Molecular Biology

Inherited Variation and Polymorphism in DNA

August 3, 2017 Cytogenetics, Laboratory Medicine, Molecular Biology, Pharmacogenetics No comments

The original Human Genome Project and the subsequent study of now many thousands of individuals worldwide have provided a vast amount of DNA sequence information. With this information in hand, one can begin to characterize the types and frequencies of polymorphic variation found in the human genome and to generate catalogues of human DNA sequence diversity around the globe. DNA polymorphisms can be classified according to how the DNA sequence varies between the different alleles.

Single Nucleotide Polymorphisms

The simplest and most common of all polymorphisms are single nucleotide polymorphisms (SNPs). A locus characterized by a SNP usually has only two alleles, corresponding to the two different bases occupying that particular location in the genome. As mentioned previously, SNPs are common and are observed on average once every 1000 bp in the genome. However, the distribution of SNPs is uneven around the genome; many more SNPs are found in noncoding parts of the genome, in introns and in sequences that are some distance from known genes. Nonetheless, there is still a significant number of SNPs that do occur in genes and other known functional elements in the genome. For the set of protein-coding genes, over 100,000 exonic SNPs have been documented to date. Approximately half of these do not alter the predicted amino acid sequence of the encoded protein and are thus termed synonymous, whereas the other half do alter the amino acid sequence and are said to be nonsynonymous. Other SNPs introduce or change a stop codon, and yet others alter a known splice site; such SNPs are candidates to have significant functional consequences.

The significance for health of the vast majority of SNPs is unknown and is the subject of ongoing research. The fact that SNPs are common does not mean that they are without effect on health or longevity. What it does mean is that any effect of common SNPs is likely to involve a relatively subtle altering of disease susceptibility rather than a direct cause of serious illness.

Insertion-Deletion Polymorphisms

A second class of polymorphism is the result of variations caused by insertion or deletion (in/dels or simply indels) of anywhere from a single base pair up to approximately 1000 bp, although larger indels have been documented as well. Over a million indels have been described, numbering in the hundreds of thousands in any one individual’s genome. Approximately half of all indels are referred to as “simple” because they have only two alleles – that is, the presence or absence of the inserted or deleted segment.

Microsatellite Polymorphisms

Other indels, however, are multiallelic due to variable numbers of the segment of DNA that is inserted in tandem at a particular location, thereby constituting what is referred to as a microsatellite. They consist of stretches of DNA composed of units of two, three, or four nucleotides, such as TGTGTG, CAACAACAA, or AAATAAATAAAT, repeated between one and a few dozen times at a particular site in the genome. The different alleles in a microsatellite polymorphism are the result of differing numbers of repeated nucleotide units contained within any one microsatellite and are therefore sometimes also referred to as short tandem repeat (STR) polymorphisms. A microsatellite locus often has many alleles (repeat lengths) that can be rapidly evaluated by standard laboratory procedures to distinguish different individuals and to infer familial relationships. Many tens of thousands of microsatellite polymorphic loci are known throughout the human genome. Finally, microsatellites are a particularly useful group of indels. Determining the alleles at multiple microsatellite loci is currently the method of choice for DNA fingerprinting used for identity testing.

Mobile Element Insertion Polymorphisms

Nearly half of the human genome consists of families of repetitive elements that are dispersed around the genome. Although most of the copies of these repeats are stationary, some of them are mobile and contribute to human genetic diversity through the process of retrotransposition, a process that involves transcription into an RNA, reverse transcription into a DNA sequence, and insertion into another site in the genome. Mobile element polymorphisms are found in nongenic regions of the genome, a small proportion of them are found within genes. At least 5000 of these polymorphic loci have an insertion frequency of greater than 10% in various populations.

Coyp Number Variants

Another important type of human polymorphism includes copy number variants (CNVs). CNVs are conceptually related to indels and microsatellites but consist of variation in the number of copies of larger segments of the genome, ranging in size from 1000 bp to many hundreds of kilobase pairs. Variants larger than 500 kb are found in 5% to 10% of individuals in the general population, whereas variants encompassing more than 1 Mb are found in 1% to 2%. The largest CNVs are sometimes found in regions of the genome characterized by repeated blocks of homologous sequences called segmental duplications (or segdups).

Smaller CNVs in particular may have only two alleles (i.e., the presence or absence of a segment), similar to indels in that regard. Larger CNVs tend to have multiple alleles due to the presence of different numbers of copies of a segment of DNA in tandem. In terms of genome diversity between individuals, the amount of DNA involved in CNVs vastly exceeds the amount that differs because of SNPs. The content of any two human genomes can differ by as much as 50 to 100 Mb because of copy number differences at CNV loci.

Notably, the variable segment at many CNV loci can include one to as several dozen genes, that thusCNVs are frequently implicated in traits that involve altered gene dosage. When a CNV is frequent enough to be polymorphic, it represents a background of common variation that must be understood if alterations in copy number observed in patients are to be interpreted properly. As with all DNA polymorphism, the significance of different CNV alleles in health and disease susceptibility is the subject of intensive investigation.

Inversion Polymorphisms

A final group of polymorphisms to be discussed is inversions, which differ in size from a few base pairs to large regions of the genome (up to several megabase pairs) that can be present in either of two orientations in the genomes of different individuals. Most inversions are characterized by regions of sequence homology at the edges of the inverted segment, implicating a process of homologous recombination in the origin of the inversions. In their balanced form, inversions, regardless of orientation, do not involve a gain or loss of DNA, and the inversion polymorphisms (with two alleles corresponding to the two orientations) can achieve substantial frequencies in the general population.

[Clinical Art][Physiology] Iron Physiology

November 3, 2016 Cytogenetics, Hematology, Molecular Biology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , , ,

screen-shot-2016-11-02-at-10-03-03-pmGlobal Iron Homeostasis

Under normal conditions, dietary iron is usually 15-25 mg daily, of which 5%-10% (1-2 mg) is absorbed through the gastrointestinal (GI) tract and the same amount lost by desquanmation of GI epithelial cells, epidermal cells of the skin, and, in menstruating women, red bood cells. The average total body content of iron in men is 35-45 mg/kg; and lower in menstruating women. Most iron (about 1800 mg) is present in hemoglobin. Men and women, respectively, have approximately 2 or 1.5 g of erythrocyte iron. Iron is stored in cells, predominantly macrophages of the spleen, bone marrow, and liver, but also in hepatocytes, as ferritin or hemosiderin (partially denatured ferritin). At steady state, the serum ferritin level is a reasonably good reflection of total body iron stores. Total storage iron is approximately 1 g in men and 600 mg in women. Additional iron is found as myoglobin in muscle and in cytochromes and other enzymes.

PS: The loss of iron

Iron is eliminated only through the loss of epithelial cells from the gastrointestinal tract, epidermal cells of the skin, and, in menstruating women, red blood cells. On the basis of long-term studies of body iron turnover, the total average daily loss of iron has been estimated at ~1 to 2 mg in normal adult men and nonmenstruating women. Although iron is a physiologic component of sweat, only a tiny amount of iron (22.5 ug/L) is lost by this route. Urinary iron excretion amounts to <0.05 mg/day and is largely accounted for by sloughed cells. Menstruating women lose an additional, highly variable amount over each menstrual cycle, from 0.006 (average) to more than 0.025 mg/kg/day.

