The blood is the body’s main transport system. Although the transport and delivery of oxygen to the cells of the tissues is carried out by specialized cells, other vital components such as nutrients, metabolites, electrolytes, and hormones are all carried in the noncellular fraction of the blood, the plasma. Some components, such as glucose, are dissolved in the plasma; others, for example, lipids and steroid hormones, are bound to carrier proteins for transport. The osmotic pressure exerted by the plasma proteins regulates the distribution of water between the blood and the tissues. Plasma proteins in conjunction with platelets maintain the integrity of the circulatory system through the process of clotting.
Blood normally circulates through endothelium-lined blood vessels. When a blood vessel is severed, a blood clot (called a thrombus, which is formed by the process of thrombosis) forms as part of hemostasis, the physiological response that stops bleeding. Blood clots also form to repair damage to the endothelial lining, in which components of the subendothelial layer become accessible to plasma proteins.
In hemostasis and thrombosis, a primary hemostatic plug, consisting of aggregated platelets and a fibrin clot, forms at the site of injury. Platelets are attached to the subendothelial layer of the vessel principally through the protein von Willebrand factor and are activated to bind fibrinogen. Fibrinogen binds the platelets to each other to form a platelet aggregate. The platelet aggregate is rapidly covered with layers of fibrin, which are formed from fibrinogen by the proteolytic enzyme thrombin.
Injury to the endothelium and exposure of tissue factor on the subendothelial layer to plasma proteins also activate the blood coagulation cascade, which ultimately activates thrombin and factor XIII. Factor XIII cross-links strands of polymerized fibrin monomers to form a stable clot (the secondary hemostatic plug). The blood coagulation cascade consists of a series of enzymes (such as factor X), which are inactive until they are proteolytically cleaved by the preceding enzyme in the cascade. Other proteins (factor V and factor VIII) serve as binding proteins, which assemble factor complexes at the site of injury. Ca2+ and γ-carboxyglutamate residues in the proteins (formed by a vitamin K–dependent process in the liver) attach the factor complexes to phospholipids exposed on platelet membranes. Consequently, thrombus formation is rapidly accelerated and localized to the site of injury.
Regulatory mechanisms within the blood coagulation cascade and antifibrinolytic mechanisms prevent random coagulation within blood vessels that might obstruct blood flow. Impairments in these mechanisms lead to thrombosis.
There are two basic assays for measuring the activity of factor VIII in a blood sample. The first is a functional (clotting) assay; the second is a coupled (to factor X activation) enzyme system leading to the cleavage of a chromogenic substrate, generating a colored product.
For the functional assay, a sample of the patient’s plasma is added to a factor VIII-deficient sample of plasma (which can be obtained commercially), and the time to generate clot formation is determined. Although clot formation is the end product of this assay, one is measuring the eventual activation of factor II to IIa, which allows clot formation to initiate.
In the automated, chromogenic assay, a sample of the unknown is mixed with purified factor IXa, Ca2+, phospholipid vesicles, the chromogenic substrate, and purified factor X. If the unknown sample contains factor VIII, the factors VIII and IXa will synergize and activate factor X to Xa. The factor Xa will cleave the chromogenic substrate, forming a colored product, whose concentration can be determined spectrophotometrically. Comparison to a standard curve of factor VIII addition enables an accurate estimate of the level of active factor VIII in the sample.
THE WAITING ROOM 
Peter K., a 6-month-old male infant, was brought to his pediatrician’s office with a painful, expanding mass in his right upper thigh that was first noted just hours after he fell down three uncarpeted steps in his home. The child appeared to be in severe distress.
A radiograph showed no fractures, but a soft tissue swelling, consistent with a hematoma (bleeding into the tissues), was noted. Peter K.’s mother related that soon after he began to crawl, his knees occasionally became swollen and seemed painful.
The pediatrician suspected a disorder of coagulation. A screening coagulation profile suggested a possible deficiency of factor VIII, a protein involved in the formation of blood clots. Peter K.’s plasma factor VIII level was found to be only 3% of the average level found in normal subjects. A diagnosis of hemophilia A was made.
I. Plasma Proteins Maintain Proper Distribution of Water between Blood and Tissues
When cells are removed from the blood, the remaining plasma is composed of water, nutrients, metabolites, hormones, electrolytes, and proteins. Plasma has essentially the same electrolyte composition as other extracellular fluids and constitutes approximately 25% of the body’s total extracellular fluid. The plasma proteins serve a number of functions, including maintaining the proper distribution of water between the blood and the tissues, transporting nutrients, metabolites, and hormones throughout the body, defending against infection, and maintaining the integrity of the circulation through clotting. Many diseases alter the amounts of plasma proteins produced and hence their concentration in the blood. These changes can be determined by electrophoresis of plasma proteins over the course of a disease.
