BLOOD
By the time a human embryo is a few mm in
length, cells need more oxygen and glucose than simple diffusion can provide
and cells produce wastes faster than diffusion can remove them. As a result, a system to
perform transport of both is needed, especially since the differentiated
cells of the body can't move towards food on their own or move
away from their wastes. The specialized
cells of the human body are also less able to protect themselves from
temperature changes, pH changes, and toxic chemicals and thus the cardiovascular
system must help control the composition of the fluid which bathes the
cells. By the end of the 3rd week the human heart beats
while other organ systems have barely begun their development.
Human blood is composed of three types of
specialized cells suspended in plasma.
The plasma is mostly water but also contains a number of proteins
which function in osmotic regulation, immune reactions, transport, and
blood clotting. Red blood cells
(erythrocytes) are enucleated cells which are packed with the oxygen-
and carbon dioxide-binding protein hemoglobin.
Diverse classes of white blood cells (leukocytes) protect the body
from microorganisms, viruses, and cancer cells using a variety of immune
mechanisms. Platelets are tiny
cell fragments which induce coagulation, platelet plugs, regenerations,
and vascular spasms at the site of an injury.
HEMOGLOBIN
Hemoglobin’s ability to transport oxygen
is essential for human life, but it is not essential for life in general. During the first half of earth’s history, there
was little if any free oxygen in the atmosphere. Bacteria relied on glycolytic
pathways long before there was sufficient oxygen in the atmosphere to
support aerobic respiration. Early
organisms did not require with molecules to transport and utilize oxygen;
these molecules could evolve over millions of years as the atmospheric
oxygen levels gradually rose. Once oxygen was
present in the atmosphere, microscopic eukaryotic organisms
utilized hemoglobin to supplement the ATP generated anaerobically
long before there were circulatory systems to carry oxygen. Even the smallest and simplest animals could
rely on diffusion with the surrounding seawater without a blood system
containing respiratory pigments, such as hemoglobin. Living things had hundreds of millions of years
to adapt to the presence of oxygen in the atmosphere and to evolve circulatory
systems to transport it.
In humans, each red blood cell carries about
250,000 molecules of hemoglobin which can transport both oxygen and carbon
dioxide. Hemoglobin is a molecule
made of two separate components: the protein globin
and the nonprotein heme
groups. Heme
belongs to a group of molecules classified as porphyrins. A wide diversity of organisms, including many
bacteria and virtually all aerobic organisms, modify the amino acid glycine to synthesize these porphyrins
which can then bind metal ions. Iron
porphyrins evolved early in the history of life, some of which
function as cytochromes in energy pathways.
When iron binds to protoporphyrin, it forms the respiratory pigment heme.
Hemoglobin is not the only pigmented molecule
capable of transporting oxygen: there are a number of recognized respiratory
pigments in animals, such as hemoglobin, hemocyanin, chlorocruorin,
and hemerythrin (Hoar, 1983). One of these pigments is related to heme (chlorocruorin which is a green
pigment in annelids) while the others are not (the red haemoerythrin of some protostomes
and the copper containing haemocyanin). Crustaceans use hemocyanin
for oxygen tranpsort in their hemolymph. Insects lack hemocyanins
but possess proteins known as hexamerins in
their hemolymph. Analysis
of newly discovered molecules such as cryptocyanin
indicate that oxygen binding and molting in many invertebrates are mediated
by members of the hemocyanin gene family which
include hemocyanins, hexamerins, cryptocyanins, and prophenoloxidase
(Terwilliger, 1999).
Hemoglobins are
heme-containing proteins which reversibly bind oxygen. Hemoglobin is not unique to higher animals with
circulatory systems: a variety of hemoglobins
are known in bacteria, fungi, higher plants, most invertebrates and all
vertebrates. All of them belong
to the same globin gene family, having evolved
from a single ancient ancestral protein.
In bacteria and yeast, multi-domain proteins combine hemoglobin
with other domains to produce proteins novel proteins such as flavohemoglobins. Bacterial flavohemoglobin
can remove NO (nitric oxide) by reacting it with oxygen to form nitrate. When oxygen is not present, flavohemoglobin removes NO by promoting the conversion of
N2O. Thus these molecules offer protection from NO
in both aerobic and anaerobic conditions.