The release of iron into the circulation is regulated by ferroportin, expressed on the basolateral GI epithelial cell surface (and on cells of the reticuloendothelial system [RES] and hepatocytes). Ferroportin is downregulated by hepcidin, and when iron is low, hepcidin is low, allowing GI iron absorption to increase and stores to be mobilized from the RES. When iron is plentiful, hepcidin levels increase and result in decreased iron absorption and RES export. Hepcidin, in turn, is downregulated by the recently described hormone erythroferrone (ERFE), produced by erythroblasts during stress erythropoiesis. Absorbed iron is transported by transferrin and taken up into cells via the transferrin receptor. Each molecule of transferrin can bind two molecules of ferric (Fe3+) iron. Transferrin-bound iron turns over as iron is used, particularly by developing red blood cells in the bone marrow. The distribution of iron is influenced by multiple factors, and under normal conditions cells maintain a pool of labile iron by controlling uptake via expression of transferrin receptors and storage via ferritin. Most cells have no mechanism for iron efflux.

Iron balance is regulated such that the amount of iron absorbed equals the amount lost. There is, however, no physiologically regulated pathway for excretion of excess iron in iron overload.

Intestinal Iron Absorption

Iron is found in food as inorganic iron and heme (iron complexed to protoporphyrin IX). The typical diet consists of 90% inorganic and 10% heme iron, though diets in the industrial world can contain up to 50% heme iron from iron-rich meats. The bioavailability of inorganic but not heme iron is influenced by multiple factors such as other dietary constituents, for example, ascorbic acid (enhanced) and phytates and polyphenols in cereals and plants (inhibited). Iron absorption is strongly inhibited by tea, and less so by coffee.

PS: Intestinal Absorption of Iron

Iron is absorbed in the duodenum, and humans and other omnivorous mammals have at least two distinct pathways for iron absorption: one for uptake of heme iron and another for ferrous (Fe2+) iron. Heme iron is derived from hemoglobin, myoglobin, and other heme proteins in foods of animal origin, representing approximately 10% to 15% iron content in the typical Western diet, although heme-derived iron accounts for 2/3 of absorbed iron in meat-eating humans. Exposure to acid and proteases present in gastric juices frees the heme from its apoprotein. Heme is taken up by mucosal cells, but the specific receptor is still unkonwn. Once heme iron has entered the cell, the porphyrin ring is enzymatically cleaved by heme oxygenase. The liberated iron then probably follows the same pathways as those used by nonheme iron. A small proportion of the heme iron may pass into the plasma intact via heme exporter protein FLVCR (feline leukemia virus, subgroup C receptor), which transfers heme onto a heme-binding protein, hemopexin. Absorption of heme iron is relatively unafected by the overall composition of the diet.

Dietary nonheme iron is largely in the form of ferric hydroxide or loosely bound to organic molecules such as phytates, oxalate, sugars, citrate, lactate, and amino acids. Low gastric pH is thought be important for the solubility of inorganic iron. Dietary constituents may also have profound effects on the absorption of nonheme iron, making the bioavailability of food iron highly variable. Ascorbate, animal tissues, keto sugars, organic acids, and amino acids enhance inorganic iron absorption, whereas phytates, polyphenols, and calcium inhibit it. Depending on various combinations of enhancing and inhibitory factors, dietary iron assimilation can vary as much as tenfold.

The rate of iron absorption is influenced by several factors, including body iron stores, the degree of erythropoietic activity, blood hemoglobin and oxygen content, and the presence of inflammation. Iron absorption increases when stores are low or when increased erythropoietic activity is required, such as during anemia or hypoxemia. Conversely, the physiologically appropriate response to iron overload is downregulation of intestinal iron absorption; this downregulation fails in patients with hereditary hemochromatosis or chronic iron-loading anemias.

Iron is absorbed in the intestine via two pathways: one for inorganic iron and the other for heme-bound iron. Little is known about heme iron absorption. Nonheme iron in the diet is largely in the form of ferric-oxyhydroxides (Fe3+, ie, rust), but the intestinal epithelial cell apical iron importer, divalent metal transporter 1 (DMT1 or SLC11A2), transports only ferrous iron (Fe2+). DMT1 is a protein with 12 predicted transmembrane segments, which is expressed on the apical surface of absorptive enterocytes. Iron must therefore be reduced to be absorbed, and this is facilitated by duodenal cytochrome B (Dcytb), a heme-dependent ferrireductase. However, since knockout mice appear to have normal metabolism, Dcytb may not be the only ferrireductase enzyme involved in absorption of nonheme ironOnce transported across the apical border of the enterocyte, iron may be stored within the cell. For this purpose, iron is oxidized to Fe3+ by the H-subunit of ferritin and stored in this form. Eventually, the cell senesces and sloughs off into the feces, and stored iron is lost to the system. Alternatively, iron may be transported across the basolateral membrane into the portal circulation via ferroportin. Ferroportin 1 (FPN1) is the only known iron exporter in mammals and, like DMT1, transports only ferrous iron. FPN1 is a multi-transmembrane segment protein expressed on the basolateral surface of enterocytes. Aslo they are expressed in other tissues involved in handling large iron fluxes including macrophages, hepatocytes, and placental trophoblast. Similar to apical iron uptake, basolateral iron efflux is aided by an enzyme that changes the oxidation state of iron. Once reduced, ferrous iron is transported across the basolateral membrane by ferroportin, then oxidized to ferric iron by hephaestin. Intestinal iron absorption is regulated by hepcidin, which binds to ferroportin, inducing its internalization and degradation.

Cellular Iron Uptake, Storage, and Recycling

Each molecule of transferrin binds two ferric (Fe3+) iron atoms. Diferric transferrin (holotransferrin) binds to the transferrin receptor (TfR1) on target cells and enters by receptor-mediated endocytosis; it is then released from the TfR1 by acidification and transported into the cytoplasm by DMT1. The TfR is recycled to the cell surface.

Most iron in erythroid cells binds protoporphyrin to form heme, which complexes with globin proteins, forming hemoglobin. Erythrocytes survive in the circulation for approximately 120 days, after which aging red blood cells are phagocytized by macrophages of the RES. Hemoglobin is catabolized and iron released to transferrin via ferroportin, or stored within the RES as ferritin or hemosiderin.

PS: Reticuloendothelial System/RES



The main form of cellular iron storage is ferritin, a complex of subunits that binds iron and renders it insoluble and redox inactive. Circulating ferritin is present in a different subunit form than cellular storage ferritin. The function of circulating ferritin is incompletely understood.

Regulation of Iron Physiology

Because the total body iron content is largely determined by the efficiency of absorption of iron, the regulation of absorption has been of great interest for many years.

Hepcidin is a 25-amino-acid peptide produced in the liver and is the major regulator of iron absorption and storage. Hepcidin regulates cellular iron egress by binding to ferroportin, leading to its internalization and degradation. In this way, elevated levels of hepcidin inhibit iron absorption from the GI tract and promote storage (inhibit release) of iron within hepatocytes and macrophages. Hepcidin production is induced by interleukin (IL)-6 and IL-1 via the JAK/STAT pathway, and can be increased more than 100-fold in inflammatory states. The dysregulation of iron balance seen in the anemia of inflammation (anemia of chronic disease) can be attributed to an inappropriate increase in hepcidin levels, which leads to decreased circulating iron. Hepcidin levels are downregulated by anemia and iron deficiency. Hepcidin agonists and antagonists are under clinical development for the treatment of disorders of inappropriately low or high hepcidin levels, respectively.

