Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 37. Normal hemostasis and coagulation

Margaret G. Fritsma, George A. Fritsma

OUTLINE

Overview of Hemostasis

Vascular Intima in Hemostasis

Anticoagulant Properties of Intact Vascular Intima

Procoagulant Properties of Damaged Vascular Intima

Fibrinolytic Properties of Vascular Intima

Platelets

Coagulation System

Nomenclature of Procoagulants

Classification and Function of Procoagulants

Plasma-Based (In Vitro) Coagulation: Extrinsic, Intrinsic, and Common Pathways

Cell-Based (In Vivo, Physiologic) Coagulation

Coagulation Regulatory Mechanisms

Tissue Factor Pathway Inhibitor

Protein C Regulatory System

Antithrombin and Other Serine Protease Inhibitors (Serpins)

Fibrinolysis

Plasminogen and Plasmin

Plasminogen Activation

Control of Fibrinolysis

Fibrin Degradation Products and D-Dimer

Objectives

After completion of this chapter, the reader will be able to:

1. List the systems that interact to provide hemostasis.

2. Describe the properties of the vascular intima in the initiation and regulation of hemostasis and fibrinolysis.

3. List the hemostatic functions of tissue factor-bearing cells and blood cells, especially platelets, in hemostasis.

4. Describe the relationships among platelet function, von Willebrand factor, and fibrinogen, and their impact on hemostasis.

5. Describe the nature, origin, and function of each of the tissue and plasma factors necessary for normal coagulation.

6. Explain the role of vitamin K in the production and function of the prothrombin group of plasma clotting factors.

7. Distinguish between coagulation pathway serine proteases and cofactors.

8. Describe six roles of thrombin in hemostasis.

9. Diagram fibrinogen structure, fibrin formation, fibrin polymerization, and fibrin cross-linking.

10. For each coagulation complex—extrinsic tenase, intrinsic tenase, and prothrombinase—identify the serine protease and the cofactor forming the complex, the type of cell involved, and the substrate(s) activated.

11. List the factors in order of reaction in the plasma-based extrinsic, intrinsic, and common pathways.

12. Describe the cell-based in vivo coagulation process and the role of tissue factor–bearing cells and platelets.

13. Show how tissue factor pathway inhibitor, the protein C pathway, and the serine protease inhibitor antithrombin function to regulate coagulation and prevent thrombosis.

14. Describe the fibrinolytic pathway, its regulators, and its products.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A pregnant woman developed a blood clot in her left leg (deep vein thrombosis, or DVT). Her mother reportedly had a history of thrombophlebitis. She had a brother who was diagnosed with a DVT following a flight from Los Angeles to Sydney, Australia.

1. Is this hemostatic disorder typical of an acquired or an inherited condition?

2. Are these symptoms most likely caused by a deficiency of a procoagulant or an inhibitor?

Hemostasis is a complex physiologic process that keeps circulating blood in a fluid state and then, when an injury occurs, produces a clot to stop the bleeding, confines the clot to the site of injury, and finally dissolves the clot as the wound heals. When hemostasis systems are out of balance, hemorrhage (bleeding) or thrombosis (pathological clotting) can be life-threatening. The absence of a single plasma procoagulant may destine the individual to lifelong anatomic hemorrhage, chronic inflammation, and transfusion dependence. Conversely, absence of a control protein allows coagulation to proceed unchecked and results in thrombosis, stroke, pulmonary embolism, deep vein thrombosis, and cardiovascular events.

Understanding the major systems of hemostasis—blood vessels, platelets, and plasma proteins—is essential to interpreting laboratory test results and to prevent, predict, diagnose, and manage hemostatic disease.

Overview of hemostasis

Hemostasis involves the interaction of vasoconstriction, platelet adhesion and aggregation, and coagulation enzyme activation to stop bleeding. The coagulation system, similar to other humoral amplification mechanisms, is complex because it translates a diminutive physical or chemical stimulus into a profound lifesaving event.1 The key cellular elements of hemostasis are the cells of the vascular intima, extravascular tissue factor (TF)–bearing cells, and platelets. The plasma components are the coagulation and fibrinolytic proteins and their inhibitors.

Primary hemostasis (Table 37-1) refers to the role of blood vessels and platelets in response to a vascular injury, or to the commonplace desquamation of dying or damaged endothelial cells. Blood vessels contract to seal the wound or reduce the blood flow (vasoconstriction). Platelets become activated, adhere to the site of injury, secrete the contents of their granules, and aggregate with other platelets to form a platelet plug. Vasoconstriction and platelet plug formation comprise the initial, rapid, short-lived response to vessel damage, but to control major bleeding in the long term, the plug must be reinforced by fibrin. Defects in primary hemostasis such as collagen abnormalities, thrombocytopenia, qualitative platelet disorders, or von Willebrand disease can cause debilitating, sometimes fatal, chronic hemorrhage.

TABLE 37-1

Primary and Secondary Hemostasis

Primary Hemostasis

Secondary Hemostasis

Activated by desquamation and small injuries to blood vessels

Activated by large injuries to blood vessels and surrounding tissues

Involves vascular intima and platelets

Involves platelets and coagulation system

Rapid, short-lived response

Delayed, long-term response

Procoagulant substances exposed or released by damaged or activated endothelial cells

The activator, tissue factor, is exposed on cell membranes

Secondary hemostasis (Table 37-1) describes the activation of a series of coagulation proteins in the plasma, mostly serine proteases, to form a fibrin clot. These proteins circulate as inactive zymogens (proenzymes) that become activated during the process of coagulation and, in turn, form complexes that activate other zymogens to ultimately generate thrombin, an enzyme that converts fibrinogen to a localized fibrin clot. The final event of hemostasis is fibrinolysis, the gradual digestion and removal of the fibrin clot as healing occurs.2

Although the vascular intima and platelets are associated with primary hemostasis, and coagulation and fibrinolysis are associated with secondary hemostasis, all systems interact in early- and late-hemostatic events. For example, platelets, although a key component of primary hemostasis, also secrete coagulation factors stored in their granules and provide an essential cell membrane phospholipid on which coagulation complexes form. The remainder of this chapter examines vascular intima, platelets, normal coagulation, coagulation control, and fibrinolysis in detail.

Vascular intima in hemostasis

The vascular intima provides the interface between circulating blood and the body tissues. The innermost lining of blood vessels is a monolayer of metabolically active endothelial cells (EC) (Box 37-1Figure 37-1).3Endothelial cells are complex and heterogeneous and are distributed throughout the body. They display unique structural and functional characteristics, depending on their environment and physiologic requirements, not only in subsets of blood vessels such as arteries versus veins but also in the various tissues and organs of the body.45 ECs play essential roles in immune response, vascular permeability, proliferation, and, of course, hemostasis.

Image 

FIGURE 37-1 Normal blood flow in intact vessels. Smooth, rhomboid endothelial surfaces promote even flow. RBCs and platelets are concentrated toward the center, WBCs roll along the endothelium. The endothelium contains or secretes several hemostasis-suppressing materials. EC, Endothelial cell; ECM, extracellular matrix; FB, fibroblast; PLT, platelet; RBC, red blood cell; SMC, smooth muscle cell; WBC, white blood cell; lines indicate collagen.

BOX 37-1

Vascular Intima of the Blood Vessel

Innermost vascular lining

Endothelial cells (endothelium)

Supporting the endothelial cells

Internal elastic lamina composed of elastin and collagen

Subendothelial connective tissue

Collagen and fibroblasts in veins

Collagen, fibroblasts, and smooth muscle cells in arteries

ECs form a smooth, unbroken surface that eases the fluid passage of blood. An elastin-rich internal elastic lamina (basement membrane) and its surrounding layer of connective tissues support the ECs. In all blood vessels, fibroblasts occupy the connective tissue layer and produce collagen. Smooth muscle cells in arteries and arterioles, but not in the walls of veins, venules, or capillaries, contract during primary hemostasis.

Anticoagulant properties of intact vascular intima

Normally, the intact vascular endothelium prevents thrombosis by inhibiting platelet aggregation, preventing coagulation activation and propagation, and enhancing fibrinolysis. Several specific anticoagulant mechanisms prevent intravascular thrombosis (; Box 37-2Figure 37-2). First, ECs are rhomboid and contiguous, providing a smooth inner surface of the blood vessel that prevents harmful turbulence that otherwise may activate platelets and coagulation enzymes. ECs form a physical barrier separating procoagulant proteins and platelets in blood from collagen in the internal elastic lamina that promotes platelet adhesion, and tissue factor in fibroblasts and smooth muscle cells that activates coagulation.

Image 

FIGURE 37-2 Anticoagulant functions of normal intact endothelial cells and procoagulant properties of endothelial cells when damaged. EC, Endothelial cells; PGI2, prostacyclin or prostaglandin I2TFPI, tissue factor pathway inhibitor; EPCR, endothelial cell protein C receptor; TPA, tissue plasminogen activator; VWF, von Willebrand factor; ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; TF, tissue factor; PAI-1, plasminogen activator inhibitor-1; TAFI, thrombin-activatable fibrinolysis inhibitor.

BOX 37-2

Anticoagulant Properties of Intact Endothelium

Composed of rhomboid cells presenting a smooth, contiguous surface

Secretes the eicosanoid platelet inhibitor prostacyclin

Secretes vascular “relaxing” factor nitric oxide

Secretes the anticoagulant glycosaminoglycan heparan sulfate

Secretes coagulation extrinsic pathway regulator tissue factor pathway inhibitor

Expresses endothelial protein C receptor

Expresses cell membrane thrombomodulin, a protein C coagulation control system activator

Secretes TPA, thereby activating fibrinolysis

ECs synthesize and secrete a variety of substances that maintain normal blood flow. Prostacyclin, a platelet inhibitor and a vasodilator, is synthesized through the eicosanoid pathway (Chapter 13) and prevents unnecessary or undesirable platelet activation in intact vessels.6 Nitric oxide is synthesized in ECs, vascular smooth muscle cells, neutrophils, and macrophages. Nitric oxide induces smooth muscle relaxation and subsequent vasodilation, inhibits platelet activation, and promotes angiogenesis and healthy arterioles.78 An important EC-produced anticoagulant is tissue factor pathway inhibitor (TFPI), which controls activation of the tissue factor pathway, also called the extrinsic coagulation pathway.

