Blood and Lymphatic System

Deep Vein Thrombosis

The normal function of a blood clot is to increase wound healing. However, when a clot is formed in a major blood vessel (often in the leg) unrelated to wound healing, it can be a major threat to life. Adventitious clot formation can occur when an individual spends long periods of time without movement, such as when sitting in a car or plane or lying in bed without much movement. Older people are especially prone. There are other factors that may contribute to accidental blood clotting: use of oral contraceptives, surgery, childbirth, massive trauma, burns, or fractures of the hips or femur. These clots (deep vein thrombosis, DVT) usually occur in vessels that carry blood to the heart. In 1 year, these types of clots will occur in one person in a thousand. There have been reports of 100,000–300,000 deaths per year in the United States and an incidence of DVT overall of up to 900,000 per year. Clots in a deep vein can break apart allowing parts of the clot ( embolus ) to travel in the bloodstream, coming to rest in the lungs or the heart. Clots can occur in the legs, hips, pelvis (iliac or femoral vein), or arms (subclavian vein). The deep veins are shown in Fig. 21.1 .

In general, the site of a clot can be detected by Doppler ultrasound scanning . Sometimes, clots can occur without swelling or pain and pulmonary emboli can be detected by magnetic resonance scanning , although it may be difficult to diagnose a clot of this type when there are no symptoms except shortness of breath on climbing stairs and weakness due to compromise of the major circulation. Pulmonary emboli may interfere with the flow of blood into the lungs and vessels in the heart can be blocked, sometimes resulting in a heart attack and death. A clot may travel beyond the lungs and the heart to the brain and cause a stroke. Hypercoagulability of the blood comes into question following a blood clot and, if it exists, hypercoagulability can result from various factors. Characteristics of deep vein clots are shown in Fig. 21.2 .

Proteins that are inhibitors of clotting may be underexpressed. Protein C is one such protein and protein S is a cofactor of protein C . Antithrombin III could also be limiting. If the levels of these proteins are low in blood, this could be the cause of hypercoagulability . There are about 2 million persons each year, globally, who have been found to be hypercoagulable. In the case of a patient with cancer, there could be adventitious blood clotting, especially if the tumor interferes with blood flow. Usually, heparin is injected to thin the blood; it has a short half-life of about 1–2 hours. Then increasing oral doses of a blood thinner, usually Coumadin ( warfarin ), is taken until the proper zone of prothrombin time of clotting is reached; this is expressed as an international normalized ratio ( INR ) and the appropriate target is usually an INR of 2.5 in an acceptable range of 2–3. The amount of oral Coumadin varies from person to person and is the result of many variables. Usually, blood samples are drawn monthly, after a patient has been stabilized, to determine that the clotting time is appropriate. In many cases the blood thinner must be taken for life; in other cases where hypercoagulability is not a problem, the Coumadin regimen may be terminated. As anticoagulation therapy extends the clotting time, excessive bleeding may become a threat. With long-term anticoagulant therapy, there is a 3% chance of a major hemorrhage in a year and 20% of these hemorrhages are fatal. If a surgical or dental procedure that may incur bleeding is needed for a patient on Coumadin, the drug can be stopped for 4–5 days prior to the procedure during which time the level of the anticoagulant in blood will have been cleared and the anticoagulant can be resumed after the procedure has been completed without danger.

Factor V Leiden

One inherited or acquired trait that predisposes to venous clots is a deficiency of the physiological anticoagulant protein C . This is a rare inherited trait that predisposes to venous clots and habitual spontaneous abortions. The most common hereditary form of hypercoagulation is the expression of factor V Leiden. Factor V Leiden is an inherited gene encoding a variant of factor V and expression of factor V Leiden makes blood more likely to clot ( thrombophilia ). Acquired forms involve elevated concentrations of factor VIII . Protein C cleaves and inactivates factor Va (activated factor V) and factor VIIIa. The activation of protein C results from the activation of thrombomodulin by thrombin . Activated protein C ( APC ) then combines with its cofactor, protein S and the complex binds to a platelet membrane that contains a receptor for APC. Once APC is on the platelet membrane, it cleaves and inactivates factor Va and factor VIIIa. The activated altered factor V (Leiden) is resistant to inactivation by protein C . The majority of patients with APC resistance express factor V Leiden . Fig. 21.3 shows the blood-clotting process and the consequence of the expression of factor V Leiden.

The use of oral contraceptives increases the chance of venous thrombosis over a normal person not using oral contraceptives by fourfold. These and other conditions are listed in Table 21.1 .