In cases of severe protein malnutrition (kwashiorkor), the concentration of the plasma proteins decreases, as a result of which the osmotic pressure of the blood decreases. As a result, fluid is not drawn back to the blood and instead accumulates in the interstitial space (edema). The distended bellies of famine victims are the result of fluid accumulation in the extravascular tissues because of the severely decreased concentration of plasma proteins, particularly albumin. Albumin synthesis decreases fairly early under conditions of protein malnutrition.
A. Body Fluid Maintenance between Tissues and Blood
As the arterial blood enters the capillaries, fluid moves from the intravascular space into the interstitial space (that surrounding the capillaries) due to Starling’s forces. The hydrostatic pressure in the arteriolar end of the capillaries (~37 mm Hg) exceeds the sum of the tissue pressure (~1 mm Hg) and the osmotic pressure of the plasma proteins (~25 mm Hg). Thus, water tends to leave the capillaries and enter extravascular spaces. At the venous end of the capillaries, the hydrostatic pressure falls to approximately 17 mm Hg while the osmotic pressure and the tissue pressure remain constant, resulting in movement of fluid back from the extravascular (interstitial) spaces and into the blood. Thus, most of the force bringing water back from the tissues into the plasma is the osmotic pressure mediated by the presence of proteins in the plasma.
In spite of the importance of albumin in the maintenance of osmotic pressure in the blood, individuals who lack albumin (analbuminemia) have only moderate edema. Apparently, the concentration of other plasma proteins is increased to compensate for the lack of albumin. The frequency of analbuminemia is <1 per million individuals.
B. The Major Serum Protein, Albumin
As indicated in Table 43.1, the liver produces a number of transport or binding proteins and secretes them into the blood. The major protein synthesized is albumin, which constitutes approximately 60% of the total plasma protein, but because of its relatively small size (69 kDa), it is thought to contribute 70% to 80% of the total osmotic pressure of the plasma. Albumin is a carrier of free fatty acids, calcium, zinc, steroid hormones, copper, and bilirubin. Many drugs also bind to albumin, which may have important pharmacologic implications. For example, when a drug binds to albumin, such binding will likely lower the effective concentration of that drug and may lengthen its lifetime in the circulation. Drug dosimetry may need to be recalculated if a patient’s plasma protein concentration is abnormal.
TABLE 43.1 Specific Plasma-Binding Proteins Synthesized in the Liver
| Albumin | Binds free fatty acids, calcium, zinc, steroid hormones, copper, bilirubin |
| Ceruloplasmin | Binds copper; appears to be more important as a copper storage pool than as a transport protein; integrates iron and copper homeostasis |
| Corticosteroid-binding globulin | Binds cortisol |
| Haptoglobin | Binds extracorpuscular heme |
| Lipoproteins | Transport cholesterol and fatty acids |
| Retinol-binding protein | Binds vitamin A |
| Sex hormone–binding globulin | Binds estradiol and testosterone |
| Transferrin | Transports iron |
| Transthyretin | Binds thyroxine (T4); also forms a complex with retinol-binding protein |
II. The Plasma Contains Proteins That Aid in Immune Defense
Two different sets of proteins aid in the immune response, the immunoglobulins, and the complement proteins. The immunoglobulins are secreted by a subset of differentiated B lymphocytes termed plasma cells and bind antigens at binding sites formed by the hypervariable regions of the proteins (see Chapter 7). Once the antibody–antigen complex is formed, it must be cleared from the circulation. The complement system participates in this function. The complement system consisting of approximately 20 proteins becomes activated in either of two ways. The first is interaction with antigen–antibody complexes, and the second, specific for bacterial infections, is through interaction of bacterial cell polysaccharides with complement protein C3b. Activation of the complement system by either trigger results in a proteolytic activation cascade of the proteins of the complement system, resulting in the release of biologically active peptides or polypeptide fragments. These peptides mediate the inflammatory response, attract phagocytic cells to the area, initiate degranulation of granulocytes, and promote clearance of antigen–antibody complexes.
