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’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’



     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 (Lyons, 2002).

     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. When human fetuses undergo hypoxia, the number of nucleated red blood cells increases (Soslau, 2005).

     Alligators have a hematocrit of 25-25% and loggerhead turtles 29-35% (Mauro, 1997).







Nonmammalian vertebrates possess nucleated red blood cells.  Although the red blood cells of adult mammals lack nuclei, the first red blood cells synthesized in the embryo are nucleated. Human fetuses increase the percentage of nucleated red blood cells in response to acidemia (Soslau, 2005).The nucleated erythrocytes of pig embryos are pictured below.
    Most of the erythrocyte precursor cells which eject their nuclei (called reticulocytes) exist only in the bone marrow, a few escape into circulation.  Human reticulocytes are depicted in the following images.


     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). 




     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).



     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. 




     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).



     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).



     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.



     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.