Download The step-by-step evolution of vertebrate blood coagulation

Birds lose factor XII.
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Frog
Human
Chicken
Platypus
Sea Squirt
Duplication leads to separate
factor XI and prekallikrein.
Amphioxus
First appearance of prekallikrein.
First appearance of factor XII.
Block duplication leads to factors VIII and IX.
Did anything happen here?
Period of invention.
The step-by-step evolution of vertebrate blood coagulation
Dr. Russell F. Doolittle
UC San Diego
a talk given on Jan 29, 2010 at
California State University Fresno
Bernstein and Kairinen (1971)
R. Williams, 1980
Blood clotting in humans is very complex. It involves
more than two dozen genetically encoded proteins.
XI
TFI
tissue factor
XIIa
Kallikrein
X
VII
XIa
TF/fVII
ProtS
IXa
VIIIa
APC
(HMWK)
Prekallikrein
IX
VIII
V
ProtC
Plasminogen
XIII
Thrombomodulin
Xa
Prothrombin
Pro-u-PA
PAI-1
Va
t-PA
Thrombin
Antithrombin
Fibrinogen
XII
XIIIa
TAFI
Fibrin
Monomer
Fibrin
u-PA
α -Antiplasmin
2
Plasmin
Cross-linked
Fibrin
Lysed
Fibrin
Mammalian blood clotting can be divided into sets of reactions.
Thrombin
Fibrinogen
Fibrin
Monomer
Fibrin
XIII
Prothrombin
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
Cross-linked
Fibrin
tissue factor
X
VII
TF/fVII
IXa
Va
VIIIa
V
IX
VIII
XIII
Xa
Prothrombin
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
Cross-linked
Fibrin
tissue factor
X
VII
XII
Kallikrein
TF/fVII
IXa
Va
(HMWK)
Prekallikrein
XIIa
VIIIa
V
IX
VIII
XIII
Xa
Prothrombin
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
Cross-linked
Fibrin
tissue factor
X
VII
XII
Kallikrein
TF/fVII
IXa
Va
(HMWK)
Prekallikrein
XIIa
VIIIa
V
IX
VIII
Plasminogen
XIII
Pro-u-PA
Xa
Prothrombin
t-PA
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
u-PA
Plasmin
Cross-linked
Fibrin
tissue factor
X
VII
XII
Kallikrein
TF/fVII
IXa
Va
(HMWK)
Prekallikrein
XIIa
VIIIa
V
IX
VIII
Plasminogen
XIII
Pro-u-PA
Xa
Prothrombin
t-PA
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
u-PA
Plasmin
Cross-linked
Fibrin
Lysed
Fibrin
PAI-1
prekallikrein
factor VII
factor XIII
factor XII
factor XI
factor VIII
factor V
factor IX
factor X
tissue factor
fibrinogen
thrombin
gelation
The Delicate Balance
protein S
thrombomodulin
tissue factor inhibitor
u-PA
t-PA
plasminogen
antithrombin3
protein C
fibrin
fluidity
It is well established that:
It is well established that:
Thrombin-clottable fibrinogen is found in all vertebrate
animals, but not in protochordates (amphioxus, tunicates,
etc.) or invertebrate animals.
It is well established that:
Thrombin-clottable fibrinogen is found in all vertebrate
animals, but not in protochordates (amphioxus, tunicates,
etc.) or invertebrate animals.
The earliest diverging vertebrates (lampreys and hagfish) have
six-chained, fully differentiated fibrinogens that polymerize
and cross-link the same as mammalian ones.
It is well established that:
Thrombin-clottable fibrinogen is found in all vertebrate
animals, but not in protochordates (amphioxus, tunicates,
etc.) or invertebrate animals.
The earliest diverging vertebrates (lampreys and hagfish) have
six-chained, fully differentiated fibrinogens that polymerize
and cross-link the same as mammalian ones.
Vitamin-K dependent factors play a role in the clotting of
lower vertebrates like the lamprey and hagfish.
It is well established that:
Thrombin-clottable fibrinogen is found in all vertebrate
animals, but not in protochordates (amphioxus, tunicates,
etc.) or invertebrate animals.
The earliest diverging vertebrates (lampreys and hagfish) have
six-chained, fully differentiated fibrinogens that polymerize
and cross-link the same as mammalian ones.
Vitamin-K dependent factors play a role in the clotting of
lower vertebrates like the lamprey and hagfish.
