Russell F. Doolittle

The Step-by-Step Evolution of Vertebrate Blood Coagulation

UC San Diego

Bernstein and Kairinen (1971)

R. Williams, 1980

Blood clotting in humans is very complex. It involves more than two dozen genetically encoded proteins.

Fibrinogen FibrinMonomer

Fibrin Cross-linkedFibrin

LysedFibrin

Thrombin

Antithrombin

Thrombomodulin

Prothrombin

Xa

TAFIXIIIa

XIII

Plasmin

Plasminogen

u-PAt-PA

PAI-1

VIIIa

XIa

IXa

XI

X

-Antiplasmin2

Va

ProtC

VIII

V

TF/fVII

VII

tissue factorTFI

APC

ProtS IX

XIIaKallikrein

XII

Prekallikrein

(HMWK)

Pro-u-PA

Fibrinogen FibrinMonomer

Fibrin

Thrombin

Mammalian blood clotting can be divided into sets of reactions.

Fibrinogen FibrinMonomer

Fibrin Cross-linkedFibrin

ThrombinProthrombin

XIIIa

XIII

Fibrinogen FibrinMonomer

Fibrin Cross-linkedFibrin

ThrombinProthrombin

Xa

XIIIa

XIII

VIIIaIXa

X

LysedFibrin

Va VIIIV

TF/fVII

VII

tissue factor

Plasmin

Plasminogen

u-PAt-PA

Pro-u-PA

IXXIIa

Kallikrein

XII

Prekallikrein(HMWK)

The Delicate Balance

thrombinfibrinogen

factor VII

factor Xfactor IXfactor Vfactor VIIIfactor XIfactor XIIfactor XIII

prekallikreinPAI-1

tissue factorplasminogen

fibrinprotein Cantithrombin3

gelation fluidity

tissue factor inhibitor

t-PAu-PA

thrombomodulinprotein S

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:

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.

Doolittle R. F. (1993) Thromb Haemost. 70:24-28.

The evolution of vertebrate blood coagulation: a case of Yin and Yang

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.

Protostomes Deuterostomes

Crustacea EchinodermataArachnidaInsecta (Protochordata) Vertebrata

Five Animal Groups with Different Types of Blood Clotting

Hagfish

Lamprey E. shark

Pufferfish Zebrafish

Frog

Lizard Chicken

Mouse Human

(70 mya)

(310 mya)

(430 mya)(500 mya)

(550 mya)

Sea Squirt

Amphioxus

(380 mya)

Platypus

Many of the clotting proteins are multi-domainedand involve various combinations of common domains.

Prothrombin

K K SPG

Protein C, factor VII.Factor IX, Factor X

SPG

K

u-PA

SPF1

F1

t-PA

E KK SPF1

E

F2

KEE

Factor XII

SPF1F2

Plasminogen

P KKKKK SP

K

Kringle SP

Serine protease

P

G

GLA

PAN

EGFFN2

FN1

FN3

FRED

CP- A

Serpin Kunitz

Carboxypep

TGDiscoidin

Sushi cc

E

E

TBHUTBBO

F9HUF9BO

F9FUAF9FUB

F10BOF10HUF10FUF7HU

F7BOF7FUB

F7FUAF7FUC

PCBOPCHU

PCFU

TBFU

Vitamin-K Dependent Factors (GLA-containing)

Thrombins

Factors IX

Factors X

Factors VII

Proteins C

*

**

*

* Gene duplication 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.

But not the hagfish.

Whole genome databases are available for many vertebrates,including human, other mammals, opossum, platypus, chickenLizard, frog, and several fish. A draft genome is available for lamprey.

My students and I have been scouring the lamprey data base,as well as those other vertebrates listed above.

Fibrinogen FibrinMonomer

Fibrin Cross-linkedFibrin

LysedFibrin

ThrombinProthrombin

Xa

XIIIa

XIII

Plasmin

Plasminogen

u-PAt-PA

VIIIaIXa

X

Va VIIIV

TF/fVII

VII

tissue factor

Pro-u-PA

IX

Human Blood Clotting

Fibrinogen FibrinMonomer

Fibrin Cross-linkedFibrin

LysedFibrin

ThrombinProthrombin

Xa

XIIIa

XIII

Plasmin

Plasminogen

u-PAt-PA

VIIIaIXa

X

Va VIIIV

TF/fVII

VII

tissue factor

Pro-u-PA

IXmissing in lamprey

Fibrinogen FibrinMonomer

Fibrin Cross-linkedFibrin

LysedFibrin

ThrombinProthrombin

Xa

XIIIa

XIII

Plasmin

Plasminogen

u-PAt-PA

X

Va V

TF/fVII

VII

tissue factor

Pro-u-PA

Lamprey System

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.

In particular, they lack the “contact phase” factors.

factor XII factor XIIa

factor XI

factor XIafactor IX factor IXa

factor X

factor Xa

prekallikrein

-kallikrein

XIIa

prothrombin thrombin

The Contact System Proteases

HGFASPE E KF1F2

factor XIISPE E KF1F2

K SP

SP

SPKKP plasminogen

SPP HGF

SPPP PP factor XI

PP PP SP PK

K K t-PAF1

u-PA

E

E

K KK

KK K K

Without exception, all of the proteins involved in mammalian blood clottingare descended from other protein families that are not involved in clotting.

