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Introduction
Our understanding of blood clotting is intimately tied to the history of civilization. With the advent of writing 5000 years ago, it could be argued that the first symbols used for blood, bleeding, or clot- ting represented the first published coagulation pathway. The ancient peoples of the world always held blood in utmost mystical esteem. Through the ages, this esteem has been transmitted to mod- ern times in the many expressions that use “blood,” such as “blood is thicker than water,” “blood of our fathers,” and others. Mysticism aside, the study of blood clotting and the development of laboratory tests for blood clotting abnormalities are historically inseparable. The workhorse tests of the modern coagulation laboratory, the prothrombin time (PT) and the acti- vated partial thromboplastin time (aPTT), are the basis for the published extrinsic and intrinsic coagulation pathways, even though it is now known that these pathways do not accurately reflect the function of blood clotting in a living organism. In this chapter, and ultimately this text- book, the many authors hope to present a clear explanation of coagulation testing and its impor- tant place in the medical armamentarium for diag- nosing and treating disease.
Constituents of the
Hemostatic System
With the evolution of vertebrates and their pres- surized circulatory system, there had to arise some method to seal the system if injured—hence the hemostatic system. Interestingly, there is nothing quite comparable to the vertebrate hemostatic sys- tem in invertebrate species. In all vertebrates stud- ied, the basic constituents of the hemostatic sys- tem appear to be conserved. Figure 1-1 illustrates the three major con- stituents of the hemostatic pathways and how they are interrelated. Each element of the hemosta-
tic system occupies a site at the vertex of an equi- lateral triangle. This representation implies that each system constituent interacts with and influ- ences all other constituents. In the normal resting state, these interactions conspire to maintain the fluidity of the blood to ensure survival of the organism. Normally, only at the site of an injury will the fluidity of the blood be altered and a blood clot form.
Endothelium
Figure 1-2 shows some of the basic properties of the endothelium. The endothelium normally pro- motes blood fluidity, unless there is an injury. With damage, the normal response is to promote coag- ulation at the wound site while containing the coagulation response and not allowing it to prop- agate beyond this site. Until recently, the dogma of blood clotting sug- gested that the single, major procoagulant func- tion of the endothelium is to make and express tis- sue factor with injury. Endothelial cells do not normally make tissue factor but may synthesize it following cytokine stimulation or acquire the material from activated monocytes in the circula- tion. Tissue factor is a glycosylated intrinsic mem- brane protein that is expressed on the surface of
Coagulation Pathway and Physiology
Jerry B. Lefkowitz, MD
Figure 1-1. Basic representation of the elements of hemostasis.
Coagulation Proteins
Platelets Endothelium
CHAPTER 1
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adventitial vascular wall cells and is exposed to flowing blood during vascular injury or endothe- lial denudation. Tissue factor, when bound to fac- tor VIIa, is the major activator of the extrinsic pathway of coagulation. Classically, tissue factor is not present in the plasma but only presented on cell surfaces at a wound site. Because tissue factor is “extrinsic” to the circulation, the pathway was thusly named. Endothelium is the major synthetic and storage site for von Willebrand factor (vWF). Von Willebrand factor is secreted from the endothelial cell both into the plasma and also abluminally into the subendothelial matrix. It is a large multimeric protein that acts as the intercellular glue binding platelets to one another and also to the suben- dothelial matrix at an injury site. In addition, the second major function of vWF is to act as a carrier protein for factor VIII (antihemophilic factor). Von Willebrand factor is synthesized in the endothelial cell as a dimeric protein, with a molecular weight of the monomer approximately 250,000 daltons. The propeptide of vWF, the N-terminal portion of the protein that is cleaved off prior to secretion or
storage, is important in directing the multimer- ization of vWF. Von Willebrand factor multimers can range in size up to 20 x 10 6 daltons. Von Willebrand factor binds to the platelet glycopro- tein Ib/IX/V receptor and mediates platelet adhe- sion to the vascular wall under shear, which is dis- cussed in more detail in chapter 2. The remaining endothelial procoagulant func- tions discussed in this brief review and listed in Figure 1-2 are all parts of the fibrinolytic system, discussed in more detail in chapter 3. The fibri- nolytic system is responsible for proteolysis and solubilization of the formed clot constituents to allow its removal. Plasminogen activator inhibitor acts to block the ability of tissue plasminogen acti- vator to turn on plasmin, the primary enzyme of fibrinolysis. The thrombin activatable fibrinolytic inhibitor (TAFI), similar to the thrombin regulato- ry enzyme protein C, is cleaved to its activated form by the thrombin/thrombomodulin complex. The endothelial cell surface receptor thrombo- modulin is a 450,000-dalton protein whose only known ligand is thrombin. Thrombin, once bound to thrombomodulin, loses some proteolytic capa-
Figure 1-2. A stylized view of endothelial functions related to procoagulation and anticoagulation. The subendothelial matrix, represented by the purple interlocking lines, is a com-
plex of many materials. The most important constituents of the subendothelial matrix related to coagulation function are collagen and von Willebrand factor.
