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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice
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Copyright 2006 Lippincott Williams & Wilkins

Colman, Robert W., Clowes, Alexander W., Goldhaber, Samuel Z.,
Marder, Victor J., George, James N.
Hemostasis and Thrombosis: Basic Principles and Clinical Practice,
5th Edition
Chapter 1
Overview of Hemostasis
Robert W. Colman
Alexander W. Clowes
James N. George
Samuel Z. Goldhaber
Victor J. Marder
Humans have evolved an intricate hemostatic system that is
designed to maintain blood in a fluid state under physiologic
conditions, but that is primed to react to vascular injury in an
explosive manner to stem blood loss by sealing the defect in the
vessel wall. Thrombosis may occur if the hemostatic stimulus is
unregulated, either because the capacity of inhibitory pathways is
impaired or, more commonly, because the capacity of the natural
anticoagulant mechanism is overwhelmed by the intensity of the
stimulus. Thrombosis may be regarded as an accident of nature
that has not had time to adapt through the lengthy process of
evolution to the advances of modern medicine, which allow
patients to survive the hemostatic challenge of major surgery and
trauma but leave them vulnerable to venous thrombosis.
The normal vascular endothelium maintains blood fluidity by
inhibiting blood coagulation and platelet aggregation and
promoting fibrinolysis (see Fig. 1-1). It also provides a protective
barrier that separates blood cells and plasma factors from highly
reactive elements in the deeper layers of the vessel wall. These
components include adhesive proteins such as collagen,
fibronectin, laminin, vitronectin, and von Willebrand factor (VWF),
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which promote platelet adhesion, and tissue factor, a membrane

protein located in smooth muscle, fibroblasts, and macrophages,
that triggers blood coagulation. After the vessel is severed, it
constricts, thereby diverting blood from the site of injury, and the
shed blood is exposed to these subendothelial structures that
stimulate hemostatic plug formation by promoting platelet
adhesion and aggregation and by activating blood coagulation.
After platelets are stimulated by subendothelial collagen, they
expose and assemble membrane glycoprotein (GP) IIb and GP IIIa,
which can then bind fibrinogen and VWF, cofactors for platelet
recruitment and aggregation. Secretion of proteins from is
mediated by thromboxane synthesis, phosphorylation of specific
proteins, and intracellular calcium translocation. Protein cofactors
such as factor V, secreted by platelets or derived from plasma,
serve as a nidus for assembling enzymecofactor complexes on
the platelet surface, thereby accelerating factor X and prothrombin
activation. The result is thrombin formation, which augments its
own production manyfold by converting factors V and VIII into
activated cofactors and stimulating platelet secretion.
This explosive cellular and molecular reaction is modulated by
endothelial cell elaboration of antithrombotic lipids (prostacyclin,
or PGI 2 ), proteins (thrombomodulin), inorganic compounds [nitric
oxide (NO)], and polysaccharides (heparan); by surface-binding
enzymes such as adenosine diphosphatase (ADPase) (CD39); and
by several plasma protease inhibitors, most importantly
antithrombin (AT) III for factors IXa, Xa, and thrombin; C1
inhibitor, for the contact system enzymes factor XIIa,
1 -antitrypsin for factor XIIf, and 2 -macroglobulin kallikrein; for
factor XIa; and 2 -macroglobulin as a general backup. A major
substrate of thrombin is fibrinogen, which, after initial hydrolysis,
forms fibrin monomers that then undergo spontaneous
polymerization to form the fibrin clot. Covalent cross-linking by
the thrombin-activated enzyme factor XIIIa increases the
resistance of the clot to fibrinolysis.
Plasminogen, a plasma zymogen, is converted to plasmin by two
plasminogen activators elaborated by endothelial cells. The
process is modulated by at least three plasminogen activator
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inhibitors. Plasmin normally does not act on fibrinogen in solution

because of the presence of However, plasmin on the surface of the
fibrin clot is protected from the inhibitor, and fibrinolysis occurs
with the formation of fibrin degradation products. Second,
plasminogen activator inhibitor (PAI-1) released by endothelial
cells and by platelets neutralizes tissue-type plasminogen
activator (tPA) and prevents early lysis of clot. Third, thrombin
activates a carboxypeptidase B proenzyme, called
thrombin-activated fibrinolytic inhibitor (TAFI), that impairs
fibrinolytic degradation of fibrin strands. Therefore, with more
active coagulation and with more complete conversion of
prothrombin to thrombin, not only is more fibrin formed from
fibrinogen, but such fibrin is further protected from fibrinolysis by
TAFI. Dissolution of the clot paves the way for the deposition of
collagen, formation of fibrous tissue, and wound healing.
ENDOTHELIUM
Normal endothelium (see Fig. 1-2) maintains blood fluidity by
producing inhibitors of blood coagulation and platelet aggregation,
modulating vascular tone and permeability, and providing a
protective envelope, thereby separating hemostatic blood
components from reactive subendothelial structures. Endothelial
cells synthesize and secrete basement membrane and extracellular
matrix, which contain adhesive proteins, collagen, fibronectin,
laminin, vitronectin, and VWF. The endothelium inhibits blood
coagulation by synthesizing and secreting thrombomodulin and
heparan sulfate onto its surface; modulates fibrinolysis by
synthesizing and secreting tPA, urokinase plasminogen activator
(uPA), and plasminogen activator inhibitors; inhibits platelet
aggregation by releasing PGI 2 and NO; and regulates vessel wall
tone by synthesizing endothelins, which induce vasoconstriction,
and PGI 2 and NO, which produce vasodilation.
Defective vascular function can lead to abnormal bleeding if the
endothelium becomes more permeable to blood cells, if the
vasoconstrictive response is impaired because of structural
abnormalities of the vessel wall or extravascular supporting
tissues, or if physiologic fibrinolysis is not controlled by the
normal production of plasminogen activator inhibitor. Bleeding
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associated with endothelial injury may be mediated by immune

complexes and viruses (1,2). Proteolytic enzymes released from
leukocytes in inflammatory states perturb endothelial cells and
alter connective tissue proteins and also could contribute to
petechial hemorrhage in vasculitic disorders (3,4). Attenuation and
fenestration of the vascular endothelium may contribute to the
hemorrhagic manifestations
P.4
of idiopathic thrombocytopenia purpura, which may respond to
prednisone therapy promptly, even before a detectable rise in
platelet count (5).
FIGURE 1-1. Overview of hemostasis. ADP, adenosine

diphosphate; ADPase, adenosine diphosphatase; Epi,
epinephrine; TxA 2 , thromboxane A 2 ; PAI, plasminogen activator
inhibitor; PGI 2 prostacyclin; NO, nitric oxide; VWF, von
Willebrand factor.
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Endothelial cells lose their nonthrombogenic protective properties

after they are stimulated by enzymes such as thrombin, hypoxia,
fluid shear stress, oxidants; cytokines such as interleukin-1, tumor
necrosis factor, and -interferon; synthetic hormones such as
desmopressin acetate and endotoxin. Synthesis of tissue factor
and PAI-1 is induced and the concentration of surface-bound
thrombomodulin is reduced by cytokines and endotoxin, whereas
desmopressin acetate results in the release of
high-molecular-weight VWF multimers from Weibel-Palade bodies
that may augment platelet adhesion to the injured vessel wall.
Endothelial cells contain receptors, termed integrins, of the very
late antigen (VLA) type, that allow binding of fibronectin ( 1 1 ),
collagen ( V 1 ), and laminin ( 3 1 , 6 1 ) and of the cytoadhesive
type, notably the vitronectin receptor ( 2 3 ) (6). The stimulated
endothelial cell synthesizes chemokines, such as monocyte
chemoattractant protein, interleukin-8, regulated on activation
normal T-cell expressed and secreted (RANTES), and GRO.
Endothelial cells also contain intercellular adhesion molecules (see
Chapter 45) such as ICAM-1 and ICAM-2, and VCAM-1, which act
as counterreceptors for leukocyte integrins (7,8). Before tight
adhesion to the endothelium, platelets and leukocytes roll, an
interaction mediated by E selectin and P selectin (which is stored
in Weibel-Palade bodies).
FIGURE 1-2. Thromboresistant properties of endothelium. The

endothelial cells express adenosine diphosphatase (ADPase),
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and synthesize prostacyclin (PGI 2 ) thrombomodulin, heparan,

and plasminogen activators, all of which inhibit hemostasis
(and thrombus formation) and contribute to the maintenance of
vascular patency. ADP, adenosine diphosphate; AT,
antithrombin; NO, nitric oxide; tPA, tissuetype plasminogen
activator; uPA, urokinase plasminogen activator.
Endothelium is heterogeneous, both metabolically and structurally