Hepcidin, in turn, is negatively regulated by the recently described hormone ERFE. ERFE is produced by erythroblasts during stress erythropoiesis, feeding back on hepcidin and reducing its levels, thus allowing iron absorption and export via ferroportin to proceed.

The Molecular Mechanism for Hepcidin Regualtion

Systematic Mechanism

The two transferrin receptors TfR1 and TfR2, and HFE, an MHC class I-like membrane protein, may serve as holotransferrin sensors. HFE can interact with both transferrin receptors, but this interaction is modulated by holotransferrin concentrations. Because HFE and holotransferrin binding sites on TfR1 overlap, increasing concentrations of holotransferrin result in displacement of HFE from TfR1, and free HFE then interacts with TfR2. TfR2 protein is further stabilized by binding of holotransferrin. The holotransferrin/HFE/TfR2 complex then stimulates hepcidin expression through an incompletely understood pathway, possibly by potentiating BMP pathway signaling. HFE, however, may also regulate hepcidin expression without complexing with TfR2. The role of HFE or TfR2 in hepcidin regulation by iron is supported by genetic evidence: HFE and TfR2 mutations in humans or mice cause hepcidin deficiency and an adult form of hemochromatosis.

screen-shot-2016-11-03-at-3-19-33-pmThe BMP pathway with its canonical signaling via Smad proteins has a central role in the regulation of hepcidin transcription. BMP receptors are tetramers of serine/threonine kinase receptors, with two type I and two type II subunits. Recent data indicate that type I subunits Alk2 and Alk3 and type II subunit ActRIIA and BMPRII are specific BMP receptors involved in iron regulation. In the liver, BMP pathway signaling to hepcidin is modulated by a coreceptor hemojuvelin and, at least in mice, by the ligand BMP6. Loss of hemojuvelin or BMP6 in mice decrease hepcidin expression and impairs hepcidin response to acute or chronic iron loading. In humans, hemojuvelin mutations result in severe hepcidin deficiency and cause juvenile hemochromatosis. It remains to be clarified how BMP receptors and hemojuvelin interact with iron-sensing molecules that regulate hepcidin expression.

Local Mechanism (intestine)

In addtition to the regulaltion by systemic signals, iron absorption is subject to local regualtion by intracellular mechanisms in duodenal enterocytes. At least two mechanisms have been described: one related to the enterocyte iron levels and the other to the hypoxia pathway.

Regulation of the synthesis of multiple proteins involved in iron physiology, including TfR1, DMT1, FPN1, and ferritin, is controlled at a posttranscriptional level by influencing mRNA stability and translation. The mRNA of these proteins contain iron response elements (IREs), conserved nucleotide sequences with a stem-loop structure that binds iron regulatory proteins (IRPs)-1 and -2. The mRNAs for ferritin, DMT1, and FPN1 have IREs in the 5' untranslated region (UTR), and the mRNA for the TfR has multiple IREs in the 3' UTR. In low-iron states, IRP-1 is in a conformation that allows it to bind to IREs; for example, it binds the 3' IRE of the TfR mRNA, stabilizing it and allowing transcription of more TfR protein, and to the 5' UTR of ferritin mRNA, decreasing translation of ferritin for iron storage. Intracellular iron induces ubiquitination and degradation of IRP-2. Iron deficiency by this mechanism upregulates IRP activity via increased IRE binding, resulting in increased cellular iron uptake and decreased iron storage.

Dcytb mRNA does not contain an IRE but is strongly upregulated in iron-deficient duodenum, indicating additional regulation of transcription of iron-related genes. Hypoxia-inducible factor (HIF) transcriptin factors may serve as important local regulators of intestinal iron absorption. Iron deficiency induced HIF signaling in duodenum of mice, and caused increased Dcytb and DMT1 expression, and an increase in iron uptake. Accordingly, targeted deletion of Hif-2alpha in the intestine resulted in dramatic decrease in the expressions of DMT1-IRE and Dcytb mRNA. In vitro studies further demonstrated that HIF-2alpha directly binds to the promoters of DMT1 and Dcytb, activating their transcription.

Summary: Factors That Affecting Hepcidin Levels

Systemic mechanisms: 1) by transferrin levels, including holotransferrin/HFE/TfR2 complex + hemojuvelin (coreceptor) + BMP6 (ligand), which increases hepcidin gene expression; 2) by erythropoiesis, however, the mechanisms by which erythropoiesis regulates hepcidin production are not well understood, but it is thought that erythroid precursors in the bone marrow secrete a factor which exerts its effect on hepatocytes and causes hepcidin supression (so the ERFE previously mentioned?); and 3) by cytokines in inflammation state, via STAT-3 pathway (already described above).

Local mechanisms: 1) posttranscriptional level by influencing mRNA stability and tanslation, via IRPs; 2) HIF's impact on cis-acting regulatory elements of DMT1 and Dcytb genes.

Iron Cycle

screen-shot-2016-11-03-at-5-12-27-pmMost functional iron in the body is not derived from daily intestinal absorption (1 to 2 mg/day) but rather from recycling of iron (20 to 25 mg/day) from senescent erythrocytes and other cells. The most important source and destination of recycled iron is the erythron. At the end of a 4-month lifespan, effete erythrocytes are engulfed by reticuloendothelial macrophages, which lyse the cells and degrade hemoglobin to liberate their iron. This process is poorly understood, but it appears to involve the action of heme oxygenase for enzymatic degradation of heme. Some of this iron may remain stored in macrophages as ferritin or hemosiderin, but most is delivered to the plasma, and the rate of iron export is determined by the hepcidin-ferroportin interaction. In plasma, iron becomes bound to transferrin, completing the cycle. A small amount of iron, probably <2 mg, leaves the plasma each day to enter hepatic parenchymal cells and other tissue. Here, the iron is stored or used for synthesis of cellular heme proteins, such as myoglobin and the cytochromes.

Macrophage Iron Recycling

Although there are many types of tissue macrophages, those that participate in the catabolism of red blood cells can be subdivided into two categories. One type, exemplified by pulmonary alveolar macrophages, is able to phagocytose erythrocytes or other cells and convert the iron they contain into storage forms, but lacks the ability to return the iron to the circulation. This type of macrophage appears to retain the iron throughout its life span. The second type of macrophage, comprising the reticuloendothelial system, acquires iron in a similar fashion but is able to return it to the plasma. The latter macrophages, found espeically in the sinuses of the spleen and the liver, play a primary role in the normal reutilization of iron from destroyed red cells, allowing completion of the iron cycle shown in Figure 23.4.

Plasma Transport

The plasma iron-binding protein, transferrin, is a glycoprotein with a molecular weight of approximately 80 kDa. Transferrin is synthesized chiefly in the liver and actively secreted by hepatocytes, but lesser amounts are made in other tissues, including the central nervous system, the ovary, the testis, and helper T lymphocytes (CD4+ subset). The rate of synthesis shows an inverse relationship to iron in stores; when iron stores are depleted, more transferrin is synthesized, and when iron stores are overfilled, the level of transferrin decreases. Transferrin keeps iron nonreactive in the circulation and extravascular fluid, delivering it to cells bearing transferrin receptors.