α1-Antiproteinase (AAP), also known as α1-antitrypsin, is the main serine protease inhibitor of human plasma. Individuals with a point mutation in exon 5 of the AAP gene, which results in a single amino acid substitution in the protein, have diminished secretion of AAP from the liver. These individuals are at increased risk for developing emphysema. When neutrophils degranulate in the lungs as part of the body’s defense against microorganisms, insufficient levels of AAP are present to neutralize the elastase and other proteases released. The excess proteolytic activity damages lung tissue, leading to loss of alveolar elasticity and the development of emphysema.
Methionine 358 of AAP is necessary for AAP binding to the proteases. Oxidation of this methionine destroys AAP’s protease-binding capacity. Chemicals within cigarette smoke lead to the oxidation of Met-358, thereby increasing the risk for emphysema through the inactivation of AAP.
Protease inhibitors in plasma serve to carefully control the inflammatory response. Activated neutrophils release lysosomal proteases from their granules that can attack elastin, the various types of collagen, and other extracellular matrix proteins. The plasma proteins α1-antiproteinase (α1-antitrypsin) and α2-macroglobulin limit proteolytic damage by forming noncovalent complexes with the proteases, thereby inactivating them. However, the product of neutrophil myeloperoxidase, HOCl, inactivates the protease inhibitors, thereby ensuring that the proteases are active at the site of infection.
III. Plasma Proteins Maintain the Integrity of the Circulatory System
Blood is lost from the circulation when the endothelial lining of the blood vessels is damaged, and the vessel wall is partly or wholly severed. When this occurs, the subendothelial cell layer is exposed, consisting of the basement membrane of the endothelial cells and smooth muscle cells. In response to the damage, a barrier (the hemostatic plug, a fibrin clot), initiated by platelet binding to the damaged area, is formed at the site of injury. Regulatory mechanisms limit clot formation to the site of injury and control its size and stability. As the vessel heals, the clot is degraded (fibrinolysis). Plasma proteins are required for these processes to occur.
Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which antibodies to platelet glycoproteins are produced. Antibody binding to platelets results in their clearance by the spleen. An early symptom of the disorder is the appearance of small red spots on the skin (petechial hemorrhages) caused by blood leakage from capillaries. Minor damage to vascular endothelial cells is constantly being caused by mechanical forces related to blood flow. In patients with ITP, few platelets are available to repair the damage.
A. Formation of the Hemostatic Plug
1. The Platelet
Platelets are nonnucleated cells in the blood whose major role is to form mechanical plugs at the site of vessel injury and to secrete regulators of the clotting process and vascular repair. In the absence of platelets, leakage of blood from rents in small vessels is common. Platelets are derived from megakaryocytes in the bone marrow. Megakaryocytes differentiate from the hematopoietic stem cell. As the megakaryocyte matures, it undergoes synchronous nuclear replication without cellular division, to form a cell with a multilobed nucleus and enlarged cytoplasm. At approximately the eight-nucleus stage, the cytoplasm becomes granular, and the platelets are budded off the cytoplasm. A single megakaryocyte gives rise to approximately 4,000 platelets.
In the nonactivated platelet (a platelet that is not involved in forming a clot), the plasma membrane invaginates extensively into the interior of the cell, forming an open membrane (canalicular) system. Because the plasma membrane contains receptors and phospholipids that accelerate the clotting process, the canalicular structure substantially increases the membrane surface area that is potentially available for clotting reactions. The platelet interior contains microfilaments and an extensive actin/myosin system. Platelet activation in response to endothelial injury causes Ca2+-dependent changes in the contractile elements, which, in turn, substantially change the architecture of the plasma membrane. Long pseudopodia are generated, increasing the surface area of the membrane as clot formation is initiated.
Platelets contain three types of granules. The first is electron-dense granules, which contain calcium, adenosine diphosphate (ADP), adenosine triphosphate (ATP), and serotonin. The second type of granule is the α-granule, which contains a heparin antagonist (heparin interferes with blood clotting; see “Biochemical Comments”), platelet-derived growth factor, β-thromboglobulin, fibrinogen, von Willebrand factor (vWF), and other clotting factors. The third type of granule is the lysosomal granule, which contains hydrolytic enzymes. During activation, the contents of these granules, which modulate platelet aggregation and clotting, are secreted.
2. Platelet Activation
Three fundamental mechanisms are involved in platelet function during blood coagulation: adhesion, aggregation, and secretion. Adhesion sets off a series of reactions termed platelet activation, which leads to platelet aggregation and secretion of platelet granule contents.
