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SUPPLEMENTARY MATERIAL
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A refined model of the genomic basis for phenotypic variation in vertebrate
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hemostasis
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Ângela M. Ribeiro1, M. Lisandra Zepeda-Mendoza2, Mads F. Bertelsen3, Annemarie T.
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Kristensen4, Erich D. Jarvis5,6, M. Thomas P. Gilbert2,7, Rute R. da Fonseca1,2,8§
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University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal
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Interdisciplinary Centre of Marine and Environmental Research – CIIMAR/CIMAR,
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen,
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Øster Voldgade 5-7, 1350, Copenhagen, Denmark
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Frederiksberg, Denmark
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4 Department
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Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark
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Department of Neurobiology, Duke University Medical Centre, Durham, NC 27710, USA
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Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
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Trace and Environmental DNA Laboratory, Department of Environment and Agriculture,
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Curtin University, Perth, Western Australia, 6102, Australia
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Centre for Zoo and Wild Animal Health, Copenhagen Zoo, Roskildevej 38, 2000
of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical
The Bioinformatics Centre, University of Copenhagen, Copenhagen, Denmark
Corresponding author
Email addresses:
AMR: ribeiro.angela@gmail.com
MLZP: lisandracady@gmail.com
MFB: MFB@zoo.dk
ATK: atk@sund.ku.dk
ED: jarvis@neuro.duke.edu
MTPG: mtpgilbert@gmail.com
RRF: fonseca@binf.ku.dk
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Supplementary Text
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1.1. Main steps of the hemostasis network:
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 The process of blood coagulation is initiated either by: a) the exposure of tissue factor and collagen
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or b) exposure of Factor XII (FXII) to negatively charged surfaces which results in the production
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of FXIIa (”a” denotes ”activated”).
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 Tissue factor forms a complex with FVII and this activates FIX to FIXa.
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 Factor XIIa then activates Factor XI, which in turn activates Factor IX that together with Factor
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VIIIa activates Factor X to Factor Xa (on the surface of platelets).
 Factor Xa in combination with Factor Va induces a thrombin (Factor II) burst, since a single FXaFVa complex can activate many prothrombin molecules.
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 The thrombin burst further mobilizes platelets to the site of the injury where they stick to collagen.
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 Thrombin further activates FV, FVIII and FXI, in a positive feedback loop.
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 Fibrinogen attaches itself to platelets.
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 Thrombin promotes the formation of fibrin from fibrinogen.
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 Fibrin stabilizes the platelet plug forming an arterial clot.
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 Once the fibrin-platelet clot has formed, the clotting process is limited to avoid thrombotic
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occlusion of vasculature.
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1.2. Other details:
 Platelets bind to the exposed subendothelium collagen in a ruptured blood vessel via alpha2beta1
integrin and GPVI.
 The binding at high shear occurs mainly through a complex formed by the glycoproteins Ib, V and
IX (GPIb, GPV and GPIX), which cross-links with collagen-bound von Willebrand factor (vWF).
 The observed lack of thrombocyte-thrombocyte adhesion is explained by the density of α2bβ3
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integrins (required for cell-cell adhesion) being at least 18-25 times lower in thrombocytes
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suggested to results from a lack of up-regulation of an ADP receptor (P2RY12) that regulates the
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α2bβ3 integrins during formation of shear-resistant platelet aggregates (avian thrombocytes do not
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respond strongly to ADP – see reference 16 in the main text).
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 LRP8 (in a complex with GPIb) is the major recruiter of FXI to the platelet surface.
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 FXI circulates in the plasma with KNG1.
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 FVIII circulates in the plasma bound to vWF.
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 FXI can be activated by FXIIa, FIIa and FVIIa.
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 The tissue factor/Factor VIIa complex can also activate Factor IX.
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 FIX recruits FVIIIa to activate FX an essential step in the intrinsic pathway.
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 The adhesion of platelets to FXI is followed by platelet activation.
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 LRP8, together with alpha-2-macroglobulin (A2M) contributes to the turnover of FXa.
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1. Supplementary Figures
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Figure S1. Percent sequence identity and coverage for all the filtered BLAST hits. Includes
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all the elements of the ortholog groups found by searching genomes and transcriptomes across
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vertebrates that matched the basic orthology assignment criteria (see Methods).