Transferrin can be measured directly using immunologic techniques; and normal concentration in the plasma is approximately 2 to 3 g/L. Alternatively, transferrin is quantified in terms of the amount of iron it will bind, a measure called the total iron-binding capacity (TIBC: normal values for plasma iron and TIBC are given in Appendix A). In the average subject, the plasma iron concentration is 100 ug/dL, and the TIBC is 300 ug/dL. Thus, only about one third of the available transferrin binding sites are occupied, leaving a large capacity to deal with excess iron. Plasma iron concentration varies over the course of the day, with the highest values in the morning and the lowest in the evening. Levels of serum transferrin are more constant, and there is no apparent diurnal variation in TIBC. General practice has been to evaluate transferrin saturation using a first morning, fasting sample to standardize the results, but this may not be helpful.

Transferrin has two homologous iron-binding domains, each of which binds an atom of trivalent (ferric) iron. The iron atoms are incorporated one at a time and appear to bind randomly at either or both of the two sites. When binding is complete, the iron lies in a pocket formed by two polypeptide loops. One mole of anion, usually carbonate or bicarbonate, is taken up, and 3 moles of hydrogen ion are released from each mole of iron bouond. There are functional differences between teh two iron-binding sites, but it is not clear that these have physiologic importance.

Under physiologic circumstances, ferric iron binds to transferrin with very high affinity, with an affinity constant of ~1-6 x 1022 M-1. The affinity of iron-transferrin interaction is pH-dependent, decreasing as pH is lowered. Other transition metals, such as copper, chromium, manganese, gallium, aluminum, indium, and cobalt, can be bound by transferrin but with less affinity than iron.

Iron Delivery to Erythroid Precursors

The biologic importance of transferrin in erythropoiesis is illustrated by abnormalities observed in patients and mice with congenital atransferrinemia. When transferrin is severely deficient, red cells display the morphologic stigmata of iron deficiency. This occurs despite the fact that intestinal iron absorption is markedly increased in response to a perceived need for iron for erythropoiesis. Nonhematopoietic tissues avidly assimilate the non-transferrin-bound metal. Similarly, mutant mice lacking tissue receptors for transferrin die during embryonic development from severe anemia, apparently resulting from ineffective iron delivery to erythroid precursor cells.

Transferrin delivers its iron to developing normoblasts and other cells by binding to specific cell-surface receptors. The transferrin receptor (TfR1) is a disulfide-linked homodimer of a glycoprotein with a single membrane-spanning segment and a short cytoplasmic segment. It is a type II membrane protein, with its N terminus located within the cell. The native molecular weight of TfR1 is ~180 kDa. Each TfR1 homodimer can bind two tranferrin molecules. Diferric transferrin is bound with higher affinity than monoferric transferrin. As a result, diferric transferrin has a competitive advantage in delivering iron to the erythroid precursors. Apotransferrin has little affinity for the receptor at physiologic pH but considerable affinity at lower pH, an important factor in intracellular iron release.

TfR1 numbers are modulated during erythroid cell maturation, reaching their peak in intermediate normoblasts. Very few TfR1 molecules are found on burst-forming-unit erythroid cells, and only slightly greater numbers are found on colony-forming-unit erythroid cells. However, by the early normoblast stage, approximately 300,000 receptors are found on each cell, increasing to 800,000 at the intermediate stages. The rate of iron uptake is directly related to the number of receptors. The number decreases as reticulocytes mature, and late in maturation, erythroid cells shed all remaining receptors by exocytosis and by proteolytic cleavage. The shed receptors, referred to as soluble transferrin receptors (sTfR) is a sensitive indicator of erythroid mass and tissue iron deficiency.

After ligand and receptor interact, iron-loaded transferrin undergoes receptor-mediated endocytosis. Specialized endocytic vesicles form, which are acidified to a pH of 5 to 6 by the influx of protons. The low pH facilitates release of iron from transferrin and strengthens the apotransferrin-receptor interaction. Released iron is reduced by an endosomal ferrireductase, STEAP3, and transferred to the cytosol by DMT1. Because DMT1 must cotransport protons with iron atoms, vesicle acidification is also important for the function of this transporter. After the iron enters the cytosol, the protein components of the endosome return to the membrane surface, where neutral pH promotes the release of apotransferrin to the plasma.

After Entering into Erythroid Precursor Cells

In the normal subject, ~80% to 90% of the iron that enters erythoid precursor cells is ultimately taken up by mitochondria and incorporated into heme. Most of the remainder is stored in ferritin. Granules of ferritin may sometimes be detected using the Prussian blue reaction. Normoblasts with Prussian blue-positive (siderotic) granules are called sideroblasts, and, if the granules persist after enucleation, the mature cells are called siderocytes. In normal individuals, approximately half of the normoblasts are sideroblasts, each containing less than five small granules.

It is intriguing that even though erythroid precursors require large amounts of iron and heme for hemoglobin synthesis, they have been found to express the heme exporter FLVCR and the iron exporter ferroportin. Studies suggest that FLVCR is necessary for survival of erythroid precursors. Because heme is toxic to cells at high concentrations, it is thought that FLVCR functions as a safety valve to prevent accumulation of excess heme early during erythroid differentiation.

Erythorid precursors also express ferroportin but its role in erythroid maturation is yet unclear. It is interesting that erythoid cells preferentially express non-IRE ferroportin transcript during the stages of rapid iron uptake, thus avoiding any iron-induced increase in ferroportin translation dependent on the IRE/IRP system.

[Physiology][Hematology] Coagulation Factors, Anticoagulation Factors, and Pathways of Hemostasis and Thrombosis

July 14, 2016 Hematology, Molecular Biology No comments , , , , , , , , , , , , , , , , , ,

The role of surfaces in coagulation and coagulation inhibition and fibrinolysis

Functionally, the relationship between clotting and surface is striking. For a process that is typically termed humoral, most of the coagulation reactions take place on biologic surfaces.

  • The presence of a phospholipid surface increases the rate of activation of prothrombin by several orders of magnitude.
  • The presence of a phospholipid surface also localizes the reaction to the site of injury and may protect the reaction from inhibitors.
  • The activation of protein C by thrombin is a reaction that occurs on a cellular surface.
  • Coagulation inhibitors such as antithrombin and heparin cofactor II are also more efficient when the reactions occur on surfaces. The typical surface in these reactions is glycosaminoglycans like heparin, heparin sulfate, and derma tan sulfate.

The Vitamin K-dependent Zymogens


Tenase/Intrinsic tenase complex: factor VIIIa-factor IXa complex

Extrinsic tenase complex: tissue factor-factor VIIa complex

Prothrombinase compelx: factor Va-factor Xa complex

These zymogens all a similar domain structure of a C-terminal serine protease domain and an N-terminal γ-carboxy glutamic acid (Gla) domain, which are connected by two epidermal growth factor (EGF)-like domains or kringle domains. The Gla domain mediates the binding of zymogens to a negatively charged lipid surface (in a calcium-dependent manner), a domain that is characteristic to the vitamin K–dependent proteins.

Screen Shot 2016-08-11 at 7.36.50 PMThe Gla domain refers to the 42-residue region located in the N-terminus of the mature protein that comprises 9 to 12 glutamic acid residues that are posttranslationally γ-carboxylated into Gla residues by a specific γ-glutamyl carboxylase in the endoplasmatic reticulum of hepatocytes. This γ-carboxylase requires oxygen, carbon dioxide, and the reduced form of vitamin K for its action, hence the name vitamin K–dependent proteins. For each Glu residue that is carboxylated, one molecule of reduced vitamin K is converted to the epoxide form. Warfarin inhibits the activity of vitamin K epoxide reductase, thereby preventing vitamin K recycling and hinibiting γ-carboxylation, which results in a heterogeneous population of circulaing undercarboxylated forms of the vitamin K-dependent proteins with reduced activity. Recognition by and interaction with γ-carboxylase is facilitated by the propeptide sequence that is located C-terminal to the signal peptide.