(9). For instance, angiotensin-converting enzyme apparently is
synthesized by most endothelial cells but is principally synthesized
by aortic endothelium, not by cardiac microvessel endothelium; on
the other hand, thromboxane is not synthesized by most
endothelial cells but is synthesized by pulmonary arterial
endothelium. Endothelial cell turnover is low under resting
conditions but varies with location. At sites of hemodynamic stress
and injury, proliferation is especially increased. Endothelial cells
contain a full array of contractile proteins, but of special
importance are the stress fibers involved in cell attachment and
maintenance of endothelial junctional apposition by vascular
endothelial (VE) cadherin and plateletendothelial cell adhesion
molecule (PECAM). Endothelial cells contain caveolae that
concentrate glycosylphosphatidyl-linked proteins such as the
urokinase receptor.
Endothelial cell permeability is influenced by the functional
adaptations that join the cells to their neighbors. Macromolecules
pass across the endothelium into the vessel wall through
P.5
patent intercellular junctions, by endocytosis, and through the
transendothelial pores. Vessel permeability is increased by
vasodilation, by induction of severe thrombocytopenia, and by
high doses of heparin. Spontaneous bleeding observed with a low
platelet count or after heparin infusion may be induced by
increased vascular permeability.
Increased fenestration and attenuation of endothelium may
account for the loss of barrier function associated with
experimental thrombocytopenia. The thrombocytopenia-induced
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extravasation of erythrocytes, manifest clinically as petechiae,

occurs principally through postcapillary venular interendothelial
channels. The loss of endothelial barrier function, associated with
extreme platelet depletion, may be related to a loss of serotonin
and norepinephrine delivered by platelets to the microvascular
milieu, as exogenous sources of either amine prevent petechial
formation in severely thrombocytopenic animals or failure of
platelets to plug gaps at intracellular junctions between retracted
endothelial cells.
Endothelial cells are highly negatively charged, a feature that may
repel the negatively charged platelets. This anionic surface, as
well as other antithrombotic properties of endothelium, could be
important in limiting the intravascular extension of the hemostatic
reaction induced by vessel injury (10). Therefore, synthesized and
released from endothelial cells close to the site of hemostatic plug
formation, could inhibit intravascular platelet aggregation
(11,12,13 and 14). Thrombomodulin and heparan sulfate, the two
endothelial surfacebound thrombin inhibitors, could limit the
intravascular spread of fibrin beyond the confines of the
hemostatic plug (15,16,17,18 and 19). Heparan sulfate, a
glycosaminoglycan, activates AT and, therefore, catalyzes the
inhibition of thrombin and factor Xa. Endothelial cellassociated
ADPase (CD39) cleaves adenosine diphosphate (ADP) to adenosine
monophosphate (AMP), thereby modulating this stimulatory
agonist (20).
Thrombomodulin binds thrombin and inhibits the ability of the
enzyme to cleave fibrinogen and activate platelets and factors Va
and VIIIa. Thrombomodulin also markedly enhances thrombin's
ability to activate protein C. Protein C binds to endothelial cell
protein C receptor, which enhances its activation (21). Protein C,
in turn, inactivates factors Va and VIIIa and enhances fibrinolysis,
probably by binding an inhibitor of plasminogen activators (22).
Thrombin bound to thrombomodulin also is inactivated by
circulating AT, a step accelerated by heparan sulfate. Protein C
activity is controlled by protein C inhibitor (PAI-3) and inhibitor
and is stimulated by protein S, a cofactor (23,24). Protein S, in
turn, is controlled by C4b, which forms a complex with it, thereby
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preventing its action (25). The enhancement of fibrinolysis by

protein C also may depend on protein S (26). Therefore, the
binding of thrombin with thrombomodulin results in the loss of the
coagulant effect and in the enhancement of thrombin's ability to
activate protein C and therefore to inhibit thrombogenesis.
The synthesis of PGI 2 by endothelial cells is stimulated by
thrombin and other stimuli, including epinephrine and trauma
(27). Other agonists, including histamine, adenosine triphosphate
(ATP), bradykinin, and acetylcholine, stimulate endothelial cell
guanylate cyclase, raising the levels of intracellular cyclic
3,5-guanosine monophosphate (cGMP), which results in the
synthesis of NO. Therefore, endothelial cells exposed to
appropriate stimuli synthesize and release two distinct mediators
of vasodilation and inhibition of platelet function (28). Stimulated
endothelial cells also synthesize a group of peptides known as
endothelins that have counterregulatory properties, including
vasoconstriction (29). Endothelial cells also elaborate plasminogen
activators, which, in the presence of fibrin, promote fibrinolysis
and can aggravate a bleeding tendency in susceptible patients.
The bleeding tendency can be controlled by synthetic and natural
fibrinolytic inhibitors (see Chapter 79). PAI-1 also is elaborated
with a different time course and in response to different stimuli
(30). Deficiency of PAI-1 causes a bleeding tendency, an indication
that unopposed physiologic fibrinolysis disrupts the hemostatic
balance.
Endothelial cells are stimulated by cytokines and other mediators
to mount a procoagulant response characterized by an increased
synthesis and release of PAI-1, release of VWF, synthesis and
availability of tissue factor, and reduction of cell
membraneassociated thrombomodulin. The postoperative and
posttraumatic fibrinolytic shutdown is associated with increased
synthesis of PAI-1 and could be mediated by cytokines elaborated
as a response to tissue damage (30). Thrombin bound to
thrombomodulin also more efficiently activates the TAFI (31). This
response protects the newly formed hemostatic plug from
premature dissolution but also may contribute to the increased
risk of postoperative venous thrombosis.
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Chapter 41 summarizes the changing focus of the function of

endothelial cells from mediating mostly antithrombotic reaction to
changes based on studies of embryonic and adult development of
endothelial cells into new vessels. These changes may be in
response to alteration of blood flow roles as well as changes
required on vascular remodeling in physiologic and pathologic
changes. An explosion of studies of both vasculogenesis
(origination of blood vessels) and angiogenesis
(neovascularization) have delineated a large number of angiogenic
stimulators and inhibitors as well as explored some of the
signaling mechanisms.
The complex interplay between mediators and countermediators
derived from the vessel wall, the endothelial lining, and even the
vasomotor regulation of arteries and veins, affects hemostasis and
wound healing. All these vascular processes act in concert with
similarly complex processes in the platelet, in plasma coagulation,
and in fibrinolytic and inhibitory pathways to maintain normal
hemostasis. Occasionally, however, the hemostatic response is
excessive and leads to intravascular thrombosis. Proteins of the
hemostatic system participate in the regulation of angiogenesis,
including plasminogen, AT, kininogen, platelet factor 4, and
collagen. Fragmented or altered forms of these proteins show
antiangiogenic activity.
PLATELETS
Platelets are produced from bone marrow megakaryocytes, a giant
cell with 8 to 32 nuclei as a result of division of nuclei without cell
division (32). Recent data shows that the fragmentation is similar
to apoptosis because it is a result of caspase activation within the
megakaryocytes (33). The dominant growth factor is
thrombopoietin, which is responsible for both DNA replication and
cytoplasmic differentiation (34). Multiple transcription factors
specific for the hematopoietic lineage are responsible (35).
The participation of platelets in hemostasis is a fundamental
component of this physiologic process. The reactions involved
include adhesion to the cut end of a blood vessel, spreading of
adherent platelets on the exposed subendothelial surface,
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secretion of stored platelet constituents (including molecules