Procoagulant Anticoagulant
von Willebrand factor
Expression of tissue factor ??
Heparin-like material
Tissue factor pathway inhibitor (TFPI)
Tissue plasminogen activator (tPA)
Thrombin
Prostacyclin (PGI 2 )
Thrombomodulin receptor
Thrombin / thrombomodulin
Thrombin / thrombomodulin
Subendothelial matrix
Endothelial lining of vessel
Protein C Activated protein C (APC)
Plasminogen activator inhibitor (PAI-1)
Thrombin activatable fibrinolytic inhibitor (TAFI)
Activated TAFI
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Figure 1-3. A representation of the original extrinsic path- way proposed in 1905.
Name Description Function
Fibrinogen (Factor I) Molecular Weight (MW) = 340, daltons (Da); glycoprotein
Adhesive protein that forms the fibrin clot
Prothrombin (Factor II) MW = 72,000 Da; vitamin K-dependent serine protease
Activated form is main enzyme of coagulation Tissue factor (Factor III) MW = 37,000 Da; also known as thromboplastin
Lipoprotein initiator of extrinsic pathway
Calcium ions (Factor IV) Necessity of Ca++ ions for coagulation reactions described in 19th century
Metal cation necessary for coagulation reactions Factor V (Labile factor) MW = 330,000 Da Cofactor for activation of prothrombin to thrombin Factor VII (Proconvertin) MW = 50,000 Da; vitamin K-dependent serine protease
With tissue factor, initiates extrinsic pathway Factor VIII (Antihemophilic factor) MW = 330,000 Da Cofactor for intrinsic activation of factor X
Factor IX (Christmas factor) MW = 55,000 Da; vitamin K-dependent serine protease
Activated form is enzyme for intrinsic activation of factor X Factor X (Stuart-Prower factor) MW = 58,900 Da; vitamin K-dependent serine protease
Activated form is enzyme for final common pathway activation of prothrombin Factor XI (Plasma thromboplastin antecedent)
MW = 160,000 Da; serine protease Activated form is intrinsic activator of factor IX Factor XII (Hageman factor) MW = 80,000 Da; serine protease Factor that nominally starts aPTT-based intrinsic pathway Factor XIII (Fibrin stabilizing factor) MW = 320,000 Da Transamidase that cross-links fibrin clot
High-molecular-weight kininogen (Fitzgerald, Flaujeac, or William factor)
MW = 110,000 Da; circulates in a complex with factor XI
Cofactor
Prekallikrein (Fletcher factor) MW = 85,000 Da; serine protease Activated form that participates at beginning of aPTT-based intrinsic pathway
Table 1-1. Coagulation Factors
clot in vivo. Although discussed more fully in the following sections, evidence suggests that factor XII and prekallikrein may not normally participate in clotting in vivo but are important in the in vitro laboratory clot assays.