There are also cases of idiopathic (without known cause) hypercoagulation that are not related to expression of factor V Leiden, to deficiencies of protein C or protein S or to other measurable factors in the blood coagulation system. In these unusual cases a factor could be identified in the future.

Protein C is a vitamin K–dependent protease that is activated by thrombin to form APC . It has similarities to other serine proteases. The receptor for APC is the endothelial protein C receptor ( EPCR ) that recognizes the γ-carboxylglutamate (Gla) domain and a phospholipid domain of protein C. The EPCR accelerates the thrombin- and thrombomodulin-dependent generation of APC. EPCR binds both protein C and APC with equal affinity. In addition to the inactivation of factors Va and VIIIa (together with thrombomodulin), APC inhibits inflammation ( Fig. 21.4 ).

The cofactor of protein C is protein S . Fig. 21.5 shows an illustration of the structural domains of protein S, protein C, thrombin, and thrombomodulin.

Blood-Clotting Mechanism

The blood-clotting system consists of a series of precursor proteins that become activated by hydrolysis through a series of proteases. A serine protease becomes activated and then activates the next protein in the cascade and this process continues until fibrinogen becomes activated forming the long and stringy fibrin that forms a clot. The formation of a blood clot is schematically illustrated in Fig. 21.6A and a scanning electron micrograph of a clot formed in a blood vessel is shown in Fig. 21.6B .

Some of the proteins in the clotting cascade are named for the original investigators of those proteins. For example, factor XII is also the Hageman factor , factor IX is also the Christmas factor , and factor X is also the Stuart factor . The blood-clotting cascade is shown in Fig. 21.7 .

Two types of events can signal the clotting pathway. In the case of a deep vein thrombus initiated by an undetermined cause within the body, the intrinsic pathway of blood-clotting is activated, whereas signals from outside the body such as trauma trigger the extrinsic pathway of blood clotting. These pathways are shown in Fig. 21.8 .

At the bottom of both pathways, fibrinogen (about 3% of the blood proteins) is soluble and in its center, it has a sticky region covered by short amino acid chains that are negatively charged. Thrombin , activated from prothrombin by factor X , is a serine protease that hydrolyzes the short charged amino acid chains to expose the sticky region and forms fibrin . Fibrin molecules, so formed, stick together to form the clot. In the normal situation, clots do not form adventitiously because of the presence of α -1-antitrypsin , a serine protease inhibitor produced by the tissues, and another inhibitor, antithrombin , that blocks the action of thrombin. In the normal situation, when a clot is to be dissolved in the process of wound healing, the protease, plasmin , a blood enzyme, is released by the activation of plasminogen, and it breaks down fibrin and dissolves the blood clot.

A deficiency of factor VIII , the antihemophilic factor , leads to a familial bleeding tendency in males. This disease is hemophilia and it affects about 1 in 5000 males. Factor VIII is the cofactor of activated factor IX in the factor X-activating complex in the blood-clotting intrinsic pathway ( Figs. 21.7 and 21.8 ). The formation, life cycle, and degradation of factor VIII are shown in Fig. 21.9 .

Blood

Blood in the circulatory system transports oxygen to the tissues and carbon dioxide back from the tissues for expiration in the lungs. In addition to the population of cells in the blood, it carries hormones, enzymes, various nutrients, and plasma proteins. To maintain a critical pH range of 6.8–7.4, it contains a pH buffering system. Blood carries water and toxic urea to the kidneys for clearance. The nutrients in blood are amino acids, glucose, vitamins, minerals, fatty acids, and glycerol. The total volume of blood is about 5 L, and it circulates through the kidneys to remove toxins from the body into the urine. A total of 45% of the blood volume is occupied by cells of which 99% are red blood cells (erythrocytes) and the remaining 1% is made up of leukocytes and platelets. The noncell volume (55%) is plasma that is 92% water and contains various ions (sodium, chlorine potassium, manganese, and calcium) and blood plasma proteins [albumin, globulins, fibrinogen, and various hormones (proteins, peptides, and steroids)]. A diagram of the human systemic circulation is shown in Fig. 21.10 . In Table 21.2 the major blood components are listed along with their sites of production and their major functions. Blood is oxygenated by the lungs and enters the left auricle through the pulmonary vein and then it is pumped to the left ventricle of the heart and then out through the aorta (reverse arrow in Fig. 21.10 ). The aorta is the major artery leading away from the heart to the liver by way of the hepatic artery , to the small intestine by way of the mesenteric artery , to the kidneys by way of the renal artery , and to the legs by way of the iliac artery . As for the upper body, oxygenated blood is carried to the arms through the subclavian artery and to the head by the carotid artery . After the tissues have been oxygenated, the deoxygenated blood travels back to the right ventricle of the heart from the legs via the iliac vein , from the kidneys via the renal vein , and from the small intestine via hepatic portal vein to the liver. The liver empties the deoxygenated blood into the hepatic vein to the right ventricle of the heart via the inferior vena cava . Deoxygenated blood from the head and the arms via the subclavian vein flows into the superior vena cava to the right ventricle of the heart. The deoxygenated blood is then pumped from the heart into the lungs through the pulmonary artery and in the lungs CO ₂ from the blood is expired and the newly oxygenated blood finally enters the aorta and is recycled. In 1 day the heart beats about 100,000 times and approximately 5 L of blood circulate three times each minute so that in 1 day the blood travels a total of 12,000 mi.

