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332 Chapter 37/Fundamental Mechanisms in Venous Thrombosis
https://t.me/med1917
also raise valid concerns regarding side effects such as the
loss of bone mineral density and alopecia.
14,15
Systemic thrombolytic therapy has not demonstrated
consistent effi cacy due to diffi culties in patient selection and
an unacceptable risk of catastrophic bleeding complica-
16,17
tions.
Catheter-directed thrombolysis in the fi rst week
following ileofemoral DVT formation has a lower incidence
of bleeding complications and has demonstrated benefi t in
terms of venous patency and a decreased incidence of
18
CVI.
The development of new therapies for the treatment of
VTE and the prevention of its sequelae will require a more
thorough understanding of venous thrombosis, propagation,
and resolution. The goals of any new therapy should be a
reduction in hemorrhagic complications and increased
effi cacy.
Infl ammation and Thrombosis
In the 1850s, Ludwig Rudolf Karl Virchow postulated
what came to be known as Virchow’s Triad of risk factors
for DVT formation: blood stasis, vessel wall injury, and
changes in the constituents of blood (hypercoagulability). A
contemporary view still benefi ts from this framework but is
informed by knowledge of genetics and molecular biology.
Mammalian physiology strikes a fi ne balance among coagulation factors, inhibitors of coagulation, and fi brinolytic
factors to optimize hemostasis while maintaining fl uidity
and end-organ oxygen delivery. There are many described
perturbations in this balance leading to hemophilic and
thrombophilic disorders. These perturbations are discussed
in detail in other chapters of this text.
A signifi cant advance from the time of Virchow occurred
with the work of Stewart et al. in 1974, who suggested a
link between vascular infl ammation and thrombosis.19 Stewart’s original hypothesis stated that the initiating factors that
promote thrombosis cause the activation of leukocytes and
platelets. She proposed that this activation leads to the localization of leukocytes and platelets to the area of injury,
resulting in amplifi cation of the thrombus. Advances in
molecular biology since 1974 have allowed the elucidation
of the relationship between infl ammatory mediators and
pathways with the coagulation cascade. Infl ammatory
mediators upregulate procoagulant factors and downregulate
natural anticoagulants while inhibiting fi brinolysis (see
Table 37.1).
mation such as the pro-infl ammatory cytokine TNFα, cytokines released locally from the area of injury such as IL-6
and IL-8, or factors localized within the growing thrombus
such as thrombin. Our research has focused on strategies to
treat venous thrombosis and prevent vein wall fi brosis via
inhibition of selectin signaling.
20
These may be systemic mediators of infl am-
TABLE 37.1 Impact of Infl ammation on Coagulation
(Adapted from Esrnon
Elevated Tissue factor
Negatively charged phospholipids
Platelet reactivity
Fibrinogen
Selectins
Diminished Thrombomodulin
Endothelial protein C receptor
Activated protein C half life
Protein Z
Vascular heparins
Fibrinolysis
20
)
SELECTIN AND MICROPARTICLE
BIOLOGY
Selectin Overview
The selectins are a well-conserved family of glycoprotein, transmembrane molecules expressed on the surface of
leukocytes, platelets, and endothelial cells. They play a
crucial role in leukocyte and platelet rolling and adhesion to
areas of vascular injury and infl ammation.21 Three selectins
have been described: L-selectin, E-selectin, and P-selectin.
P-selectins are stored as transmembrane proteins in preformed cytoplasmic granules mobilized by activated platelets and endothelial cells. E-selectin is inducible on vascular
endothelium, and L-selectins are expressed by nearly all
leukocytes.
The selectin family has a unique extracellular structure
consisting of an amino terminal calcium dependent lectin
domain, an epidermal growth factor-like domain, and two
to nine consensus repeat sequences (CRS) that are homologous to complement binding domains. They also possess a
lipophilic transmembrane domain and a short cytoplasmic
tail. The variable structural length of consensus repeat
sequences determines which complementary ligand carbohydrates the conserved lectin and epidermal growth factor
domains it will interact with.22 L-selectin has two consensus
repeat sequences, E-selectin six consensus repeat sequences
and P-selectin nine consensus repeat sequences.
