; Hietakangas, Ville; Heino, Tapio I.; Saarma, Mart (2012)
  • Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S.; Zusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres (2016)
    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.
  • Gorvin, C.M.; Hannan, F.M.; Cranston, T.; Valta, Helena; Mäkitie, Outi; Schalin-Jäntti, Camilla; Thakker, R.V. (2018)
    G-protein subunit -11 (G(11)) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca-i(2+)) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function G(11) mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous G(11) germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of G(11), which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant G(11) proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 G(11) protein to impair CaSR-mediated Ca-i(2+) and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the G(11) cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 G(11) mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser G(11) mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel G(11) germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the G(11) hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. (c) 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
  • Tirkkonen, Taneli; Pakarinen, Jaakko; Rintala, Elina; Ali-Vehmas, Terhi; Marttila, Harri; Peltoniemi, Olli A. T.; Makinen, Johanna (2010)
  • Guryanov, Ivan; Korzhikov-Vlakh, Viktor; Bhattacharya, Madhushree; Biondi, Barbara; Masiero, Giulia; Formaggio, Fernando; Tennikova, Tatiana; Urtti, Arto (2021)
    The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, C-alpha-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.
  • Przybyla, Beata; Pinomäki, Anne; Petäjä, Jari; Joutsi-Korhonen, Lotta; Strandberg, Karin; Hillarp, Andreas; Öhlin, Ann-Kristin; Ruutu, Tapani; Volin, Liisa; Lassila, Riitta (2017)
    Background Allogeneic stem cell transplantation (SCT) enhances coagulation via endothelial perturbation and inflammation. Role of natural anticoagulants in interactions between coagulation and inflammation as well as in acute graft-versus-host disease (GVHD) are not well known. The purpose of this study was to define changes in natural anticoagulants over time in association with GVHD. Patients and methods This prospective study included 30 patients who received grafts from siblings (n = 19) or unrelated donors (n = 11). Eight patients developed GVHD. Standard clinical assays were applied to measure natural anticoagulants, represented by protein C (PC), antithrombin (AT), protein S (PS), complex of activated PC with its inhibitor (APC-PCI) and by markers of endothelial activation: Factor VIII coagulant activity (FVIII: C) and soluble thrombomodulin (s-TM) at 6-8 time points over three months. Results Overall, PC, AT and FVIII: C increased in parallel after engraftment. Significant correlations between PC and FVIII: C (r = 0.64-0.82, p Conclusion The coordinated activation of natural anticoagulants in our longitudinal study indicates the sustained ability of adaptation to endothelial and inflammatory activation during allogenic SCT treatment. The suboptimal control of coagulation by natural anticoagulants at early stage of SCT may contribute to onset of GVHD.