It was long ago predicted that some factors would not
play a role in the clotting of lower vertebrates.
The evolution of vertebrate blood coagulation: a case of Yin and Yang
Doolittle R. F. (1993) Thromb Haemost. 70:24-28.
How did blood clotting become so complex?
How did blood clotting become so complex?
Why is it so complex?
How did blood clotting become so complex?
Why is it so complex?
How can we find out?
How did blood clotting become so complex?
Why is it so complex?
How can we find out?
We can start to answer these questions by
examining clotting in more primitive creatures.
Five Animal Groups with Different Types of Blood Clotting
Protostomes
Insecta
Crustacea
Deuterostomes
Arachnida
Echinodermata
(Protochordata)
Vertebrata
Five Animal Groups with Different Types of Blood Clotting
Protostomes
Insecta
Crustacea
Deuterostomes
Arachnida
Echinodermata
(Protochordata)
Vertebrata
Pufferfish
Lamprey
Zebrafish
E. shark
Hagfish
Mouse
Lizard
Human
Chicken
Platypus
Frog
(70 mya)
Sea Squirt
Amphioxus
(310 mya)
(380 mya)
(430 mya)
(500 mya)
(550 mya)
Many of the clotting proteins are multi-domained
and involve various combinations of common domains.
P
F1
PAN
FN1
G
GLA
K
Kringle
F2
E
FN2
EGF
G
SP
K
K
Serine protease
SP
P
K
K
K
Prothrombin
G
E
E
E
F1
E
Factor XII
Carboxypep
FRED
TG
CP- A
Discoidin
Serpin
Kunitz
Sushi
cc
K
SP
Plasminogen
SP
Protein C, factor VII.
Factor IX, Factor X
F2
K
FN3
K
SP
F1
E
K
K
SP
t-PA
F1
K
u-PA
SP
Vitamin-K Dependent Factors (GLA-containing)
TBHU
TBBO
TBFU
F9HU
F9BO
Factors IX
F9FUA
F9FUB
F10BO
F10HU Factors X
F10FU
F7HU
F7BO
Factors VII
F7FUB
F7FUA
F7FUC
PCBO
PCHU
Proteins C
PCFU
*
*
*
*
* Gene duplication
Thrombins
HU = human
BO = bovine
FU = puffer fish
Today it is possible to find out what clotting factors a creature
has by computer searching of whole genome databases.
Today it is possible to find out what clotting factors a creature
has by computer searching of whole genome databases.
Whole genome databases are available for many vertebrates,
including human, other mammals, opossum, platypus, chicken
Lizard, frog, and several fish. A draft genome is available for lamprey.
Today it is possible to find out what clotting factors a creature
has by computer searching of whole genome databases.
Whole genome databases are available for many vertebrates,
including human, other mammals, opossum, platypus, chicken
Lizard, frog, and several fish. A draft genome is available for lamprey.
But not the hagfish.
Today it is possible to find out what clotting factors a creature
has by computer searching of whole genome databases.
Whole genome databases are available for many vertebrates,
including human, other mammals, opossum, platypus, chicken
Lizard, frog, and several fish. A draft genome is available for lamprey.
But not the hagfish.
My students and I have been scouring the lamprey data base,
as well as those other vertebrates listed above.
Human Blood Clotting
tissue factor
X
VII
TF/fVII
IXa
Va
VIIIa
V
IX
VIII
Plasminogen
XIII
Pro-u-PA
Xa
Prothrombin
t-PA
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
u-PA
Plasmin
Cross-linked
Fibrin
Lysed
Fibrin
tissue factor
X
VII
TF/fVII
IXa
Va
VIIIa
V
missing in lamprey
IX
VIII
Plasminogen
XIII
Pro-u-PA
Xa
Prothrombin
t-PA
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
u-PA
Plasmin
Cross-linked
Fibrin
Lysed
Fibrin
Lamprey System
tissue factor
X
VII
TF/fVII
Va
V
Plasminogen
XIII
Pro-u-PA
Xa
Prothrombin
t-PA
Thrombin
XIIIa
Fibrinogen
Fibrin
Monomer
Fibrin
u-PA
Plasmin
Cross-linked
Fibrin
Lysed
Fibrin
Lampreys have a simpler clotting system than other vertebrates.
Lampreys have a simpler clotting system than other vertebrates.
(We’re anxious to find out what the hagfish has!)