Finally, ECs synthesize and express on their surfaces inhibitors of thrombin formation, thrombomodulin, facilitated by endothelial protein C receptor (EPCR), and heparan sulfate. EPCR binds protein C, and thrombomodulin catalyzes the activation of the protein C pathway. The protein C pathway downregulates coagulation by digesting activated factors V and VIII, thereby inhibiting thrombin formation. Heparan sulfate is a glycosaminoglycan that enhances the activity of antithrombin, a serine protease inhibitor.9 The pharmaceutical anticoagulant heparin, manufactured from porcine gut tissues, resembles EC heparan sulfate in its antithrombin activity. Heparin is used extensively as a therapeutic agent to prevent propagation of the thrombi that cause coronary thrombosis, strokes, deep vein thromboses, and pulmonary emboli.

Procoagulant properties of damaged vascular intima

Although the intact endothelium has anticoagulant properties, when damaged, the vascular intima promotes coagulation. First, any harmful local stimulus, whether mechanical or chemical, induces vasoconstriction in arteries and arterioles (; Table 37-2Figure 37-2). Smooth muscle cells contract, the vascular lumen narrows or closes, and blood flow to the injured site is minimized. Although veins and capillaries do not have smooth muscle cells, bleeding into surrounding tissues creates extravascular pressure on the blood vessel, effectively minimizing the escape of blood.

TABLE 37-2

Procoagulant Properties of the Damaged Vascular Intima

Structure

Procoagulant Property

Smooth muscle cells in arterioles and arteries

Induce vasoconstriction

Exposed subendothelial collagen

Binds VWF and platelets

Damaged or activated ECs

Secrete VWF Secrete adhesion molecules: P-selectin, ICAMs, PECAMs

Exposed smooth muscle cells and fibroblasts

Tissue factor exposed on cell membranes

ECs in inflammation

Tissue factor is induced by inflammation

ECs, endothelial cells; ICAMs, Intercellular adhesion molecules; PECAMs, platelet endothelial cell adhesion molecules; VWF, von Willebrand factor.

Second, the subendothelial connective tissues of arteries and veins are rich in collagen, a flexible, elastic structural protein that binds and activates platelets. Some connective tissue degeneration occurs naturally in aging, which leads to an increased bruising tendency.

Third, ECs secrete von Willebrand factor (VWF) from storage sites called Weibel-Palade bodies when activated by vasoactive agents such as thrombin. VWF is a large multimeric glycoprotein that is necessary for platelets to adhere to exposed subendothelial collagen in arterioles.10

Fourth, on activation, ECs secrete and coat themselves with P-selectin, an adhesion molecule that promotes platelet and leukocyte binding.11 ECs also secrete immunoglobulin-like adhesion molecules calledintercellular adhesion molecules (ICAMs) and platelet endothelial cell adhesion molecules (PECAMs) that further promote platelet and leukocyte binding.12

Finally, subendothelial smooth muscle cells and fibroblasts support the constitutive membrane protein tissue factor.13 Physiologically, EC disruption exposes tissue factor in subendothelial cells and activates the coagulation system through contact with plasma factor VII. In pathological conditions, tissue factor may also be expressed on bloodborne monocytes during inflammation and sepsis and by tissue factor–positive microparticles derived from membrane fragments of activated or apoptotic vascular cells and possibly on the surface of some ECs.14 Activation of the TF:VIIa:Xa complex within the circulation is limited by TFPI.

Fibrinolytic properties of vascular intima

ECs support fibrinolysis (Figure 37-2), the removal of fibrin to restore vessel patency, with the secretion of tissue plasminogen activator (TPA). During thrombus formation, both TPA and plasminogen bind to polymerized fibrin. TPA activates fibrinolysis by converting plasminogen to plasmin, which gradually digests fibrin and restores blood flow. ECs also regulate fibrinolysis by providing inhibitors to prevent excessive plasmin generation. ECs, as well as other cells, secrete plasminogen activator inhibitor 1 (PAI-1), a TPA control protein that inhibits plasmin generation and fibrinolysis.15 Another inhibitor of plasmin generation, thrombin-activatable fibrinolysis inhibitor (TAFI), is activated by thrombin bound to EC membrane thrombomodulin.16 Elevations in PAI-1 or TAFI can slow fibrinolysis and increase the tendency for thrombosis.

Although the significance of the vascular intima in hemostasis is well recognized, there are few valid laboratory methods to assess the integrity of ECs, smooth muscle cells, fibroblasts, and their collagen matrix.17The diagnosis of blood vessel disorders is often based on clinical symptoms, family history, and laboratory tests that rule out platelet or coagulation disorders.

Platelets

Platelets are produced from the cytoplasm of bone marrow megakaryocytes (Chapter 13).18 Although platelets are only 2 to 3 μm in diameter on a fixed, stained peripheral blood film, they are complex, metabolically active cells that interact with their environment and initiate and control hemostasis.19

At the time of an injury, platelets adhere, aggregate, and secrete the contents of their granules ().Table 37-32021 Adhesion is the property by which platelets bind nonplatelet surfaces such as subendothelial collagen (Figures 37-3 and 37-4). Further, VWF links platelets to collagen in areas of high shear stress such as arteries and arterioles, whereas platelets may bind directly to collagen in damaged veins and capillaries. VWF binds platelets through their glycoprotein GP Ib/IX/V membrane receptor.22 The importance of platelet adhesion is underscored by bleeding disorders such as Bernard-Soulier syndrome, in which the platelet GP Ib/IX/V receptor is absent, and von Willebrand disease, in which VWF is missing or defective.

Image 

FIGURE 37-3 Platelet adhesion. Upon desquamation of endothelial cells (EC ), platelets (PLT  ) adhere to the internal elastic lamina or extracellular matrix (ECM) and fill in until new ECs grow. FB, Fibroblast; PL, phospholipid; RBC, red blood cell; SMC, smooth muscle cell; TF, tissue factor; VWF, von Willebrand factor; WBC, white blood cell; lines indicate collagen.

Image 

FIGURE 37-4 Interaction of platelets (PLT  ), von Willebrand factor (VWF ), and collagen. In arterioles and arteries, where blood flows rapidly, platelets adhere by binding VWF. The larger VWF multimers form a fibrillar carpet on which the platelets assemble. Though a protective mechanism, a white clot consisting of PLTs and VWF may occlude the vessel, causing acute myocardial infarction, stroke, or peripheral artery disease. EC, Endothelial cell; ECM, extracellular matrix; SMC, smooth muscle cell; FB, fibroblast; RBC, red blood cell; WBC, white blood cell; lines indicate collagen.

TABLE 37-3

Platelet Function

Function

Characteristics

Adhesion: platelets roll and cling to nonplatelet surfaces

Reversible; seals endothelial gaps, some secretion of growth factors, in arterioles VWF is necessary for adhesion

Aggregation: platelets adhere to each other

Irreversible; platelet plugs form, platelet contents are secreted, requires fibrinogen

Secretion: platelets discharge the contents of their granules

Irreversible; occurs during aggregation, platelet contents are secreted, essential to coagulation

Aggregation is the property by which platelets bind to one another (Figure 37-5). When platelets are activated, a change in the GP IIb/IIIa receptor allows binding of fibrinogen, as well as VWF and fibronectin.23Fibrinogen binds to GP IIb/IIIa receptors on adjacent platelets and joins them together in the presence of ionized calcium (Ca2+). Fibrinogen binding is essential for platelet aggregation, as evidenced by bleeding and compromised aggregation in patients with afibrinogenemia or in patients who lack the GP IIb/IIIa receptor (Glanzmann thrombasthenia). In in vitro platelet aggregation studies, the most commonly used agonists to induce aggregation are thrombin (or thrombin receptor activation peptide, TRAP), arachidonic acid, adenosine diphosphate (ADP), collagen, and epinephrine, which bind to their respective platelet membrane receptors.24

Image 

FIGURE 37-5 Platelet aggregation. In veins and venules, the bulky “red clot” consists of platelets (PLT ), von Willebrand factor (VWF ), fibrin, and red blood cells (RBC). Though a protective mechanism, the red clot may occlude the vessel, causing venous thromboembolic disease. EC, Endothelial cell; ECM, extracellular matrix; FB, fibroblast; WBC, white blood cell; SMC, smooth muscle cell; lines indicate collagen.

Platelets secrete the contents of their granules during adhesion and aggregation, with most secretion occurring late in the platelet activation process. Platelets secrete procoagulants, such as factor V, VWF, factor VIII, and fibrinogen, as well as control proteins, Ca2+, ADP, and other hemostatic molecules. See Table 37-4 for a summary of the contents of platelet α-granules and dense bodies (dense granules).

TABLE 37-4

Platelet Granule Contents

Platelet α-Granules

Platelet Dense Granules (Dense Bodies)

Large Molecules

Small Molecules

β-Thromboglobulin

Adenosine diphosphate (activates neighboring platelets)

Factor V

Adenosine triphosphate

Factor XI

Calcium

Protein S

Serotonin (vasoconstrictor)

Fibrinogen

 

VWF

 

Platelet factor 4 (heparin inhibitor)

 

Platelet-derived growth factor

 

During activation, ADP and Ca2+ activate phospholipase A2, which converts membrane phospholipid to arachidonic acid. Cyclooxygenase converts arachidonic acid into prostaglandin endoperoxides. In the platelet, thromboxane synthetase converts prostaglandins into thromboxane A2, which causes Ca2+ to be released and promotes platelet aggregation and vasoconstriction (Figure 37-6). Aspirin acetylation permanently inactivates cyclooxygenase, blocking thromboxane A2 production and causing impairment of platelet function (aspirin effect).25

Image 

FIGURE 37-6 Arachidonic acid and aspirin effect. Phospholipase A2 converts membrane phospholipids to arachidonic acid during platelet activation. Arachidonic acid is converted to prostaglandin endoperoxides (PGG2/PGH2) by cyclooxygenase, then to thromboxane A2 (TXA2). TXA2 causes release of Ca2+, which promotes platelet aggregation and vasoconstriction. Aspirin permanently blocks the action of cyclooxygenase-1 and TXA2 synthesis, impairing platelet function.