Leukocyte adhesion to endothelium is controlled by
the binding of vascular selectins to leukocyte expressed glycoproteins or glycolipids containing the tetrasaccharide
sialyl-Lewisx (sialic acid, galactose, fucose and N-acetylgalactosamine).22 The ligands for P-selectin (PSGL-1) and
E-selectin (not well defi ned) are found on leukocytes, and
the ligands for L-selectin (CD34, MAdCAM-1) are found
on endothelial cells. Each ligand functions by presenting
O-linked saccharide chains to the calcium dependent lectin
domain on the corresponding selectin. Our research has
focused on modulating the P-selectin:PSGL-1 signaling axis
with the therapeutic goals of preventing DVT formation,
Selectin and Microparticle Biology 333
https://t.me/med1917
treating established DVTs and preventing chronic venous
insuffi ciency.
Selectin Biology
P-selectin is stored in the alpha granules of platelets and
in the Weibel-Palade bodies of endothelial cells (EC).23
Exposure to an activating stimulus such as thrombin results
in rapid translocation of P-selectin to the cell surface,
avoiding the need for transcription or translation.24 Eselectin is upregulated after the initiation of thrombosis in a
transcription-dependent fashion. P-selectin can be secreted
into the circulation as a component of EC and plateletderived microparticles (MP) or, in small quantities, as a free,
alternatively spliced version lacking a transmembrane
domain.25 These two forms of soluble P-selectin are elevated
in humans in association with atherosclerosis and thrombosis and are predictive of future adverse cardiovascular
events, including myocardial infarction and stroke.
Soluble P-selectin levels are also elevated at times of overwhelming systemic thrombosis and consumption, such as
disseminated intravascular coagulation and heparin-induced
thrombocytopenia.
29
P-selectin plays a critical role in the initial adhesion and
rolling of platelets and leukocytes to areas of injury and
infl ammation via the P-selectin receptor, P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin also plays a prominent
role in hemostasis and thrombosis through PSGL-1 signaling in leukocytes and platelets and GPIbα in platelets. Pselectin defi cient mice demonstrate a hemophilic phenotype
with marked bleeding tendencies.30 Mice with a deletion of
the transmembrane domain of P-selectin have elevated
levels of circulating P-selectin resulting in thrombophilia.
P-selectin based therapy has been demonstrated to correct a
mouse model of hemophilia.
31
In animal models of DVT, we, and others, have demonstrated that P-selectin expression regulates fi brin deposition
and thrombus size.
32,33
We have also demonstrated that
P-selectin and E-selectin deletions are associated with
fi brin content.
34
In a primate model of stasis-induced DVT,
P-selectin blocking antibodies or antibodies blocking the Pselectin receptor, PSGL-1, inhibit thrombosis and promote
recanalization.
35,36
In animal models we have found that Pselectin blockade with monoclonal antibodies has been found
to be as effective as low molecular weight heparin in promoting thrombus resolution and preventing vein wall thrombosis, all without the risk of hemorrhagic complications.
We have also demonstrated that P-selectin inhibition is
an effective treatment for established primate and rodent
iliofemoral DVT through augmentation of fi brinolytic
38
activity.
The fi ndings of an improvement in spontaneous
thrombolysis in animals in which P-selectin is inhibited by
26–28
37
rPSGL-Ig are similar to results found in primate, porcine,
and rat models of arterial and venous thrombosis using P-
36,39–41
selectin inhibition.
A reduction in the fi brin content of
thrombi formed in the presence of P-selectin inhibition is
likely contributory, as leukocyte-platelet interactions leading
to fi brin deposition are P-selectin dependent.
33
Microparticles
Microparticles (MP) are small (less than 1 micrometer,
about the size of a bacterium) phospholipid vesicles that are
shed from a variety of cell types including platelets, leukocytes, and endothelial cells.
constituent of blood and can be isolated from plasma by
ultracentrifugation. Microparticles lack DNA and RNA but
are protein rich. All circulating blood cells, platelets, and
endothelial cells are capable of releasing MP in a calciumdependent fashion. The protein expression profi le of MP
depends on the mother cell of origin and the conditions
infl uencing their production. The protein and phospholipid
content of MP determines the biologic activity of MP with
regard to hemostasis and other physiologic processes. Subpopulations of MP rich in tissue factor (TF) and phosphatidylserine, two critical components of the coagulation
cascade, have been identifi ed.
of infl ammation once thought to be soluble actually are
carried by MP.
