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Coagulation Proteins
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- Platelets Endothelium
stituents of the hemostatic pathways and how
they are interrelated. Each element of the hemosta- Figure 1-1. Basic representation of the elements of hemostasis.
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I HEMOSTASIS PHYSIOLOGY
Procoagulant Anticoagulant
Thrombin / thrombomodulin
Expression of Prostacyclin
tissue factor ?? (PGI2)
von Willebrand
factor
Heparin-like
material
Thrombin
Thrombomodulin
receptor
Subendothelial matrix
Figure 1-2. A stylized view of endothelial functions related plex of many materials. The most important constituents of
to procoagulation and anticoagulation. The subendothelial the subendothelial matrix related to coagulation function are
matrix, represented by the purple interlocking lines, is a com- collagen and von Willebrand factor.
adventitial vascular wall cells and is exposed to storage, is important in directing the multimer-
flowing blood during vascular injury or endothe- ization of vWF. Von Willebrand factor multimers
lial denudation. Tissue factor, when bound to fac- can range in size up to 20 x 106 daltons. Von
tor VIIa, is the major activator of the extrinsic Willebrand factor binds to the platelet glycopro-
pathway of coagulation. Classically, tissue factor is tein Ib/IX/V receptor and mediates platelet adhe-
not present in the plasma but only presented on sion to the vascular wall under shear, which is dis-
cell surfaces at a wound site. Because tissue factor cussed in more detail in chapter 2.
is “extrinsic” to the circulation, the pathway was The remaining endothelial procoagulant func-
thusly named. tions discussed in this brief review and listed in
Endothelium is the major synthetic and storage Figure 1-2 are all parts of the fibrinolytic system,
site for von Willebrand factor (vWF). Von discussed in more detail in chapter 3. The fibri-
Willebrand factor is secreted from the endothelial nolytic system is responsible for proteolysis and
cell both into the plasma and also abluminally into solubilization of the formed clot constituents to
the subendothelial matrix. It is a large multimeric allow its removal. Plasminogen activator inhibitor
protein that acts as the intercellular glue binding acts to block the ability of tissue plasminogen acti-
platelets to one another and also to the suben- vator to turn on plasmin, the primary enzyme of
dothelial matrix at an injury site. In addition, the fibrinolysis. The thrombin activatable fibrinolytic
second major function of vWF is to act as a carrier inhibitor (TAFI), similar to the thrombin regulato-
protein for factor VIII (antihemophilic factor). Von ry enzyme protein C, is cleaved to its activated
Willebrand factor is synthesized in the endothelial form by the thrombin/thrombomodulin complex.
cell as a dimeric protein, with a molecular weight The endothelial cell surface receptor thrombo-
of the monomer approximately 250,000 daltons. modulin is a 450,000-dalton protein whose only
The propeptide of vWF, the N-terminal portion of known ligand is thrombin. Thrombin, once bound
the protein that is cleaved off prior to secretion or to thrombomodulin, loses some proteolytic capa-
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Coagulation Pathway and Physiology 1
bilities but gains the ability to either activate TAFI bin inactivates the proteinases inefficiently, due to
or protein C. Relevant to this discussion is that the conformational inaccessibility of the arginine-ser-
activated form of TAFI catalyzes the removal of ine bond. Inhibition is accelerated approximately
lysine residues from the fibrin clot, making it less 1000-fold by the binding of heparin to arginine
recognizable as a substrate for plasmin and hence residues in the D-helix of antithrombin, with a
more persistent. resultant conformational change and exposure of
The endothelium and ultimately the entire ves- the reactive center.
sel are normally geared to maintain blood fluidity
in the absence of injury. To that end, the endothe- Platelets
lium possesses a variety of functions whose ulti- Platelets are discoid anucleate subcellular frag-
mate goal is to promote blood fluidity. Although ments that can vary in size up to 3 µm in diameter.
