GERBI ALICE, DE CAROLI MARIO, MOLA VIRGINIA
DEFINITION
The normal haemostasis is the result of several processes which, if properly regulated, serve two important functions: keeping the blood in a fluid state in normal vessels and induce a haemostatic plug quickly and well localized at the site of damage to the vessel . This haemostatic plug is a temporary formation, under physiological conditions, necessary to allow wound repair mechanisms repair the lesion.
Coagulation is the process by which blood forms clots. It is an important part of haemostasis, the cessation of blood loss from a damaged vessel, wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel. Disorders of coagulation can lead to an increased risk of bleeding (haemorrhage) or obstructive clotting (thrombosis).
Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium lining of the vessel itself. Platelets immediately form a plug at the site of injury; this is called primary haemostasis. Secondary haemostasis occurs simultaneously: Proteins in the blood plasma, called coagulation factors or clotting factors, respond in a complex cascade to form fibrin strands, which strengthen the platelet plug.
The coagulation factors are chemically heterogeneous, circulating in the blood or released from the tissues at the time of the vessel injury. Today, we know 12 factors numbered with Roman numerals (I to XIII, excluding VI, which does not exist), and two factors that correspond to prekallikrein and kininogen. They are mostly synthesized by the liver, some of them (II, VII, IX, X) through a synthesis that depends strictly on vitamin K. They are chemically heterogeneous substances, circulating in the blood or liberated from the tissues at the time of lesion of the vessel, whose activity is required for the normal development of blood clotting.
FACTOR V: Factor V is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor. Deficiency leads to predisposition for hemorrhage, while there are some mutations that predispose for thrombosis. The mutations of the gene that codes for Factor V are four and the altered proteins that results are known as Factor V Leiden, Factor V Cambridge, Factor V Hong Kong and Factor V HR2. The first three of them give coagulation disorders since they lose their ability to be inactivated by Activated Protein C (APC), while the last one, Factor V HR2, gives the same disorder only in association with the Factor V Leiden. All the four of them result then dangerous because lead to hypercoabulability disorders and deep vein thrombosis.
The most common and best understood of these mutations is Factor V Leiden.
FACTOR VIII: Factor VIII (FVIII) is an essential blood-clotting protein, also known as anti-haemophilic factor (AHF). In humans, factor VIII is encoded by the F8 gene. Defects in this gene result in haemophilia A, a recessive X-linked coagulation disorder.
Blood coagulation
Screening di polimorfismi genetici associati al rischio di insorgenza di patologie cardiovascolari
PATHOPHYSIOLOGY
In the normal person, factor V functions as a cofactor to allow factor Xa to activate an enzyme called thrombin. Thrombin in turn cleaves fibrinogen to form fibrin, which polymerizes to form the dense meshwork that makes up the majority of a clot. Activated protein C (aPC) is a natural anticoagulant that acts to limit the extent of clotting by cleaving and degrading factor V.
Factor V Leiden is an autosomal dominant condition that exhibits incomplete dominance and results in a factor V variated that cannot be as easily degraded by aPC (activated Protein C). The gene that codes the protein is referred to as F5. Mutation of this gene - a single nucleotide polymorphism (SNP) - is located in exon 10. As a missense substitution it changes a protein's amino acid from arginine to glutamine. Depending on the chosen start the position of the nucleotide variant is either at position 1691 or 1746. It also affects the amino acid position for the variant, which is either 506 or 534. Since this amino acid is normally the cleavage site for aPC, the mutation prevents efficient inactivation of factor V. When factor V remains active, it facilitates overproduction of thrombin leading to generation of excess fibrin and excess clotting.