involved in hemostasis and wound healing), and formation of large
platelet aggregates (36). In addition, platelet membrane sites
become available for adsorption and concentration of clotting
factors, and plasma coagulation is accelerated, resulting in the
formation of a fibrin network that reinforces the otherwise friable
platelet plug. The firm plateletfibrin clot subsequently retracts
into a smaller volume, a process that is also platelet-dependent.
Platelets do not adhere to normal vascular endothelial cells, but an
area of endothelial disruption (e.g., the cut end of a divided blood
vessel) provides binding sites for the adhesive protein, VWF,
through the platelet GP Ib/IX/V complex, and fibrinogen as well as
fibronectin through integrin receptors (37). These adhesive
proteins are thought to participate
P.6
in the formation of a bridge from platelets to subendothelial
connective tissue, although this may be an oversimplification.
The importance of these events is illustrated by the occurrence of
hemorrhage in Bernard-Soulier disease, in which patients lack GP
Ib/IX, or in von Willebrand disease, in which VWF is decreased or
defective. At high-shear rates (i.e., > 800 per second),
comparable to those found in arteries in the microvasculature,
plasma VWF is required for normal adhesion of platelets to
subendothelium, perhaps as a bridge between platelets and the
fibrillar surface (38). At low-shear rates, adhesion of platelets to
subendothelium is normal in patients with these disorders,
suggesting that other proteins can substitute for the action of
VWF, at least to some extent. Signaling through GP Ib/IX/V
activates GP IIb/IIIa without involvement of other receptors (39).
Other adhesive events that are involved include interactions of
collagen with the platelet GP Ia/IIa (40) followed by activation of
intracellular signaling pathways by platelet GP VI (41).
Abnormalities in either of these platelet receptors for collagen
cause bleeding defects.
Once adherent to subendothelium, platelets spread out on the
surface, and additional platelets, delivered by the flowing blood,
adhere first to the basal layer of adherent platelets and eventually
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to one another, forming a mass of aggregated platelets. A crucial

event in platelet aggregation is induction of a change in the
disposition of surface membrane GP IIb/IIIa (42), which acquires
the capacity to bind fibrinogen, as well as VWF, fibronectin, and
vitronectin (43). Fibrinogen appears to be the most important in
aggregation by virtue of its divalent structure, possibly allowing it
to form a bridge from platelet to platelet and thereby mediating
aggregation.
The current paradigm for bidirectional signaling has been well
summarized (44). Inside-out signaling from the cytoplasmic tail of
this integrin to the ligand recognition site results in
conformational changes leading to increased ligand affinity. An
increase in the number of surface receptors is derived from fusion
of the platelet membranes with the plasma membranes. Shuttling
of GP IIb/IIIa between these two membranes is responsible for
acquisition of fibrinogen from plasma.
Several other integrins in the platelet membrane act as receptors
for adhesive plasma proteins. These heterodimers, such as the
vitronectin receptor, are present on the surface of both blood cells
and endothelial cells (45). Whereas VWF and collagen can interact
with resting platelets, fibrinogen forms a high-affinity bond only
with an integrin GP IIb/IIIa on activated platelets (46). In the
congenital disorder Glanzmann thrombasthenia, the GP IIb/IIIa
complex is deficient, and the associated defect in fibrinogen
binding results in a bleeding tendency (47). Likewise, the bleeding
in congenital afibrinogenemia is caused in part by an abnormality
of platelet aggregation.
Interaction with GP receptors in the platelet membrane also is a
feature of participation in platelet aggregation by fibronectin and
thrombospondin. The interaction of the latter with GP IV may act
to stabilize platelet aggregates.
Of the many platelet agonists whose ability to induce aggregation
and secretion has been studied in vitro, those having the greatest
physiologic relevance are the proteolytic enzyme thrombin, ADP,
collagen, arachidonic acid, and epinephrine. Epinephrine is the
only one of these that does not result in a detectable change in
platelet shape.
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Specific receptors exist on the platelet surface for these agonists

(see Fig. 1-3) (48). Many of the receptoragonist complexes
interact in the platelet membrane with coupling proteins that
hydrolyze guanosine triphosphate, the G proteins. Evidently, some
interact with target proteins coupled to ion-permeable channels in
the platelet membrane, modulating ion flux, especially the inward
movement of ionized calcium. Others are linked to protein tyrosine
kinases (TK) that phosphorylate other sites on the receptor
protein itself. Accompanying these biochemical events are visible
effects, such as the disappearance of the equatorial band of
microtubules that normally maintain the platelet's discoid shape,
centralization of storage granules, and formation of pseudopodia.
Stimulatory agonists lead to activation of phospholipase C, which
cleaves phosphatidylinositol bisphosphate (PIP 2 ) to form inositol
triphosphate (IP 3 ) and diacylglycerol. IP 3 reacts with receptors on
calcium storage organelles known as the dense tubular system,
analogous to the sarcoplasmic reticulum of muscle, leading to
mobilization of ionized calcium and increasing its cytoplasmic
concentration (49). Familial abnormalities in a G protein (50) and
a phospholipase C (51) have been shown to lead to mild
hemorrhagic disorders.
Many processes involved in platelet activation are calcium
dependent, including phosphorylation of the light chain of myosin
by a specific kinase enzyme and liberation of arachidonic acid from
membrane phospholipids by the enzyme phospholipase A 2 (52,53).
Phospholipase liberates arachidonic acid from phosphatidylcholine
and probably other phospholipids. Arachidonic acid is converted by
the enzyme cyclooxygenase to prostaglandin endoperoxides and
ultimately to the potent platelet agonist thromboxane as well as to
stable prostaglandins such as prostaglandin The latter inhibits
platelet activation and, in a negative feedback system, may serve
to modulate platelet activities. A reactive serine in cyclooxygenase
is alkylated by aspirin, inactivating the enzyme permanently; this
accounts for the substantial pharmacologic action on platelets of
this widely used drug. High concentrations of intracellular calcium
(occurring, for example, after thrombin stimulation) lead to
activation of a calciumdependent neutral cysteine protease
(calpain), which may participate in remodeling of cytoskeletal
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proteins, cleavage of receptor proteins (54), and thrombin-induced

activation of platelets.
Diacylglycerolsuch as IP 3 , a product of the action of
phospholipase Cactivates a ubiquitous enzyme, protein kinase C,
in platelets (55). Protein kinase C phosphorylates (among other
substrates) pleckstrin, a 47-kDa protein that is a marker for
activation of the kinase. Phosphatases provide a negative
feedback, reducing the elevation of ionized calcium by IP 3 (56).
Diacylglycerol may be responsible for the alleged
calcium-independent reactions occurring during platelet
activation, or may act together with ionized calcium to activate
protein kinase C and stimulate secretion (57).
Platelets contain several classes of granules in which intracellular
constituents are sequestered, including dense bodies (containing
serotonin, ATP, ADP, pyrophosphate, and calcium), -granules
[containing fibrinogen, VWF, factor V, highmolecular-weight
kininogen (HK), fibronectin, 1 -antitrypsin, (A5)b-thromboglobulin,
platelet factor 4, and platelet-derived growth factor], and
lysosomes (containing a variety of acid hydrolases) (58).
Centralization of these granules after stimulation of platelets
results from activation of the platelet cytoskeletal contractile
apparatus; polymerization of filamentous actin and
phosphorylation of myosin are prominent reactions in platelets
responding to receptor-mediated stimulation. In the presence of
elevated cytoplasmic calcium, this leads to fusion of the granular
envelope with the lining membranes of intracellular canaliculi and
to external secretion of the granule contents.
ADP is thought to react with three receptors. The first, is an
ADP-operated calcium channel with little functional effect. P2Y 1
mediates shape change by activating phospholipase C, whereas
P2Y 12 decreases stimulated adenylate cyclase activity and reduces
platelet cyclic 3,5-adenosine monophosphate (cAMP) (59). Both
P2Y 1 and P2Y 12 are required for aggregation (see Chapter 32)
(60). A molecular defect in P2Y 1 2 results in hemorrhagic defect
(61). The 2 -adrenergic receptor responsible for epinephrine
interaction with platelets has been cloned and sequenced, and a
thromboxane A 2 and PGH 2 receptor has been demonstrated in
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binding studies (62,63). All