47
Lipid rafts are sphingolipid ordered, cholesterol-rich
microdomains fl oating within the more fl uid cell surface
bilayer (the “fl uid mosaic”).48 These rafts allow for the concentration of receptor clusters, G-proteins, adaptor proteins,
and downstream kinases to facilitate signal transduction
through the concentration of the proximal machinery of cell
signaling pathways.49 Apoptotic blebs and microparticles are
derived from raft-rich regions of the plasma membrane,
resulting in high concentrations of cholesterol, negatively
charged phospholipids, and protein complexes, as compared
with the cell of origin.
Microparticles are rich in the external leafl et aminophospholipids phosphatidylethanolamine and phosphatidylserine
providing a critical, negatively charged substrate for coagulation. Under quiescent conditions, membrane asymmetry is
maintained by the active transport of nonthrombotic, choline
phospholipids to the outer leafl et in exchange for aminophospholipids by the enzymes fl ippase and fl oppase. Cellular activation results in the induction of scramblase activity
and the inhibition of fl ippase. This results in a loss of asymmetry and induction of a procoagulant phenotype.
to mobilize aminophospholipids to the outer membrane
leafl et due to defective phospholipid translocation results in
a bleeding tendency manifested as Scott syndrome.
Rafts and raft-derived MP can concentrate tissue factor
(TF) in cavaolae where it is stored with tissue factor pathway
42–44
Microparticles are a normal
45,46
Several circulating markers
50
Inability
51
334 Chapter 37/Fundamental Mechanisms in Venous Thrombosis
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FIGURE 37.1 The role of microparticles in thrombus amplifi cation (Adapted from Myers et al
inhibitor (TFPI).52 Fusion of MP with activated platelets
results in decryption of TF and the initiation of thrombosis.53
Monocytes concentrate TF and PSGL-1 in rafts. Monocytederived MP deliver TF to areas of injury and infl ammation
by binding to P-selectin mobilized to the surface of activated
platelets and EC, resulting in the generation of fi brin (see
Figure 37.1).45 Through the depletion of cholesterol in the
raft regions of platelets and leukocytes, statin drugs may
decrease the risk of thrombosis through raft architectural
disruption. Cholesterol-depleted platelets and leukocytes are
less adherent and may release less thrombotic MP.
54
The P-selectin receptor, PSGL-1, is expressed on leukocytes and platelets as well as on their derived microparticles.
MP coexpressing TF and leukocyte markers have been
shown to accumulate in growing thrombi in a PSGL-1:
P-selectin dependent fashion.
55,56
P-selectin:PSGL-1 interactions also stimulate the production of thrombogenic MP
from leukocytes, particularly monocytes.
31,57
These prothrombotic MP express TF and possess a phosphatidylserine
rich anionic surface capable of assembling prothrombinase,
tenase, and factor V/Va.58 Hrachinova et al. treated mice with
hemophilia A with P-sel-Ig to generate prothrombotic MP.
This normalized tail bleeding times and systemic coagulation times through augmentation of the extrinsic coagulation
pathway.31 This strategy may result in new therapies for
hemophilia A patients with alloantibodies. Human pericardial MP expressing TF have been demonstrated to increase
59
thrombosis in a rat venous stasis model.
Venous stasis and
ischemia results in the upregulation of vascular P-selectin,
which localizes prothrombotic MP to the area of stasis and
promotes DVT formation (see Figure 37.1).
60–62
61
).
In some vascular models of injury, P-selectin dependent
TF accumulation and fi brin deposition begins within the
fi rst 20 seconds of injury, before leukocyte rolling occurs,
suggesting that a major function of selectins in venous
thrombogenesis is independent of leukocyte rolling and
extravasation. P-selectin:PSGL-1 interactions are critical for
the localization of prothrombotic MP to areas of injury and
infl ammation.
63,64
The relative importance of MP-born TF in
thrombosis appears to vary according to the nature and
scope of the injury. MP appear to play a critical role in
stasis-induced thrombosis and small vascular injuries. Chou
et al. demonstrated that leukocyte-derived TF contributed
signifi cantly to thrombus formation after laser injury of
the microvessels in the cremaster muscle, a small injury
model. Day et al. demonstrated that leukocyte-derived TF
did not contribute signifi cantly to thrombosis following
treatment of the carotid artery with ferric chloride, a
dramatic, somewhat artifi cial and large-scale injury. It is
likely that MP-bearing tissue factor contribute to the initial
thrombus formation and amplifi cation that occurs in
the minutes following small scale vascular injuries (see
Figure 37.2).