Chapter 13 provides an in-depth description of platelet structure and function. Platelet disorders are considered in detail in Chapters 40 and 41.

The platelet membrane is the key surface for coagulation enzyme-cofactor-substrate complex formation.26 Platelets supply Ca2+, the membrane phospholipid phosphatidylserine, procoagulant factors, and receptors. Coagulation is initiated on tissue factor–bearing cells (such as fibroblasts) with the formation of the extrinsic tenase complex TF:VIIa:Ca2+, which activates factors IX and X and produces enough thrombin to activate platelets and factors V, VIII, and XI in a feedback loop. Coagulation is then propagated on the surface of the platelet with the formation of the intrinsic tenase complex (IXa:VIIIa:phospholipid:Ca2+) and the prothrombinase complex (Xa:Va:phospholipid:Ca2+), ultimately generating a burst of thrombin at the site of injury. See subsequent text for more details.

Erythrocytes, monocytes, and lymphocytes also participate in hemostasis. Erythrocytes add bulk and structural integrity to the fibrin clot; there is a tendency to bleed in anemia. In inflammatory conditions, monocytes and lymphocytes, and possibly ECs, provide surface-borne tissue factor that may trigger coagulation. Leukocytes also have a series of membrane integrins and selectins that bind adhesion molecules and help stimulate the production of inflammatory cytokines that promote the wound-healing process.27

Coagulation system

Nomenclature of procoagulants

Plasma transports at least 16 procoagulants, also called coagulation factors. Nearly all are glycoproteins synthesized in the liver, although monocytes, ECs, and megakaryocytes produce a few (Table 37-5Figure 37-7). Eight are enzymes that circulate in an inactive form called zymogens. Others are cofactors that bind, stabilize, and enhance the activity of their respective enzymes. The sequence of activation is shown in Figure 37-8. During clotting, the procoagulants become activated and produce a localized thrombus. In addition, there are plasma glycoproteins that act as controls to regulate the coagulation process. See subsequent text for more details.

Image 

FIGURE 37-7 Procoagulants (zymogens), cofactors, and anticoagulants (control proteins). HMWK, High-molecular-weight kininogen; TFPI, tissue factor pathway inhibitor; ZPI, protein Z-dependent protease inhibitor.

Image 

FIGURE 37-8 Simplified coagulation pathway. Exposed tissue factor (TF  ) activates factor VII, which activates factors IX and X. Factor IXa:VIIIa complex also activates X, and the factor Xa:Va complex activates prothrombin (factor II). The resulting thrombin cleaves fibrinogen to form fibrin and activates factor XIII to stabilize the clot. Thrombin also activates factors V, VIII, XI, and platelets. In vitro exposure to negatively charged surfaces activates the contact factors XII, pre-kallikrein (pre-K  ) and high-molecular-weight kininogen (HMWK  ), which activate factor XI.

TABLE 37-5

Plasma Procoagulants: Function, Molecular Weight, Plasma Half-Life, and Plasma Concentration

Factor

Name

Function

Molecular Weight (Daltons)

Half-Life (Hours)

Mean Plasma Concentration

I*

Fibrinogen

Thrombin substrate, polymerizes to form fibrin

340,000

100–150

200–400 mg/dL

II*

Prothrombin

Serine protease

71,600

60

10 mg/dL

III*

Tissue factor

Cofactor

44,000

Insoluble

None

IV*

Ionic calcium

Mineral

40

NA

8–10 mg/dL

V

Labile factor

Cofactor

330,000

24

1 mg/dL

VII

Stable factor

Serine protease

50,000

6

0.05 mg/dL

VIII

Antihemophilic factor

Cofactor

260,000

12

0.01 mg/dL

VWF

von Willebrand factor

Factor VIII carrier and platelet adhesion

600,000–20,000,000

24

1 mg/dL

IX

Christmas factor

Serine protease

57,000

24

0.3 mg/dL

X

Stuart-Prower factor

Serine protease

58,800

48–52

1 mg/dL

XI

Plasma thromboplastin antecedent (PTA)

Serine protease

143,000

48–84

0.5 mg/dL

XII

Hageman factor

Serine protease

84,000

48–70

3 mg/dL

Prekallikrein

Fletcher factor, pre-K

Serine protease

85,000

35

35–50 μg/mL

High-molecular-weight kininogen

Fitzgerald factor, HMWK

Cofactor

120,000

156

5 mg/dL

XIII

Fibrin-stabilizing factor (FSF)

Transglutaminase, transamidase

320,000

150

2 mg/dL

Platelet factor 3

Phospholipids, phosphatidylserine, PF3

Assembly molecule

Released by platelets

* These factors are customarily identified by name rather than Roman numeral.

From Greenberg DL, Davie EW: The blood coagulation factors: their complementary DNAs, genes, and expression. In Colman RW, Marder VJ, Clowes, AM, et al, editors: Hemostasis and thrombosis: basic principles and clinical practice, ed 5, Philadelphia, 2006, Lippincott Williams & Wilkins, pp. 21-58.

In 1958 the International Committee for the Standardization of the Nomenclature of the Blood Clotting Factors officially named the plasma procoagulants using Roman numerals in the order of their initial description or discovery.28 When a procoagulant becomes activated, a lowercase a appears behind the numeral; for instance, activated factor VII is VIIa. Both zymogens and cofactors become activated in the coagulation process.

We customarily call factor I fibrinogen and factor II prothrombin, although occasionally they are identified by their numerals. The numeral III was given to tissue thromboplastin, a crude mixture of tissue factor and phospholipid. Now that the precise structure of tissue factor has been described, the numeral designation is seldom used. The numeral IV identified the plasma cation calcium (Ca2+); however, calcium is referred to by its name or chemical symbol, not by its numeral. The numeral VI was assigned to a procoagulant that later was determined to be activated factor V; VI was withdrawn from the naming system and never reassigned. Factor VIII, antihemophilic factor, is a cofactor that circulates linked to a large carrier protein, VWF. Prekallikrein (pre-K), also called Fletcher factor, and high-molecular-weight kininogen (HMWK), also called Fitzgerald factor, have never received Roman numerals because they belong to the kallikrein and kinin systems, respectively, and their primary functions lie within these systems. Platelet phospholipids, particularly phosphatidylserine, are required for the coagulation process but were given no Roman numeral; instead they were once called collectively platelet factor 3.

Classification and function of procoagulants

The plasma procoagulants may be serine proteases or cofactors, except for factor XIII, which is a transglutaminase ().Table 37-629 Serine proteases are proteolytic enzymes of the trypsin family and include the procoagulants thrombin (factor IIa); factors VIIa, IXa, Xa, XIa, and XIIa; and pre-K.30 Each member has a reactive seryl amino acid residue in its active site and acts on its substrate by hydrolyzing peptide bonds, digesting the primary backbone, and producing smaller polypeptide fragments. Serine proteases are synthesized as inactive zymogens consisting of a single peptide chain. Activation occurs when the zymogen is cleaved at one or more specific sites by the action of another protease during the coagulation process.

TABLE 37-6

Plasma Procoagulant Serine Proteases

Inactive Zymogen

Active Protease

Cofactor

Substrate

Prothrombin (II)

Thrombin (IIa)

Fibrinogen, V, VIII, XI, XIII

VII

VIIa

Tissue factor

IX, X

IX

IXa

VIIIa

X

X

Xa

Va

Prothrombin

XI

XIa

IX

XII

XIIa

High-molecular-weight kininogen

XI

Prekallikrein

Kallikrein

High-molecular-weight kininogen

XI

The procoagulant cofactors that participate in complex formation are tissue factor, located on membranes of fibroblasts and smooth muscle cells, and soluble plasma factors V, VIII, and HMWK. The remaining components of the coagulation pathway are fibrinogen, factor XIII, phospholipids, calcium, and VWF (). Fibrinogen is the ultimate substrate of the coagulation pathway. When hydrolyzed by thrombin, fibrinogen forms the primary structural protein of the fibrin clot, which is further stabilized by factor XIII.Box 37-331

BOX 37-3

Other Plasma Procoagulants

Fibrinogen

Factor XIII

Phospholipids

Calcium

VWF

Calcium is required for the assembly of coagulation complexes on platelet or cell membrane phospholipids. Serine proteases bind to negatively charged phospholipid surfaces, predominantly phosphatidylserine, through positively charged calcium ions. Activation is a localized cell-surface process, limited to the site of injury and controlled by regulatory mechanisms. If zymogen activation is uncontrolled and generalized, the condition is called disseminated intravascular coagulation (DIC), a serious, often life-threatening condition (Chapter 39).

The molecular weights, plasma concentrations, and plasma half-lives of the procoagulants are given in Table 37-5. These essential pieces of clinical information assist in the interpretation of laboratory tests, monitoring of anticoagulant therapy, and design of effective replacement therapies in deficiency-related hemorrhagic diseases. For example, factor VIII has a short half-life of 12 hours, so replacement therapy for hemophilic individuals who are deficient in factor VIII is administered every 12 hours. For most factors, the level that achieves hemostatic effectiveness is 25% to 30%. This is the minimum level that must be maintained to prevent bleeding in factor-deficient patients. Therapy for a hemophilic patient is designed to maintain the factor level above 30%. A higher level may be desirable, such as in a patient preparing for surgery. The half-life is also important in monitoring anticoagulant therapy, especially warfarin (Coumadin), because even though factor VII becomes reduced in 6 hours, the reduction of prothrombin takes 4 to 5 days. Therefore, the full effect of warfarin is not realized until approximately 5 days after therapy has begun.

Vitamin k–dependent prothrombin group

Prothrombin (factor II), factors VII, IX, and X and the regulatory proteins protein C, protein S, and protein Z are vitamin K–dependent (). These are named the Table 37-7prothrombin group because of their structural resemblance to prothrombin. All seven proteins have 10 to 12 glutamic acid units near their amino termini. All except proteins S and Z are serine proteases when activated; S and Z are cofactors.