P.7
these receptoragonist interactions result in unmasking of
functional fibrinogen-binding sites by outside-in signaling through
G proteins.
During activation, platelets expose receptors for specific plasma
clotting factors, particularly activated factor V (Va), which may be
either secreted and expressed by the platelet or bound from
plasma. This acquired receptor, in conjunction with anionic
phospholipids exposed on activated platelets, also functions as a
binding site for factor Xa and thereby provides an efficient
catalytic environment for the conversion of prothrombin to
thrombin by factor Xa (64). An analogous system appears to exist
for the binding of factor IXa and conversion of factor X to Xa on
platelets.
Platelet activation and its effects are modulated by several
regulatory substances, of which the most important is cAMP (65).
Like virtually all other animal cells except human red cells,
platelets contain adenylate cyclase, the enzyme that converts ATP
to cAMP. Its action is powerfully stimulated by the arachidonic acid
products prostaglandin in platelets and (prostacyclin) in
endothelial cells. Platelets also contain cyclic nucleotide
phosphodiesterases that cleave cAMP to AMP, modulating
intracellular cAMP concentration (66). The major cAMP
phosphodiesterase in platelets, PDE3A, is inhibited by cGMP.
Therefore, compounds that increase cGMP also inhibit platelet
activation. cAMP stimulates a protein kinase that mediates
phosphorylation of an ATP-dependent calcium-pumping system
that removes calcium from the cytosol. In sufficient concentration,
cAMP inhibits not only platelet aggregation, secretion, and shape
change but adhesion to surfaces as well.
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FIGURE 1-3. Platelet function. Adhesion to endothelial cells is

mediated by glycoprotein (GP) Ib, which binds von Willebrand
factor (VWF) on the endothelial cells. Aggregation is mediated
by GP IIb/IIIa bridged to GP IIb/IIIa on another platelet by
fibrinogen. Various agonists such as adenosine diphosphate
(ADP) and platelet activating factor (PAF) are pictured as
interacting with specific receptors and activating
phospholipase C, probably through G proteins. This enzyme
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catalyzes the cleavage of phosphatidyl inositol bisphosphate

(PIP 2 ) to IP 3 , which mobilizes Ca 2 + from the dense tubular
system to activate myosin light chain kinase (MLCK), which
phosphorylates myosin light chain (MLC). Ca 2 + also activates
phospholipase A 2 (PLA 2 ) to release arachidonic acid from
phospholipids, which is in turn converted by cyclooxygenase
(CO) to PGG 2 and PGH 2 , and then by thromboxane synthetase
(TS) to thromboxane A 2 .The other product of the cleavage of
PIP 2 is diacylglycerol (DAG), which stimulates protein kinase C
(PKC) to phosphorylate the intracellular protein P47 to
P47-PO 4 . The latter, thromboxane and MLC-PO 4 , together
stimulate secretion of products of the dense - and lysosomal
granules. Platelet coagulant activity is generated by
coagulation factors, shown in roman numerals form tenase
(VIII, IXa, Ca 2 + ) and prothrombinase (Va, Xa, Ca 2 + ), on the
platelet external membrane phospholipid to convert
prothrombin (II) to thrombin (IIa). (Courtesy of Dr. A. Koneti
Rao.)
Other checks on unbridled platelet activation exist on the surface

of endothelial cells, including an ADP-destroying ectoenzyme
(ADPase), and thrombomodulin, a powerful thrombin inhibitor.
Endothelial cells, when stimulated by agonists such as ATP,
produce NO, a potent vasodilator that inhibits platelet function by
raising platelet cGMP (67). There is evidence to indicate that
platelets themselves have the capacity to form NO from L-arginine
and that this results in a rise in the concentration of cGMP, which
is a powerful intracellular regulator of platelet activity (68).
COAGULATION
Although it has been traditional (and useful for in vitro laboratory
testing) to divide the coagulation system into intrinsic and
extrinsic pathways, such a division does not occur in vivo because
tissue factorfactor VIIa complex is a potent activator of both
factor IX and factor X.
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Extrinsic System
The principal initiating pathway of in vivo blood coagulation is the
extrinsic system, which involves components from both the
P.8
blood and vascular elements (see Chapter 5). The crucial
component is tissue factor, an intrinsic membrane protein
composed of a single polypeptide chain that functions as a
cofactor to factor VIII in the intrinsic system, and to factor V in
the final common pathway (see Fig. 1-4). Tissue factor pathway
inhibitor (TFPI) is a protein that in association with factor Xa
inhibits the tissue factorfactor VII complex (69,70). Tissue factor
synthesis in macrophages and endothelial cells is induced by
endotoxin and by such cytokines as interleukin-1 and tumor
necrosis factor (71,72).
The major plasma component of the extrinsic pathway is factor
VII, one of a group of vitamin Kdependent proteins (including
factors IX and X, prothrombin, and protein C) synthesized as
prozymogens and converted (activated) to serine proteases by a
limited number of proteolytic cleavages (see Fig. 1-5). Protein S,
also a vitamin Kdependent protein, is a cofactor rather than a
zymogen. Common to these proteins are unique -glutamyl
carboxyl acid (Gla) residues at the N-terminal end of the molecule
that require vitamin K for proper synthesis by hepatocytes. This
postribosomal modification of the protein is required for calcium
binding, one calcium with the two carboxyl groups of a Gla
residue, thereby serving as a bridge for protein binding to the
phospholipid surface.
Both factor IX and factor X are activated by the TFFVIIa complex
and by factor Xa itself. The active form is designated g-glutamyl
factor VIIa and represents approximately 1% of total factor VII.
Interaction between the intrinsic and extrinsic pathways occurs at
several levels of the clotting cascade. The zymogen factor VII
itself has minimal but definite protease activity and is capable of
autoactivation. It converts factor VII to VIIa and then activates it,
thereby displaying both positive and negative feedback effects.
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FIGURE 1-4. The clotting cascade. The central precipitating

event is considered to involve tissue factor (TF), which, under
physiologic conditions, is not exposed to the blood. With
vascular or endothelial cell injury, TF acts in concert with
activated factor VIIa and phospholipid (PL) to convert factor IX
to IXa and factor X to Xa. The intrinsic pathway includes
contact activation of factor XI by the XIIaactivated
high-molecular-weight kininogen (HKa) complex. It should be
noted that the contact system contributes to fibrinolysis and
bradykinin formation in vivo. Factor XIa also converts factor IX
to IXa, and factor IXa, in turn, converts factor X to Xa, in
concert with factor VIIIa and PL (the tenase complex).
However factor Xa is formed, it is the active catalytic
ingredient of the prothrombinase complex, which includes
factor Va and PL and converts prothrombin to thrombin.
Thrombin cleaves fibrinopeptides (FPA, FPB) from fibrinogen,
allowing the resultant fibrin monomers to polymerize, and
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converts factor XIII to XIIIa, which cross-links (XL) the fibrin

clot. Thrombin accelerates the process (interrupted lines) by its
potential to activate factors V and VIII, but continued
proteolytic action also dampens the process by activating
protein C, which degrades factor Va and VIIIa. Thrombin
activation of factor XI to XIa is a proposed pathway. Natural
plasma inhibitors retard clotting: C1-inhibitor (C1 INH)
neutralizes factor XIIa, tissue factor pathway inhibitor (TFPI)
blocks factor VIIa/TF, and antithrombin III (ATIII) blocks
factors IXa and Xa and thrombin. Arrows, active enzymes;
filled rectangles, sites of inhibitor action; dashed lines,
feedback reactions.
The factor VIIatissue factor enzyme complex, which assembles on