Von Willebrand factor is a large multimeric glycoprotein with a subunit molecular weight of 220,000 Da. Its size in the circulation ranges between 500 and 20,000 kDa, and its role in circulation is to stabilize factor VIII and protect it from degradation. The HMW forms are concentrated in the endothelium of blood vessels and are released in response to stress hormones and endothelial damage. HMW vWF released by the endothelium is cleaved by a specific metalloprotease in the serum, reducing the size of the circulating vWF. Large vWF multimers are more effective at forming complexes with platelets than are small vWF multimers.
vWF deficiency is the most common cause of inherited bleeding disorders. Both platelet adherence and the clotting cascade are affected because levels of factor VIII are low. In the absence of vWF, factor VIII is rapidly cleared from the system. The vWF gene is large, covering approximately 180 kb, and contains 52 exons. Multiple mutations are known, with varying clinical presentations.
The adhesion step refers primarily to the platelet-subendothelial interaction that occurs when platelets initially adhere to the sites of blood vessel injury (Fig. 43.1). Blood vessel injury exposes collagen, subendothelial matrix-bound vWF, and other matrix components. vWF is a protein synthesized in endothelial cells and megakaryocytes, and it is located in the subendothelial matrix, in specific platelet granules, and in the circulation bound to factor VIII. The platelet cell membrane contains glycoproteins (GPs) that bind to collagen and to vWF, causing the platelet to adhere to the subendothelium. Binding to collagen by GPIa (integrin α2β1) causes the platelet to change its shape from a flat disk to a spherical cell. The latter extrudes long pseudopods, which promote platelet/platelet interactions. Binding of subendothelial vWF by GPIb causes changes in the platelet membrane that expose GPIIb/IIIa (integrin αIIbβ3)-binding sites to fibrinogen and vWF.
FIGURE 43.1 Adhesion of platelets to the subendothelial cell layer. (1) GPIa initially binds to the exposed collagen, which results in changes in the three-dimensional configuration of the complex, allowing GPIb to bind to von Willebrand factor (Vwf) (2). (3) This second binding event exposes the GPIIb/GPIIIa complex, which also can bind to vWF and fibrinogen.
Mutations in GPIb cause a bleeding disorder known as Bernard–Soulier syndrome. Platelet aggregation is affected because of the inability of GPIb to adhere to subendothelial vWF.
Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by the formation in the circulation of microclots (microthrombi) consisting of aggregated platelets. The microthrombi collect in the microvasculature and damage red blood cells, resulting in hemolytic anemia. They also damage vascular endothelium, exposing collagen and releasing HMW vWF, promoting more platelet aggregation. The subsequent depletion of platelets renders the patient susceptible to internal hemorrhage. Mortality in untreated TTP can approach 90%.
Familial TTP, which is a less common cause of TTP, is associated with mutations in the vWF-specific metalloprotease, although not all individuals with defective protease develop TTP. Sporadic cases of TTP are associated with the development of an antibody to the metalloprotease.
The initial adherence of platelets sets off a series of reactions (platelet activation) that results in more platelets being recruited and aggregated at the site of injury. After initial adherence, some of the platelets release the contents of their dense granules and their α-granules, with ADP release being of particular importance because ADP is a potent platelet activator. ADP released from the platelets and from damaged red blood cells binds to a receptor on the platelet membrane, which leads to the further unmasking of GPIIb/IIIa-binding sites. Aggregation of platelets cannot take place without ADP stimulation because ADP induces swelling of the activated platelets, promoting platelet/platelet contact, and adherence.
Fibrinogen is a protein that circulates in the blood and is also found in platelet granules. It consists of two triple helices held together with disulfide bonds (Fig. 43.2). Binding of fibrinogen to activated platelets is necessary for aggregation, providing, in part, the mechanism by which platelets adhere to one another. Cleavage of fibrinogen by thrombin (a protease that is activated by the coagulation cascade) produces fibrin monomers that polymerize and, together with platelets, form a “soft clot.” Thrombin itself is a potent activator of platelets, through binding to a specific receptor on the platelet surface.
FIGURE 43.2 Cleavage of fibrinogen results in clot formation. A. Fibrinogen, the precursor protein of fibrin, is formed from two triple helices joined together at their N-terminal ends. The α,β-peptides are held together by disulfide bonds, and the γ-peptides are joined to each other by disulfide bonds. The terminal α,β-peptide regions, shown in red, contain negatively charged glutamate and aspartate residues that repel each other and prevent aggregation. B. Thrombin, a serine protease, cleaves the terminal portions of fibrinogen that contain negative charges. The fibrin monomers can then aggregate and form a “soft” clot. The soft clot is subsequently cross-linked by another enzyme.