Figure 1-2 portrays a typical endothelium, evi- They arise from the megakaryocyte in the bone
dence is accumulating suggesting that endotheli- marrow and circulate in blood at a platelet count
um from different vessels and from various parts that ranges from 200,000 to 400,000/µL. The
of the vascular tree is not homogeneous. platelet has a complex ultrastructure that includes
The anticoagulant functions of the endothelium many different surface receptors, several types of
inhibit all aspects of the coagulation pathways. storage granules, and a network of actin and
Tissue factor pathway inhibitor (TFPI) is a 33,000- myosin filaments. At the site of an injury, the
dalton low-concentration inhibitor of the extrinsic platelets contact extracellular matrix components,
pathway that is secreted into the plasma by the causing a series of metabolic changes that result in
endothelium. This material rapidly inhibits the tis- the formation of a platelet plug. These metabolic
sue factor/factor VIIa complex. Prostacyclin changes are usually termed aggregation.
(PGI2), a prostaglandin produced by the endothe- Ultimately, this platelet plug is stabilized by the
lial-specific cyclooxygenase enzyme system, is a formation of a fibrin clot. Platelet structure and
potent inhibitor of platelet aggregation. Tissue function are described in chapter 2.
plasminogen activator (tPA) is the main enzymat-
ic activator of the potent fibrinolytic enzyme plas- Coagulation Proteins
min. The fibrinolytic system is discussed in more Table 1-1 lists the proteins involved in the forma-
detail in chapter 3. tion of the fibrin clot. Factors II, VII, IX, and X (as
Once the enzyme thrombin is bound to throm- well as proteins C, S, and Z) are the zymogen
bomodulin, it also gains the ability to activate pro- forms of vitamin K-dependent serine proteases.
tein C to activated protein C (APC). APC, along Vitamin K is a necessary cofactor for a post-trans-
with its cofactor protein S, phospholipid surface, lational modification that adds a carboxyl group to
and calcium ions, acts to down-regulate thrombin the 10 to 12 glutamic acid residues in the amino
generation by proteolyzing the two protein cofac- terminal portion of each of these proteins. The
tors in the coagulation pathways, factor Va and vitamin K-dependent proteins utilize these clus-
factor VIIIa. Finally, the last anticoagulant proper- ters of γ-carboxyl glutamic acid (gla) residues to
ty of endothelium listed in Figure 1-2 is the pres- adhere to phospholipid surfaces and assemble
ence of the heparin-like material on the surface. multimolecular coagulation complexes. Without
Heparan sulfate is a glycosaminoglycan that is this important post-translational modification, the
attached to the luminal surface of the endothelium assembly of cell-based coagulation complexes is
by a protein backbone. The cell-surface heparan impaired, leading to ineffective fibrin formation.
sulfate acts as a cofactor for one of the main direct Another reason to understand this biochemical
inhibitors of many of the coagulation enzymes, fact is that the most commonly used oral anticoag-
antithrombin. Antithrombin is a 58,000-dalton ser- ulant, warfarin, exerts its anticoagulant effect by
pin (serine protease inhibitor) that has a five- inhibiting this modification and ultimately pro-
stranded central β-sheet (the A-sheet), together ducing dysfunctional vitamin K-dependent fac-
with a heparin-binding D-helix and a mobile reac- tors. These dysfunctional factors affect coagula-
tive site loop containing an Arginine393-Serine394 tion tests, and the warfarin anticoagulant effect is
bond that resembles the substrate for thrombin therefore monitored by the clot-based PT assay.