the activated monocyte or perturbed endothelial cell, has two
principal substrates, factor IX (see Chapter 7) and factor X (see
Chapter 10), both vitamin Kdependent proteins. Cleavage of
either protein results in a serine protease, factor IXa or Xa, that
remains membrane-bound. Its Gla residues facilitate further
reactions if appropriate cofactors are present. The required
cofactor for factor IXa to catalyze the conversion of factor X to
factor Xa is factor VIII (see Chapter 8), whereas that for Xa
conversion of prothrombin to thrombin is factor V (see Chapter 9).
Factor VIII exists in plasma mostly as a noncovalent complex with
VWF, and its coagulant function is to accelerate factor IXa
conversion of factor X to Xa. The absence of factor VIII or IX
underlies the hemophilia syndromes, classic hemophilia A and
hemophilia B, which produce identical hemorrhagic states. Perhaps
the similarity of the hemorrhagic condition with either factor VIII
or IX deficiency results from a lack in each case of a
P.9
proper tenase complex that is crucial for factor X activation (Fig.
1-4). The severity of the clinical disorder reflects the
concentration of factor VIII or IX. The most severe clinical
disease, manifest by spontaneous joint hemorrhage
(hemarthroses), occurs with factor VIII or factor IX levels of 0%
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to 1% (see Chapter 50). At factor levels of 5% to 30%, symptoms

may be mild or even nonexistent, except in serious trauma such as
surgery, and activity above 30% usually suffices for normal
hemostasis. The presence of more than twice as much factor VIII
or IX in healthy persons as is present in carriers (mean 50%)
indicates that clotting proteins usually are present in excess and
that deficiencies must be relatively severe to produce clinically
significant effects.
FIGURE 1-5. Tentative structures of human prothrombin,

factor IX, factor X, and protein C. The Ys refer to the
-carboxyglutamic acid residues present in the Gla domains.
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The open diamonds refer to potential N-linked carbohydrate

chains. Cleavage sites and the enzymes involved during the
activation of each protein are identified by solid arrows. Amino
acids involved in catalysis (H, D, S) also are identified.
Proposed disulfide bonds have been placed by analogy to those
in bovine prothrombin and epidermal growth factor.
The direct conversion of factor X to factor Xa by factor VIIatissue

factor complex bypasses the need for factor VIII or IX.
Nonetheless, a congenital deficiency of factor VII or X produces a
similar hemorrhagic condition, and distinguishing one from the
other requires the determination of specific coagulation factor
activities (see Chapter 58). A clinically definable decrease in
tissue factor has not been described.
Intrinsic System
Parallel with the extrinsic system is the intrinsic system, which
could be defined as coagulation initiated by components entirely
contained within the vascular system. This pathway results in the
activation of factor IX by a novel dimeric serine protease, factor
XIa (Fig. 1-4), providing a pathway independent of factor VII for
blood coagulation. However, an important difference
P.10
exists between these two pathways in the clotting cascade.
Whereas the activation of factor IX by XIa requires only the
presence of ionized calcium, the activation of factor IX by VIIa
requires calcium and the protein cofactor, tissue factor, embedded
in a cell membrane (lipid bilayer).
The role of the contact system proteins (see Chapter 6) in
initiation of the intrinsic pathway of coagulation in hemostasis is
questionable, because only a deficiency of factor XI is associated
with a hemorrhagic tendency. These proteins participate instead in
the initiation of the inflammatory response, complement
activation, fibrinolysis, angiogenesis (73), and kinin formation
(74), and studies show that kininogen is an anticoagulant protein
in vivo (75). The mechanism may be the inhibition of binding of
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low concentrations of thrombin to platelet GP Ib/IX (76). The

contact system is involved when blood interacts with a foreign
surface, as in cardiopulmonary bypass. The zymogen factor XII
(Hageman factor) is the first protein in the series of tightly
regulated reactions (Fig. 1-4) and binds to negatively charged
surfaces such as kaolin, dextran sulfate, and sulfatides. The heavy
chain of factor XII binds to the surface, allowing a large increase
in local concentration of the enzyme, autoactivation, and action on
its substrates, prekallikrein and factor XI, to form kallikrein and
factor XIa (77). In most coagulation enzymes, the light chain
contains the active site residues serine, histidine, and aspartic
acid, homologous to the archetypal serine protease chymotrypsin,
whereas the heavy chain contains binding regions to surfaces,
phospholipids, cell membrane, and connective tissue, which define
the unique role of each coagulation proteolytic enzyme.
The assembly of cofactor, enzyme, and substrate is a recurrent
theme in blood coagulation, resulting in maximal efficiency and
speed of the molecular reactions, especially as a phospholipid or
cell membrane provides the surface for efficient positioning of
interacting enzyme complexes with proenzyme substrates.
Kallikrein cleaves HK to liberate the nonapeptide bradykinin, and
the remaining kinin-free kininogen (activated HK) binds at least
10-fold better to surface than to the intact procofactor, thereby
allowing more prekallikrein to associate with the urokinase
receptor on the endothelial cell (78), enhance fibrinolysis (79),
and inhibit angiogenesis (73).
Negative feedback regulation is characteristic of the coagulation
system. One such reaction is factor XIa cleavage of the light chain
of HK, which contains the coagulant activity, thereby destroying
its cofactor activity and allowing factor XIa to dissociate from the
activating surface (80). Similarly, thrombin activates factors V and
VIII, but conversion of protein C to activated protein C leads to
the destruction of factors Va and VIIIa. Although deficiency of any
of the three proteins involved in the contact system pathway
results in slow generation of thrombin and a prolonged in vitro
partial thromboplastin time, their effect in vivo appears to be
unrelated or the opposite. HK is an antithrombotic protein after
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endothelial injury (75), and a deficiency may predispose to

thrombosis. Factor XII deficiency has been implicated as a risk
factor in venous and perhaps arterial thrombosis (81), so it may
be a natural anticoagulant. Only a deficiency of factor XI may
result in a hemorrhagic disorder. Even factor XI deficiency results
only in a mild disorder of hemostasis in half of the affected
individuals, and it is likely that blood coagulation in vivo is
initiated by factor IX or X through TFVIIa mechanisms.
Coagulation Common Pathway

Once factor Xa is formed by either the extrinsic or the intrinsic
pathway, it converts prothrombin to thrombin (see Chapter 10). As
with the other vitamin Kdependent factors (Fig. 1-5),
prothrombin has distinct functional domains devoted to calcium
binding to phospholipids (10 Gla residues at the N-terminal
portion). This region resembles epidermal growth factor containing
-hydroxyaspartic acid or asparagine, which can bind Ca 2+ a
region for cofactor (factor V) interaction, an activation peptide
region, and a portion containing the catalytic center. Elevated
prothrombin levels due to a mutation in the untranslated region,
G20120A, result in a common genetic cause of the
hypercoagulable state (82). After appropriate cleavage of
prothrombin by factor Xa, the N-terminal Gla portion is removed,
and the resultant two-chain thrombin molecule detaches from the
phospholipid surface. The interaction of the four components of
the prothrombinase complex (factor Xa, factor V, phospholipid,
and calcium) provides a markedly increased rate of prothrombin
activationmore than 300,000-fold more than that achievable with
only the enzyme (factor Xa) and substrate (prothrombin). Factor V
that participates in this prothrombinase complex on the platelet
membrane probably is supplied as the result of its secretion from
platelet or fusion with the plasma membrane, and it serves as a
receptor for factor Xa binding to the activated platelet (83).
Because of this involvement of platelets, the bleeding
manifestations of factor V deficiency may resemble those of
qualitative platelet disorders. Alternative pathways for
prothrombin activation by factor Xa independent of factor V have
been described in malignant cells, hypoxic endothelial cells, and
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macrophages (84,85 and 86).