B. The Blood Coagulation Cascade
Thrombus (clot) formation is enhanced by thrombin activation, which is mediated by the complex interaction that constitutes the blood coagulation cascade. This cascade (Fig. 43.3) consists primarily of proteins that serve as enzymes or cofactors, which function to accelerate thrombin formation and localize it at the site of injury. These proteins are listed in Table 43.2. All of these proteins are present in the plasma as proproteins (zymogens). These precursor proteins are activated by cleavage of the polypeptide chain at one or more sites. The key to successful and appropriate thrombus formation is the regulation of the proteases that activate these zymogens.
TABLE 43.2 Proteins of Blood Coagulation
| FACTOR | DESCRIPTIVE NAME | FUNCTION/ACTIVE FORM |
| Coagulation Factors | ||
| I | Fibrinogen | Fibrin |
| II | Prothrombin | Serine protease |
| III | Tissue factor | Receptor and cofactor |
| IV | Ca2+ | Cofactor |
| V | Proaccelerin, labile factor | Cofactor |
| VII | Proconvertin | Serine protease |
| VIII | Antihemophilia factor A | Cofactor |
| IX | Antihemophilia factor B, Christmas factor | Serine protease |
| X | Stuart–Prower factor | Serine protease |
| XI | Plasma thromboplastin antecedent | Serine protease |
| XIII | Fibrin-stabilizing factor | Ca2+-dependent transglutaminase |
| Regulatory Proteins | ||
| Thrombomodulin | Endothelial cell receptor binds thrombin | |
| Proteins C and S | Activated by thrombomodulin-bound thrombin; is a serine protease cofactor; binds activated protein C | |
FIGURE 43.3 The blood coagulation cascade. Activation of clot formation occurs through interlocking pathways, termed the intrinsic and extrinsic pathways. The intrinsic pathway is activated when factor XI is converted to factor XIa by thrombin. The extrinsic pathway (external damage, such as a cut) is activated by tissue factor. The reactions designated by “PL, Ca” are occurring through cofactors bound to phospholipids (PL) on the platelet and blood vessel endothelial cell surface in a Ca2+-coordination complex. Factors XIa, IXa, VIIa, Xa, and thrombin are serine proteases. Note the positive feedback regulation of thrombin on the activation of proteases earlier in the cascade sequence (indicated by the dashed lines).
The proenzymes (factors VII, XI, IX, X, and prothrombin) are serine proteases that, when activated by cleavage, cleave the next proenzyme in the cascade. Because of the sequential activation, great acceleration and amplification of the response are achieved. That cleavage and activation have occurred are indicated by the addition of an “a” to the name of the proenzyme (e.g., factor IX is cleaved to form the active factor IXa).
The cofactor proteins (tissue factor, factors V and VIII) serve as binding sites for other factors. Tissue factor is not related structurally to the other blood coagulation cofactors and is an integral membrane protein that does not require cleavage for active function. Factors V and VIII serve as procofactors, which, when activated by cleavage, function as binding sites for other factors.
Two additional proteins that are considered part of the blood coagulation cascade, protein S and protein C, are regulatory proteins. Only protein C is regulated by proteolytic cleavage, and when it is activated, it is itself a serine protease.
Additionally, in response to collagen and thrombin, platelets release vasoconstrictors. Serotonin is released from the dense granules of the platelets, and the synthesis of thromboxane A2 is stimulated. This reduces blood flow to the damaged area. Platelet-derived growth factor, which stimulates proliferation of vascular cells, is also released into the environment surrounding the damage.
The utilization of an active-site serine to cleave a peptide bond is common to a variety of enzymes referred to as serine proteases. Serine proteases are essential for activating the formation of a blood clot from fibrin. Fibrin and many of the other proteins involved in blood coagulation are present in the blood as inactive precursors or zymogens, which must be activated by proteolytic cleavage. Thrombin, the serine protease that converts fibrinogen to fibrin, has the same aspartate–histidine–serine catalytic triad found in chymotrypsin and trypsin.
Thrombin is activated by proteolytic cleavage of its precursor protein, prothrombin. The sequence of proteolytic cleavages leading to thrombin activation requires factor VIII, the blood clotting protein that is deficient in Peter K.
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