and other serine proteases such as factor Xa, factor Since the identification of all the factors listed in
IXa, and factor XIa. Once thrombin cleaves the Table 1-1, accumulating epidemiologic evidence
bond, the protease is inhibited by a covalent link has called into question whether some of these fac-
to the antithrombin. In its native state, antithrom- tors truly participate in the formation of a fibrin
5
I HEMOSTASIS PHYSIOLOGY
Fibrinogen (Factor I) Molecular Weight (MW) = 340,000 Adhesive protein that forms the fibrin clot
daltons (Da); glycoprotein
Prothrombin (Factor II) MW = 72,000 Da; vitamin K-dependent Activated form is main enzyme of
serine protease coagulation
Tissue factor (Factor III) MW = 37,000 Da; also known as Lipoprotein initiator of extrinsic pathway
thromboplastin
Calcium ions (Factor IV) Necessity of Ca++ ions for coagulation Metal cation necessary for coagulation
reactions described in 19th century reactions
Factor VII (Proconvertin) MW = 50,000 Da; vitamin K-dependent With tissue factor, initiates extrinsic
serine protease 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 Activated form is enzyme for intrinsic
serine protease activation of factor X
Factor X (Stuart-Prower factor) MW = 58,900 Da; vitamin K-dependent Activated form is enzyme for final common
serine protease pathway activation of prothrombin
Factor XI (Plasma thromboplastin MW = 160,000 Da; serine protease Activated form is intrinsic activator of
antecedent) 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
Prekallikrein (Fletcher factor) MW = 85,000 Da; serine protease Activated form that participates at
beginning of aPTT-based intrinsic pathway
clot in vivo. Although discussed more fully in the essentially made “thrombokinase” with calcium
following sections, evidence suggests that factor ions. This “thrombokinase” was prepared from a
XII and prekallikrein may not normally participate saline extract of rabbit brain with the addition of
in clotting in vivo but are important in the in vitro calcium. The more modern nomenclature for this
laboratory clot assays. material is thromboplastin. The basis for Dr.
Quick’s assay was that adding calcium ions with
The Extrinsic Pathway and the PT
The first description of the extrinsic pathway Prothrombin
was reported by Dr. Paul Morawitz in 1905. Dr.
Thrombokinase Calcium
Morawitz produced a hemostasis model incorpo-
rating all of the scientific information of his day.
Thrombin
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 Fibrinogen Fibrin clot
use today. Dr. Quick, using the classic four-compo- Figure 1-3. A representation of the original extrinsic path-
nent extrinsic pathway model of Dr. Morawitz, way proposed in 1905.
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Coagulation Pathway and Physiology 1
an excess of thromboplastin to anticoagulated In the early 1960s, a new synthesis of all hemo-
plasma was a direct measure of the prothrombin stasis knowledge was put together, and the PT or
amount in the plasma—hence the name of the extrinsic and aPTT or intrinsic coagulation path-
assay, prothrombin time. Only in the 1950s and ways were published. This provided a framework
early 1960s, with the discovery of additional coag- of how many of the proteins listed in Table 1-1
ulation factors, did the true nature of the extrinsic interact to form a blood clot. This pathway is illus-
pathway become known. This is discussed in trated in Figure 1-4. Although there have been
more detail below in the section, “The PT and some modifications since the original papers,
aPTT Pathways.” these are the pathways with which most workers
in hemostasis are familiar. From the initial publica-
The Intrinsic Pathway and the aPTT tion, accumulating epidemiologic evidence sug-
Dr. Quick, in his first publication, observed that gests this formulation of the intrinsic and extrinsic
his new PT assay was not sensitive to the hemo- hemostasis pathways might not be a correct repre-
philic defect. Patients with the symptoms of hemo- sentation of blood clotting in vivo. For example,
philia did not usually have an abnormal PT. patients deficient in factor XII, prekallikrein, or
Evidence had been accumulating that the four- high-molecular-weight kininogen do not have a
component extrinsic pathway model of blood clot- bleeding or thrombotic phenotype. All of these
ting was not complete. The plasma had the poten- proteins are present at the start of the intrinsic
tial to clot without the addition of an extrinsic pathway, and deficiencies of each factor can cause
material. The thromboplastin, thrombokinase, or a significantly prolonged aPTT assay. Logically, it
what we now call tissue factor was not always would make sense that a deficiency of a factor at
needed to make blood clot, especially in vitro. the start of a pathway would cause bleeding
Therefore, it appeared that plasma had within it or pathology. However, all evidence suggests this is
intrinsic to it all the factors necessary to cause not so. The intrinsic and extrinsic pathways as
blood clotting. they have existed since their inception are based
In 1953, Drs. Langdell, Wagner, and Brinkhous on in vitro testing. The in vivo function appears to
published a paper detailing a clot-based assay that be different. It is important to be familiar with
was sensitive to the defect in hemophilic plasma. the older pathway model because the PT and the
Instead of using a complete tissue extract, such as aPTT are still useful as diagnostic tests.