Blood coagulation proteins can be grouped according to shared
properties, activities, or localization. For instance,
phospholipid-oriented enzymes require vitamin Kdependent
carboxylation of glutamic acid residues at their N-terminal
domains, and procofactors with no enzymatic activity share an
ability to facilitate attachment and interaction of clotting factors
on biological surfaces. Another grouping of factors includes those
that serve as a substrate for thrombin: for instance, cofactors V
and VIII (activated, then inactivated), protein C (activated),
prothrombin (cleaved to prethrombin), protein S (inactivated),
factor XIII (to form active fibrin-stabilizing factor), and fibrinogen
(liberating the fibrinopeptides). Further, deficiency of factor VIII
or IX produces the same clinical disorder by virtue of their
cooperation in the tenase complex. In addition, factor V,
fibrinogen, and the adhesive proteins fibronectin, VWF, and
thrombospondin are all stored in platelet -granules.
The mapping of the entire human genome has uncovered new
relationships of old pairings of coagulation proteins. Mutations in
the LMANI gene (87) leads to defects in the processing of both
factor V and VIII in the ERGogli subcellular system, explaining
the combined deficiency of these coagulation cofactors. Deficiency
of vitamin K epoxide reductase (88) leads to warfarin resistance of
factor II, VII, IX, and X. Another important new hemostatic gene
recently discovered (89) is ADAMTS, which controls the proteolytic
breakdown of VWF mutimers and ADAMTS deficiency, as associated
with thrombotic thrombocytopenic purpura (TTP).
Plasma proteolytic inhibitors (see Chapters 11, 13, 19, and 21)
serve to limit and control the extent and speed of both blood
coagulation and fibrinolytic reactions (see Table 1-1).
Inhibition of Coagulation
The major inhibitor of the contact system is C1 inhibitor, which
accounts for 95% of the plasma inhibitory capacity for factor XIIa
and more than 50% toward kallikrein; however, hereditary
deficiency of C1 inhibitor results in angioedema rather than
bleeding (90). 1 -Antitrypsin is the major inhibitor of factor XIa,
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but a more critical role is its inhibition of neutrophil elastase;

inhibitor deficiency results in emphysema due to the unopposed
effects of elastase in the lung alveoli (91).
TABLE 1-1 PLASMA PROTEASE INHIBITORS OF CONTACT

SYSTEM COAGULATION AND FIBRINOLYSIS
Plasma
concentration
Inhibitor
Molecular
weight
(daltons)
Major target
enzymes
g/mL
nM
1 -Protease
inhibitor
2,500
45,000
55,000
Factor XI,
elastase
Antithrombin III
290
4,700
62,000
Factor Xa,
thrombin
2 -Macroglobulin
2,500
3,400
725,000
Kallikrein,
plasmin,
thrombin
C1 inhibitor
240
2,300
105,000
Activated
factor XII,
kallikrein
2 -Antiplasmin
70
1,050
67,000
Plasmin
Heparin cofactor
II
40
600
65,000
Thrombin
Plasminogen
activator
10
200
50,000
Tissue
plasminogen
inhibitor-1
activator,
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(PAI-1)
Protein C
urokinase
10
53,000
inhibitor (PAI-3)
Tissue factor
pathway
inhibitor
Protein C,
kallikrein
0.1
0.25
40,000
Factor VIIa
(tissue
factor),
factor Xa
(tissue
factor)
P.11
ATIII is the major inhibitor of factors IXa, Xa, and thrombin.
Although enough ATIII is present to neutralize three times the
total amount of thrombin that could form in the blood, a decrease
to 40% to 50% predisposes to thrombotic disorders. That
congenital ATIII deficiency is associated with a strikingly increased
risk of venous thromboembolism indicates that inhibitors play a
major regulatory role and that a delicate balance exists between
the procoagulant and anticoagulant forces. The catalytic-site
serine of thrombin reacts with an arginine in the active center of
ATIII to form a covalent inactive complex. The inhibition produced
by ATIII is potentiated by heparin, a sulfated polysaccharide with
the highest negative charge of any naturally occurring polymer
and a close relative to the heparan sulfate that exists on the
endothelial surface (Fig. 1-2). Heparin binds to a basic group in
ATIII to increase its rate of inactivation of thrombin. Once
thrombin is bound to fibrin, it is resistant to ATIII and even more
so to ATIIIheparin complex (92,93). Heparin cofactor II is a
serpin (serine protease inhibitor) that selectively inactivates
thrombin (not factor Xa) in the presence of heparin or dermatan
sulfate (94).
Factor Xa also has a specific inhibitor, Z protease inhibitor, the
action of which is markedly enhanced by protein Z, a vitamin
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Kdependent protein, in a phospholipid and manner

Ca 2 + -dependent (95).
2 -Macroglobulin is a secondary or backup inhibitor for many
plasma coagulant and fibrinolytic enzymes, including kallikrein,
thrombin, and plasmin. Because enzymes trapped in the cage
structure of this inhibitor exhibit some activity, complexes may
serve as a repository of enzymatic activity that is protected
against other inhibitors. No clinical disorder of severe deficiency
has been described. is the primary inhibitor of plasmin acting to
prevent a systemic fibrinogenolytic response to noxious stimuli,
limiting the fibrinolytic response to thrombi in the affected region
and allowing hemostatic plugs to remain intact until healing is
complete (96). In the absence of hemostatic plugs dissolve before
healing has occurred and a hemorrhagic state results (97). A
deficiency in PAI-1 also results in a hemorrhagic tendency (98).
Protein C inhibitor also is a serpin that can inactivate protein C
and thereby function as a potential procoagulant molecule (99).
FIBRIN FORMATION AND FIBRINOLYSIS

Thrombin acts on multiple substrates, including fibrinogen; factors
XIII, V, and VIII; platelet membrane GP V; protein S; and protein
C (see Fig. 1-6). In so doing, thrombin occupies a central role in
the process of hemostatic plug formation, influencing its form,
rate of formation, and limitation. Its potentiating effect on factors
VIII and V produces an increase in the tenase and prothrombinase
complexes (Fig. 1-4), resulting in a burst of thrombin activity and
fibrin strand formation. When thrombin hydrolyzes factor V too
slowly because of a point mutation at a cleavage site, the result is
the most common genetic cause of thrombosis factor V Le id e n
(100). Thrombin also helps to recruit platelets into the hemostatic
plug, depending on the relative influences of intrinsic or extrinsic
clotting systems that are operative. When coagulation starts
principally on the altered platelet surface (intrinsic system),
thrombin formation is slower than when the extrinsic coagulation
system is initiated by exposure to tissue factor, a membrane
protein found in macrophages, activated endothelial cells, and
tumor cells. In the latter situation, thrombin might have a greater
influence on platelet aggregation.
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The formation of fibrin strands represents the second phase in

hemostasis (the first being the primary platelet aggregate). The
precursor of fibrin is fibrinogen, a large glycoprotein of Mr
340,000 present in high concentration in both plasma and platelet
granules that interacts with other proteins, including factor XIII,
2 -plasmin fibronectin, inhibitor, plasminogen, and plasminogen
activator (101). The location and surface concentration of these
modifying proteins influence the orderly process of fibrin
formation, cross-linking, and fibrin lysis. Thrombin binds to the
fibrinogen central domain and liberates fibrinopeptides A and B,
resulting in fibrin monomer and polymer formation (102).
Progressive lengthening of the polymer chain occurs by a
half-overlap, side-to-side approximation of fibrin monomer
molecules (see Fig. 1-7), and the two-stranded protofibrils
interact laterally to form long, thin fibrin strands or short, broad
sheets of fibrin (103,104). Although the degree of lateral strand
association probably contributes to the tensile strength of the
P.12
clot, its resistance to plasmin degradation is influenced mainly by
cross-linking mediated by factor XIIIa (105). In addition, factor
XIIIa, by linking 2 -plasmin inhibitor to fibrin, may protect the
clot against fibrinolysis. Factor XIII exists in plasma as a
four-chain precursor molecule of Mr 320,000, and after thrombin
activation, the enzyme (with calcium) induces cross-linking of the
fibrin polymer (106). Covalent isopeptide bonds form between
lysine donors and glutamine receptors, with two cross-linked
rapidly to form - dimers; chains are crosslinked more slowly,
each with two other such chains, to form a polymer network
(107,108). In mature forms, the fibrin fiber contains
approximately 100 protofibrils, with a somewhat random pattern
of branching that links the fibers together.
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FIGURE 1-6. The multitude of actions of thrombin, resulting in

procoagulant tendencies, potentiation of ongoing reactions,
feedback inhibition, and limitation of clotting.
The fibrin mesh binds the platelets together and contributes to