All fish have a simpler clotting system than tetrapods.
All fish have a simpler clotting system than tetrapods.
In particular, they lack the “contact phase” factors.
The Contact System Proteases
prekallikrein
XIIa
α-kallikrein
factor XII
factor XI
factor XIIa
factor X
factor XIa
factor IX
factor IXa
factor Xa
prothrombin
thrombin
E
F1
E K
SP
u-PA
K
K
SP
t-PA
F2 E
F1
E
K
SP
factor XII
E
F1
E
K
SP
HGFA
K K K K K
SP
K K K K
SP
HGF
SP
PK
F2
P
P
P
P
P
P
P
P
P
P
SP
plasminogen
factor XI
Without exception, all of the proteins involved in mammalian blood clotting
are descended from other protein families that are not involved in clotting.
Without exception, all of the proteins involved in mammalian blood clotting
are descended from other protein families that are not involved in clotting.
The backbone of clotting, like many other extracellular processes, is
limited proteolysis, especially employing serine proteases. Hundreds of
serine proteases--all evolutionarily related--are found in animals.
Without exception, all of the proteins involved in mammalian blood clotting
are descended from other protein families that are not involved in clotting.
The backbone of clotting, like many other extracellular processes, is
limited proteolysis, especially employing serine proteases. Hundreds of
serine proteases--all evolutionarily related--are found in animals.
There is also a full complement of serine protease inhibitors, members
of a widely spread family called “serpins.”
Without exception, all of the proteins involved in mammalian blood clotting
are descended from other protein families that are not involved in clotting.
The backbone of clotting, like many other extracellular processes, is
limited proteolysis, especially employing serine proteases. Hundreds of
serine proteases--all evolutionarily related--are found in animals.
There is also a full complement of serine protease inhibitors, members
of a widely spread family called “serpins.”
Factor V (or factor 5) and factor VIII (factor 8) are descended from
ferroxidase enzymes that can be traced back to bacteria.
Without exception, all of the proteins involved in mammalian blood clotting
are descended from other protein families that are not involved in clotting.
The backbone of clotting, like many other extracellular processes, is
limited proteolysis, especially employing serine proteases. Hundreds of
serine proteases--all evolutionarily related--are found in animals.
There is also a full complement of serine protease inhibitors, members
of a widely spread family called “serpins.”
Factor V (or factor 5) and factor VIII (factor 8) are descended from
ferroxidase enzymes that can be traced back to bacteria.
Fibrinogen is a multi-domain protein, the globular portions of which
have numerous relatives throughout the animal kingdom.
Occurrence of Genes for Contact Phase Proteases and Some Paralogs
Factor XI
Human
Opossum
Platypus
Chicken
Green Lizard
Frog
Zebra Fish
Puffer Fish
Lamprey
Yes
Yes
No
No
No
No
No
No
No
Prekallikrein Factor XII
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
Yes
Yes
No
No
No
HGFA HGF Plasminogen t-PA
Yes
Yes
Yes
Yes
Yes
Yes
?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Updated from Ponczek, Gailani & Doolittle, 2008
Chromosomal locations of factor XII and HGFA
Jawless Fish
Fish Amphibians
Reptiles
Birds
Mammals
monotremes
marsupials
eutherians
4
3
1
2
1,2,3 = gene duplications 4
= gene deletion
Updated from Ponczek, Gailani & Doolittle, 2008
Chromosomal locations of prekallikrein and factor XI
Pufferfish
Lamprey
Hagfish
Sea Squirt
Amphioxus
E. shark
Zebrafish
Mouse
Lizard
Frog
Chicken
Platypus
Human
Pufferfish
Lamprey
Zebrafish
E. shark
Hagfish
Mouse
Lizard
Frog
Sea Squirt
Amphioxus
Period of invention.
Chicken
Platypus
Human
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Chicken
Frog
Sea Squirt
Amphioxus
Did anything happen here?
Period of invention.
Platypus
Human
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Frog
Human
Chicken
Platypus
Sea Squirt
Amphioxus
Block duplication leads to factors VIII and IX.
Did anything happen here?
Period of invention.
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Frog
Human
Chicken
Platypus
Sea Squirt
Amphioxus
First appearance of factor XII.
Block duplication leads to factors VIII and IX.
Did anything happen here?
Period of invention.
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Frog
Human
Chicken
Platypus
Sea Squirt
Amphioxus
First appearance of prekallikrein.