In the ancient earth (and in the communities of deep sea vents),
NO would have been far more abundant than oxygen.
Hemoglobin is an ancient molecule (given
its distribution across both prokaryotes and eukaryotes) that probably
first evolved in a world without much oxygen.
Hemoglobin is similar in structure to peroxidase
which reacts with dangerous forms of oxygen to protect cells. In nematodes, hemoglobin can function both as
a peroxidase and to remove NO. This is interesting
since it is possible that the original hemoglobin molecules might have
had a function other than the transport of oxygen (such as the removal
of NO) and that its ability to transport oxygen might have developed through
transitional stages involved in the removal of oxygen in organisms sensitive
to it. (Hausladen,
2001). So many plants are known to possess hemoglobin
molecules that it is possible that all plants possess hemoglobins
(Zhu, 1992; Anderson, 1996).
There
are a variety of hemoglobins known in invertebrates.
Some are made of a single polypeptide chain with one heme
group (as in dipterans), multi-subunit proteins with two heme
groups per subunit (each subunit is 30-40 kd
[kilodaltons] and the entire protein may be 250-800 kd; known primarily from crustaceans), multi-subunit proteins
with multiple heme groups (8 to 20 per subunit;
known in crustaceans and mollusks), and multi-subunit proteins in which
not all subunits contain heme and subunits can
be united by disulfide bones (this group includes the chlorocruorins
of annelids and erythrocruorins). Some hemoglobins in
invertebrates function inside cells; others are extracellular
(Goodman, 1988). Annelids which
live in deep sea hydrothermal vents have adapted their extracellular
hemoglobin molecules to transport hydrogen sulfide (Bailly,
2003).
Before the evolution of jawed vertebrates,
two duplications of the ancestral hemoglobin gene occurred. The first duplication gave rise to myoglobin and hemoglobin; the subsequent duplication of hemoglobin
gene gave rise to alpha and beta globin genes.
Tandem duplications of the alpha and beta genes gradually gave
rise to the alpha and beta gene families located on human chromosomes
16 and 11 respectively. Some of
the genes in these families are expressed only in the embryo; others are
no longer functional. Some of the
pseudogenes in humans correspond to functioning
genes in other groups of organisms. Eta hemoglobin
apparently was an embryonic hemoglobin in the
ancestors of eutherian mammals. In artiodactyls (deer, cows, giraffes, etc.)
it is still a functional gene. In
primates, eta is a nonfunctional pseudogene. Rodents no longer have any trace of the eta hemoglobin gene. (Online
Mendelian Inheritance in Man).
On human chromosome 16, there is a cluster
of hemoglobin genes resulting from duplications of an ancestral gene which
form the genes of the alpha hemoglobin family.
5’-----zeta-----zeta
pseudogene----alpha pseudogene---alpha
2---alpha 1---theta----3’
On human chromosome 11, there is a cluster
of hemoglobin genes resulting from duplications of an ancestral gene which
form the genes of the beta hemoglobin family.
5’---epsilon------gammma G------gamma A--------eta------------delta----------beta----3’
RED BLOOD CELLS
In humans, hemoglobin is carried through
the blood by red blood cells. In
most invertebrates, the respiratory pigment floats freely in the plasma. Pigmented circulating cells do not have to be
limited to a circulatory system and often occur in the coelom
of coelomates. Some circulating cells in nemertine worms, the most primitive organisms which possess
a circulatory system, actually contain hemoglobin and thus appear red
(Turbeville, 1986, p. 128).
At least one of the polychaete worms,
Magelona, possesses
its respiratory pigment inside circulating cells, similar to vertebrate
blood cells (Barrington, p. 213).
In hemichordates, the blood pigment is located
outside blood cells. (Benito, form Harrison 1997, p. 61) In contrast, most pigments in tunicates are
in the hemocytes which occur in blood vessels
or in connective tissue spaces (Burighel, from
Harrison, 1997). In Amphioxus, there are few if any blood cells. (Ruppert, from Harrison, 1997, p. 445-52). In hagfish, hemoglobin is contained in red blood
cells although the amount of hemoglobin in each cell is low (Tufts, 1998).