62
Microparticles are not only prothrombotic but also appear
to inhibit fi brinolysis. Platelet activator inhibitor-1 (PAI-1)
is stored in the α-granules of quiescent platelets.65 PAI-1 is
a potent inhibitor of tissue plasminogen activator (tPA) and
urokinase-type plasminogen activators, which are largely
66
responsible for the initiation of fi brinloysis.
Upon activa-
tion, MP shed from platelets express PAI-1, and these MP
are localized to the growing thrombus via P-selectin-PSGL1 interactions. In this manner, platelet microparticles are not
Thrombosis Resolution and Vein Wall Fibrosis 335
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FIGURE 37.2 Proposed role of tissue factor in vessel wall injury (Adapted from Polgar et al
only prothrombotic but also inhibit fi brinolysis, delaying
thrombus resolution.
67
Translating P-selectin and
Microparticle Research
Traditional anticoagulation is contraindicated in many
patients and results in signifi cant hemorrhagic complications.
Patients on optimal therapy have a 20% incidence of thrombus recurrence or extension. The new, infl ammatory perspective on VTE has opened up a line of attack along the axis of
selectin biology. Presently, we are conducting rodent and
primate research on the prophylactic and therapeutic use of
compounds that inhibit P-selectin or its receptor, PSGL-1, and
that can be delivered by multiple routes for use in VTE.
D-dimers are a plasmin breakdown product of insoluble,
cross-linked fi brin. Although D-dimers are elevated in
patients with VTE, this fi nding has a specifi city of only 50
to 70%. In current practice D-dimer is useful only when
negative in association with patients with low clinical assessment scores.
The abundance of evidence supporting an important role
for P-selectin and MP in thrombosis led us to hypothesize
that they may be reliable clinical indicators of an ongoing,
thrombotic event. We have found that a panel of blood tests
including D-dimer, P-selectin, and MP levels is more sensi-
68
62
).
tive and specifi c for the detection of DVT than D-dimer
alone, though still less sensitive than duplex ultrasound
(US). This panel would be most useful in locations or at
times when duplex US is unavailable. As our knowledge of
microparticle subtypes and protein expression improves, it
is likely that even more defi nitive tests for the detection of
thrombotic events and assessment of overall cardiovascular
risk will become available.
THROMBOSIS RESOLUTION AND
VEIN WALL FIBROSIS
The resolution of a deep vein thrombus parallels the traditional model of wound healing with a predictable orchestration of leukocyte infi ltration, the elaboration of profi brotic
growth factors and collagen deposition. This process is
required for thrombus resolution and neovascularization but
also results in vein wall fi brosis, valvular incompetency, and
subsequent CVI. There is indirect clinical evidence that the
length of time the thrombus is in contact with the vein wall
is proportional to the magnitude of the fi brotic response.
Early, aggressive anti-coagulation and catheter-directed
thrombolysis result in earlier thrombus resolution and a
reduction in the risk of CVI.
336 Chapter 37/Fundamental Mechanisms in Venous Thrombosis
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Neutrophils are the fi rst cell type to infi ltrate the thrombus and play an essential role in thrombus resolution.69 Neutropenia is associated with larger thrombi in a rat model of
stasis DVT and treatment with IL-8, a neutrophil stimulatory
and chemotactic cytokine, accelerates thrombus resolu-
70,71
tion.
Mice lacking the receptor for the IL-8 analogs KC
and MIP-2 have larger and less organized thrombi due to the
lack of neutrophil infi ltration.
72
Monocyte and monocyte-derived macrophage infi ltration
follows the neutrophil phase. Monocyte infi ltration peaks on
day 8 after formation of a stasis-induced DVT in animal
models and is dependent on the elaboration of monocyte
chemoattractant protein-1 (MCP-1) from the site of injury.
Exogenous introduction of MCP-1 into a newly formed
thrombus accelerates recanalization and resolution.73 Experimental over-expression of MCP-1 accelerates thrombus
resolution, and we have found that strategies that inhibit
monocyte infi ltration also inhibit thrombus resolution.
As venous thrombi resolve they demonstrate increased
urokinase-type plasminogen activator (u-PA) and tissue
plasminogen activator (t-PA) activity.74 The in vivo expression of u-PA and t-PA activity is monocyte dependent.75 A
series of experiments utilizing bone marrow transplantation
strategies in knock-out mice have revealed that monocyte
expressed u-PA plays a more critical role in thrombus resolution as compared to t-PA.76 Monocytes further degrade fi brin
via receptor-mediated lysosomal activity.77 Modulation of
monocyte infi ltration and the fi brinolytic pathway are potential targets for the treatment of VTE and prevention of
CVI.