TABLE 37-7

Vitamin K–Dependent Coagulation Factors

Procoagulants

Regulatory Proteins

Prothrombin (II)

Protein C

VII

Protein S

IX

Protein Z

X

 

Vitamin K is a quinone found in green leafy vegetables () and is produced by the intestinal organisms Box 37-4Bacteroides fragilis and Escherichia coli. Vitamin K catalyzes an essential posttranslational modification of the prothrombin group proteins: γ-carboxylation of amino-terminal glutamic acids (Figure 37-9). Glutamic acid is modified to γ-carboxyglutamic acid when a second carboxyl group is added to the γ carbon. With two ionized carboxyl groups, the γ-carboxyglutamic acids gain a net negative charge, which enables them to bind ionic calcium (Ca2+). The bound calcium enables the vitamin K–dependent proteins to bind to negatively charged phospholipids to form coagulation complexes.

Image 

FIGURE 37-9 Vitamin K (K ) posttranslational γ-carboxylation of coagulation factors II (prothrombin), VII, IX, and X, and control proteins C, S, and Z. Vitamin K hydroxyquinone transfers a carboxyl (COO) group to the γ carbon of glutamic acid (Glu), creating γ-carboxyglutamic acid (Gla). The negatively charged pocket formed by the two carboxyl groups attracts ionic calcium, which enables the molecule to bind to phosphatidylserine. Vitamin K hydroxyquinone is oxidized to vitamin K epoxide by carboxylase in the process of transferring the carboxyl group but is subsequently reduced to the hydroxyquinone form by epoxide reductase. Warfarin suppresses epoxide reductase, which slows the reaction and prevents γ-carboxylation. “Des-carboxy” proteins are unable to participate in coagulation. There are typically 10 to 12 γ-carboxyglutamic acid molecules near the amino terminus of the vitamin K–dependent factors.

BOX 37-4

Food Sources High in Vitamin K

Kale

Spinach

Turnip greens

Collards

Mustard greens

Swiss chard

Brussels sprouts

Broccoli

Asparagus

Cabbage

Green onions

Lettuce: Boston, romaine, or Bibb

Avocado

Cauliflower

Parsley, fresh

In vitamin K deficiency or in the presence of warfarin, a vitamin K antagonist, the vitamin K–dependent procoagulants are released from the liver without the second carboxyl group added to the γ carbon. These are called des-γ-carboxyl proteins or proteins in vitamin K antagonism (PIVKAs). Because they lack the second carboxyl group, they cannot bind to Ca2+ and phospholipid, so they cannot participate in the coagulation reaction. Vitamin K antagonism is the basis for oral anticoagulant (warfarin, Coumadin) therapy (Chapter 43).

Vitamin K–dependent procoagulants are essential for the assembly of three membrane complexes leading to the generation of thrombin (). Each complex is composed of a vitamin K–dependent serine protease, its non-enzyme cofactor, and CaFigure 37-102+, bound to the negatively charged phospholipid membranes of activated platelets or tissue factor–bearing cells. The initial complex, extrinsic tenase, is composed of factor VIIa and tissue factor, and it activates factors IX and X, which are components of the next two complexes, intrinsic tenase and prothrombinase, respectively (Table 37-8). Intrinsic tenase is composed of factor IXa and its cofactor VIIIa; it also activates factor X much more efficiently than the TF:VIIa complex. Prothrombinase is composed of factor Xa and its cofactor Va; this converts prothrombin to thrombin in a multistep hydrolytic process that releases thrombin and a peptide fragment called prothrombin fragment 1.2 (F 1.2). Prothrombin fragment 1.2 in plasma is thus a marker for thrombin generation.

Image 

FIGURE 37-10 Coagulation complexes. Coagulation complexes form on TF-bearing cells (TF:VIIa) and on platelet phospholipid membranes (IXa:VIIIa and Xa:Va). Each complex consists of a vitamin K–dependent serine protease, a cofactor, and Ca2+, bound to the cell membrane. Extrinsic tenase complex is factor VIIa and tissue factor (TF) on the membrane of a TF-bearing cell. This complex activates both factors IX and X. Intrinsic tenase complex, which is factor IXa and its cofactor VIIIa on platelet membranes, activates factor X also. Prothrombinase complex is factor Xa and its cofactor Va, bound to the surface of platelets. Prothrombinase cleaves prothrombin to the active enzyme, thrombin.

TABLE 37-8

Coagulation Complexes

Complex

Components

Activates

Extrinsic tenase

VIIa, tissue factor, phospholipid, and Ca2+

IX and X

Intrinsic tenase

IXa, VIIIa, phospholipid, and Ca2+

X

Prothrombinase

Xa, Va, phospholipid, and Ca2+

Prothrombin

Cofactors in hemostasis

Procoagulant cofactors are tissue factor, factor V, factor VIII, and HMWK. Coagulation control cofactors are thrombomodulin, protein S, and protein Z ().Table 37-932 Thrombomodulin is also a cofactor in control of fibrinolysis. Each cofactor binds its particular serine protease. When bound to their cofactors, serine proteases gain stability and increased reactivity.

TABLE 37-9

Hemostasis Cofactors

Cofactor

Function

Binds

Tissue factor

Procoagulant

VIIa

V

Procoagulant

Xa

VIII

Procoagulant

IXa

High-molecular-weight kininogen

Procoagulant

XIIa, prekallikrein

Thrombomodulin

Control (Protein C) Antifibrinolytic (TAFI)

Thrombin Thrombin

Protein S

Control

Protein C, TFPI

Protein Z

Control

ZPI

Tissue factor is a transmembrane receptor for factor VIIa and is found on extravascular cells such as fibroblasts and smooth muscle cells, but under normal conditions, it is not found on blood vessel ECs.33Vessel injury exposes blood to the subendothelial tissue factor–bearing cells and leads to activation of coagulation through VIIa. Tissue factor is expressed in high levels in cells of the brain, lung, placenta, heart, kidney, and testes. In inflammatory conditions and sepsis, leukocytes and other cells can also express tissue factor and initiate coagulation.34

Factors V and VIII are soluble plasma proteins. Both are activated by thrombin and inactivated by protein C. Factor V is a glycoprotein circulating in plasma and also present in platelet α-granules. During platelet activation and secretion, platelets release partially activated factor V at the site of injury. Factor Va is a cofactor to Xa in the prothrombinase complex in coagulation. The prothrombinase complex accelerates thrombin generation more than 300,000-fold compared to Xa alone.35 As described below, thrombomodulin-bound thrombin activates protein C, which inactivates Va to Vi. Therefore, factor V is both activated and then ultimately inactivated by the generation of thrombin, as is factor VIII. Factor VIII is a cofactor to factor IX, which together form the intrinsic tenase complex, discussed in the next section. High-molecular-weight kininogen is a cofactor to factor XIIa and prekallikrein in the intrinsic contact factor complex, a mechanism for activating coagulation in conditions where foreign objects such as mechanical heart valves or bacterial membranes and/or high levels of inflammation are present.

Thrombomodulin, a transmembrane protein constitutively expressed by vascular ECs, is a thrombin cofactor. Together, thrombomodulin and thrombin activate protein C, a coagulation regulatory protein, and thrombin activatable fibrinolysis inhibitor (TAFI), a fibrinolysis inhibitor. In one of many examples of negative feedback regulation in coagulation, once thrombin is bound to thrombomodulin, it loses its procoagulant ability to activate factors V and VIII, and, through activation of protein C, leads to destruction of factors V and VIII, thus suppressing further generation of thrombin.

Both protein S and protein C are cofactors in the regulation and control of coagulation, discussed later in this chapter. Protein S is a cofactor to protein C, as well as TFPI. Protein Z is a cofactor to Z-dependent protease inhibitor (ZPI).

Factor VIII and von willebrand factor

Factor VIII has a molecular mass of 260,000 Daltons and is produced primarily by hepatocytes, but also by microvascular ECs in lung and other tissues.36 Free factor VIII is unstable in plasma; it circulates bound to VWF. During coagulation, thrombin cleaves factor VIII from VWF and activates it. Factor VIIIa binds to activated platelets and forms the intrinsic tenase complex with factor IXa and Ca2+. Like factor Va, factor VIIIa is also inactivated by protein C.

Factor VIII and factor IX are the two plasma procoagulants whose production is governed by genes carried on the X chromosome. Hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency) are therefore sex-linked disorders occurring almost exclusively in males. Males with hemophilia A have diminished factor VIII activity but normal VWF levels.37 Factor VIII is a cofactor, but its importance in hemostasis cannot be overstated, as evidenced by the severe bleeding and symptoms associated with hemophilia A.

Factor VIII deteriorates more rapidly than the other coagulation factors in stored blood. In thawed component plasma, the factor VIII level drops to approximately 50% after 5 days.38 Treatment for hemophilia bleeding episodes consists of replacement therapy transfused according to the 12-hour half-life of factor VIII.

VWF is a large multimeric glycoprotein that participates in platelet adhesion and transports the procoagulant factor VIII. VWF is composed of multiple subunits of 240,000 Daltons each.39 The subunits are produced by ECs and megakaryocytes, where they combine to form multimers that range from 600,000 to 20,000,000 Daltons.40 VWF molecules are stored in α-granules in platelets and in Weibel-Palade bodies in ECs. The molecules are released from storage into the plasma, and they circulate at a concentration of 7 to 10 μg/mL. ECs release ultralarge multimers of VWF into plasma, where they are normally degraded into smaller multimers by a VWF-cleaving protease, ADAMTS-13 (a disintegrin and metalloproteinase with a thrombo spondin type 1 motif, member 13), in blood vessels with high shear stress. In thrombotic thrombocytopenic purpura (TTP), inherited or acquired defective ADAMTS-13 enzyme activity is associated with the presence of ultralarge VWF multimers in plasma, resulting in platelet aggregation and microvascular thrombosis.41

VWF has receptor sites for both platelets and collagen () and helps to bind platelets to exposed subendothelial collagen during platelet adhesion, especially in arteries and arterioles where the flow of blood is faster. The primary platelet surface receptor for VWF is GP Ib/IX/V. Arginine-glycine-aspartic acid (RGD) sequences in VWF also bind a second platelet integrin, GP IIb/IIIa, during platelet aggregation. Figure 37-11

Image 

FIGURE 37-11 Von Willebrand factor (VWF  )–factor VIII complex. Factor VIII circulates covalently bound to VWF. VWF provides three other active receptor sites: VWF binds to collagen and binds to glycoprotein Ib/IX/V to support platelet adhesion and binds to glycoprotein IIb/IIIa to facilitate platelet aggregation.