In
general, the red blood cells of lower vertebrates are smaller than those
of higher vertebrates (Torrey). A number of genes found to control hematopoeisis in fish (including some which cause blood cell
numbers to be significantly reduced or absent), belong to the same GATA
family of transcription factors which is important in mammalian hematopoeisis. Mutations
in GATA1 disrupt the formation of red blood cells in both fish and mammals
(
Turtle red blood cells are biochemically similar to those of mammals in their degree of anaerobic
metabolism, use of the pentose phosphate metabolic pathway, and low oxygen
use. The red blood cells of reptiles
such as turtles can be used as models of intermediates between the erythrocytes
of invertebrates and those of mammals.
Invertebrate red blood cells are nucleated and can derive energy
from amino acids more rapidly than from carbohydrates. Occasionally reptilian erythrocytes eject their
nuclei to form circulating cells known as hematogones
(Mauro, 1997).
In mammals,
the nucleus of red blood cells is ejected before the cells enter circulation,
allowing mammals to fit more red blood cells (and thus more hemoglobin)
in each milliliter of blood. Because
mammalian red blood cells cannot divide in circulation, the bone marrow
must produce millions of them per second (Torrey).
Some marsupials have some circulating nucleated red blood cells as adults
(Stonehouse, 1977) and all vertebrates, including humans, possess
embryonic nucleated red blood cells originating from the yolk sac.
Alligators have a hematocrit
of 25-25% and loggerhead turtles 29-35% (Mauro, 1997).
FISH
FROG
TURTLE BLOOD
BLOOD GROUPS
The existence of different blood groups in
a species seems to be a typical condition of vertebrate species ranging
from sharks to teleosts to birds and mammals
(Hoar, 1983).
The proteins whose variants result in the
Rh blood groups are significant factors in blood transfusions,
the possible maternal immune reaction against fetuses, and even
as a potential mechanism for reproductive isolation of different human
populations. Red blood cells can possess an Rh complex on their surface which composed of two Rh proteins, 2 RhAG proteins, duffy glycoprotein, and several other
components. The blood RH proteins
are encoded by two genes RHCE and RHD; other members of the Rh
family in humans include RHAG, RHGK, and RHBG genes. Humans express two members of the Rh gene family on red blood cells and 2 on white blood cells.
The gene duplication which resulted in the two genes expressed
on red blood cells predated the African ape lineage.
The two proteins expressed on red blood cells produce the D antigen
and the other produces the C/c and E/e antigens.
Rh negative individuals lack the gene
producing the D antigen. Rarely, humans without either Rh gene are born but they suffer from anemia. Rh also expressed
in human (and mouse) kidneys, brain, and other tissues. Rh genes known in protozoans, sponges, worms,
flies, fish and amphibians (Huang, 2001; Westhoff,
1999). Although the function
of these glycoproteins is not entirely known,
they are homologous to ammonium transporters in bacteria, fungi, plants,
and invertebrates and RhAG and RhGK
have been shown to function in ammonium transport (Okuda, 2002).
CARBONIC ANHYDRASE
Most vertebrates depend on an exchange between
chloride and bicarbonate ions for the carbon dioxide transport of red
blood cells. In jawless fish, this
ability is extremely limited. Unlike
lampreys, hagfish red blood cells do not transport additional carbon dioxide
in deoxygenated blood compared with oxygenated blood and there is no measurable
Haldane effect. Hagfish
red blood cells possess both hemoglobin and carbonic anhydrase
but seem to lack the anion exchanger which transports bicarbonate (in
exchange for chloride) into the blood plasma.
Thus, hagfish seem to lack the processes for carbon dioxide transport
found in all other vertebrates (Peters, 2000).
Lungfish blood a significant
Haldane effect while sharks do not (Hoar, 1983). Hagfish red blood cells have a less significant
role in transporting carbon dioxide, accounting for 30% of that which
can be lost at the gills as opposed to 65% in lampreys (Tufts, 1998).