Transport of Oxygen

Hemoglobin in red blood cells is the oxygen transporter in blood. The counterpart to hemoglobin in muscle is myoglobin , a small globular monomeric protein, like hemoglobin (heterodimeric tetramer), that binds oxygen. The kinetics of oxygen binding by the two proteins is different, as shown in Fig. 21.11 .

Myoglobin is a monomeric globulin (binds one heme) and binds oxygen in a linear fashion, whereas hemoglobin is a tetramer (two α-globins+two β-globins+four hemes) ( Fig. 21.12 ) and displays allosteric kinetics in which an initial lag occurs as the oxygen concentration is increased.

When about 10 Torr (10 mmHg) of oxygen pressure is reached, the rate of oxygen binding increases until saturation is reached. The iron in heme must be in the ferrous state (Fe ²⁺ ) to bind oxygen. The allosteric kinetics of oxygen binding to hemoglobin can be explained in the following way. The first molecule of the oxygen ligand binds with a low affinity; however, the binding of oxygen alters the conformation of hemoglobin such that the succeeding molecules of oxygen bind with increased affinity reflecting a conformational change in the other hemoglobin subunits. Therefore there could be two forms of hemoglobin, one with low affinity for oxygen and the other with higher affinity for oxygen. In the higher affinity conformation of hemoglobin, oxygen would bind with high affinity to all four subunits. Fig. 21.13 shows two possible explanations. In the symmetry model the two forms of hemoglobin exist in equilibrium and the oxygen ligands all bind to the four subunits with equally high affinity to the high-affinity conformation. In the sequential model the first oxygen ligand binds to one subunit and alters its conformation to the high-affinity form. The succeeding oxygen molecules perform similarly until the four hemoglobin subunits are all in the high-affinity conformation. The purpose of cooperativity (allosterism) between the four oxygen-binding sites is that 1.7 times as much oxygen can be delivered to the tissues compared to a protein in which the oxygen-binding sites were independent of each other.

The differences between the three-dimensional structures of the fully oxygenated form ( R form ; for “relaxed”) of hemoglobin compared to deoxyhemoglobin ( T form ; for “taut”) have been solved. This difference results from a rotation of about 15° between the two α–β dimers (α-globin dimer and β-globin dimer). This rotation alters the bonds between the side chains of the two dimers, causing the heme molecule to alter its position. In deoxyhemoglobin (T structure) the iron atom is displaced from the plane of the porphyrin ring. This movement of the iron atom makes it harder for oxygen to bind to it and, thus, the affinity of oxygen for the T form is reduced. Conversely, in the R form, the iron atom is directly in the plane of the porphyrin ring and, in this position, is more able to bind oxygen; thus the R form has an increased affinity for oxygen. The high oxygen pressure of the environment in the lungs causes the shift from the T to the R form. In the low oxygen environment of the tissues, on the other hand, the transformation of the R form to the T form occurs. Hemoglobin, therefore, is able to adjust its structure in the presence or relative absence of oxygen . This change of state is visualized in Fig. 21.14 .

The situation in uncontrolled diabetes , for example, can produce another alteration of the hemoglobin molecule. In this case, blood glucose circulates at a high level and, as a result, hemoglobin can become glycosylated . Residues of glucose bind to amino acid residues of hemoglobin. Elevated levels of glycated hemoglobin (HbA1c) are diagnostic of diabetes. Levels above 10% of hemoglobin, HbA0, that are glycated are indicative of poor metabolic control of carbohydrates and the level of HbA1c can be used to monitor treatment. In the situation where their high glucose levels are maintained in blood, other proteins as well as hemoglobin can be glycated, including insulin . Normally, there may be some glycated insulin in blood but in diabetes the level of glycated insulin rises by two to threefold. This alteration may affect 10% of the circulating insulin. In the glucose tolerance test where insulin lowers the level of blood glucose over time, glycated insulin has this activity but decreased by a factor of 20%–40%. However, glycated insulin binds to the insulin receptor normally, so that its negative effect may occur at some step other than receptor binding. Elevated levels of HbA1c can indicate kidney problems, as well. Experimentally, hemoglobin is highly sensitive to damage by glucose that can result in destruction of the heme and the release of the iron atom. Hemoglobin is primarily glycated on valine and lysine residues in vivo. A representation of the reaction of glucose with the terminal amino group of a lysine residue (of hemoglobin) is shown in Fig. 21.15 .