the prothrombin time thromboplastin reagent,
their assay used only a partial extract. Hence these Newer Coagulation Model
researchers called their assay a partial thrombo- Figure 1-5 illustrates a newer model of coagula-
plastin time (PTT). In this group’s initial assay, the tion. In this figure, thrombin is depicted as the cen-
activator necessary to cause clotting was added ter of the coagulation universe; all aspects of
separately from the PTT reagent and calcium ions. hemostasis feed into the regulation and control of
Other workers modified the assay, adding an acti- thrombin generation, which in turn forms the
vator to the PTT reagent, producing the modern definitive clot at the site of an injury. Of note, the
activated thromboplastin time assay. figure lacks several proteins normally considered
part of the classic intrinsic coagulation pathway:
The PT and aPTT Pathways factor XII and prekallikrein. These proteins are not
The PT and aPTT assays were developed based on currently thought to be important for in vivo coag-
theories and specific testing needs, without com- ulation activation. Although high-molecular-
plete knowledge of all the proteins involved in weight kininogen deficiency may not be associat-
coagulation. In the period from 1935 and the ed with a bleeding diathesis, it is still part of the
inception of the PT until the early 1970s, all of the new coagulation pathway as it circulates in plas-
procoagulant proteins involved in forming a fibrin ma bound to factor XI. Also important in this
clot were identified. Many of these factors were newer concept is that the majority of the steps in
identified because patients were found with defi- the coagulation cascade take place by the forma-
ciency states. Some of these patients had congeni- tion of multimolecular coagulation protein com-
tal bleeding disease, while others presented with plexes on phospholipid cell surfaces.
an abnormal prolongation in the PT and/or the This new coagulation model has extrinsic and
aPTT. It became clear that a fresh model of coagu- intrinsic pathway limbs, but the in vivo process of
lation other than the classic extrinsic pathway was hemostasis is thought only to be initiated by cell-
needed. based tissue factor expressed at an injury site.
7
I HEMOSTASIS PHYSIOLOGY
Negatively charged surface Kallikrein Tissue factor + Factor VIIa Factor VII
Factor VIIIa
Factor X Factor Xa
Factor II Thrombin
Factor XIIIa
Fibrin polymer
Figure 1-4. A model of the classic extrinsic and intrinsic pholipids are necessary for most reactions except the activa-
coagulation pathways. For the sake of clarity, calcium ions and tion of factor XII and the activation of prekallikrein.The cofac-
phospholipids, two important cofactors for most coagulation tor for the activation of prekallikrein, high-molecular-weight
reactions, have been omitted from the figure. Ca++ and phos- kininogen, has also been omitted from the figure for clarity.
Tissue factor binds to factor VII or VIIa in a 1:1 the conversion of prothrombin to thrombin by
complex. Limited proteolysis leads to a tissue fac- cleavage of an activation peptide, prothrombin F1.2.
tor/factor VIIa complex that can activate factor X The generation of a small amount of thrombin
or factor IX to activated serine proteases through initiated by extrinsic means appears to be enough
cleavage of an activation peptide. Once the path- to start the coagulation mechanism and, if condi-
way commences, the tissue factor/factor VIIa acti- tions are right, the expansion of thrombin genera-
vation of factor X is rapidly shut down by an tion through an intrinsic mechanism. The intrinsic
inhibitor produced by endothelial cells, tissue fac- limbs of the pathway include activation of factor
tor pathway inhibitor (TFPI). The newly activated XI to factor XIa by thrombin, with the ultimate
factor IXa then binds to its cofactor, factor VIIIa, on generation of more thrombin using factor IXa and
a phospholipid surface to form the tenase complex factor VIIIa to activate factor X. This pathway
that results in the activation of factor X to factor Xa. model also explains the mechanism of how two of
The activation of factor X to Xa starts the final, the important cofactors, factor V and factor VIII,
common pathway for thrombin activation. Factor are activated by thrombin. It was known that both
Xa combines with the cofactor, factor Va, together factor V and factor VIII had to be partially prote-
with calcium on phospholipid surfaces to form the olyzed or activated to participate in the formation
prothrombinase complex. This complex then effects of a blood clot, but the mechanism was never
heretofore part of the coagulation model.