Carbonic anhydrase
(CA) is an enzyme which converts carbon dioxide and water into carbonic
acid which then dissociates to form bicarbonate.
In humans, 70% of the carbon dioxide transport in the blood is
in the form of bicarbonate (23% is bound to hemoglobin and 7% is dissolved
in solution). Carbonic anhydrase
existed long before vertebrate circulatory systems, given that it is known
from bacteria and plants (Hoar, 1983).
The diverse carbonic anhydrase genes
in living organisms, including the multiple genes which can be expressed
in mammals, belong to one gene family (Tufts, 2003).
Rates of CA activity suggest that there have been increases in
the catalytic efficiency of CA in the ancestors of agnathans
and in the ancestors of higher vertebrates (Tufts, 2003).
ICEFISH
The development of polar environments in
the southern ocean is
a relatively new phenomenon, dropping from about 20oC
to –2oC in the past 55 million years. While most fish went extinct, teleosts of the group Notothenioidei
adapted to this environment, reducing the amount of hemoglobin in their
blood to compensate for the thickening of fluids which occurs at lower
temperatures. Some have reduced
the number of different hemoglobins produced
(fish in general typically produce multiple hemoglobins,
perhaps as a way of adapting to different environments). Some notothenioids
produce 5 different hemoglobins while others
produce two or one. One family,
Channichthyidae, completely lacks hemoglobin. The blood of white-blooded notothenioids can only carry 10% of the oxygen of the red
blood of other notothenioids. The icefishes (Channichthyidae) suffered a deletion of b hemoglobin at the base of their clade
after which the a
genes were inactivated. Traces
of the a globin genes, but not the
b globin genes, still remain.
This family has scaleless skin, allowing them
to perform cutaneous gas exchange. Oxygen concentration in Antarctic waters is
high and oxygen demands of the fish are low. (Hays,
1996; Bargelloni, 1998).
One study found that myoglobin
was absent from the oxidative skeletal muscle and auricle of 8 species
of icefishes (Channichthyidae). Of these 8 species, five possessed myoglobin in their heart’s ventricles (and one produced the
mRNA but not the protein). All
of the species possessed a myoglobin gene and
analysis suggests that the loss of myoglobin
expression occurred multiple times within this family (Sidell,
1997).
Some larval eels and deep sea fish also
lack hemoglobin.
PLATELETS
AND BLOOD COAGULATION
In animals ranging from worms and echinoderms
to humans, vascular spasms of blood vessels reduce blood flow to wounded
areas. In some invertebrates, this
response is sufficient to stop the bleeding. Once animals evolved hard body coverings or
higher blood pressures, such mechanisms were no longer sufficient. In animals which attempt to plug wounded areas,
the most primitive use only blood cells which temporarily agglutinate
but which never disintegrate or degranulate. In some arthropods, mollusks, and echinoderms,
cells may fuse . Many arthropods produce proteins which form
a clot at the wound site although this process is different from that
observed in vertebrates. These
clotting factors may be released from Hardy’s
explosive cells and coagulocytes (Hoar, 1983).
All vertebrates possess both red and white
blood cells. Platelets, however,
do not exist in nonmammals; instead blood clotting
is performed by leukocytes referred to as thrombocytes.
(Jiang, 2003). The blood of most vertebrates possesses thrombocytes, which can burst to release clotting factors.
In mammals, the cells which produce clotting factors are restricted
to the bone marrow but they release cell fragments called platelets which
circulate and induce coagulation (Torrey). Human platelets
are depicted in the following images (as the small purple cells).