The serine protease domains of the vitamin K-dependent proteins are highly homologous, as they bear a chymotrypsin-like fold and display trypsin-like activity.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Activates TAFI, platelet, fibrinogen, FV, FVIII, FXI, FXIII, and protein C

Activated by prothrombinase complex

Inhibited by serpins (enhanced by glycosaminoglycans like heparin)

Screen Shot 2016-05-20 at 2.19.12 PMProthrombin is composed of fragment 1 (F1: Gla and kringle 1), fragment 2 (F2: kringle 2), and the serine protease domain. The primary function of kringle 1 and kringle 2 domain is to be bound by prothrombinase complex. PS: Gla and kringle, kringle 2, and serine protease.

Prothrombin is proteolytically activated by the prothrombinase complex that cleaves at Arg271 and Arg320, both of which are necessary to generate procoagulant α-thrombin (IIa). Thrombin's main function is to induce the formation of a fibrin clot by removing fibrinopeptides A and B from fibrinogen to form fibrin monomers, which then spontaneously polymerize. The dynamic structural conformation of thrombin allows for binding to diverse ligands, and the subsequent ligand-indued conformational stabilization, known as thrombin allostery, regulates and controls thrombin activity. Thrombin also is able to cleave a wide variety of substrates with high specificity (TAFI, platelet, FV, FVIII, FXI, FXIII, protein C).

The physiologic inhibitors of thrombin are the serine protease inhibitors (serpins) antithrombin, heparin cofactor II, protein C inhibitor, and protease nexin 1, with antithrombin being the primary plasma inhibitor. For all four serpins, the rate of thrombin inhibition can be accelerated by glycosaminoglycans, such as heparin, through mutual binding to the serpin and thrombin, which ensures rapid inhibition of thrombin at the intact endothelial cell surface where heparin-like glycosaminoglycans are found.


Activates FIX, FX (in the form of extrinsic tenase)

Activated by Xa, thrombin, IXa, and XIIa

Inhibited by TFPI; antithrombin (only in the presence of heparin)

Screen Shot 2016-05-20 at 2.38.51 PMFactor VII consists of a Gla domain with 10 Gla residues, two EGF-like domains, a connecting region, and the serine protease domain.

Factor VII is proteolytically activated once it has formed a high-affinity complex with its cofactor tissue factor (there are small amount of VIIa in the circulation by unknown mechanism). A number of coagulation proteases including factor Xa, thrombin, IXa, and XIIa are capable of cleaving factor VII at Arg152 to generate factor VIIa, with factor Xa being considered the most potent and physiologically relevant activator of factor VII. Autoactivation can also occur, which is initiated by minute amounts of preexisting factor VIIa.

The extrinsic tenase complex activates both FIX and X.

The extrinsic tenase complex is inhibited by the tissue factor pathway inhibitor (TFPI). Antihrombin (only in the presence of heparin) also can inhibits the extrinsic tenase complex.


Aactivates FX in the form of intrinsic tenase

Activated by extrinsic tenase, factor XIa

Inhibited by antithrombin (enhanced by heparin)

Screen Shot 2016-05-20 at 3.08.29 PMFactor IX consists of a Gla domain, two EGF-like domains, a 35-residue activation peptide, and the serine protease domain. The Gla domain contains 12 Gla residues, of which the 11th and 12th Gla (Glu36 and Glu40) are not evolutionary conserved in other vitamin K-dependent proteins and are not essential for normal factor IX function.

Limited proteolysis of factor IX at both Arg145 and Arg180 by either the extrinsic tenase or factor XIa results in the release of the activation peptide and generation of factor IXa.

Factor IXa has a low catalytic efficiency as a result of impaired access of substrates to the active site that results from steric and repulsion. Reversible interaction with the cofactor VIIIa on anionic membranes and subsequent factor  X binding leads to rearrangement of the regions surrounding the active site and proteolytic factor X activation.

The primary plasma inhibitor of factor IXa is the serpin antihrombin, and this inhibition is enhanced by heparin, which induces a conformational change in antithrombin that is required for simultaneous active site and exosite interactions with factor IXa.


Activates prothrombin (prothrombinase complex); FV, VII, and VIII

Activated by extrinsic tenase; intrinsic tenase

Inhibited by antithrombin (enhanced by heparin); TFPI

Factor X is a two-chain zymogen consisting of a light chain which comprises the Gla domain with 11 Gla residues and the EGF domain, and a heavy chain that consists of a 52-residue activation peptide and the serine protease domain. The two chains are linked via a disulfide bond.

Factor X is activated by intrinsic tenase or extrinsic tenase, following cleavage at Arg194 in the heavy chain. After activated, Xa reversibly associates with its cofactor Va on an anionic membrane surface in the presence of calcium ions to form prothrombinase, the physiologic activator of prothrombin. Factor Xa is also involved in the proteolytic activation of FV, FVII, and VIII.

Further autocatalytic cleavage at Arg429 near the C-terminus of the factor Xa heavy chain leads to release of a 19-residue peptide, yielding the enzymatically active factor Xaβ. Plasmin-mediated cleavage of factor Xa at adjacent C-terminal Arg or Lys residues also results in the generation of factor Xaβ and factor Xaβ derivatives. While the coagulation activity is eliminated in the factor Xaβ derivatives, they are capable of interacting with the zymogen plasminogen and enhance its tissue plasminogen activator-mediated conversion to plasmin, thereby promoting fibrinolysis.

A primary plasma inhibitor of factor Xa is the serpin antithrombin, and this inhibition is enhanced by heparin. Another potent factor Xa inhibitor is TFPI, which inhibits both the extrinsic tenase-factor Xa complex as well as free factor Xa, for which protein S function as a cofactor.

Protein C

Inactivates FVa, FVIIIa (cofactor protein S, with calcium and surface) 

Activated by thrombin-thrombomodulin complex (enhanced by EPCR and PF4)

Inhibited by heparin-dependent serpin protein C inhibitor and by PAI-1

Protein C is synthesized as a single-chain precursor and during intracellular processing amino acids Lys146-Arg147 are excised. The zymogen consists of a light chain comprising the Gla domain and the EGF domains, which is linked via a disulfide bond to the heavy chain that consists of the activation peptide and the serine protease domain.

Screen Shot 2016-05-24 at 8.45.18 PMProtein C is proteolytically activated by alpha-thrombin in complex with the endothelial cell surface protein thrombomodulin following cleavage at Arg169. The activation peptide is released and the mature serine protease activated protein C (APC) is formed. Activation of protein C is enhanced by its localization on the endothelial surface through association with the endothelial cell protein C receptor (EPCR). Also, protein C activation is accelerated by platelet factor 4 (PF4), which is secreted by activated platelets. Upon interaction with the Gla domain of protein C, PF4 modifies the conformation of protein C, thereby enhancing its affinity for the thrombomodulin-thrombin complex.

APC consists of the disulfide-linked light chain comprising the Gla and EGF domains and the catalytic heavy chain. In complex with its cofactor protein S, APC proteolytically inactivates factors Va and VIIIa in a calcium- and membrane-dependent manner. Intact factor V has been reported to function as a cofactor for the inactivation of factor VIIIa in the presence of protein S.