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Extracellular domain
TM domain
Cytoplasmic tail
GP1balpha
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Figure S2. Multispecies alignment of GP1balpha. The amino acid sequences were aligned with MAFFT1 using the Blosum62
score matrix as implemented in Geneious v7.0 (Biomatters Ltd). The absence of the cytoplasmic tail of GP1balpha is notorious in
archosaurs (birds and crocodiles) and turtles. Four birds are included in the depiction . Species acronyms are reported in Table
S2. TM domain: trans-membrane domain.
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Katoh, Kazutaka; Kuma, Kei-ichi; Toh, Hiroyuki; Miyata, Takashi (2005). "MAFFT version 5: improvement in accuracy of multiple sequence alignment".
Nucleic Acids Research 33 (2): 511–8
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Reptilia
Aves
FXI
Mammalia
KLK
Amphibia
Osteichthyes
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Figure S3. FastTree [38] phylogeny of the FXI-KLK genes. The duplication that gave rise to
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KLKB1 seems to have occurred in therian lineage that includes placentals (‘HUMA’: human;
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‘BOTA’: cattle) and marsupials (‘MODO’: opossum). Prototheria (‘ORAN’: platypus), as other
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vertebrates, have a single FXI-KLK gene (other species acronyms in Table S2).
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Figure S4. FastTree [38] phylogeny of the detected FXII orthologs, noting its absence in
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birds.
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2. Supplementary Tables
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Table S1. Protein names and ID according to the KEGG pathway database: coagulation network ko04610
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and extracellular matrix receptor interaction ko4512.
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Functional
Component
Subendothelium
components
Platelet
Plasma Proteins
Ensembl IDs
ENST00000261405
ENST00000329125
ENST00000366425
ENST00000323007
ENST00000307395
ENST00000306052
ENST00000311907
ENST00000360256
ENST00000218099
ENST00000375559
ENST00000264692
ENST00000253496
ENST00000511608
ENST00000447445
ENST00000225698
ENST00000403558
ENST00000318602
ENST00000393399
ENST00000205948
Protein name
Acronym
KEGG
ID
von Willebrand Factor
vWF
K03900
Glycoprotein Ib subunit alpha
Glycoprotein Ib subunit beta
Glycoprotein V
Glycoprotein IX
Low density lipoprotein receptor-related
protein 8
Coagulation Factor II, Thrombin
Coagulation Factor VIII
Coagulation Factor IX
Coagulation Factor X
Coagulation Factor XI
Coagulation Factor XII
Plasma Kallikrein
Kininogen
Complement Component 1 Binding Protein
Serpin G1
Alpha-2-macroglobulin
Prolylcarboxypeptidase or Angiotensinase C
or
ApoH, beta2glycoprotein I
GPIb
GPIb
GPV
GPIX
K06261
K06262
K06260
K06263
LRP8
7804
FII
FVIII
FIX
FX
FXI
FXII
KLK
HK
CC1Q
SERPING1
A2M
PRCP
ApoH
K01313
K03899
K01321
K01314
K01323
K01328
K03910
K03898
K15414
K04001
K03910
K01285
--
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Table S2. Vertebrate species for which genomic data was compiled and the corresponding searched
databases.