The Extrinsic Pathway and the PT
The first description of the extrinsic pathway was reported by Dr. Paul Morawitz in 1905. Dr. Morawitz produced a hemostasis model incorpo- rating all of the scientific information of his day. Figure 1-3 illustrates a version of this model. In 1935, Dr. Armand Quick published his method for the prothrombin time—with minor variations, the same laboratory test that is still in use today. Dr. Quick, using the classic four-compo- nent extrinsic pathway model of Dr. Morawitz,
essentially made “thrombokinase” with calcium ions. This “thrombokinase” was prepared from a saline extract of rabbit brain with the addition of calcium. The more modern nomenclature for this material is thromboplastin. The basis for Dr. Quick’s assay was that adding calcium ions with
Prothrombin
Thrombin
Fibrinogen Fibrin clot
Thrombokinase Calcium
Coagulation Pathway and Physiology 1
an excess of thromboplastin to anticoagulated plasma was a direct measure of the prothrombin amount in the plasma—hence the name of the assay, prothrombin time. Only in the 1950s and early 1960s, with the discovery of additional coag- ulation factors, did the true nature of the extrinsic pathway become known. This is discussed in more detail below in the section, “The PT and aPTT Pathways.”
The Intrinsic Pathway and the aPTT
Dr. Quick, in his first publication, observed that his new PT assay was not sensitive to the hemo- philic defect. Patients with the symptoms of hemo- philia did not usually have an abnormal PT. Evidence had been accumulating that the four- component extrinsic pathway model of blood clot- ting was not complete. The plasma had the poten- tial to clot without the addition of an extrinsic material. The thromboplastin, thrombokinase, or what we now call tissue factor was not always needed to make blood clot, especially in vitro. Therefore, it appeared that plasma had within it or intrinsic to it all the factors necessary to cause blood clotting. In 1953, Drs. Langdell, Wagner, and Brinkhous published a paper detailing a clot-based assay that was sensitive to the defect in hemophilic plasma. Instead of using a complete tissue extract, such as the prothrombin time thromboplastin reagent, their assay used only a partial extract. Hence these researchers called their assay a partial thrombo- plastin time (PTT). In this group’s initial assay, the activator necessary to cause clotting was added separately from the PTT reagent and calcium ions. Other workers modified the assay, adding an acti- vator to the PTT reagent, producing the modern activated thromboplastin time assay.
The PT and aPTT Pathways
The PT and aPTT assays were developed based on theories and specific testing needs, without com- plete knowledge of all the proteins involved in coagulation. In the period from 1935 and the inception of the PT until the early 1970s, all of the procoagulant proteins involved in forming a fibrin clot were identified. Many of these factors were identified because patients were found with defi- ciency states. Some of these patients had congeni- tal bleeding disease, while others presented with an abnormal prolongation in the PT and/or the aPTT. It became clear that a fresh model of coagu- lation other than the classic extrinsic pathway was needed.
In the early 1960s, a new synthesis of all hemo- stasis knowledge was put together, and the PT or extrinsic and aPTT or intrinsic coagulation path- ways were published. This provided a framework of how many of the proteins listed in Table 1- interact to form a blood clot. This pathway is illus- trated in Figure 1-4. Although there have been some modifications since the original papers, these are the pathways with which most workers in hemostasis are familiar. From the initial publica- tion, accumulating epidemiologic evidence sug- gests this formulation of the intrinsic and extrinsic hemostasis pathways might not be a correct repre- sentation of blood clotting in vivo. For example, patients deficient in factor XII, prekallikrein, or high-molecular-weight kininogen do not have a bleeding or thrombotic phenotype. All of these proteins are present at the start of the intrinsic pathway, and deficiencies of each factor can cause a significantly prolonged aPTT assay. Logically, it would make sense that a deficiency of a factor at the start of a pathway would cause bleeding pathology. However, all evidence suggests this is not so. The intrinsic and extrinsic pathways as they have existed since their inception are based on in vitro testing. The in vivo function appears to be different. It is important to be familiar with the older pathway model because the PT and the aPTT are still useful as diagnostic tests.