The backbone of clotting, like many other extracellular processes, islimited proteolysis, especially employing serine proteases. Hundreds ofserine proteases--all evolutionarily related--are found in animals.

There is also a full complement of serine protease inhibitors, membersof a widely spread family called “serpins.”

Factor V (or factor 5) and factor VIII (factor 8) are descended fromferroxidase enzymes that can be traced back to bacteria.

Fibrinogen is a multi-domain protein, the globular portions of whichhave numerous relatives throughout the animal kingdom.

Occurrence of Genes for Contact Phase Proteases and Some Paralogs

Factor XI Prekallikrein Factor XII HGFA HGF Plasminogen t-PA

Human Yes Yes Yes Yes Yes Yes YesOpossum Yes Yes Yes Yes Yes Yes YesPlatypus No Yes Yes Yes Yes Yes YesChicken No Yes No Yes Yes Yes Yes Green Lizard No Yes Yes Yes Yes Yes Yes Frog No Yes Yes Yes Yes Yes YesZebra Fish No No No ? Yes Yes YesPuffer Fish No No No Yes Yes Yes YesLamprey No No No Yes Yes Yes Yes

Updated from Ponczek, Gailani & Doolittle, 2008

Chromosomal locations of factor XII and HGFA

3

Jawless Fish Fish Amphibians Reptiles Birds

monotremes marsupials eutherians

Mammals

1 2

4

Updated from Ponczek, Gailani & Doolittle, 2008

1,2,3 = gene duplications 4 = gene deletion

Chromosomal locations of prekallikrein and factor XI

Duplication leads to separatefactor XI and prekallikrein.

Sea Squirt

Hagfish

LampreyE. shark

Pufferfish Zebrafish

Frog

Lizard Chicken

Mouse Human

Period of invention.

Amphioxus

Block duplication leads to factors VIII and IX.First appearance of factor XII.

First appearance of prekallikrein.

Birds lose factor XII.

Did anything happen here?

Platypus

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 .

E E

K K

KK

K

Protein C, factor VII.Factor IX, Factor X

4-Kringle Protease

KKKK SP

Prothrombin

K K SPG

G

SPG K

u-PA

SP

t-PA

E KK SPF1

EF1

EF2

KEE

Factor XII

SPF1F2

KK

KF1 E

Plasminogen

P KKKKK SP

P K

E

E

E E

or HGFA

P

HGF

P KKKK SP

Updated from Jiang & Doolittle, 2003

Prekallikrein, Factor XI

P P SPPP

P

K

+4

-5

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 clottingwas available in the form of domains in the common ancestor ofvertebrates and protochordates.

The number of components increases as one moves up the evolutionary scale from the jawless fish to mammals. Even among mammals somerecently evolved features are apparent.

Reasonable scenarios can be presented that show a step-by-stepdevelopment 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

Gene

Time Duplication

MB FB

Species diverge

Gene Duplication

MB LB

MA LA

divergeSpecies

Newmarket, New Hampshire, May, 1962

K

u-PA

SPF1KEE

Factor XII

SPF1F2

Prothrombin

K K SPG

Plasminogen

P KKKKK SP

K

KringleSP

Serine protease

G

GLA

P

PAN

F2

FN2

F1

FN1

Protein C, factor VII.Factor IX, Factor X

SPG

t-PA

E KK SPF1

EE

EGF

E

fibrinogen -------------> fibrin + fibrinopeptidesthrombin

fibrin ----------------> cross-linked fibrinfactor XIIIa

plasmin

lysed fibrin (fragments D and E, etc.)

plasminogen ------------> plasmint-PA

prothrombin

fibrininactive t-PA -----------------> active t-PA

“thrombocytes”

cell clot

(pro)thrombin

tissue factor

A Simple System (n = 2 plus cells)

(tissue factor, prothrombin, thrombocytes)

thrombin

(pro)thrombin

K K SPG

This can’t be! Prothrombin has kringles;Tissue factor interacts with EGF domains.

Unless prothrombin originally hadEGF domains!

fibrinogen

fibrin

(pro)thrombin

tissue factor

Another Simple System (n = 3)

(tissue factor, prothrombin, fibrinogen)

thrombin

(pro)thrombin

K K SPG

This can’t be! Prothrombin has kringles.Tissue factor interacts with EGF domains.

Unless prothrombin originally hadEGF domains!

Hagfish

LampreyDogfish

Pufferfish Zebrafish

Frog

Lizard Chicken

Mouse Human

(70 mya)

(310 mya)

(400 mya)(500 mya)

(540 mya)

Sea Squirt

Amphioxus

(380 mya)

invertebrates

jawless fishes

jawed fishes

primates

myoglobin

Fir

st A

ppea

ranc

e

Mill

ion

Yea

rs

Hemoglobins

-200

-400

-600

-800

X

VII

P

V

Xa

VIIa

TF

T T

Va

fibrinogen fibrin

Lamprey

IX

VII

X

IXa

VIIa

TF

T

VIIIa

fibrinogen

fibrin

P T

Xa

Va

V

T

Other Vertebrates

VIII

The simultaneous doubling of two interacting gene products is consistent with the 2R hypothesis.

Other VertebratesLamprey

X

VII

PVIIa

TF

Va

Tfibrinogen fibrin

T

V

Xa

IX

IXa

T

VIIIa

X

VII

PVIIa

TF

Va

Tfibrinogen fibrin

T

V

Xa

VIII*