First appearance of factor XII.
Block duplication leads to factors VIII and IX.
Did anything happen here?
Period of invention.
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Frog
Human
Chicken
Platypus
Sea Squirt
Duplication leads to separate
factor XI and prekallikrein.
Amphioxus
First appearance of prekallikrein.
First appearance of factor XII.
Block duplication leads to factors VIII and IX.
Did anything happen here?
Period of invention.
Birds lose factor XII.
Pufferfish
Lamprey
Zebrafish
E. shark
Mouse
Lizard
Hagfish
Frog
Human
Chicken
Platypus
Sea Squirt
Duplication leads to separate
factor XI and prekallikrein.
Amphioxus
First appearance of prekallikrein.
First appearance of factor XII.
Block duplication leads to factors VIII and IX.
Did anything happen here?
Period of invention.
In the amphioxus genome:
Lots of genes for fibrinogen-related domains (FREDs), but none for
multi-domained fibrinogen.
Some genes for proteases with sequences that resemble thrombin or factor X,
but no domainal arrangements that correspond to these factors..
There is a gene for a tranglutaminase that is 39% identical with factor XIII,
but it lacks a thrombin-activation site.
A similar situation exists in sea squirt.
In the amphioxus genome:
Lots of genes for fibrinogen-related domains (FREDs), but none for
multi-domained fibrinogen.
Some genes for proteases with sequences that resemble thrombin or factor X,
but no domainal arrangements that correspond to these factors..
There is a gene for a tranglutaminase that is 39% identical with factor XIII,
but it lacks a thrombin-activation site.
A similar situation exists in sea squirt.
There are no bona fide clotting factor genes in the protochordate genomes .
P
K
K
K
K
P
K
SP
P
F1
G
K
K
SP
HGF
4-Kringle Protease
K
K
K
E
K
K
K
P
K
K
K
K
K
SP
Plasminogen
+4 P
G
K
K
SP
F1
E
K
Prothrombin
E
K
G
E
E
K
-5 K
SP
t-PA
E
F2
K
P
E
E
Protein C, factor VII.
Factor IX, Factor X
F2
E
F1
E
F1
Factor XII
or HGFA
K
SP
E
P
P
SP
Prekallikrein,
Factor XI
K
SP
P
K
E
K
SP
u-PA
Updated from Jiang & Doolittle, 2003
Summary
Genomic sequence data are making it possible to reconstruct the
individual events that have led to the complex system of blood clotting
observed in mammals.
Summary
Genomic sequence data are making it possible to reconstruct the
individual events that have led to the complex system of blood clotting
observed in mammals.
The raw material for all the many proteins involved in blood clotting
was available in the form of domains in the common ancestor of
vertebrates and protochordates.
Summary
Genomic sequence data are making it possible to reconstruct the
individual events that have led to the complex system of blood clotting
observed in mammals.
The raw material for all the many proteins involved in blood clotting
was available in the form of domains in the common ancestor of
vertebrates and protochordates.
The number of components increases as one moves up the evolutionary
scale from the jawless fish to mammals. Even among mammals some
recently evolved features are apparent.
Summary
Genomic sequence data are making it possible to reconstruct the
individual events that have led to the complex system of blood clotting
observed in mammals.
The raw material for all the many proteins involved in blood clotting
was available in the form of domains in the common ancestor of
vertebrates and protochordates.
The number of components increases as one moves up the evolutionary
scale from the jawless fish to mammals. Even among mammals some
recently evolved features are apparent.
Reasonable scenarios can be presented that show a step-by-step
development of the process. Whole genome duplications may have played
a role in expanding the inventory of similar proteins.
Acknowledgements
Yong Jiang
Michel Ponczek
Justin Nand
Sung Hong
Da-Fei Feng
David Gailani (Vanderbilt)
My friend, the lamprey (Petromyzon marinus)
MA
FA
MB
MB
FB
MA
LB
LA
Species
diverge
Gene
Duplication
Time
Species
diverge
Gene Duplication
Newmarket, New Hampshire, May, 1962
G
F1
F2
E
K
PAN
GLA
FN1
FN2
EGF
Kringle
G
K
P
K
SP
P
K
SP
K
Prothrombin
G
E
E
E
F1
K
K
K
SP
Plasminogen
SP
F1
Protein C, factor VII.