Factor VIII participates in blood coagulation. It is released into the bloodstream by the endothelial cells of the vascular networks: glomerular and tubular cells and liver sinusoidal. It is a cofactor for factor IXa which, in the presence of Ca+2 and phospholipids forms a complex that converts factor X to the activated form Xa. The factor VIII gene produces two alternatively spliced transcripts. Transcript variant 1 encodes a large glycoprotein, isoform a, which circulates in plasma and associates with von Willebrand factor in a noncovalent complex. This protein undergoes multiple cleavage events. Transcript variant 2 encodes a putative small protein, isoform b, which consists primarily of the phospholipid binding domain of factor VIIIc. This binding domain is essential for coagulant activity. After activation by thombin (factor IIa), it dissociates from the complex and interacts with factor IXa in the coagulation cascade. Factor VIII assumes the role of cofactor of Factor IXa in the activation of factor X, which in turn, with its cofactor factor Va, activates more thombin. Thrombin cleaves fibrinogen to fibrin which polymerizes to give crosslink (with factor XIII) in a blood clot.
Factor V Is an Anticoagulant Cofactor for Activated Protein C during Inactivation of Factor Va,2010
Factor VIII,2012
REGULATION AND CORRELATIONS
It is known, at this point, that disorders of both factors lead to changes in coagulation ability. Although among the known clotting factors, factors VIII and V are exceptional in not possessing enzymatic activity, the malfunctioning of the two are not related one to the other.
Since calcium is required for the activation of these two factors, FVIII in particular, it is plausible to think that their activity may be correlated to calcium absorption and with, for example, Vitamin's D activity in this regulation.
Studies have been made on the effects of calcium as an additional cofactor on the activation of FVIII by thrombin. The conclusions are that calcium is required for this activation, but higher levels of the cation do not necessarily increase the results of this reaction increasing the prtoein’s procoagulant activity. There is, in fact, a definite stoichiometry ratio between the two elements, and too high levels of calcium can actually decrease thrombin power on FVIII activation. Thus, the effects of calcium and the recurrence of Hemophilia A, for instance, cannot be directly correlated.
Disorders in FVIII production can be associated instead to pulmonary hypertension. It is known, in fact, that this coagulation factor can be synthesized not only by the endothelial cells present in the hepatic sinusoid, but also by those in pulmonary microvascular circulation and in several other vascular beds. Pulmonary hypertension could lead in this way to disorders in FVIII synthesis, also related to problems with Von Willebrand Factor, protein to which FVIII is strictly related.
Factor VIII: structure and function in blood clotting. 1984
Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin. 1977
Endothelial Cell Processing and Alternatively Spliced Transcripts of Factor VIII: Potential Implications for Coagulation Cascades and Pulmonary Hypertension. 2010
GENETICS
The gene for factor V is located on the first chromosome (1q23). It is genomically related to the family of multicopper oxidases, and is homologous to coagulation factor VIII. The gene spans 70 kb, consists of 25 exons, and the resulting protein has a relative molecular mass of approximately 330kDa.
The gene for factor VIII is located on the tenth chromosome (Xq28). It presents an interesting primary structure, as another gene is embedded in one of its introns.
EPIDEMIOLOGY
It is estimated that about 3% of the world population has factor V Leiden, and most of the people with this predisposing factor will not manifest clinical problems of thrombosis. In North Americait has been shown a prevalence of approximately 5% of Caucasians, while the variation is less common among Hispanics and among African Americans and is very rare among Asians.
In Europe about 6-8% of the population presents this mutation.
Up to 25-30% of patients with deep vein thrombosis or pulmonary embolism has this polymorphism.
Inheriting one or two copies of the mutated gene brings to a different risk of developing thrombosis: it is estimated that having one copy ( heterozygous ) increases the risk by two to eight times, while having two copies ( homozygous ) - then one from each parent - increases the risk by twenty to eighty times. Considering that the risk of developing a thrombosis in total population is approximately 1 out of 1000 a year, the presence of a copy of the gene of factor V Leiden increases the risk between 1 out of 250 and 1 out of 125. Having two copies leads the risk up to 1 out of 12. The condition of homozygosity is necessarily more rare than heterozygosity, and it is estimated to be about 1% of people with factor V Leiden.
Women with factor V Leiden have an increased risk of deep vein thrombosis and pulmonary embolism during pregnancy. In addition, there may be a small increase in the risk of preeclampsia, or low birth weight, miscarriage or fetal death.
In literature it is reported that the incidence of hemophilia A is equal to 20 out of 100.000 born, without variations in different populations and races while the average value of prevalence ranges from 12.8 out of 100,000 males in the more developed countries to 6.6 out of 100,000 in the rest of the world.