A third site on the VWF molecule binds collagen, and a fourth site binds the plasma procoagulant cofactor, factor VIII. VWF is decreased in von Willebrand disease (VWD), a relatively common disorder that occurs in 1% to 2% of the general population. Because factor VIII depends on VWF for stability, individuals with VWD who have diminished VWF also have diminished factor VIII activity levels. Typically, factor VIII levels decrease to hemorrhagic levels (less than 30%) only in severe VWD. The level of VWF also varies in people according to their ABO blood type. Group O individuals have lower levels of VWF than other ABO types.42 VWF is an acute phase protein, as is factor VIII, and levels increase in pregnancy, trauma, infections, and stress.

Factor XI and the contact factors

The “contact factors,” also called intrinsic accessory pathway proteins, are factor XII, high-molecular-weight kininogen (HMWK, Fitzgerald factor), and prekallikrein (pre-K, Fletcher factor). They are so named because they are activated by contact with negatively charged foreign surfaces. Factor XIIa transforms pre-K, a glycoprotein that circulates bound to HMWK, into its active form kallikrein, which cleaves HMWK to form bradykinin. Factor XII and pre-K are zymogens that are activated to become serine proteases; HMWK is a nonenzymatic cofactor.

The contact factor complex (HMWK:pre-K:FXII) activates factor XI; factor XIa is an activator of factor IX (Figure 37-8). Deficiencies of factor XII, HMWK, or pre-K do not cause clinical bleeding disorders. However, deficiencies do prolong laboratory tests and necessitate investigation. Factor XII is activated in vitro by negatively charged surfaces such as nonsiliconized glass, kaolin, or ellagic acid in partial thromboplastin time (PTT) reagents. In vivo, foreign materials such as stents or valve prostheses may activate contact factors to cause thrombosis.

Factor XI is activated by the contact factor complex and, more significantly, by thrombin during coagulation generated from tissue factor activation. Factor XIa activates factor IX, and the reaction proceeds as described previously. Deficiencies of factor XI (Rosenthal syndrome) usually result in mild and variable bleeding.43 Factor XI supplements or boosts factor IX activation, so deficiencies of factor XI are less severe clinically than deficiencies of the other factors such as IX or VIII.

Thrombin

The primary function of thrombin is to cleave fibrinopeptides A and B from the α and β chains of the fibrinogen molecule, triggering spontaneous fibrin polymerization (). In addition, thrombin amplifies the coagulation mechanism by activating cofactors V and VIII and factor XI by a positive feedback mechanism (Figure 37-12Figure 37-8). Thrombin also activates factor XIII, which forms covalent bonds between the D domains of the fibrin polymer to cross-link and stabilize the fibrin clot. Thrombin also initiates aggregation of platelets. Thrombin bound to thrombomodulin activates the protein C pathway to suppress coagulation, and it activates TAFI to suppress fibrinolysis. Thrombin, therefore, plays a role in coagulation (fibrin), in platelet activation, in coagulation control (protein C), and in controlling fibrinolysis (TAFI). Because of its multiple autocatalytic functions, thrombin is considered the key protease of the coagulation pathway.

Image 

FIGURE 37-12 Fibrinogen domains and cleavage by thrombin. Each molecule of fibrinogen or fibrin has three “domains”: two D domains (the carboxyl ends of the molecule) and one E domain (the central portion of the molecule), as shown in this diagram. Thrombin (THR) cleaves fibrinopeptides A and B from the alpha and beta chains in the E domain.

Fibrinogen structure and fibrin formation, factor XIII

Fibrinogen is the primary substrate of thrombin, which converts soluble fibrinogen to insoluble fibrin to produce a clot. Fibrinogen is also essential for platelet aggregation because it links activated platelets through their GP IIb/IIIa platelet fibrinogen receptor. Fibrinogen is a 340,000 Dalton glycoprotein synthesized in the liver. The normal plasma concentration of fibrinogen ranges from 200 to 400 mg/dL, the most concentrated of all the plasma procoagulants. Fibrinogen is an acute phase reactant protein, whose level increases in inflammation, infection, and other stress conditions. Platelet α-granules absorb, transport, and release abundant fibrinogen.44

The fibrinogen molecule is a mirror-image “trinodular” dimer, each half consisting of three nonidentical polypeptides, designated Aα, Bβ, and γ, united by disulfide bonds (). The six N-terminals assemble to form a bulky central region called the E domain. The carboxyl terminals assemble at the outer ends of the molecule to form two D domains.Figure 37-1345

Image 

FIGURE 37-13 Structure of fibrinogen. Fibrinogen is a trinodular structure composed of three pairs (Aα, Bβ, and γ) of disulfide-bonded polypeptide chains. The central node is known as the E domain. Thrombin cleaves small peptides, A and B, from the α and β chains in this region to form fibrin. The central nodule is joined by supercoiled α-helices to the terminal nodules known as the D domains.  Source: (From McKenzie SB, Williams JL: Clinical laboratory hematology, ed 2, Upper Saddle River, NJ, 2009, Pearson, p 653.)

Thrombin cleaves fibrinopeptides A and B from the protruding N-termini of each of the two α and β chains of fibrinogen, reducing the overall molecular weight by 10,000 Daltons. The cleaved fibrinogen is called fibrin monomer. The exposed fibrin monomer α and β chain ends (E domain) have an immediate affinity for portions of the D domain of neighboring monomers, spontaneously polymerizing to form fibrin polymer(Figure 37-14).

Image 

FIGURE 37-14 Formation of a stabilized fibrin clot. Thrombin cleaves fibrinopeptides A and B to form fibrin monomer. Fibrin monomers polymerize due to the affinity of thrombin-cleaved positively charged E domains for negatively charged D domains of other monomers. Factor XIIIa catalyzes the covalent cross-linking of γ chains of adjacent D domains to form an insoluble stable fibrin clot.

Thrombin also activates factor XIII, a heterodimer whose α subunit is produced mostly by megakaryocytes and monocytes, and whose β subunit is produced in the liver.46 Factor XIIIa covalently crosslinks fibrin polymers to form a stable insoluble fibrin clot. Factor XIIIa is a transglutaminase that catalyzes the formation of covalent bonds between the carboxyl terminals of γ chains from adjacent D domains in the fibrin polymer. These bonds link the ε-amino acid of lysine moieties and the γ-amide group of glutamine units. Multiple cross-links form to provide an insoluble meshwork of fibrin polymers linked by their D domains, providing physical strength to the fibrin clot. Factor XIIIa reacts with other plasma and cellular structural proteins and is essential to wound healing and tissue integrity. Cross-linking of fibrin polymers by factor XIIIa covalently incorporates fibronectin, a plasma protein involved in cell adhesion, and α2-antiplasmin, rendering the fibrin mesh resistant to fibrinolysis. Plasminogen, the primary serine protease of the fibrinolytic system, also becomes covalently bound via lysine moieties, as does TPA, a serine protease that ultimately hydrolyzes and activates bound plasminogen to initiate fibrinolysis.

Plasma-based (in vitro) coagulation: Extrinsic, intrinsic, and common pathways

In the past, two coagulation pathways were described, both of which activated factor X at the start of a common pathway leading to thrombin generation (). The pathways were characterized as cascades in that as one enzyme became activated, it in turn activated the next enzyme in sequence. Most coagulation experts identified the activation of factor XII as the primary step in coagulation because this factor could be found in blood, whereas tissue factor could not. Consequently, the reaction system that begins with factor XII and culminates in fibrin polymerization has been called the Figure 37-15intrinsic pathway. The coagulation factors of the intrinsic pathway, in order of reaction, are XII, pre-K, HMWK, XI, IX, VIII, X, V, prothrombin (II), and fibrinogen. The laboratory test that detects the absence of one or more of these factors is the activated partial thromboplastin time (APTT or PTT; Chapter 42). We now know that the contact factors XII, pre-K, and HMWK do not play a significant role in in vivo coagulation with trauma-type injuries, although their deficiencies prolong the in vitro laboratory tests of the intrinsic pathway, in particular, the PTT.

Image 

FIGURE 37-15 Plasma-based in vitro coagulation: intrinsic, extrinsic, and common pathways. In the intrinsic pathway, the contact factors XII, prekallikrein (pre-K ), and high-molecular-weight kininogen (HMWK ) are activated and proceed to activate factors XI, IX, VIII, X, and V and prothrombin, which converts fibrinogen to fibrin. In the extrinsic pathway, exposed tissue factor (TF ) on subendothelial cells activates factor VII, which activates factors X, V, and prothrombin, cleaving fibrinogen to fibrin. Both the intrinsic and extrinsic pathways converge with the activation of factor X, so factors X, V, prothrombin, and fibrinogen are called the common pathway.

Formation of TF:VIIa has since proven to be the primary in vivo initiation mechanism for coagulation. Because tissue factor is not present in blood, the tissue factor pathway has been called the extrinsic pathway. This pathway includes the factors VII, X, V, prothrombin, and fibrinogen. The test used to measure the integrity of the extrinsic pathway is the prothrombin time test (PT; Chapter 42).

The PT and PTT are assays often used in tandem to screen for coagulation factor deficiencies. Factor VIII and factor IX are not considered to be part of the extrinsic pathway, because the PT fails to identify their absence or deficiency. But clearly, the IXa:VIIIa complex in the intrinsic pathway is crucial to the activation of factor X. Deficiencies of either one of these components—factor VIII in hemophilia A, or factor IX in hemophilia B—can result in severe and life-threatening hemorrhage.

The two pathways have in common factor X, factor V, prothrombin, and fibrinogen; this portion of the coagulation pathway is often called the common pathway. These designations—intrinsic, extrinsic, and common—are used extensively to interpret in vitro laboratory testing and to identify factor deficiencies; however, they do not adequately describe the complex interdependent reactions that occur in vivo.