Newer Coagulation Model
Figure 1-5 illustrates a newer model of coagula- tion. In this figure, thrombin is depicted as the cen- ter of the coagulation universe; all aspects of hemostasis feed into the regulation and control of thrombin generation, which in turn forms the definitive clot at the site of an injury. Of note, the figure lacks several proteins normally considered part of the classic intrinsic coagulation pathway: factor XII and prekallikrein. These proteins are not currently thought to be important for in vivo coag- ulation activation. Although high-molecular- weight kininogen deficiency may not be associat- ed with a bleeding diathesis, it is still part of the new coagulation pathway as it circulates in plas- ma bound to factor XI. Also important in this newer concept is that the majority of the steps in the coagulation cascade take place by the forma- tion of multimolecular coagulation protein com- plexes on phospholipid cell surfaces. This new coagulation model has extrinsic and intrinsic pathway limbs, but the in vivo process of hemostasis is thought only to be initiated by cell- based tissue factor expressed at an injury site.
Coagulation Pathway and Physiology 1
Figure 1-5. A newer model of the coagulation pathway. For the sake of clarity, Ca++^ and phospholipids have been omitted from the figure. These two cofactors are necessary for all of the reactions listed in the figure that result in the activation of prothrombin to thrombin. The pathway is initiated by an extrinsic mechanism that generates small amounts of factor Xa, which in turn activate small amounts of thrombin.The tis- sue factor/factor VIIa proteolysis of factor X is quickly inhibit-
ed by tissue factor pathway inhibitor (TFPI).The small amounts of thrombin generated from the initial activation feedback to create activated cofactors, factors Va and VIIIa, which in turn help to generate more thrombin. Tissue factor/factor VIIa is also capable of indirectly activating factor X through the acti- vation of factor IX to factor IXa. Finally, as more thrombin is created, it activates factor XI to factor XIa, thereby enhancing the ability to ultimately make more thrombin.
Factor VII Factor VIIa + Tissue factor
Factor X
Factor Xa + Factor Va
Prothrombin
Thrombin activatable fibrinolytic inhibitor (TAFI) activation
Protein C activation
Platelet aggregation
Endothelial cell effects
Factor IXa + Factor VIIIa Factor VIII
Fibrinogen Fibrin monomer
Factor XIII
Fibrin polymer
Cross-linked clot
Factor XIIIa
Thrombin
Factor IX Factor XIa
Factor XI
Factor V
Activation of factors V and VIII by thrombin results in a further burst of coagulation activity through increased activity of the tenase and pro- thrombinase complexes. Fibrinogen is the ultimate substrate protein of the coagulation cascade and forms the principal structural protein of the fibrin clot. Fibrinogen, produced in the liver, is a dimer composed of three pairs of protein chains, Aα, Bβ, and γ, that are disulfide-linked at their N-terminal ends. Fibrinogen, as viewed by molecular imaging tech- niques, is composed of three globular domains, a central E domain flanked by two identical D
domains (Figure 1-6). Thrombin cleaves small peptides, termed fibrinopeptides A and B, from the Aα and Bβ chains, respectively, to form a fibrin monomer. These monomers assemble into protofibrils in a half-staggered, side-to-side fash- ion that is stabilized by noncovalent interactions between fibrin molecules. The protofibrils lateral- ly associate into thicker fibrin fibers and form the fibrin clot. This clot, however, is not stable and ultimately will come apart if not covalently cross- linked. Thrombin activates factor XIII to the trans- glutaminase enzyme factor XIIIa. Factor XIIIa, act- ing upon the glutamic acid and lysine side chains
I HEMOSTASIS PHYSIOLOGY
in the fibrin amino acid sequence, creates covalent bonds between fibrin monomer γ chains, creating a stable clot. In addition, factor XIIIa can covalent- ly cross-link a variety of other materials into the forming fibrin clot, including plasminogen and antiplasmin. This property of factor XIIIa is impor- tant for the penultimate purpose of the clot: wound healing and tissue repair. Finally, Figure 1-5 alludes to the fact that there are many properties of thrombin other than the formation of the fibrin clot. Thrombin has direct effects on the other constituents of the coagulation triad: platelets and endothelial cells. Additionally, thrombin participates in its own downregulation. In the next section, some of these coagulation reg- ulatory processes will be mentioned.