APC is primarily inhibited by the the heparin-dependent serpin protein C inhibitor and by plasminogen activator inhibitor-1 (PAI-1).

The Procoagulant Cofactors V and VIII

Factors V and VIII both function as cofactors in coagulation and dramatically enhance the catalytic rate of their macromolecular enzyme complexes, resulting in the generation of thrombin (via prothrombinase) and factor Xa (via intrinsic tenase), respectively. Apart from their functional equivalence, they also share similar gene structure, amino aacid sequences, and protein domain structures.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Accelerates the ability of FXa to rapidly convert prothrombin to thrombin

Activated by thrombin (principal activator), FXa (primarily in initiation phase)

Inhibited by APC

FV and factor V-short interact with full-length TFPI

Approximately 20% percent of the total factor V in blood is stored in the alpha-granules of platelets. Although it was originally thought that megakaryocytes synthesize factor V, studies in humans indicate that platelet factor V originates from plasma through endocytic uptake. Platelet factor V is modified intracellularly such that it is functionally unique compared to its plasma-derived counterpart. It is partially activated, more resistant to inactivation by APC, and has several different posttranslational modifications.

Screen Shot 2016-05-29 at 8.22.28 PMFactor V has an A1-A2-B-A3-C1-C2 domain structure. The two C-type domains belong to the family of discoidin domains, which are generally involved in cell adhesion, and share approximately 55 percent sequence identity with the factor VIII C domains. The C domain mediate binding to the anionic phospholipid surface, thereby localizing factor V to the site of injury and facilitating interaction with factor Xa and prothrombin. In contrast, large central B domain of factor V shows weak homology to the factor VIII B domain or to any other known protein domain.

Factor V undergoes extensive postranslational modifications, including sulfation, phosphorylation, and N-linked glycoslation. Sulfation at sites in teh A2 and B domain are involved in the thrombin mediated proteolytic activation of factor V. Phosphorylation at Ser692 in the A2 domain enhances the APC-dependent inactivation of the cofactor Va.

Sequential proteolytic cleavage of the procofactor factor V at Arg709, Arg1018, and Arg1545 in the B domain results in release of the inhibitory constraints exerted by the B domain and in the generation of the heterodimeric cofactor Va, where maximal cofactor activity correlates with cleavage at Arg1545. Thrombin has generally been recognized to be the principal activator of factor V. However, recent findings suggest that in the initiation phase of coagulation factor V is primarily activated by factor Xa. Factor Xa initially cleaves factor V at Arg1018, followed by proteolysis at Arg709 and Arg1545.

Factor Va is composed of a heavy chain comprising the A1-A2 domains and the A3-C1-C2 light chain, which are noncovalently associated via calcium ions. Factor Va is a nonenzymatic cofactor within the prothrombinase complex that greatly accelerates the ability of factor Xa to rapidly convert prothrombin to thrombin.

APC catalyzes the inactivation of factor Va by cleavage at the main sites Arg306 and Arg506, upon which the cleaved A2 fragment dissociates and factor Va can no longer associated with factor Xa.

Both factor V and an alternativel spliced isoform of factor V (factor V-short), which lacks the major part of the B domain (residues 756 to 1458) and normally circulates in low abundance, interact with full-length TFPI (TFPIalpha), most likely through the acidic B domain region. The linkage of factor V and TFPIalpha is considered to attenuate the bleeding phenotype in factor V-deficient patients, as the low TFPIalpha levels in these patients allow the residual platelet factor to be sufficient for coagulation. Conversely, increased factor V-short expression caused by an A2440G mutation in the factor V gene leads to a dramatic increase in plasma TFPIalpha, resulting in a bleeding disorder.


Accelerates the ability of FIXa to rapidly convert FX to FXa

Activated by thrombin (principal) and FXa (also principal)

Activity downregulated spontaneously, by APC, FXa or FIXa

Factor VIII (antihemophilic factor) was first discovered in 1937, but it was not until 1979 that its purification by Tuddenham and coworkers led to the molecular identification of the protein. The mature factor VIII procofactor comprises 2332 amino acids and circulates in a high-affinity complex with its carrier protein VWF at a concentration of approximately 0.7 nM and a circulatory half-life of 8 to 12 hours. Complex formation with VWF protects factor VIII from proteolytic degradation, premature ligand binding, and rapid clearance from the circulation.

The primary source of factor VIII is the liver, but extrahepatic synthesis of factor VIII also occurs. While contradictory evidence exists on the cellular origin of both hepatic and extrahepatic factor VIII synthesis, recent studies in mice support that endothelial cells from many tissues and vascular beds synthesize factor VIII, with a large contribution from hepatic sinusoidal endothelial cells. This is consistent with observations on factor VIII expression in human endothelial cells from the liver and lung.

Screen Shot 2016-05-29 at 8.22.42 PMThe A1-A2-B-A3-C1-C2 domain structure of factor VIII shares significant homology with factor V except in the B domain region. In contrast to factor V, factor VIII B domain is dispensable for procoagulant activity. The C-terminal regions of the A1 and A2 domians and the N-terminal portion of the A3 domain contain short segments of 30 to 40 negatively charged residues known as the a1, a2, and a3 regions. Interaction with VWF is faciliated by the a3 region and C1 domain. The C domains mediate binding to the anionic phospholipid surface, thereby localizing factor VIII to the site of injury and facilitating interaction with factor IXa and factor X.

Factor VIII is heavily glycosylated and the majority of the N-linked glycosylation sites are found in the B domain, which mediate interaction with the chaperons calnexin and calreticulin and, in part, with the LMAN1-MCDF2 receptor complex. Sulfation of tyrosin residues is required for optimal activation by thrombin, maximal activity in complex with factor IXa, and maximal affinity of factor VIIIa for VWF.

Thrombin and factor Xa are the principal activators of the procofactor VIII and generate the cofactor VIIIa through sequential proteolysis at Arg740, Arg372, and Arg1689. The heterotrimeric factor VIIIa is composed of the A1, A2, and the A3-C1-C2 light chain subunits. The A1 and A3-C1-C2 subunites are noncovalently linked through calcium ions, whereas A2 is associated with weak affinity primarily by electrostatic interactions. Once activated, factor VIIIa functions as a cofactor for factor IXa in the phospholipid-dependent conversion of factor X to factor Xa. The rapid and spontaneous loss of factor VIIIa activity is attributed to A2 domain dissociation from the heterotrimer. Additional proteolysis by APC, factor Xa, or factor IXa also results in the downregulation of factor VIIIa cofactor activity.

The Soluble Cofactors Protein S and Von Willebrand Factor

Protein S

Protein S is a vitamin K-dependent single-chain GP of 635 amino acids that circulates with a plasma half-life of 42 hours. Part of the total protein S pool circulates in a free form at a concentration of 150 nM, whereas the majority (~60%; 200 nM) circulates bound to the complement regulatory protein C4b-binding protein (C4BP). Protein S is primarily synthesized in the liver by hepatocytes, in addition to endothelial cells, megakaryocytes, testicular Leydig cells, and osteoblasts.

The protein structure of protein S differs from the other vitamin K-dependent proteins as it lacks a serine protease domain and, consequently, is not capable of catalytic activity. Protein S is composed of a Gla domain comprising 11 Gla residues, a thrombin-sensitive region (TSR), four EGF domains, and a C-terminal sex hormone-binding globulin (SHBG)-like region that consists of two laminin G-type domains. The SHBG-like domain is involved in the interaction with beta-subunit of C4BP.