The stoppage of bleeding in vertebrates makes use of both blood cells (thrombocytes/platelets) and an array of proteins. These proteins are not unique to humans, nor is there only one possible cascade. Primitive vertebrates possess simpler cascades and the components of the cascade can vary in vertebrate groups. Jawless fish have a simple clotting system, consisting of TF, prothrombin, and fibrinogen. (Davidson, 2003; Frost 2000). Teleosts possess both intrinsic and extrinsic pathways, prothrombin, factor X, protein C, antithrombin, and heparin cofactor II (Jagadeeswaran, 1999). In fact, teleost fish possess proteins which are homologous to most of the 26 mammalian proteins involved in coagulation. (Some mammalian proteins seem to be absent in fish and several fish proteins exist as multiple copies). Most birds lack clotting factor IX. Ostriches also lack factors VII, X, XI, and XII. Caimans lack additional clotting factors; only factors I, II, and X have been reported. Ostrich clotting mechanisms seem to be intermediate between those of reptiles and those of higher birds (Frost, 1999).
Thrombocytes in bony fish aggregate when stimulated by ADP, collagen, epinephrine, and thrombin. Thrombocytes in turtles aggregate when stimulated by thrombin and those of birds aggregate in response to arachidonic acid, collagen, serotoin and thrombin. Ancestral amniotes appear to have possessed a response to limit blood loss using interactiosn with von Willebrand factor, a thromobcyte aggregation pathway involving collagen and thrombin, a high level of plasma fibrinogen (ten times the level of mammals), and the extrinsic coagulation pathway (Soslau, 2005).
All of these proteins appear to have evolved
from ancestral proteins which were not involved in a coagulation cascade.
The tunicate genome possesses gene family members (and the functional
domains) of almost all of these proteins, none of them are truly homologous,
indicating that the evolution of the vertebrate coagulation cascade occurred
after the separation of the urochordate lineage from that of vertebrates (Jiang, 2003).
A) FIBRINOGEN
The blood of some invertebrates can clot
in response to injury, although this response does not occur through the
clotting factor cascade observed in vertebrates. The invertebrate coagulogen
is not related to fibrinogen, the clotting protein of vertebrates (coagulogen is instead similar to nerve growth factor). However, there are fibrinogen-like molecules,
in both vertebrates and invertebrates, including a group known as lectins (Xu and Doolittle, 1990). Invertebrate lectins
can recognize carbohydrate groups on bacteria and cause the agglutination
of bacteria (Adema, 1997; Gokudan,
1999; Kairies, 2001).
Vertebrates, including humans, also possess homologues of fibrinogen
which function in innate immunity (called ficolins)
which recognize carbohydrate groups on bacteria. Human ficolins bind
the same molecules as some invertebrate lectins
(such as tachylectin 5A) and are more closely
related to tachylectins than to fibrinogen (Kairies,
2001). Interestingly, the von
Willebrand factor (also involved in coagulation) is homologous
to invertebrate lectins (Adema, 1997). Thus,
it seems that the protein that vertebrates use to form clots at a wound
site is a modified version of ancestral proteins which were used to agglutinate
bacteria (which would have been common at wound sites).
Vertebrate fibrinogen is composed of several
subunits, a, b,
and g in all vertebrates studied
(including lampreys). These subunits
are homologous, suggesting that a common ancestral gene duplicated to
produce the a gene and the precursor
of the b-g genes which duplicated subsequently. The ancestral b and g fibrinogen gene seems to have resulted from gene fusion
with an a-like
fibrinogen gene (the N-terminus) with a second gene homologous to cytotactin and pT49 in humans (the C-terminus) (Weissbach, 1990).
B)
THROMBIN
Thrombin belongs to a family of proteins
known as serine proteases. This
is an ancient gene family, including eubacterial
digestive enzymes and the vertebrate digestive enzymes trypsin
and chymotrypsin.
Most of these proteins possess the amino acid proline
at residue 225 in the protein. However,
in vertebrates, some of these proteins possess the amino acid serine at
residue 225. The change in some
of the serine proteases needed to acquire a function in coagulation seems
to stem from one ancestral mutation changing the amino acid at residue
225 (Guinto,1998; Dang, 1996). This change enabled the binding of sodium
and novel protein functions. Some
serine proteases in blood (such as plasmin and
clotting factor XIa) possess proline
at site 225 while others such as thrombin, clotting factor Xa
(involved in clotting), and complement protein C1r (involved in immunity)
possess serine. Mutations at site
225 drastically affect the function of thrombin (changing ligand
recognition up to 60,000 fold).