their attachment to the vessel wall, mediated by binding to
platelet receptor glycoproteins and by interactions with other
adhesive proteins such as thrombospondin, fibronectin, and
platelet fibrinogen (released from platelet granules but probably
otherwise equivalent to plasma fibrinogen) (109). After
attachment to platelet-binding sites, these proteins may serve as
molecular bridges between plasma proteins and the platelet
interior, between platelets and the vessel wall, and between
plasma
P.13
fibrin fibers and the subendothelial matrix. For instance,
fibronectin is cross-linked by factor XIIIa to fibrin, and its
separate binding site for collagen could serve to bridge fibrin to
the vessel wall (110,111). VWF also could serve as a bridge
between platelet membrane GP Ib (or IIb/IIIa) and a
subendothelial matrix component (112). Additionally, the platelet
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membrane GP IIb/IIIa could join plasma fibrinogen (or -granule

fibrinogen) to intracellular actin, thereby mediating clot retraction
and vessel wall constriction (113).
FIGURE 1-7. Fibrinogen and thrombin-induced fibrin monomer

and polymer formation, factor XIIIainduced fibrin
cross-linking, and plasmininduced cross-linked fibrin
degradation. The curly lines represent coiled coils between
central and terminal domains, and the double horizontal lines
represent cross-link sites induced by factor XIIIa between
chains of two contiguous fragment D-domains. The central and
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two terminal domains of fibrinogen are included in the

fragment E- and D-domains, respectively. The fibrinopeptides
are indicated as small vertical lines connected to the central
(E) domain of fibrinogen and are absent from the fibrin
monomer molecules after thrombin action. Plasmin action is
depicted here as limited to cleavage of the coiled coils between
center (E) and terminal (D) domains, to yield the complexes
noted at the bottom. These complexes consist of two
noncovalently bound fragments (e.g., fragments DD and E in
the DDE complex).
The potential for hemorrhagic or thrombotic disease that results

from derangements in fibrinogen structure, concentration, or
interaction with thrombin or factor XIII, is great and varied. It
could, for instance, manifest as a poorly polymerizing protein, by
slow or absent liberation of a fibrinopeptide, or as an inadequately
cross-linked fibrin (114). The latter situation similarly could be
produced by an absent or faulty factor XIII molecule, which could
contribute to both a hemorrhagic condition and inadequate wound
healing. The most common acquired disorders of fibrinogen are
those of consumption, the disseminated intravascular coagulation
(DIC) syndromes, which may reflect excessive or inappropriate
coagulation or proteolytic degradation of plasma fibrinogen and
can result in a variety of hemorrhagic and thrombotic
manifestations, depending to a great extent on the underlying
pathologic process (115).
Several distinct mechanisms for controlling and localizing
hemostasis exist, including the effects of vascular flow and
hemodilution, proteolytic feedback by thrombin, inhibition by
plasma proteins and endothelial celllocalized activation of an
inhibitory enzyme (protein C), and fibrinolysis. First, the
hemostatic plug is exposed to the disruptive pull of blood flow,
and small clumps of platelets that are inadequately attached to the
main body of platelets or to the vessel wall can be washed free
into the blood. Second, thrombin that is present in the hemostatic
plug and that has already contributed to its formation by
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potentiating the activation of factors V and VIII ultimately

inactivates these same cofactors (Fig. 1-6) in the presence of
thrombomodulin, a membrane protein of endothelial cells. This
complex effect of thrombin is an exquisite yet simple example of
self-dampening that effectively limits the growth of the
fibrin-platelet plug. Third, soluble activated coagulant proteins
such as factor Xa or thrombin may diffuse away from the clot, to
be bound to inhibitory plasma proteins that destroy or at least
markedly decrease their coagulant potential. Principal among
these inhibitors is ATIII, which forms a tight complex not only with
thrombin but also with other serine protease coagulant proteins
and with the fibrinolytic enzyme plasmin. However, although
thrombin can be readily inactivated by ATIII in solution,
thrombin's catalytic site is inaccessible to the inhibitor while the
enzyme is bound to fibrin, and it may retain the ability to cleave
fibrinopeptides even in the presence of heparin. Fourth, thrombin
that diffuses into the endothelial cell surface may bind to a
specific receptor, thrombomodulin, thereby setting into motion
another restraint on local coagulation. As previously stated, the
thrombinthrombomodulin complex serves as a receptor for the
vitamin Kdependent protein C (Figs. 1-2 and 1-5), which is
activated and released from the endothelial cell surface. Activated
protein C reacts with factors V and VIII to destroy their coagulant
properties, thereby limiting the effect of thrombin. Patients with
deficiencies of protein C, protein S (a cofactor of protein C), and
ATIII have been described in whom the hemostatic process is not
effectively limited and in whom there is a lifelong tendency for
pathologic thromboembolic disease.
The last mechanism for limiting clot formation is fibrinolysis, which
also constitutes a repair mechanism, along with endothelial cell
regrowth and vessel recanalization. Fibrinolysis resembles the
cascade mechanism of clotting factor activation in that it involves
zymogen-to-enzyme conversions, feedback potentiation and
inhibition, and a finely tuned balance with inhibitors. The inactive
precursor protein is plasminogen, present in plasma at twice the
molar concentration of inhibitor. During the initial period of
hemostatic plug formation, platelets and endothelial cells release
plasminogen activator inhibitors that facilitate fibrin formation
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(116). However, in response to a poorly understood but precisely

timed and orchestrated sequence of stimuli, endothelial cells
liberate tissue plasminogen activator (117). Both tissue
plasminogen activators and prourokinase have the capacity to
convert plasminogen (especially a plasminogen molecule bound to
fibrin) to the serine protease-active form, plasmin (118).
As with thrombin feedback that leads to accelerated factor Xa
formation, plasmin also exerts positive feedback by cleavage of an
activation peptide from plasminogen (connecting Gluplasminogen
to Lys-plasminogen), rendering it more susceptible to surface
binding and subsequent activation by plasminogen activators.
Perhaps more critical is the markedly heightened reactivity of
plasminogen after it is bound to fibrin by lysinebinding sites
located on its kringle structures. Lipoprotein A with multiple
kringles and histidine-rich glycoprotein also modulates
fibrinplasminogen interactions by inhibiting plasminogen binding
to fibrin (119,120).
Although only a small proportion of plasma plasminogen is bound
to fibrin during particulate clot formation, this is sufficient to
influence subsequent physiologic fibrinolysis (121). The process is
a balanced one, however, because 2 -plasmin inhibitor also is
bound to the fibrin, covalently attached by factor XIIIa action
(122). The relative proportions and positions of profibrinolytic
plasminogen and plasminogen activator molecules and
antifibrinolytic 2 -plasmin inhibitor molecules on the fibrin strand
influence the timing and degree of clot dissolution. Clinical
derangements related to the molecular disorders include a
hemorrhagic disorder due to deficient or defective 2 -plasmin
inhibitor and PAI-1 (123).
Studies have elucidated an important connection between the
coagulation and fibrinolytic pathways by virtue of
thrombinthrombomodulin mediation of both protein C and TAFI
activation. Whereas activation of protein C leads to inactivation of
factors Va and VIIIa and curtailment of further clot formation,
TAFI activation promotes stabilization of fibrin and therefore
persistence of formed fibrin clots. The mode of action of TAFI is to
cleave C-terminal lysine residues from fibrin, thereby preventing
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plasminogen, plasmin, or tissue-type plasminogen activators (tPA)