Class
Osteichthyes
Amphibia
Reptilia
Aves
Species
Dario rerio
Takifugo rubripes
Tetraodon nigroviridis
Xenopus tropicalis
Ambystoma mexicanum
Ambystoma tigrinum
Annolis carolinensis
Thamnophis elegans
Podarcis sp
Emuys orbicularis
Caretta caretta
Chelonoids nigra
Chelonia mydas
Pelodiscus sinensis
Alligator mississipiensis
Caiman crocodilus
Gavialis gangeticus
Crocodylus porosus
Acanthisitta chloris
Anas platyrhynchos
Apaloderma vittatum
Aptenodytes forsteri
Balearica regulorum
Buceros rhinoceros
Calypte anna
Caprimulgus carolinensis
Cariama cristata
Cathartes aura
Chaetura pelagica
Charadrius vociferus
Chlamydotic macquenii
Columba livia
Colius striatus
Corvus brachyrhynchos
Cuculus canorus
Egretta garzetta
Eurypyga helias
Falco peregrinus
Fulmarus glacialis
Gallus gallus
Gavia stellata
Geospiza fortis
Haliaeetus albicilla
Leptosomus discolor
Manacus vitellinus
Meleagris gallopavo
Melopsittacus undulatus
Merops nubicus
Mesitornis unicolor
Common name
Zebrafish
Tiger puffer
Green Spotted Puffer
Western clawed frog
Axolotl
Tiger salamander
Carolina anole
Western Terrestrial Garter Snake
Wall lizards
European pond turtle
Loggerhead sea turtle
Galápagos giant tortoise
Green sea turtle
Chinese softshell turtle
American alligator
Spectacled caiman
Gharial
Saltwater crocodile
Rifleman
Mallard
Bar-tailed trogon
Emperor penguin
Grey crowned crane
Rhinoceros hornbill
Anna's hummingbird
Chuck-will's-widow
Red-legged Seriema
Turkey vulture
Chimney swift
Killdeer
MacQueen's bustard
Rock dove
Speckled mousebird
American crow
Common cuckoo
Little egret
Sunbittern
Peregrine falcon
Northern Fulmar
Red junglefowl
Red-throated loon
Medium ground finch
White-tailed eagle
Cuckoo roller
Golden-collared manakin
Wild turkey
Budgerigar
Northern carmine bee-eater
Brown mesite
Acronym
ZFIS
FUGU
TETR
FROG
MEXI
TIGR
ANOC
SNAKE
PODA
EMYS
CARET
CHELO
CHEM
PELS
ALLI
CAIMA
GAVI
PORO
ACACH
ANAPL
APAVI
APTFO
BALRE
BUCRH
CALAN
CAPCA
CARCR
CATAU
CHAPE
CHAVO
CHLMA
COLLI
COLST
CORBR
CUCCA
EGRGA
EURHE
FALPE
FULGL
GALGA
GAVST
GEOFO
HALAL
LEPDI
MANVI
MELGA
MELUN
MERNU
MESUN
Source
a
a
a
a
b
b
d
e
f
f
f
c
c
g
g
g
g
c
h
c
c
c
c
c
c
c
c
c
c
c
i
c
c
c
c
c
c
c
j
c
c
c
c
c
k
l
c
c
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Class
Mammalia
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4
5
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Species
Nestor notabilis
Nipponia nippon
Ophisthocomus hoazin
Pelecanus crispus
Phalacrocorax carbo
Phaethon lepturus
Phoenicopterus ruber
Picoides pubescens
Podiceps cristatus
Pterocles guturalis
Pygoscelis adeliae
Struthio camelus
Taeniopygia guttata
Tauraco erythrolophus
Tinamus guttatus
Tyto alba
Ornithorhynchus anatinus
Monodelphis domestica
Tursiops truncatus
Bos taurus
Homo sapiens
Common name
Kea
Crested ibis
Hoatzin
Dalmatian pelican
Great cormorant
White-tailed tropicbird
American flamingo
Downy woodpecker
Great crested grebe
Yellow-throated sandgrouse
Adélie penguin
Ostrich
Zebra finch
Red-crested turaco
White-throated tinamou
Barn owl
Platypus
Gray short-tailed opossum
Dolphin
Cattle
Human
Acronym
NESNO
NIPNI
OPHHO
PELCR
PHACA
PHALE
PHORU
PICPU
PODCR
PTEGU
PYGAD
STRCA
TAEGU
TAUER
TINGU
TYTAL
ORAN
MODO
DOLP
BOTA
HOMO
Source
c
c
c
c
c
c
c
c
c
c
c
c
m
c
c
c
a
a
a
a
a
a -ENSEMBL
b - Ambystoma database v4.0: http://www.ambystoma.org
c - C 2014. Science; Bird Phylogenomics Project - http://phybirds.genomics.org.cn
d -Schwartz et al 2010; Snake database: http://eco.bcb.iastate.edu/
e - Bird Phylogenomics Project - http://phybirds.genomics.org.cn
f - Chiari et al. 2012 BMC Biology 10:65
g - St. Johns et al. 2012; ICGWGC: International Crocodilian Genomes Working Group Consortium;
http://www.crocgenomes.org;
h - Huang et al. 2013 Nat Genet.
i - Shapiro et al. 2013 Science
j - International Chicken Genome Sequencing Consortium 2004 Nature
k - Dalloul et al. 2010 PLoS Biol.
l - Ganapathy et al. 2013 GigaScience
m - Warren et al. 2010 Nature
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