The actions of infi ltrating leukocytes are critical for
thrombus resolution but also result in the elaboration of
profi brotic cytokines including TGF-β, RANTES, and MCP-
1.78 The kinetics of leukocyte invasion into the thrombus are
duplicated in the vein wall with an initial wave of neutrophil
invasion followed by monocyte infi ltration. Elastinolysis
occurs early, resulting in diminished vein wall compliance.
Collagen I and collagen III deposition follow, further stiffening the vein wall.
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937.
CHAPTER
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38
Congenital and Acquired
Hypercoagulable Syndromes
JOCELYN A. SEGALL and TIMOTHY K. LIEM
INTRODUCTION
A fi ne balance exists between anticoagulant, procoagulant, and fi brinolytic factors. Intravascular thrombosis represents a shift in this balance, and may occur as the result
of many factors in conjunction with a congenital or acquired
abnormality in coagulation. An understanding of the differing hypercoagulable syndromes is important to appreciate
the complexity of hemostasis and the factors that may offset
normal clotting and anticoagulant mechanisms (see Figure
38.1). In addition, methods of prophylaxis and treatment of
venous thromboembolism are increasingly being stratifi ed
based, in part, on the presence or absence of a thrombophilic
state. The presence of a hypercoagulable state does not
imply that the patient will have thrombosis of a vessel. It
does suggest that the individual is at a higher risk for thrombosis especially when the other factors of Virchow’s triad
(endothelial injury and stasis) are involved.
1
CONGENITAL VS. ACQUIRED
HYPERCOAGULABLE STATES
Some congenital hypercoagulable states place the individual at higher risk for thrombosis than others. Most genetic
abnormalities in existence have clinically imperceptible
consequences.
ple genetic abnormalities, increasing their risk of thrombosis.4 The common congenital hypercoagulable disorders are
listed in Table 38.1.
Many causes exist for acquired hypercoagulable states
and some congenital hypercoagulable states may exist as
acquired states as well. For instance, Protein C and S defi ciencies may occur secondary to decreased protein produc-
2,3
Additionally, some individuals have multi-
tion from liver failure, sepsis, and/or malnutrition and
increased protein loss secondary to nephrotic syndrome and
infl ammatory states.5 In addition, hyperhomocysteinemia
may occur because of enzymatic defects or because of defi ciencies in Vitamins B6 and B12 and folate. The acquired
hypercoagulable disorders are listed in Table 38.2.
THE CONGENITAL
HYPERCOAGULABLE DISORDERS
Antithrombin Defi ciency
Antithrombin is a serine protease inhibitor of thrombin
and also inhibits factors IXa, Xa, XIa, and XIIa. Thrombin
is irreversibly bound by antithrombin and prevents thrombin’s action on fi brinogen, on factors V, VIII, and XIII, and
on platelets.6 This anticoagulant is synthesized in the liver
and endothelial cells, and has a half-life of 2.8 days.7 Antithrombin defi ciency has a prevalence of 1 : 5000 with more
than 100 genetic mutations and an autosomal dominant
inheritance pattern.8 Homozygotes typically die in utero
whereas heterozygotes typically have an antithrombin level
that is 40 to 70% of normal.
Antithrombin defi ciency is associated with lower extremity venous thrombosis as well as mesenteric venous thrombosis, and there are two clinical types. Individuals with Type
I defi ciency have a reduced number and function of antithrombin, and individuals with Type II have normal production but a reduction in function. Additionally, the heparin
binding site of the antithrombin may be mutated.9 The risk
of thrombosis increases as the functional antithrombin activity decreases to less than 80% of normal levels. The highest
risk for thrombosis occurs when the activity is less than 60%
of normal.
1
The Vein Book
339
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Copyright © 2006, Elsevier Inc.
340 Chapter 38/Congenital and Acquired Hypercoagulable Syndromes
https://t.me/med1917
IX
XII
XI
XIIa
IX
XIa
Ca • VIIIa • PL
X
IXa
Xa
Ca • Va • PL
IIa II
fibrinogen fibrin
Antithrombin·heparin
Cross-linked fibrin
A
FIGURE 38.1 The coagulation cascade (light grey) and the sites of action for the natural anticoagulants (black). PC
= protein C, APC = activated protein C. Protein S (PS) is a cofactor for the inhibition of factors V and VIII. TFPI (tissue
factor pathway inhibitor) levels increase several fold in response to heparin. TFPI binds to factor VIIa, inhibiting the
conversion of factor X to Xa, and factor IX to IXa.