HbA1c demonstrates an altered oxygen saturation curve, indicating that glycation can modify the conversion of oxyhemoglobin to deoxyhemoglobin and HbA1c has a greater affinity for oxygen than HbA0 and a decreased tendency to dissociate oxygen. This is represented by a shift in the oxygenation curve ( Fig. 21.11 ) to the left. The result is that the affinity of HbA1c for oxygen is increased as represented by tighter binding and consequently, there is a decrease in the delivery of oxygen to the tissues, all of which increases the risk of coronary artery disease in diabetics and overall poor outcome. Normally, red blood cells have the capacity to deglycosylate proteins but this activity is overrun by glucose when it is at sustained elevated levels in the circulation.

In addition, there are several possible genetic mutations in the hemoglobin molecule that result in serious consequences ( Table 21.3 ).

Glycation of Proteins

Carboxymethyl lysine (CML) is only one of several possible advanced glycation end products (AGEs). Glycation of proteins can occur nonenzymatically and the full range of these products is listed in Table 21.4 . CML is indicated at the bottom of the table under monolysyl adducts. AGEs are known to be increased in diabetes (e.g., diabetic retinopathy), renal failure (e.g., glomerulosclerosis), and aging. Binding of AGEs to their receptor ( r eceptor of AGEs) on the surface of endothelial cells (cells that line ducts or openings) causes the expression of proteins, the actions of which result in increased permeability between endothelial cells ( Fig. 21.16A ). Among these proteins are interleukin-6 and monocyte chemoattractant protein-1 that are cytokines.

Table 21.4

Advanced Glycation End Products.

Source: Reproduced from Wautier, J.-C., Schmidt, A.M., 2004. Protein glycation—a firm link to endothelial cell dysfunction. Circ. Res. 95, 233–238. https://doi.org/10.1161/01.RES.0000137876.28454.64 .

AGE Products
Bis(lysyl)imidazolium cross-links
GOLD (glyoxal-derived lysine dimer)
MOLD (methylglyoxal-derived lysine dimer)
DOLD (3-deoxy-glucosone-derived lysine dimer)
Hydroimidazolones
G-H (glyoxal-derived hydroimidazolone)
MG-H (methylglyoxal hydroimidazolone)
3DG-H (3-deoxy-glucosone hydroimidazolone)
Monolysyl adducts
N ε -carboxymethyl-lysine (CML)
N ε -carboxyethyl-lysine (CEL)
Pyrraline

In the case of HbA1c in diabetes, the end product is attached to the β-chain of hemoglobin (through a Schiff base that is formed nonenzymatically through an Amadori rearrangement ) as 1-deoxy-1-fructosyl residue of the N-terminal valine amino group. This sugar occurs through a rearrangement of the Schiff base adduct of glucose with an N-terminal lysine residue to form fructosamine that can degrade. A high level of HbA1c in blood is characteristic of diabetes. This HbA1c causes an increase in reactive free radicals within the red blood cell that can lead to blood cell aggregation and an increase in blood viscosity generating impaired blood flow . Proteins also can interact with oxidation products and these can arise from dysfunction in the mitochondria, the site of β-oxidation of fatty acids. Enzyme proteins that interact with AGEs may be inactivated.

The glycation of serum albumin is important in this context. Human serum albumin (HSA) has many sites on its surface that can be glycated as shown in Fig. 21.16B . There are multiple glycation sites identified on HSA, including Lys, Arg, and Cys residues. Amino groups on the surface of the protein serve as the glycation sites and these are most commonly Lys residues. N-terminal Lys residues are glycated nonenzymatically. Nonenzymatic glycation represents 30% of the total glycation of HSA in vivo. Glycation of Lys199 represents 5% of the total (see Fig. 21.16B ). Glycation of HSA affects its ability to bind fatty acids that causes enhanced hyperactivation of platelets and the oxidation of arachidonate, which are characteristics in type 2 diabetes.

Low-density lipoproteins can be glycated. This result in the appearance of neo-epitopes (new antigenic sites) developed that evoke an autoimmune response .