8
Coagulation Pathway and Physiology 1
Factor XI
Prothrombin
Thrombin activatable
fibrinolytic inhibitor
(TAFI) activation
Protein C activation
Thrombin
Factor XIII
Platelet aggregation
Factor XIIIa
Fibrin polymer
Cross-linked clot
Figure 1-5. A newer model of the coagulation pathway. For ed by tissue factor pathway inhibitor (TFPI).The small amounts
the sake of clarity, Ca++ and phospholipids have been omitted of thrombin generated from the initial activation feedback to
from the figure. These two cofactors are necessary for all of create activated cofactors, factors Va and VIIIa, which in turn
the reactions listed in the figure that result in the activation of help to generate more thrombin. Tissue factor/factor VIIa is
prothrombin to thrombin. The pathway is initiated by an also capable of indirectly activating factor X through the acti-
extrinsic mechanism that generates small amounts of factor vation of factor IX to factor IXa. Finally, as more thrombin is
Xa, which in turn activate small amounts of thrombin.The tis- created, it activates factor XI to factor XIa, thereby enhancing
sue factor/factor VIIa proteolysis of factor X is quickly inhibit- the ability to ultimately make more thrombin.
Activation of factors V and VIII by thrombin domains (Figure 1-6). Thrombin cleaves small
results in a further burst of coagulation activity peptides, termed fibrinopeptides A and B, from
through increased activity of the tenase and pro- the Aα and Bβ chains, respectively, to form a fibrin
thrombinase complexes. monomer. These monomers assemble into
Fibrinogen is the ultimate substrate protein of protofibrils in a half-staggered, side-to-side fash-
the coagulation cascade and forms the principal ion that is stabilized by noncovalent interactions
structural protein of the fibrin clot. Fibrinogen, between fibrin molecules. The protofibrils lateral-
produced in the liver, is a dimer composed of three ly associate into thicker fibrin fibers and form the
pairs of protein chains, Aα, Bβ, and γ, that are fibrin clot. This clot, however, is not stable and
disulfide-linked at their N-terminal ends. ultimately will come apart if not covalently cross-
Fibrinogen, as viewed by molecular imaging tech- linked. Thrombin activates factor XIII to the trans-
niques, is composed of three globular domains, a glutaminase enzyme factor XIIIa. Factor XIIIa, act-
central E domain flanked by two identical D ing upon the glutamic acid and lysine side chains
9
I HEMOSTASIS PHYSIOLOGY
Aα FpA γ
Bβ FpB Bβ
γ Aα
FpA FpB
D E D Fibrinogen
XIII
Thrombin FpA + FpB
D E D Fibrin monomer
D E D D E D
Cross-linked
XIIIa D E D fibrin polymer
D E D D E D
Figure 1-6. Fibrinogen is an abundant plasma protein cleavage of fibrinopeptides A (FpA) and B (FpB) by throm-
that is a dimer of the Aα, Bβ, and γ chains connected by bin. The fibrin monomer assembles in a half-staggered over-
disulfide bonds. The fibrinogen dimer is composed of two lap with adjoining fibrin monomers and is then covalently
flanking D globular domains with a central E domain. cross-linked into a fibrin polymer by the transamidase fac-
Fibrinogen forms the main structure of the fibrin clot, after tor XIIIa.
10
Coagulation Pathway and Physiology 1
Tissue factor pathway inhibitor Molecular Weight (MW) = 33,000 daltons Inhibits the tissue factor/factor VIIa complex
(TFPI) (Da)
Protein C MW = 62,000 Da; vitamin K-dependent Activated form cleaves coagulation cofactors
serine protease Va and VIIIa
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I HEMOSTASIS PHYSIOLOGY
12