Cell-based (in vivo, physiologic) coagulation

An intricate combination of cellular and biochemical events function in harmony to keep blood liquid within the veins and arteries, to prevent blood loss from injuries by the formation of thrombi, and to reestablish blood flow during the healing process.47 As noted above, the series of cascading proteolytic reactions traditionally known as the extrinsic and intrinsic coagulation pathways do not fully describe how coagulation occurs in vivo. These pathways are not distinct, independent, alternative mechanisms for generating thrombin but are actually interdependent. For example, a deficiency of factor VII in the extrinsic pathway can cause significant bleeding, even when the intrinsic pathway is intact. Similarly, deficiencies of factors VIII and IX may cause severe bleeding, regardless of the presence of a normal extrinsic pathway.48

In addition to procoagulant and anticoagulant plasma proteins, normal physiologic coagulation requires the presence of two cell types for formation of coagulation complexes: cells that express tissue factor (usually extravascular) and platelets (intravascular) ().Figure 37-1649 Operationally, coagulation can be described as occurring in two phases: initiation, which occurs on tissue factor–expressing cells and produces 3% to 5% of the total thrombin generated, and propagation, occurring on platelets, which produces 95% or more of the total thrombin.50

Image 

FIGURE 37-16 Cell-based in vivo physiologic coagulation. VIIa binds to tissue factor (TF ) and activates both factors X and IX. Cell-bound factor Xa combines with Va and generates a small amount of thrombin (Thr), which activates platelets, V, VIII, and XI and begins fibrin formation. Factor IXa, activated by both TF:VIIa and XIa, combines with factor VIIIa on the platelet surface to activate X, which forms prothrombinase (Xa:Va) and produces a burst of thrombin.

Initiation

In vivo, the principle mechanism for generating thrombin is begun by formation of the extrinsic tenase complex, rather than the intrinsic pathway. The initiation phase refers to extrinsic tenase complex formation and generation of small amounts of factor Xa, factor IXa, and thrombin (Figure 37-16).

Damage to the endothelium spills blood and platelets into the extravascular tissue and triggers a localized response. The magnitude of the response depends largely on the extent of the injury: how large the bleed is, how much tissue is damaged, and how many platelets are available.

About 1% to 2% of factor VIIa is present normally in blood in the activated form, but it is inert until bound to tissue factor51 and is unaffected by TFPI and other inhibitors. Fibroblasts and other subendothelial cells provide tissue factor, a cofactor to factor VIIa. Factor VIIa binds to tissue factor on the membrane of subendothelial cells, and the extrinsic tenase complex TF:VIIa is formed.

TF:VIIa activates low levels of both factor IX and factor X. Minute amounts of thrombin are generated by membrane-bound Xa and Xa:Va prothrombinase complexes. Factor Va comes from the activation of plasma factor V by thrombin, by platelets if there has been an injury, or by noncoagulation proteases.52

Coagulation complexes bound to cell membranes are relatively protected from inactivation by most inhibitors. However, if Xa: Va dissociates from the cell, it is rapidly inactivated by the protease inhibitors TFPI, antithrombin, and protein Z–dependent protease inhibitor (ZPI) until a threshold of Xa:Va activity is reached.

Even though the amount of thrombin generated in this phase is minute, platelets, cofactors, and procoagulants become activated; fibrin formation begins; and the initial platelet plug is formed. The low level of thrombin generated in the initiation phase (1) activates platelets through cleavage of protease activated receptors PAR-1 and PAR-4; (2) activates factor V released from platelet α-granules; (3) activates factor VIII and dissociates it from VWF; (4) activates factor XI, the intrinsic accessory procoagulant that activates more factor IX; and (5) splits fibrinogen peptides A and B from fibrinogen and forms a preliminary fibrin network.

Cleavage of fibrinopeptides occurs at the end of the initiation phase and beginning of the propagation phase. In most clot-based coagulation assays, this is the visual endpoint of the assay.45 It occurs with only 10 to 30 nmol/L of thrombin, or approximately 3% of the total thrombin generated.

Propagation

More than 95% of thrombin generation occurs during propagation. In this phase the reactions occur on the surface of the activated platelet, which now has all the components needed for coagulation. Large numbers of platelets adhere to the site of injury, localizing the coagulation response. Platelets are activated at the site of injury by both the low-level thrombin generated in the initiation phase and by adhering to exposed collagen (Figure 37-16). They are sometimes referred to as COAT-platelets: platelets partially activated by collagen and thrombin.53

These partially activated COAT-platelets have a higher level of procoagulant activity than platelets exposed to collagen alone. They also provide a surface for formation and amplification of intrinsic tenase and prothrombinase complexes.

The cofactors Va and VIIIa activated by thrombin in the initiation phase bind to platelet membranes and become receptors for Xa and IXa. IXa generated in the initiation phase binds to VIIIa on the platelet membrane to form the intrinsic tenase complex IXa:VIIIa. More factor IXa is also generated by platelet-bound factor XIa. This intrinsic tenase complex activates factor X at a 50- to 100-fold higher rate than the extrinsic tenase complex.49 Factor Xa binds to Va to form the prothrombinase complex, which activates prothrombin and generates a burst of thrombin. Thrombin cleaves fibrinogen into a fibrin clot, activates factor XIII to stabilize the clot, binds to thrombomodulin to activate the protein C control pathway, and activates TAFI to inhibit fibrinolysis.

Since coagulation depends on the presence of both tissue factor–bearing cells and activated platelets, clotting is localized to the site of injury. Protease inhibitors and intact endothelium prevent clotting from spreading to other parts of the body.

It may be helpful operationally to think of the extrinsic or tissue factor pathway as occurring on the tissue factor–bearing cell and the intrinsic pathway (minus factors XII, HMWK, and pre-K) as occurring on the platelet surface. However, these are not separate and redundant pathways; they are interdependent and occur in parallel until blood flow has ceased and termination by control mechanisms takes place.

Both platelets and tissue factor–bearing cells are essential for physiologic coagulation. Deficiencies of any of the key proteins of coagulation complex formation and activity (VII, IX, VIII, X, V, or prothrombin) compromise thrombin generation and manifest as significant bleeding disorders.

Coagulation regulatory mechanisms

Inhibitors and their cofactors regulate serine proteases and cofactors in the coagulation system. They also provide feedback loops to maintain a complex and delicate balance between thrombosis and abnormal bleeding. These inhibitors, or natural anticoagulants, function to slow the activation of procoagulants and suppress thrombin production. They ensure that coagulation is localized and is not a systemic response, and they prevent excessive clotting or thrombosis. The principal regulators are TFPI, antithrombin (AT), and activated protein C, the endpoint of the protein C pathway. Acquired or inherited deficiencies of these proteins may be associated with increased incidence of venous thromboembolic disease, as the hemostatic balance is shifted more toward coagulation than termination of the activated pathway. illustrates coagulation mechanism regulatory points. Characteristics of these and other coagulation regulatory proteins are summarized in Figure 37-17Table 37-10.

Image 

FIGURE 37-17 Coagulation pathway showing regulatory points. TFPI, Tissue factor pathway inhibitor; AT, antithrombin; APC, activated protein C; ZPI, protein Z–dependent protease inhibitor.

TABLE 37-10

Coagulation Regulatory Proteins

Name

Function

Molecular Mass (Daltons)

Half-Life (Hours)

Mean Plasma Concentration

Tissue factor pathway inhibitor

With Xa, binds TF:VIIa

33,000

Unknown

60–80 ng/mL

Thrombomodulin

EC surface receptor for thrombin

450,000

Does not circulate

None

Protein C

Serine protease

62,000

7–9

2–6 μg/mL

Protein S

Cofactor

75,000

Unknown

20–25 μg/mL

Antithrombin

Serpin

58,000

68

24–40 mg/dL

Heparin cofactor II

Serpin

65,000

60

30–70 μg/mL

Z-dependent protease inhibitor

Serpin

72,000

Unknown

1.5 μg/mL

α1-Protease inhibitor (α1-antitrypsin)

Serpin

60,000

Unknown

250 mg/dL

α2-Macroglobulin

Serpin

725,000

60

150–400 mg/dL

Serpin, Serine protease inhibitor; EC, endothelial cell.

Tissue factor pathway inhibitor

TFPI is a Kunitz-type serine protease inhibitor and is the principal regulator of the tissue factor pathway. The Kunitz-2 domain binds to and inhibits factor Xa, and Kunitz-1 binds to and inhibits VIIa:TF.54 TFPI is synthesized primarily by ECs and is also expressed on platelets. In the initiation of coagulation, factor VIIa and tissue factor combine to activate factors IX and X. TFPI inhibits coagulation in a two-step process by first binding and inactivating Xa. The TFPI:Xa complex then binds to TF:VIIa, forming a quaternary complex and preventing further activation of X and IX (Figure 37-18).5556 Alternatively, TFPI may bind to Xa in the TF:VIIa:Xa complex and inactivate Xa and TF:VIIa. TFPI provides feedback inhibition, because it is not actively engaged until coagulation is initiated and factor X is activated. Protein S, the cofactor of activated protein C (APC), is also a cofactor of TFPI and enhances factor Xa inhibition by TFPI tenfold.57-59 Because of the inhibitory action of TFPI, the TF:VIIa:Xa reaction is short-lived. Once TFPI shuts down extrinsic tenase and Xa, additional Xa and IXa production shifts to the intrinsic pathway.60 Propagation of coagulation occurs as factor X is activated by IXa:VIII and more factor IX is activated by factor XIa.

Image 

FIGURE 37-18 Tissue factor pathway inhibitor. TFPI binds the complex of tissue factor (TF ) and factors VIIa and Xa in a Xa-dependent feedback mechanism. A, TFPI first binds to factor Xa and inactivates it. B, The TFPI:Xa complex then binds and inactivates TF:VIIa, preventing more activation of Xa. Alternatively, TFPI may bind directly to Xa and VIIa in the TF:VIIa:Xa complex.

Protein C regulatory system

During coagulation, thrombin propagates the clot as it cleaves fibrinogen and activates factors V, VIII, XI, and XIII. In intact normal vessels, where coagulation would be inappropriate, thrombin avidly binds the EC membrane protein thrombomodulin and triggers an essential coagulation regulatory system called the protein C anticoagulant system.61 The protein C system revises thrombin’s function from a procoagulant enzyme to an anticoagulant. EC protein C receptor (EPCR) is a transmembrane protein that binds both protein C and APC adjacent to the thrombomodulin-thrombin complex and augments the action of thrombin-thrombomodulin at least fivefold in activating protein C to a serine protease (Figure 37-19).6263 APC dissociates from EPCR and binds its cofactor, free plasma protein S. The stabilized APC-protein S complex hydrolyzes and inactivates factors Va and VIIIa, slowing or blocking thrombin generation/coagulation.