Apart from gamma-carboxylation of Glu residues, protein S is posttranslationally modified via N-glycosylation in the second laminin G-type domain of the SHBG-like region. beta-Hydroxylation of Asp95 or Asn residues in each EGF domain allows for calcium binding that orients the four EGF domains relative to each other.

Free protein S serves as a cofactor for APC in the proteolytic inactivation of FVa and FVIIIa. Interaction of protein S with APC on a negatively charged membrane surface alters the location of the APC active site relative to FVa, which accounts for the selective protein S-dependent rate enhancement of APC cleavage at Arg306 in FVa. C4BP-bound protein S also exerts a similar stimulatory effect on Arg306 cleavage, albeit to lower extent, whereas it inhibits the initial APC-mediated FVa cleavage at Arg506, resulting in an overall inhibition of FVa inactivation. Cleavage of the TSR by thrombin and/or FXa results in a loss of APC-cofactor activity. Protein S also functions as a cofactor for TFPIalpha in the inhibition of factor Xa, which is mediated by the SHBG-like region in protein S.


VWF is a large multimeric GP that is required for normal platelet adhesion to components of the vessel wall and that serves as a carrier for factor VIII. It is exclusively synthesized in megakaryocytes and endothelial cells and stored in specialized organelles in platelets and endothelial cells. VWF multimers circulate at a concentration of 10 nM with a half-life of 8 to 12 hours. Clearance of VWF multimers is mainly mediated by macrophages from the liver and spleen.

Large VWF multimers are cleaved by the plasma protease ADAMTS-13. This cleavage produces the smaller size VWF multimers that circulate in plasma. Reduced ADAMTS-13 activity is linked to various microangiopathies with increased platelet activity.

The precursor protein of VWF is composed of a 22-residue signal peptide and of a proVWF protein comprising 2791 amino acids that has 14 distinct domains. Upon translocation to the ER, the signal peptide is cleaved off, and the proVWF dimerizes in a tail-to-tail fashion through cysteines in its cysteine knot (CK) domain. During transit through the Golgi apparatus, proVWF dimers multimerize in a head-to-head fashion through the formation of disulfide bonds between cysteine residues in the D3 domain. At the same time, D1 and D2 domains are cleaved off as a single fragment to form the VWF propeptide (741 amino acids), while the remaining domains comprising 2050 amino acid residues and up to 22 carbohydrate chains form mature VWF. In the trans-Golgi network, the VWF propeptide promotes mature VWF to assemble into high-molecular-weight multimers. These multimers subsequently aggregate into tubular structures that are packaged into alpha-granules in megakaryocytes and into Weibel-Palade bodies in endothelial cells.

Upon exocytosis from Weibel-Palade bodies and at high shear rates, multimeric VWF unrolls from a globular to a filamental conformation, up to many microns long, which becomes a high-affinity surface for the platelet GPIb-V-IX complex. Large VWF multimers are more active than smaller multimers, which is explained by the fact that the former contain multiple domains that support the interactions between platelets, endothelial cells, and subendothelial collagen. VWF binds to matrix collagens via its A1 and A3 domains. The A1 domain also mediates binding to platelet GPIb, which is required for the fast capture of platelets. Platelet adhesion to VWF is further supported by VWF immobilization on surface and by high shear stress.

VWF complexes with factor VIII through the first 272 residues in the N-terminal region of the mature VWF protein subunit, thereby protecting factor VIII from proteolytic degradation, premature ligand binding, and rapid dlearance from the circulation.

Factor XI and The Contact System


Activates FIX with cofactor HK (calicum-dependent, phospholipid-independent)

Activated by FXIIa, thrombin, and FXIa

Inhibited by nexin 1, nexin 2, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin

Screen Shot 2016-06-11 at 2.57.05 PMFactor XI is synthesized in the liver and secreted as a single-chain zymogen of 607 amino acids. In the circulation, FXI is found as a homodimer at a concentration of 30 nM with a plasma half-life of 60 to 80 hours. All FXI homodimers circulate in complex with high-molecular-weight kininogen (HK). HK is thought to mediate binding of factor XI to negatively charged surfaces, thereby facilitating factor XI activation.

Each FXI subunit comprises four apple domians and a serine protease domain. The apple domains are structured by three disulfide bonds and form a disk-like platform on which the serine protease domain rests. The dimerization of two FXI subunits is mediated by interactions between the two apple 4 domains that involve a disulfide bond between the Cys321 residues, hydrophobic interactions, and a salt bridge, of which only the latter two are required for dimerization. The domain structure of FXI is highly similar to that of the monomer prekallikrein (PK), the zymogen of the protease kallikrein, which also circulates in complex with HK.

FXI does not bear a Gla domain and thus does not require gamma-carboxylation to exert its procoagulant activity.

Activation of a FXI subunit to FXIa proceeds through proteolysis at Arg369 in the N-terminal region of the serine protease domain and yields two-chain activated factor XIa. There are several catalysts capable of FXI activation, which include the contact factor XIIa, thrombin, or factor XIa itself in the presence of negatively charged surfaces. FXI must be a dimer to be activated by FXIIa, whereas thrombin and factor XIa lack this requirement.

Following activation of FXI, binding sites for the substrate FIX become available in the apple 3 domain and serine protease domian of factor XIa. FXIa proteolytically activates FIX to factor FIXa in a calcium-dependent but phospholipid-independent manner. Both forms of the FXIa dimer as well as monomeric FXIa activate FIX in a similar manner.

Accumulating evidence supports the notion that FXIIa-dependent activation of FXI is not essential to normal hemostasis, but is important in pathologic thrombus formation. Thrombin-mediated activation of FXI, on the other hand, seems most significant under conditions of low tissue factor and is assumed to enhance clot stability thorugh thrombin-activation of TAFI.

FXIa function is regulated by the serpins protease nexin 1, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin. Platelets also contain a FXIa inhibitor, the Kunitz-type inhibitor protease nexin 2.

The Contact System: FXII, Prekallikrein, and High-Molecular Weight Kininogen

FXII, HK, and PK are part of the contact system in blood coagulation, which is triggered following contact activation of FXII mediated via negatively charged surface.


Activates FXII

Activated by FXIIa

PK is synthesized in the liver, circulates as a zymogen, and is highly homologous to FXI. Conversion into the serine protease proceeds through limited proteolysis by FXIIa, and the generated kallikrein reciprocally activates more FXII.


HK, which is also synthesized in the liver, is a nonenzumatic cofactor that circulates in complex with FXI, which then activates FIX.

Contact system

The contact system is at the basis of the activated partial thromboplastin time (APTT) assay that is widely used in clinical practice. In this clinical laboratory test, the negatively charged surface is provided by reagents. FXIIa activates FXI, which then activates FIX. Despite HK and PK being required for a normal APTT, they appear to be dispensable for coagulation in vivo. Individuals who are deficient in any of these factors do not have a bleeding tendency, even after significant trauma or surgery.


Activates FXI, PK

Activated by negatively charged surface (platelet polyphosphate, microparticles derived from platelets and erythrocytes, RNA, and colalgen), kallikrein

Inhibited by serpin C1 inhibitor, antithrombin, and PAI-1

FXII, which is homologous to plasminogen activators, consists of an N-terminal fibronectin type I domain, an EGF-like domain, a fibronectin type II domain, a second EGF-like domain, a kringle domain, a proline-rich region, and a C-terminal serine protease domain. The proline-rich region is unique to FXII, as it is not found in any of the other serine proteases.