TABLE 38.1 The Most Common Congenital
Hypercoagulable Disorders
Congenital hypercoagulable disorders
Antithrombin Defi ciency
Protein C Defi ciency
Protein S Defi ciency
Factor V Leiden
Prothrombin G20210A Polymorphism
Hyperhomocysteinemia
Dysfi brinogenemia and Abnormal Fibrinogens
VIIa
Ca • TF • Xa
IX
XII XIIa
X
XI
IX
XIa
Ca • VIIIa • PL
X
IXa
Xa
Ca • Va • PL
VIIa
Ca • TF • Xa
IIa II
fibrinogen fibrin
PC
Thrombomodulin
B
oral contraceptive use, estrogen replacement, trauma,
surgery, or infection.
Antithrombin defi ciency should be suspected in a patient
with spontaneous thrombosis, in a patient who cannot be
anticoagulated adequately on heparin or in a patient who
develops thrombosis while on heparin. To detect this defi ciency, antithrombin levels should be measured when the
patient has not been exposed to heparin.
Protein C and Protein S Defi ciency
PS
PS
APC
Cross-linked fibrin
12
13,14
Heparin
·TFPI
X
TABLE 38.2 Acquired Hypercoagulable Disorders
Acquired hypercoagulable disorders
Heparin Induced Thrombocytopenia/ Hyperfi brinogenemia
Heparin Induced Thrombocytopenia and Nephrotic Syndrome
Thrombosis Syndrome Renal Failure
Lupus Anticoagulant/Antiphospholipid Vasculitis
Antibody Syndrome Malignancy
Smoking Thrombocythemia
Warfarin Homocysteinemia
Pregnancy Sepsis
Oral Contraceptive Pills/Hormone Obesity
Replacement Therapy Immobility
Mechanical Injury/Trauma/Surgery
Diabetes Mellitus
Hyperlipidemia
Polycythemia vera
The most common presentation in those with antithrombin defi ciency is deep venous thrombosis with or without
pulmonary embolism.
10
The frequency of thromboembolism
is unusual before the late teenage years and plateaus around
the age of 40.11 Thromboembolism may occur spontaneously
but is often precipitated by other factors such as pregnancy,
Protein C is a vitamin K dependent anticoagulant protein
that, once activated by thrombin, will inactivate factors Va
and VIIIa, thereby inhibiting the generation of thrombin.4
Additionally, activated protein C stimulates the release of tPA. It is produced in the liver and is the dominant endogenous anticoagulant with an eight-hour half-life. Protein C
defi ciency has a prevalence of 1 in 200–300 with more than
150 mutations and an autosomal dominant inheritance.
4,7
Similar to antithrombin defi ciency, protein C defi ciency has
two types: Type I, which is associated with decreased production and function and Type II, which is associated with
a low functional level.8 Type I defi ciency predominates.
Protein S is also a vitamin K dependent anticoagulant
protein that is a cofactor to activated protein C. The actions
of protein S are regulated by complement C4b binding
protein and only the free form of protein S serves as an
activated protein C cofactor.15 Additionally, protein S appears
to have independent anticoagulant function by directly
inhibiting procoagulant enzyme complexes.
7,16
The prevalence of protein S defi ciency is about 1 : 500 with an autosomal dominant inheritance. Three types of protein S
defi ciencies exist and include Type I, which is associated
with low levels of free and total protein S antigen and
The Congenital Hypercoagulable Disorders 341
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decreased activated protein C activity; Type II, which has
normal levels of protein S antigen but low levels of activated
protein C cofactor activity; and Type III, which has normal
to low levels of total protein S, low free protein S, and an
increased proportion of protein S bound to complement
C4b.8 In addition, many patients with protein S defi ciency
also have resistance to activated protein C, which may be
the reason for the thrombosis.
1
Clinically, protein C and S defi ciencies are essentially
identical. With homozygous protein C and S defi ciencies,
infants typically will succumb to purpura fulminans, a state
of unrestricted clotting and fi brinolysis. In heterozygotes,
venous thromboses may occur at an early age especially in
the lower extremity.17 Thrombosis may also occur in mesenteric, renal, and cerebral veins. Protein C and S defi ciencies usually become clinically evident when the levels of
these proteins are less than 50% of normal.