Factor IX, Factor X
F2
Serine protease
E
Factor XII
K
E
K
K
SP
t-PA
SP
F1
K
u-PA
SP
thrombin
fibrinogen -------------> fibrin + fibrinopeptides
prothrombin
thrombin
fibrinogen -------------> fibrin + fibrinopeptides
prothrombin
thrombin
fibrinogen -------------> fibrin + fibrinopeptides
factor XIIIa
fibrin ----------------> cross-linked fibrin
prothrombin
thrombin
fibrinogen -------------> fibrin + fibrinopeptides
factor XIIIa
fibrin ----------------> cross-linked fibrin
plasmin
lysed fibrin (fragments D and E, etc.)
prothrombin
thrombin
fibrinogen -------------> fibrin + fibrinopeptides
factor XIIIa
fibrin ----------------> cross-linked fibrin
plasmin
lysed fibrin (fragments D and E, etc.)
t-PA
plasminogen ------------> plasmin
prothrombin
thrombin
fibrinogen -------------> fibrin + fibrinopeptides
factor XIIIa
fibrin ----------------> cross-linked fibrin
plasmin
lysed fibrin (fragments D and E, etc.)
t-PA
plasminogen ------------> plasmin
fibrin
inactive t-PA -----------------> active t-PA
A Simple System (n = 2 plus cells)
(tissue factor, prothrombin, thrombocytes)
tissue factor
“thrombocytes”
(pro)thrombin
thrombin
cell clot
G
K
K
SP
(pro)thrombin
A Simple System (n = 2 plus cells)
(tissue factor, prothrombin, thrombocytes)
tissue factor
“thrombocytes”
(pro)thrombin
thrombin
cell clot
G
K
K
SP
(pro)thrombin
This can’t be! Prothrombin has kringles;
Tissue factor interacts with EGF domains.
A Simple System (n = 2 plus cells)
(tissue factor, prothrombin, thrombocytes)
tissue factor
“thrombocytes”
(pro)thrombin
thrombin
cell clot
G
K
K
SP
This can’t be! Prothrombin has kringles;
Tissue factor interacts with EGF domains.
(pro)thrombin
Unless prothrombin originally had
EGF domains!
Another Simple System (n = 3)
(tissue factor, prothrombin, fibrinogen)
tissue factor
fibrinogen
(pro)thrombin
thrombin
fibrin
G
K
K
SP
(pro)thrombin
Another Simple System (n = 3)
(tissue factor, prothrombin, fibrinogen)
tissue factor
fibrinogen
(pro)thrombin
thrombin
fibrin
G
K
K
SP
(pro)thrombin
This can’t be! Prothrombin has kringles.
Tissue factor interacts with EGF domains.
Another Simple System (n = 3)
(tissue factor, prothrombin, fibrinogen)
tissue factor
fibrinogen
(pro)thrombin
thrombin
fibrin
G
K
K
SP
This can’t be! Prothrombin has kringles.
Tissue factor interacts with EGF domains.
(pro)thrombin
Unless prothrombin originally had
EGF domains!
Lamprey
Pufferfish
Zebrafish
Dogfish
Hagfish
Mouse
Lizard
Human
Chicken
Frog
(70 mya)
Amphioxus
Sea Squirt
(310 mya)
(380 mya)
(400 mya)
(500 mya)
(540 mya)
Hemoglobins
α
β
primates
Million Years
-200
-400
jawed fishes
jawless fishes
-600
invertebrates
-800
First Appearance
myoglobin
VII
VII
V
TF
TF
X
P
VIIa
X
IX
Xa
fibrinogen
Va
Xa
T
T
T
IXa
VIIIa
Va
V
T
VIIa
fibrin
P
VIII
fibrinogen
T
fibrin
Lamprey
Other Vertebrates
VII
VII
V
TF
TF
X
P
VIIa
X
IX
Xa
fibrinogen
Va
fibrin
fibrinogen
Xa
T
T
T
IXa
VIIIa
Va
V
T
VIIa
P
VIII
T
fibrin
Lamprey
Other Vertebrates
The simultaneous doubling of two interacting gene products is consistent with the 2R hypothesis.
VII
VII
V
TF
T
VIIa
X
V
TF
T
P
VIIa
Va
Va
X
Xa
Xa
IXa
VIIIa
T
*
IX
VIII
T
T
fibrinogen
Lamprey
P
fibrin
fibrinogen
fibrin
Other Vertebrates