Image 

FIGURE 37-19 Protein C pathway. After binding thrombomodulin (TM ), thrombin activates protein C (PC), bound by endothelial cell protein C receptor (EPCR). Free protein S (PS) [not bound to C4b binding protein (C4bBP)] binds and stabilizes activated protein C (APC). The APC/protein S complex digests and inactivates factors Va ( Vi, inhibited factor V ) and VIIIa ( VIIIi, inhibited factor VIII).

Protein S, the cofactor that binds and stabilizes APC, is synthesized in the liver and circulates in the plasma in two forms. About 40% of protein S is free, but 60% is covalently bound to the complement control protein C4b-binding protein (C4bBP).64 Bound protein S cannot participate in the protein C anticoagulant pathway; only free plasma protein S can serve as the APC cofactor. Protein S-C4bBP binding is of particular interest in inflammatory conditions because C4bBP is an acute phase reactant. When the plasma C4bBP level increases, additional protein S is bound, and free protein S levels become proportionally decreased, which may increase the risk of thrombosis. Chronic acquired or inherited protein C or protein S deficiency or mutations of protein C, protein S, or factor V compromise the normal downregulation of factors Va and VIIIa and may be associated with recurrent venous thromboembolic disease (Chapter 39). Underscoring the importance of the protein C regulatory system, neonates who completely lack protein C have a massive thrombotic condition called purpura fulminans and die in infancy unless treated with protein C replacement and anticoagulation.6566

Antithrombin and other serine protease inhibitors (serpins)

Antithrombin (AT) was the first of the coagulation regulatory proteins to be identified and the first to be assayed routinely in the clinical hemostasis laboratory.67 Other members of the serpin family include heparin cofactor II, protein Z-dependent protease inhibitor (ZPI), protein C inhibitor, α1-protease inhibitor (α1-antitrypsin), α2-macroglobulin, α2-antiplasmin, and PAI-1.68

AT is a serine protease inhibitor (serpin) that binds and neutralizes serine proteases, including thrombin (factor IIa) and factors IXa, Xa, XIa, XIIa, prekallikrein, and plasmin.69 Heparin cofactor II is a serpin that primarily inactivates thrombin. AT and heparin cofactor II both require heparin for effective anticoagulant activity. In vivo, heparin is available from endothelium-associated mast cell granules or as EC heparan sulfate, a natural glycosaminoglycan that activates AT, although not to the same intensity as therapeutic unfractionated heparin. AT’s activity is accelerated 2000-fold by binding to heparin and is the basis for the anticoagulant activity of pharmaceutical heparin. Therapeutically, heparin is administered as unfractionated heparin, low-molecular-weight heparin, or heparin pentasaccharide. Unfractionated heparin consists of chains of greater than 18 sugar units and accelerates inactivation of thrombin through heparin-dependent conformational changes and bridging mechanisms (Figure 37-20). With low-molecular-weight and pentasaccharide heparins lacking long polysaccharide chains for thrombin inactivation, AT preferentially inactivates factor Xa (Chapter 43).

Image 

FIGURE 37-20 Unfractionated heparin potentiates antithrombin-thrombin reaction. Antithrombin (AT  ) attaches to a specific pentasaccharide sequence in unfractionated heparin. The thrombin binding site for heparin is adjacent to the AT site. The AT is sterically modified to covalently bind and inactivate the thrombin active protease site. Thrombin and AT, covalently bound, release from heparin and form measurable plasma thrombin-antithrombin (TAT  ) complexes, useful as a marker of coagulation activation.

In vivo, antithrombin covalently binds thrombin, forming an inactive thrombin-antithrombin complex (TAT), which is then released from the heparin molecule. Laboratory measurement of TAT is used as an indicator for thrombosis, since it measures both the generation of thrombin and its inhibition.

ZPI, in the presence of its cofactor, protein Z, is a potent inhibitor of factor Xa.7071 ZPI covalently binds protein Z and factor Xa in a complex with Ca2+ and phospholipid. Protein Z is a vitamin K–dependent plasma glycoprotein that is synthesized in the liver. Although protein Z has a structure similar to that of the other vitamin K–dependent proteins (factors II, VII, IX, and X and protein C), it lacks an activation site and, like protein S, is nonproteolytic. Protein Z increases the ability of ZPI to inhibit factor Xa 2000-fold.72 ZPI also inhibits factor XIa, in a separate reaction that does not require protein Z, phospholipid, and Ca2+. The inhibition of factor XIa is accelerated twofold by the presence of heparin.

Protein C inhibitor is a nonspecific, heparin-binding serpin that inhibits a variety of proteases, including APC, thrombin, factor Xa, factor XIa, and urokinase. It is found not only in plasma but also in many other body fluids and organs. Depending on its target, it can function as an anticoagulant (inhibits thrombin), as a procoagulant (inhibits thrombin-thrombomodulin and APC), or as a fibrinolytic inhibitor.

The serpins α1-protease inhibitor and α2-macroglobulin are able to inhibit serine proteases reversibly. See Table 37-11 and the section on fibrinolysis for further information on α2-antiplasmin and PAI-1.

TABLE 37-11

Proteins of the Fibrinolysis Pathway

Name

Function

Molecular Mass (Daltons)

Half-Life

Mean Plasma Concentration

Plasminogen

Plasma serine protease, plasmin digests fibrin/fibrinogen

92,000

24–26 hr

15–21 mg/dL

Tissue plasminogen activator

Serine protease secreted by activated endothelium, activates plasminogen

68,000

Unknown

4–7 μg/dL

Urokinase

Serine protease secreted by kidney, activates plasminogen

54,000

Unknown

Plasminogen activator inhibitor-1

Secreted by endothelium, inhibits tissue plasminogen activator

52,000

1 hr

14–28 mg/dL

α2-Antiplasmin

Inhibits plasmin

51,000

Unknown

7 mg/dL

Thrombin-activatable fibrinolysis inhibitor

Suppresses fibrinolysis by removing fibrin C-terminal lysine binding sites

55,000

8–10 min

5 μg/mL

Fibrinolysis

Fibrinolysis, the final stage of coagulation (), begins a few hours after fibrin polymerization and cross-linking. Two activators of fibrinolysis, TPA and UPA, are released in response to inflammation and coagulation. Fibrinolytic proteins assemble on fibrin during clotting. Plasminogen, plasmin, TPA, UPA, and PAI-1 become incorporated into the fibrin clot as they bind to lysine through their “kringle” loops, thereby concentrating and localizing them to the fibrin clot. Fibrinolysis is the systematic, accelerating hydrolysis of fibrin by bound plasmin. TPA and UPA activate fibrin-bound plasminogen several hours after thrombus formation, degrading fibrin and restoring normal blood flow during vascular repair. Again, there is a delicate balance between activators and inhibitors. Excessive fibrinolysis can cause bleeding due to Figure 37-21 premature clot lysis before wound healing is established, whereas inadequate fibrinolysis can lead to clot extension and thrombosis.

Image 

FIGURE 37-21 Fibrinolysis pathway and inhibitors. Plasminogen and tissue plasminogen activator (TPA) are bound to fibrin during coagulation. TPA converts bound plasminogen to plasmin, which slowly digests fibrin to form fibrin degradation products (FDPs) X, Y, D, E, and D-D (D-dimer). D-dimer is produced from cross-linked fibrin. Free plasmin is neutralized by α2-antiplasmin. TPA is neutralized by plasminogen activator inhibitor-1 (PAI-1). Thrombin-activatable fibrinolysis inhibitor (TAFI ) inhibits fibrinolysis by cleaving lysine residues on fibrin, preventing the binding of plasminogen, plasmin, and TPA.

Plasminogen and plasmin

Plasminogen is a 92,000 Dalton plasma zymogen produced by the liver (Table 37-11).7374 It is a single-chain protein possessing five glycosylated loops termed kringles. Kringles enable plasminogen, along with activators TPA and UPA, to bind fibrin lysine molecules during polymerization (Figure 37-22). This fibrin-binding step is essential to fibrinolysis. Fibrin-bound plasminogen becomes converted into a two-chain active plasmin molecule when cleaved between arginine at position 561 and valine at position 562 by neighboring fibrin-bound TPA or UPA. Plasmin is a serine protease that systematically digests fibrin polymer by the hydrolysis of arginine-related and lysine-related peptide bonds.75 Bound plasmin digests clots and restores blood vessel patency. Its localization to fibrin through lysine binding prevents systemic activity. As fibrin becomes digested, the exposed carboxy-terminal lysine residues bind additional plasminogen and TPA, which further accelerates clot digestion.7677 Free plasmin is capable of digesting plasma fibrinogen, factor V, factor VIII, and fibronectin, causing a potentially fatal primary fibrinolysis. However, plasma α2-antiplasmin rapidly binds and inactivates any free plasmin in the circulation.

Image 

FIGURE 37-22 Schematic diagram of the action of fibrinolytic proteins. A, Tissue plasminogen activator (TPA) activates plasminogen to the serine protease, plasmin. TPA is inhibited by plasminogen activator inhibitor-1 (PAI-1). α2-Antiplasmin (AP) rapidly inactivates free plasmin. B, Fibrinolytic proteins TPA, plasminogen, and plasmin bind to fibrin C-terminal lysine (Lys) during clotting. Thrombin activatable fibrinolysis inhibitor (TAFI ) inhibits fibrinolysis by removing the C-terminal Lys from fibrin, thereby reducing binding of fibrinolytic proteins. AP N-terminus is bound to fibrin by FXIIIa. The AP C-terminus Lys competes with fibrin C-terminus Lys to bind plasmin and inactivates it.