Plasma protein C and S concentrations may be obtained
to diagnose defi ciencies of these proteins. Antigen and activity levels of protein C are measured, whereas for protein S,
only antigen levels are measured. These measurements
should be made prior to starting anticoagulation therapy
with either heparin or warfarin.
8,13,18
Factor V Leiden Mutation and Activated
Protein C Resistance
Factor V is a glycoprotein synthesized in the liver. With
Factor V Leiden, a point mutation occurs when arginine is
substituted by glutamine at position 506. This point mutation causes the activated Factor V to be resistant to inactivation by activated protein C thus causing a procoagulant state.
The mutation appears almost exclusively in the Caucasian
population and inheritance is autosomal dominant. The relative risk of a thromboembolic event in a heterozygous carrier
is increased fi ve- to seven-fold over the general population
and increased up to 80-fold in a homozygous carrier.4 The
risk of thrombosis also increases with combined genetic
defects and/or additional acquired risk factors and will
exceed the sum of the separate risks.
Clinically, patients may present with deep venous thrombosis in the lower extremities, or less commonly in the portal
vein, cerebral vein, or superfi cial venous system. Laboratory
testing for the diagnosis of activated protein C (APC) resistance and Factor V Leiden may be performed using functional clotting based assays or by genetic testing. Factor V
Leiden is the most common cause for APC resistance. Other
less common causes include Factor V Cambridge, HR2 haplotype, Factor V Hong Kong, and Factor V Liverpool. The
functional clotting-based assays include a modifi ed aPTT,
which dilutes the patient’s plasma in factor V defi cient
plasma or incorporates dilute Russell’s viper venom in the
assay. The presence of APC resistance is then determined by
measuring the aPTT in the presence and absence of activated
15
protein C. This modifi ed test may be used in the presence
of heparin, warfarin, and lupus anticoagulant. The genetic
test relies on DNA amplifi cation using PCR and is the most
reliable test.
18
Prothrombin G20210 Polymorphism
Prothrombin (Factor II) is a zymogen synthesized in the
liver and dependent on vitamin K. When prothrombin is
activated, it forms thrombin (Factor IIa). A single mutation
where adenine is substituted for guanine occurs at the 20210
position. The mechanism for increased thrombotic risk is not
well understood, but individuals with this genetic variant
have supranormal levels of prothrombin. The mutation is
inherited as an autosomal dominant trait and is associated
with both arterial and venous thrombosis. Like Factor V
Leiden, this mutation occurs almost exclusively in the Caucasian population. Individuals with the prothrombin gene
variant are typically heterozygous.7 Heterozygosity confers
a two-fold increase in the risk of thrombosis, and homozygosity confers approximately a 10-fold increase in the risk
of thrombosis.
13
Clinically, patients may present with deep venous thrombosis of the lower extremity, cerebral venous thrombosis, as
well as arterial thrombosis. The risk of thrombosis increases
in the presence of other genetic coagulation defects and with
acquired risk factors.
1,7
Detection of the prothrombin G20210
polymorphism is by genetic analysis alone as no correlation
exists between functional prothrombin levels and those individuals with the genetic mutation.
13
Hyperhomocysteinemia
Homocysteine is an amino acid formed during the metabolism of methionine and may be elevated secondary to
inherited defects in two enzymes that are part of the conversion of homocysteine to cysteine. The two enzymes involved
are N5,N10–methylene tetrahydrofolate reductase (MTHFR)
or cystathionine beta-synthase. Hyperhomocysteinemia has
been shown to increase the risk of atherosclerosis, atherothrombosis, and venous thrombosis.
Elevated plasma homocysteine levels cause various dysfunctions of endothelial cells leading to a prothrombotic
state. With the oxidation of homocysteine, superoxide radicals are formed, which cause endothelial damage, smooth
muscle proliferation, and activation of platelets and leukocytes. Additionally, hyperhomocysteinemia augments factor
V and VII activity and decreases the activation of protein C,
indirectly stimulates platelet aggregation, decreases the production of endothelium-derived nitric oxide, and interferes
with the binding of t-PA.
In patients with unexplained venous thromboembolism,
homocysteine levels should be measured. Levels may be
measured by obtaining a fasting plasma homocysteine or
1,19
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