from binding to fibrin, and secondarily, inhibition of fibrinolysis.
Clinical conditions of decreased coagulation, such as classic
hemophilia, not only are deficient in thrombin and fibrin formation
but, by virtue of low TAFI formation, allow fibrinolysis to proceed
relatively unimpeded. The combination of less fibrin and more lysis
contributes to the bleeding seen in patients with factor VIII
deficiency. Similarly, patients with deficiency of contact-induced
coagulation also appear to have decreased TAFI activation,
perhaps by inadequate completion of clotting after initial fibrin
formation. On the other hand, patients with deficiency of protein C
manifest a thrombotic tendency by virtue of a failure of feedback
inhibition of factors Va and VIIIa by thrombin. The predilection to
thrombosis also may have a contribution by excessive TAFI
formation due to continuously high production of thrombin. In this
case, not only are thrombin and fibrin formed but such fibrin is
rendered resistant to plasmin lysis by TAFI.
The enhanced fibrinolysis seen with activation of the plasma
kallikreinkinin system has now been explained. When HK is
cleaved by kallikrein, bradykinin is released, which stimulates
release of tPA. The cleaved HK (HKa) binds to endothelial
urokinase plasminogen [urokinase plasminogen activator receptor
(uPAR)] (78). The event places HKa [in complex with prekallikrein
(77)] in position to bind to domains 2 and 3 of uPAR in close
proximity to prourokinase bound to domain 1
P.14
of uPAR, thereby enhancing the possibility of activation of
prourokinase to urokinase. PK is converted to kallikrein by the
endothelial cell membrane protease prolylcarboxypeptidase (124).
This localization allows kallikrein to efficiently activate
prourokinase to urokinase (125) without inhibition by plasma
serpins such as C1 inhibitor. Direct evidence for this is provided
by a study showing that HKPK interaction is required (79) for
plasmin formation by prourokinase on the endothelial cell
membrane.
Once plasmin is produced locally on the hemostatic plug, the
potential for fibrin degradation exists. By an intricate balance of
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the simultaneous forces of coagulation and platelet aggregation,

inhibition of coagulation, profibrinolytic and antifibrinolytic
reactions, and cellular mechanisms for both coagulation and lysis
(in leukocytes as well as in platelets and endothelial cells), the
clot is gradually reduced. The neutral serine protease (elastase)
released from the primary granules of neutrophils also contributes
to the local fibrinolytic potential (126). The surface of the clot
may be removed first, revealing fresh surfaces that are
progressively attacked until the process is completed (127).
During hemostatic plug or thrombus dissolution, solubilized fibrin
degradation products are liberated into the circulation, some of
which represent unique cross-linked derivatives such as D-dimer
that can be distinguished from fibrinogen degradation products
(128). The circulating degradation products serve as diagnostic
markers of thrombin or factor XIIIa, or both, plus plasmin action
that reflects prior clot formation and ongoing fibrinolysis. The
surface of a clot and circulating fibrin derivatives may possess a
small but significant amount of active thrombin that could serve to
propagate the coagulant process elsewhere in the circulation
(129). Active plasmin molecules also may be released into the
circulation during fibrinolysis, but just as free thrombin is
neutralized by ATIII, plasmin is extremely susceptible in solution
to inhibitor neutralization by inhibitor (130). This latter reaction
serves to limit fibrinogenolysis to the region of the clot, just as
ATIII serves to prevent disseminated coagulation by the spread of
a regional hemostatic process.
When hemostatic plug formation is defective (e.g., in hemophilia),
naturally occurring fibrinolysis may aggravate bleeding;
conversely, the use of epsilon aminocaproic acid aids in
hemostasis. This mechanism also may apply in bleeding after
dextran infusion, 2 -antiplasmin deficiency, and factor XIII
deficiency. In the latter case, the lack of cross-links leads to
increased susceptibility to plasmin and to failure to cross-link
antiplasmin to the fibrin clot, which also may lead to increased
fibrinolysis and hemorrhage.
The entire process involving endothelial cells and platelets,
clotting factors and adhesive proteins, and inhibitory mechanisms
04/05/2006 09:59 p.m.
36 de 51
of clotting, fibrinolysis, and platelet aggregation serves to promote

the right balance and location of hemostasis and recovery. This
highly developed system of checks and balances allows a rapid and
efficient hemostatic response to bleeding but avoids a
thrombogenic response away from the site of injury or persisting
beyond the time of its physiologic need. Derangement of any
portion of the intricate process can produce an imbalance,
sometimes only slight, with a resultant hemorrhagic or thrombotic
clinical disorder. A further complicating feature of this delicate
balance is therapeutic intervention, which must be carefully
regulated to correct a hemostatic defect without upsetting the
balance too far and thereby leading to thrombosis.
References
1. Cines DB, Tomaski A, Tannenbaum S. Immune
endothelial-cell injury in heparin-associated thrombocytopenia.
N Engl J Med 1987;316(10):581589.
2. MacGregor RR, Friedman HM, Macarak EJ, et al. Virus
infection of endothelial cells increases granulocyte adherence. J
Clin Invest 1980;65(6): 14691477.
3. Janoff A, Sloan B, Weinbaum G, et al. Experimental
emphysema induced with purified human neutrophil elastase:
tissue localization of the instilled protease. Am Rev Respir Dis
1977;115(3):461478.
4. LeRoy EC, Ager A, Gordon JL. Effects of neutrophil elastase
and other proteases on porcine aortic endothelial prostaglandin
I2 production, adenine nucleotide release, and responses to
vasoactive agents. J Clin Invest 1984;74:10031010.
5. Kitchens CS. The anatomical basis of purpura. Prog Hemost
Thromb 1982;5:211210.
6. Hynes RO. Integrins: a family of cell surface receptors. Cell
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1987;48(4):549554.
7. Dustin ML, Garcia Aguilar J, Hibbs ML, et al. Structure and
regulation of the leukocyte adhesion receptor LFA-1 and its
counterreceptors, ICAM-1 and ICAM-2. Cold Spring Harb Symp
Quant Biol 1989;54(Pt 2):753765.
8. Hession C, Osborn L, Goff D, et al. Endothelial leukocyte
adhesion molecule 1: direct expression cloning and functional
interactions. Proc Natl Acad Sci U S A 1990;87(5):16731677.
9. Rosenberg RD, Aird WC. Vascular-bedspecific hemostasis
and hypercoagulable states. N Engl J Med
1999;340(20):15551564.
10. Ofosu FA, Buchanan MR, Anvari N, et al. Heparin, heparan
sulfate and dermatan sulfate. Ann N Y Acad Sci 1989;556:123.
11. Marcus AJ, Weksler BB, Jaffe EA. Enzymatic conversion of
prostaglandin endoperoxide H2 and arachidonic acid to
prostacyclin by cultured human endothelial cells. J Biol Chem
1978;253(20):71387141.
12. Moncada S, Vane JR. The role of prostacyclin in vascular
tissue. Fed Proc 1979;38(1):6671.
13. Weiss HJ, Turitto VT. Prostacyclin (prostaglandin I2, PGI2)
inhibits platelet adhesion and thrombus formation on
subendothelium. Blood 1979;53(2):244250.
14. Weksler BB, Marcus AJ, Jaffe EA. Synthesis of prostaglandin
I2(prostacyclin) by cultured human and bovine endothelial
cells. Proc Natl Acad Sci U S A 1977;74(9):39223926.
15. Busch C, Owen WG. Identification in vitro of an endothelial
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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Copyright 2006 Lippincott Williams & Wilkins

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

which promote platelet adhesion, and tissue factor, a membrane

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

inhibitors. Plasmin normally does not act on fibrinogen in solution

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

associated with endothelial injury may be mediated by immune

FIGURE 1-1. Overview of hemostasis. ADP, adenosine

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Endothelial cells lose their nonthrombogenic protective properties

FIGURE 1-2. Thromboresistant properties of endothelium. The

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

and synthesize prostacyclin (PGI 2 ) thrombomodulin, heparan,

Endothelium is heterogeneous, both metabolically and structurally

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

extravasation of erythrocytes, manifest clinically as petechiae,

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

preventing its action (25). The enhancement of fibrinolysis by

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Chapter 41 summarizes the changing focus of the function of

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

secretion of stored platelet constituents (including molecules

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

to one another, forming a mass of aggregated platelets. A crucial

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Specific receptors exist on the platelet surface for these agonists

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

proteins, cleavage of receptor proteins (54), and thrombin-induced

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

binding studies (62,63). All

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

FIGURE 1-3. Platelet function. Adhesion to endothelial cells is

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

catalyzes the cleavage of phosphatidyl inositol bisphosphate

Other checks on unbridled platelet activation exist on the surface

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

FIGURE 1-4. The clotting cascade. The central precipitating

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

converts factor XIII to XIIIa, which cross-links (XL) the fibrin

The factor VIIatissue factor enzyme complex, which assembles on

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

to 1% (see Chapter 50). At factor levels of 5% to 30%, symptoms