Plasminogen activation

Tissue plasminogen activator (TPA)

ECs secrete TPA, which hydrolyzes fibrin-bound plasminogen and initiates fibrinolysis. TPA, with two glycosylated kringle regions, forms covalent lysine bonds with fibrin during polymerization and localizes at the surface of the thrombus with plasminogen, where it begins the digestion process by converting plasminogen to plasmin. Circulating TPA is bound to inhibitors such as PAI-1 and is cleared from plasma. Synthetic recombinant TPAs mimic intrinsic TPA and are a family of drugs used to dissolve pathologic clots that form in venous and arterial thrombotic disease.

Urokinase plasminogen activator (UPA)

Urinary tract epithelial cells, monocytes, and macrophages secrete another intrinsic plasminogen activator called urokinase plasminogen activator. UPA circulates in plasma at a concentration of 2 to 4 ng/mL and becomes incorporated into the mix of fibrin-bound plasminogen and TPA at the time of thrombus formation. UPA has only one kringle region, does not bind firmly to fibrin, and has a relatively minor physiologic effect. Like TPA, purified UPA preparations are used to dissolve thrombi in myocardial infarction, stroke, and deep vein thrombosis.

Control of fibrinolysis

Plasminogen activator inhibitor 1 (PAI-1)

PAI-1 is the principal inhibitor of plasminogen activation, inactivating both TPA and UPA and thus preventing them from converting plasminogen to the fibrinolytic enzyme plasmin. PAI-1 is a single-chain glycoprotein serine protease inhibitor and is produced by ECs, megakaryocytes, smooth muscle cells, fibroblasts, monocytes, adipocytes, hepatocytes, and other cell types.7879 Platelets store a pool of PAI-1, accounting for more than half of its availability and for its delivery to the fibrin clot. PAI-1 is present in excess of the TPA concentration in plasma, and circulating TPA normally becomes bound to PAI-1. Only at times of EC activation, such as after trauma, does the level of TPA secretion exceed that of PAI-1 to initiate fibrinolysis. Binding of TPA to fibrin protects TPA from PAI-1 inhibition.80 Plasma PAI-1 levels vary widely. PAI-1 deficiency has been associated with chronic mild bleeding due to increased fibrinolysis. PAI-1 is an acute phase reactant and is increased in many conditions, including metabolic syndrome, obesity, atherosclerosis, sepsis, and stroke.79 Increased PAI-1 levels correlate with reduced fibrinolytic activity and increased risk of thrombosis.

α2-antiplasmin

α2-Antiplasmin (AP) is synthesized in the liver and is the primary inhibitor of free plasmin. AP is a serine protease inhibitor with the unique characteristic of both N- and C-terminal extensions.81 During thrombus formation, the N-terminus of AP is covalently linked to fibrin by factor XIIIa (Figure 37-22).82 The C-terminal contains lysine, which is capable of reacting with the lysine-binding kringles of plasmin. Free plasmin produced by activation of plasminogen can bind either to fibrin, where it is protected from AP because its lysine-binding site is occupied, or to the C-terminus of AP, which rapidly and irreversibly inactivates it. Thus AP with its C-terminal lysine slows fibrinolysis by competing with lysine residues in fibrin for plasminogen binding and by binding directly to plasmin and inactivating it.

The therapeutic lysine analogues, tranexamic acid and ε-aminocaproic acid, are similarly antifibrinolytic through their affinity for kringles in plasminogen and TPA. Both inhibit the proteolytic activity of plasmin.

Thrombin-activatable fibrinolysis inhibitor

TAFI is a plasma procarboxypeptidase synthesized in the liver that becomes activated by the thrombin-thrombomodulin complex. This is the same complex that activates the protein C pathway; however, the two functions are independent. Activated TAFI functions as an antifibrinolytic enzyme. It inhibits fibrinolysis by cleaving exposed carboxy-terminal lysine residues from partially degraded fibrin, thereby preventing the binding of TPA and plasminogen to fibrin and blocking the formation of plasmin (Figure 37-22).83 In coagulation factor–deficient states, such as hemophilia, decreased thrombin production may reduce the activation of TAFI, resulting in increased fibrinolysis that contributes to more bleeding. Conversely, in thrombotic disorders, increased thrombin generation may increase the activation of TAFI. The resulting decreased fibrinolysis may contribute further to thrombosis. TAFI also may play a role in regulating inflammation and wound healing.84

Fibrin degradation products and D-dimer

Plasmin cleaves fibrin and produces a series of identifiable fibrin fragments: X, Y, D, E, and D-D ().Figure 37-2385 Several of these fragments inhibit hemostasis and contribute to hemorrhage by preventing platelet activation and by hindering fibrin polymerization. Fragment X is described as the central E domain with the two D domains (D-E-D), minus some peptides cleaved by plasmin. Fragment Y is the E domain after cleavage of one D domain (D-E). Eventually these fragments are further digested to individual D and E domains.

Image 

FIGURE 37-23 Degradation of fibrinogen and fibrin by plasmin. Plasmin systematically degrades fibrinogen and fibrin by digestion of small peptides and cleavage of D-E domains. From fibrinogen, fragment X consists of a central E domain with two D domains (D-E-D); further cleavage produces fragment Y (D-E), with eventual degradation to D and E domains. From cross-linked fibrin, plasmin digestion produces fragment complexes from one or more monomers. D-dimer consists of two D domains from adjacent monomers that have been cross-linked by factor XIIIa in the process of fibrin formation (thrombosis).

The D-D fragment, called D-dimer, is composed of two D domains from separate fibrin molecules cross-linked by the action of factor XIIIa. Fragments X, Y, D, and E are produced by digestion of either fibrin or fibrinogen by plasmin, but D-dimer is a specific product of digestion of cross-linked fibrin only and is therefore a marker of thrombosis and fibrinolysis—that is, thrombin, factor XIIIa, and plasmin activation.

The various fragments may be detected by quantitative or semiquantitative immunoassay to reveal fibrinolytic activity. D-dimer is separately detectable by monoclonal antibody for D-dimer antigen, using a wide variety of automated quantitative laboratory immunoassays and other formats including point-of-care tests performed on whole blood.8687 The D-dimer immunoassay is used to identify chronic and acute DIC and to rule out venous thromboembolism in suspected cases of deep venous thrombosis or pulmonary embolism.

Summary

• The vascular intima, platelets, tissue factor–bearing cells, and coagulation and fibrinolytic proteins interact to maintain hemostasis.

• Intact vascular intima prevents coagulation through synthesis of prostacyclin, nitric oxide, TFPI, thrombomodulin, and heparan sulfate.

• Damaged intima promotes coagulation by vasoconstriction, exposure of tissue factor and collagen, and secretion of VWF and other adhesion molecules.

• Platelets function in primary and secondary hemostasis through adhesion, aggregation, and secretion of granular contents.

• Platelets adhere to collagen through VWF and use fibrinogen to aggregate.

• Most coagulation factors are produced in the liver.

• The plasma factors of the prothrombin group (prothrombin; factors VII, IX, and X; protein C; protein S; and protein Z) require vitamin K in their production.

• Plasma coagulation factors include trypsin-like enzymes called serine proteases and cofactors that stabilize the proteases. Factor XIIIa is a transamidase.

• The extrinsic pathway of coagulation consists of the membrane receptor tissue factor and coagulation factors VII, X, V, II, and I. The PT is a screening test for these factors.

• The intrinsic pathway factors are XII, pre-K, HMWK, XI, IX, VIII, X, V, II, and I. The PTT is a screening test for these factors.

• Activation of coagulation pathways produces thrombin, which converts fibrinogen to a fibrin polymer. Thrombin also activates platelets and factors V, VIII, XI, and XIII, and binds to thrombomodulin to activate protein C and TAFI.

• Fibrinogen is cleaved by thrombin to form first fibrin monomer, then fibrin polymer, and finally, when acted on by factor XIIIa, cross-linked fibrin.

• In vivo, coagulation is initiated on tissue factor–bearing cells. TF:VIIa activates factors IX and X, generating enough thrombin to activate platelets and factors V, VIII, and XI. Coagulation proceeds on activated platelet phospholipid membranes with the formation of IXa:VIIIa and Xa:Va complexes, which produces a burst of thrombin that cleaves fibrinogen to fibrin.

• The coagulation pathway is regulated by TFPI, APC, and the serpins, including antithrombin and ZPI. These control proteins prevent thrombosis and confine clotting to the site of injury.

• The fibrinolytic pathway digests the thrombus. Plasminogen is converted to plasmin by TPA. Plasmin degrades fibrin to fragments X, Y, D, and E, and D-dimer. Control proteins are PAI-1, α2-antiplasmin, and TAFI.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. What intimal cell synthesizes and stores VWF?

a. Smooth muscle cell

b. Endothelial cell

c. Fibroblast

d. Platelet

2. What subendothelial structural protein triggers coagulation through activation of factor VII?

a. Thrombomodulin

b. Nitric oxide

c. Tissue factor

d. Thrombin

3. What coagulation plasma protein should be assayed when platelets fail to aggregate properly?

a. Factor VIII

b. Fibrinogen

c. Thrombin

d. Factor X

4. What role does vitamin K play for the prothrombin group factors?

a. Provides a surface on which the proteolytic reactions of the factors occur

b. Protects them from inappropriate activation by compounds such as thrombin

c. Accelerates the binding of the serine proteases and their cofactors

d. Carboxylates the factors to allow calcium binding

5. What is the source of fibrinopeptides A and B?

a. Plasmin proteolysis of fibrin polymer

b. Thrombin proteolysis of fibrinogen

c. Proteolysis of prothrombin by factor Xa

d. Plasmin proteolysis of cross-linked fibrin

6. What serine protease forms a complex with factor VIIIa, and what is the substrate of this complex?

a. Factor VIIa, factor X

b. Factor Va, prothrombin

c. Factor Xa, prothrombin

d. Factor IXa, factor X

7. What protein secreted by endothelial cells activates fibrinolysis?

a. Plasminogen

b. TPA

c. PAI-1

d. TAFI

8. What two regulatory proteins form a complex that digests activated factors V and VIII?

a. TFPI and Xa

b. Antithrombin and protein C

c. APC and protein S

d. Thrombomodulin and plasmin

9. Coagulation factor VIII circulates bound to:

a. VWF

b. Factor IX

c. Platelets

d. Factor V

10. Most coagulation factors are synthesized in:

a. The liver

b. Monocytes

c. Endothelial cells

d. Megakaryocytes

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