When blood clots, several
clotting factors and several proteins involved in the regulation of coagulation
perform a reaction converting the amino acid glutamate to g-carboxyglutamate (Gla) after translation.
Not only is this reaction essential for blood clotting (where it
was first discovered), it also has other functions in vertebrates and
occurs in several bone proteins, for example.
This reaction and the enzyme which catalyzes it (g-glutamyl carboxylase) were thought
to be found only in vertebrates. It
is now known in insects and molluscs as well
where g-carboxylation
of glutamate has several roles, such as the production of venom peptides. The g-glutamyl carboxylase gene is conserved between mammals (including humans),
insects, and molluscs. In fact, the correspondence of intron/exon boundaries is surprisingly homologous and eight
of the introns appear to have predated the split
in coelomate lineages in the Precambrian. (Bandyopadhyay,
2002).
Tissue Factor (TF) serves as a cell membrane
attachment (tether) for one of the protease enzymes of the clotting cascade
(clotting factor VII). It is homologous
to cytokine receptors –receptors for erythropoeitin,
interleukins, colony stimulating factor, interferon,
and several hormones. This group
belongs to the immunoglobulin superfamily which
is one of the largest protein families in the animal kingdom. Before vertebrates evolved a coagulation cascade
involving TF, receptors-related to TF were already present on cell membranes
and their functions included the response to infection (such as might
occur after a wound) (Bazan, 1990).
A number of clotting factors share structural
domains. Coagulation factor VII
interacts with tissue factor to initiate the extrinsic pathway for coagulation
and is known to exist in zebrafish. The zebrafish domain
structure of Factor VII possesses the shared domains found in coagulation
factors VII, IX, X and protein C (Sheehan, 2001). Within the protease superfamily
of genes, one family of related proteins includes clotting factor IX,
factor X, factor VII, and protein C. Factors
VII and X remain linked on chromosome 13.
Clotting factor XII, tissue plasminogen
activator, and urokinase are related proteases
as are Clotting Factors VIII and V (Banfield,
1994).
Some of the enzymes used in clotting also
serve other roles in vertebrates, such as immune reactions and development.
Thrombin is expressed in the brain where it is involved in G-protein
signal transduction cascades involved in development and prothrombin
functions in neutrophil chemotaxis. Factor Xa can function
as a growth factor. Coagulen (in protostomes) is partially
homologous to nerve growth factor (Krem, 2002).
Thrombin can stimulate chemotaxis of monocytes and neutrophils in wound repair and promotes differentiation in
macrophages (Banfield, 1992).
At the present time, comparative genetic
analysis suggests that the complex vertebrate clotting cascade evolved
from simpler mechanisms, many of which were already involved in agglutinating
material at a wound site as part of the immune response. The timing of the adoption of this immune mechanism
to functions specifically in blood coagulation coincides with the proposed
rounds of genome duplication which occurred early in the evolution of
vertebrates.
PLASMA PROTEINS
In animals with an open circulatory system,
there is no distinction between blood and lymph, so the term haemolymph is used. Annelids,
echinoderms, and mollusks may have a plasma protein concentration of 1
mg/ml compared to the 30-75 mg/ml found in higher vertebrates.
In some invertebrates, the high plasma concentrations
of oxygen-binding respiratory pigments (such as hemocyanin
in cephalopods) contributes to plasma protein levels of 100-150
mg/ml. Respiratory pigments are located inside blood
cells in vertebrates. (Hoar, 1983, p. 456)
Albumin is the major plasma protein in mammals
but it is present in lower amounts in most teleost
fish and is absent from the plasma in jawless fish, cartilaginous fish,
primitive actinopterygians, and primitive teleosts. It probably
became more important in tetrapods as an adaptation
to terrestrial environments. (Hoar, 1983).
Some insects and plants produce antifreeze
glycoproteins (AFGPs). Some notothenioid
fishes and some northern cod produce AGFPs which
not only decrease the formation of ice crystals,
they help protect cell membrane integrity (Hays, 1996).
The evolution of white
blood cells and acquired immunity is discussed with the lymphatic system.