Regulatory Mechanisms
When a clot is formed, there has to be some mech- anism to limit the clot to the site of an injury and ultimately to remove the clot when that injury has healed. The clot removal system, or fibrinolysis pathway, consists of the zymogen plasminogen, a variety of activators, and several inhibitors. Primary among these activators is tissue plas- minogen activator (tPA), a product of endothelial cells. The fibrinolytic pathway is discussed in detail in chapter 3. The most important coagulation proteins that are involved in regulation of thrombin generation are summarized in Table 1-2. Tissue factor path- way inhibitor has been mentioned in both the “Endothelium” and “Newer Coagulation Model”
Figure 1-6. Fibrinogen is an abundant plasma protein that is a dimer of the Aα, Bβ, and γ chains connected by disulfide bonds. The fibrinogen dimer is composed of two flanking D globular domains with a central E domain. Fibrinogen forms the main structure of the fibrin clot, after
cleavage of fibrinopeptides A (FpA) and B (FpB) by throm- bin.The fibrin monomer assembles in a half-staggered over- lap with adjoining fibrin monomers and is then covalently cross-linked into a fibrin polymer by the transamidase fac- tor XIIIa.
Aα FpA
Bβ FpB
γ Aα
Bβ
γ
D D
FpA FpB
E
D E D
D E D
D E D D E D
D E D D E D
Fibrinogen
Thrombin FpA + FpB
XIII
XIIIa
Fibrin monomer
Cross-linked fibrin polymer
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Endothelium Aird WC. Spatial and temporal dynamics of the endothelium. J Thromb Haemost. 2005:1392-1406. Epub 2005 May 9. Carrell RW, Perry DJ. The unhinged antithrombins. Brit J Haematol. 1996;93:253-257. Eilertsen KE, Osterud B. Tissue factor: (patho)physiolo- gy and cellular biology. Blood Coagul Fibrinolysis. 2004;15:521-538. Jin L, Abrahams JP, Skinner R, et al. The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci USA. 1997;94;14683-14688. Michiels C. Endothelial cell functions. J Cell Physiol. 2003;196(3):430-443. Price GC, Thompson SA, Kam PC. Tissue factor and tissue factor pathway inhibitor. Anaesthesia. 2004; 59(5):483-492. Steffel J, Luscher TF, Tanner FC. Tissue factor in cardio- vascular diseases: molecular mechanisms and clini- cal implications. Circulation. 2006;113(5):722-731. Van de Wouwer M, Conway EM. Novel functions of thrombomodulin in inflammation. Crit Care Med. 2004;32(suppl 5):S254-S261. Verhamme P, Hoylaerts MF. The pivotal role of the endothelium in haemostasis and thrombosis. Acta Clin Belg. 2006;61(5):213-219. Coagulation Proteins Butenas S, Orfeo T, Brummel-Ziedins KE, Mann KG. Tissue factor in thrombosis and hemorrhage. Surgery. 2007;142(suppl 4):S2-14. Davie, EW, Kulman, JD. An overview of the structure and function of thrombin. Semin Thromb Haemost. 2006;32(suppl 1):3-15. Duga S, Asselta R, Tenchini ML. Coagulation factor V. Int J Biochem Cell Biol. 2004;36(8):1393-1399. Laurens N, Koolwijk P, de Maat MP. Fibrin structure and wound healing. J Thromb Haemost. 2006;4(5):932-
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