Factor V and thrombotic disease,2002
Epidemiologia dell’emofilia a nel mondo e in Italia,2011
SYMPTOMS
There is a delicate balance at work to ensure that there is enough - but not too much - clotting ability in the blood. Too little clotting ability leads to bleeding problems, whereas too much clotting ability (thrombophilia) can lead to excessive blood clot formation. The state of this balance between bleeding and clotting differs from person to person, and many things can upset the balance (Figure). Because the natural anticoagulants are required to help to stop the clotting process, deficiencies of one of these substances can upset this balance and lead to thrombophilia. The most important natural anticoagulants are protein C, protein S, and antithrombin III.
The excess of coagulation caused by the alteration of factor V is almost entirely restricted to the veins, where it may show up as a deep vein thrombosis. The venous thrombus, if it breaks, it can embolize: it means that fragments of the clot can travel through the blood up to the right side of the heart and reach the lungs, where they can get stuck in the pulmonary vessels and cause a pulmonary embolism. Depending on the size and on the position of the clot, this is manifested by dyspnea, chest pain, palpitations and may have complications such as shock and cardiac arrest. Women with this anomaly also have an increased risk of miscarriage and fetal death. On the opposite side, it is very rare that this variant can cause arterial thrombi and, accordingly, strokes or heart attacks; it is more common, however, the transient ischemic attack. The risks are greater for those who have both mutated alleles (homozygous) compared to those who have only one. In addition, since this alteration is manifested by an incomplete dominance, subjects with the same variation may experience different degrees of risk.
The most common conditions associated with thrombophilia are venous thrombosis and pulmonary embolism (PE), often defined collectively as venous thromboembolism. Deep vein thrombosis usually shows up in the legs with pain, swelling and redness of the limb. It can lead to a condition of heaviness and swelling due to the damage to the valves in the veins. However it can also occur in various parts of the body: in the cerebral veins, in the liver (portal vein thrombosis and hepatic vein thrombosis), in the mesenteric vein, in the kidney and in the arms’ veins. It is not yet clear whether thrombophilia increases the risk of arterial thrombosis (which is one of the causes of myocardial infarction).
Deficiency of protein C in infants can cause purpura fulminans, a severe coagulation disorder that leads to tissues’ death and bleedings into the skin and in other organs. This condition has also been reported in adults. Protein C and protein S deficiencies were also associated with increased skin necrosis or with the beginning of treatments with anticoagulants such as warfarin or other similar drugs.
High levels of factor VIII are associated with increased risks for deep vein thrombosis and pulmonary embolism.
Haemophilia A, also known as royal hemophilia, is due to the absence or reduced activity of coagulation factor VIII, which may be entirely absent or have an insufficient activity (the pathological condition occurs when the percentage of activity of the factor VIII is less than about 25%). The gene for factor VIII maps at the end of the long arm of chromosome X. There may be various types of mutations: deletions, insertions, missense mutations and nonsense mutations.
The manifestations classify hemophilia into three types depending on the more or less marked absence of the factor in question:
- Moderate: bleeding joints (hemarthrosis) or early muscle bleeding, severe epistaxis, persistent gengivorragia, persistent hematuria;
- Major: bleeding joints or advanced muscle bleeding, neck, tongue and pharynx hematoma, head trauma without neurological deficiencies, trauma without evident bleedings, severe abdominal pain, gastrointestinal bleeding;
- Very Serious: intracranial bleeding, major trauma with bleeding, surgeries with bleeding, retroperitoneal bleeding.
The relationship between FV Leiden and pulmonary embolism,2002
Hemophilia A,2011
PATHOGENESIS
Cofactors and Regulators
Various substances are required for the proper functioning of the coagulation cascade:
- Calcium and phospholipid (a platelet membrane constituent) are required for the tenase and prothrombinase complexes to function. Calcium mediates the binding of the complexes via the terminal gamma-carboxy residues on FXa and FIXa to the phospholipid surfaces expressed by platelets, as well as procoagulant microparticles or microvesicles shed from them. This process takes place also thanks to FVIII which is used as a cofactor for FIXa in the activation of FX to FXa. Calcium is also required at other points in the coagulation cascade.