Limited proteolysis by kallikrein at Arg353 in FXII yields the activated two-chain alpha-FXIIa. Once activated, alpha-FXIIa activates FXI to FXIa. Furthermore, alpha-FXIIa activates PK, thereby contributing to its own feedback activation. Also FXI is known to acquire alpha-FXIIa activity upon contact with a negatively charged surface, the latter inducing a conformation change in FXII. This conformational change induces a limited amount of proteolytic activity in FXII, known as auto-activation. Furthermore, the surface-induced active conformation of FXII is suggested to enhance the proteolytic conversion to alpha-FXIIa. The fibronectin type I and II domains, EGF-2, the kringle domain, and the proline-rich region are reported to conribute to interaction with a negatively charged surface.

Further cleavage of alpha-FXIIa by kallikrein at Arg334 and Arg343 in the light chain results in the generation of beta-FXIIa, which comprises a nine-residue heavy-chain fragment that is disulfide-linked to the light chain. Given the absence of the heavy chain, beta-FXIIa does not interact with anionic surfaces. Even though beta-FXIIa is still capable of activating PK, it is no longer activates FXI.

The serpin C1 inhibitor is the main plasm inhibitor of alpha-FXIIa and beta-FXIIa. In addition, antithrombin (AT) and PAI-1 also inhibit FXIIa activity.

Revised Model of Coagulation

Screen Shot 2016-07-14 at 6.11.20 PM


Hemostasis is the process through which bleeding is controlled at a site of damaged vascular endothelium and represents a dynamic interplay between the subendothelium, endotheliumcirculating cells, and plasma proteins. The hemostatic process often is divided into three phases: the vascular, platelet, and plasma phases. Although it is helpful to divide coagulation into these phases for didactic purposes, in vivo, they are intimately linked and occur in a continuum.

The vascular phase is mediated by the release of locally active vasoactive agents that result in vasoconstriction at the site of injury and reduced blood flow. Vascular injury exposes the underlying subendothelium and procoagulant proteins, including von Willebrand factor (vWF), collagenn and tissue factor (TF) that then come into contact with blood.

During the platelet phase, platelets bind to vWF incorporated into the subendothelial matrix thorugh their expression of glycoprotein Iba (GPIbalpha). Platelets bound to vWF form a layer across the exposed subendothelium, a process termed platelet adhesion, and subsequently are activated via receptors, such as collagen receptors integrin alpha2beta1 and glycoprotein (GPVI), resulting in calcium mobilization, granule release, activation of the fibrinogen receptor, integrin alpha(IIb)b3, and subsequent platelet aggregation.

The plasma phase of coagulation can be further subdivided into initiation, priming, and propagation. Initiation begins when vascular injury also leads to exposure of TF in the subendothelium and on damaged endothelial cells. TF binds to the small amounts of circulating activated factor VII (FVIIa), resulting in formation of teh TF:FVIIa complex (extrinsic tenase complex); this complex binds to and activates factor X (FX) to activated FX (FXa). The TF:FVIIa:FXa complex converts a small amount of prothrombin (factor II/FII) to thrombin (activated factor II/FIIa). This small amount of thrombin is able to initiate coagulation and generate an amplification loop by cleaving factor VIII (FVIII) from vWF, activating clotting factors FVIII, XI (FXI), and platelets, which result in exposure of membrane phospholipids and release of partially activated factor V (FVa). At the end of the initiation and priming phases, the platelet is primed with an exposed phospholipid surface with bound activated cofactors (FVa and FVIIIa).

During the propagation phase, FIXa, generated either by the action of FXIa on the platelet surface or TF-VIIa complex on the TF bearing cell, bind to its cofactor, FVIIIa, to form the potent intrinsic tenase complex. FX is then bound and cleaved by the intrinsic tenase complex (FIXa:FVIIIa) leading to large amounts of FXa, which in association with its cofactor, FVa, forms the prothrombinase complex on the activated platelet surface. The prothrombinase complex (FXa:FVa) then binds and cleaves prothrombin leading to an ultimate burst of thrombin sufficient to convert fibrinogen to fibrin and to result in subsequent clot formation. The formed clot is stabilized by the thrombin-mediated activation of factor XIII (FXIII), which acts to cross-link fibrin, and thrombin-activatable fibrinolysis inhibitor (TAFI), which acts to remove lysine residues from the fibrin clot, thereby limiting plasmin binding. Utimately, the clot undergoes fibrinolysis, resulting in the restoration of normal blood vessel architecture. The fibrinolytic process is initiated by the release of tissue plasminogen activator (tPA) near the site of injury. tPA converts plasminogen to plasmin, which (via interactions with lysine and arginine residues on fibrin) cleaves the fibrin into dissolvable fragments.


Hemostasis – Plasma Phase – Initiation

  • FX > FXa via TF:FVIIa complex
  • FII > FIIa (small amount) via TF:FVIIa:FXa complex

Hemostasis – Plasma Phase – Priming

  • FVIII:vWF complex > FVIII + vWF via FIIa (small amount)
  • FVIII > FVIIIa via FIIa (small amount)
  • FXI > FXIa via FIIa (small amount)
  • Platelet > activated platelet via FIIa (small amount)
  • Activated platelet > partially activated FV via release

Hemostasis – Plasma Phase – Propagation

  • FIX > FIXa via FXIa or TF:VII complex
  • FX > FXa (large amount) via FIXa:FVIIIa complex
  • FII > FIIa (large amount) via FXa:FVa complex
  • FXIII > FXIIIa via FIIa (large amount)

Regulation of Hemostasis

Both the hemostatic and fibrinoglytic processes are regulated by inhibitors that limit coagulation at the site of injury and quench the reactions, thereby preventing systemic activation and pathologic propagation of coagulation. The hemostatic system has three main inhibitory pathways: antihrombin (AT), the protein C:protein S complex, and tissue factor pathway inhibitor (TFPI).

AT/Antithrombin target at thrombin and FXa

AT (antithrombin) released at the margins of endothelial injury binds in a 1:1 complex with thrombin, inactivating thrombin not bound by the developing clot. Antithrombin also rapidly inactivates FXa; thus, any excess FXa generated by the TF:VIIa complex during initiation is inactivated and unable to migrate to the activated platelet surface.

Thrombomodulin, protein C and S, target at FVa and FVIIIa

Excess free thrombin at the clot margins binds to thrombomodulin, a receptor expressed on the surface of intact endothelial cells that when complexed with thrombin activates protein C; activated protein C complexes with its cofactor protein S and inactivates FVa and FVIIIa.

Tissue factor pathway inhibitor/TFPI, target at TF:FVIIa and FXa

TFPI is a protein produced by endothelial cells that inhibits the TF:FVIIa complex and FXa. Binding to FXa is required for the inhibitory effect on TF:FVIIa. This negative feedback results in reduced subsequent thrombin generation and quenching of fibrin generation. The action of both AT and TFPI inhibits FXa during the initiation phase leading to dependence of platelet-surface FXa generation during the propagation phase for adequate hemostasis.


The fibrinolytic system also includes two inhibitors, principally plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin (alpha2AP), which inhibit tPA and plasmin, respectively.