- Vitamin K is an essential factor to a hepatic gamma-glutamyl carboxylase that adds a carboxyl group to glutamic acid residues on factors II, VII, IX and X, as well as Protein S, Protein C and Protein Z. In adding the gamma-carboxyl group to glutamate residues on the immature clotting factors Vitamin K is itself oxidized. Another enzyme, Vitamin K epoxide reductase, (VKORC) reduces vitamin K back to its active form. Vitamin K epoxide reductase is pharmacologically important as a target of anticoagulant drugs warfarin and related coumarins such asacenocoumarol, phenprocoumon, and dicumarol. These drugs create a deficiency of reduced vitamin K by blocking VKORC, thereby inhibiting maturation of clotting factors. Vitamin K deficiency from other causes (e.g., in malabsorption) or impaired vitamin K metabolism in disease (e.g., in hepatic failure) lead to the formation of PIVKAs (proteins formed in vitamin K absence) which are partially or totally non-gamma carboxylated, affecting the coagulation factors' ability to bind to phospholipid.
- Protein C is a major physiological anticoagulant. It is a vitamin K-dependent serine protease enzyme that is activated by thrombin into activated protein C (APC). Protein C is activated in a sequence that starts with Protein C and thrombin binding to a cell surface protein thrombomodulin. Thrombomodulin binds these proteins in such a way that it activates Protein C. The activated form, along with protein S and a phospholipid as cofactors, degrades FVa and FVIIIa. Quantitative or qualitative deficiency of either may lead to thrombophilia (a tendency to develop thrombosis). Impaired action of Protein C (activated Protein C resistance), for example by having the "Leiden" variant of Factor V or high levels of FVIII also may lead to a thrombotic tendency.
- Antithrombin is a serine protease inhibitor (serpin) that degrades the serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa. It is constantly active, but its adhesion to these factors is increased by the presence of heparan sulfate (a glycosaminoglycan) or the administration of heparins (different heparinoids increase affinity to FXa, thrombin, or both). Quantitative or qualitative deficiency of antithrombin (inborn or acquired, e.g., in proteinuria) leads to thrombophilia.
Vitamin K Deficiency,2012
DIAGNOSIS
Tests for Blood Clotting Disorders
The following tests may help determine if you have a blood clotting disorder:
• Antiphospholipid Antibody Test. A blood test can reveal the presence of antiphospholipid antibodies. These proteins can cause your blood vessels to function irregularly. They can narrow the blood vessels, making them more susceptible to blockage, or cause blood clots to form.
• Antithrombin Test. This blood test measures the amount of antithrombin. Low levels of this protein increase the risk of a life-threatening blood clot (thrombotic episode).
• Factor V Leiden Mutation. This blood test determines the presence of Factor V Leiden. It may be ordered if you have an unexplained thrombotic episode. It also may be ordered if you suffer a clot in an unusual part of the body, such as the liver or kidneys.
• Lupus Anticoagulant. This blood test verifies the presence of a protein that interferes with the blood-clotting process. It is ordered if you have an unexplained thrombotic episode or if the time it takes your blood to clot is abnormally long.
• Prothrombin 20210. This test is a genetic screening for the presence of a gene mutation that increases the amount of thrombin.
• Protein C and Protein S Tests. These tests measure the amounts of proteins that help to control blood clotting.
Coagulation laboratory test,2012
THERAPY
The therapy for deficiency of coagulation factors treatment varies depending on the severity and the type of symptoms, that is if you have a disorder of excess or defect of coagulation.
For those suffering from increased coagulation are used therapies such as:
• Warfarin
• Heparin
• Low molecular weight Heparin
In cases of hemophilia, treatment involves replacing the coagulation factor that is too low or missing. This is known as replacement therapy, and it can be used to prevent bleeding or to stop bleeding when it occurs. Concentrated coagulation factor that are used in treating this condition may come from donated blood or from lab-produced clotting factors. Other options include a synthetic hormone known as desmopressin and antifibrinolytic drugs.
Aticoagulant therapy
Hemophilia-Treatments and Resources,2007
