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Diagnostic approach to hypercoagulable states

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Bratisl Lek Listy 2006; 107 (8): 292 295

TOPICAL REVIEW

Diagnostic approach to hypercoagulable states

Remkova A
1st Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava,
Slovakia. anna.remkova@fmed.uniba.sk
Abstract
The primary hypercoagulable states are inherited thrombotic disorders, resulting from mutations in
genes encoding a plasma protein component of one of the coagulation mechanisms. The anticoagulant
pathways most frequently involved include antithrombin III, protein C, and protein S deficiencies and
activated protein C (APC) resistance. Around 80 % of all individuals with APC resistance carry a
mutation of the factor V gene. Abnormalities in procoagulant pathways mostly concern elevated levels
and/or function of procoagulant factors. Elevation in plasma prothrombin (FII) levels is associated with
a FII gene mutation. Hyperhomocysteinemia as a risk factor for thrombosis is determined by genetic
or dietary factors. The acquired or secondary hypercoagulable states consist of a heterogeneous group
of disorders with an increased risk for developing thrombosis. Many underlying conditions (e.g., malignancies) may induce changes in the coagulation system. The risk of thrombosis is increased in individuals with antiphospholipid antibodies. They are found in about one-half of patients with systemic
lupus erythematosus, but also in the course of infections, neoplastic diseases, and sometimes in apparently normal subjects. A definite molecular abnormality of hypercoagulable states can be identified in
the special coagulation laboratory, using two types of molecular risk factors for thrombosis (genetic
factors and abnormal laboratory phenotypes). Its recognition is useful for a prevention and/or therapy
of thrombosis (Tab. 4, Ref. 10).
Key words: coagulation factors, hypercoagulation, thrombophilia, thrombosis, antiphospholipid syndrome.
The pathogenesis of venous and arterial thrombosis is complex and multifactorial. Virchow (1865) identified three risk factors of thrombosis: damage to the vessel wall, reduction in the
blood flow and alterations in the blood components. The interactions between these factors play role in the activation of hemostatic system and thrombus formation.
The function of hemostatic system depends on complex reactions among its components: the platelets, vessel wall, coagulation factors with their inhibitors, and the fibrinolytic proteins
with their inhibitors. Disturbance in the balance between coagulation and fibrinolysis is prone to thrombosis. Patients with a
propensity to develop thrombosis are frequently labeled as having a hypercoagulable or prethrombotic state. In humans,
several of the conditions that predispose to thrombosis involve
the hypercoagulable states. Hypercoagulable state is a disorder
of the blood coagulation, with a shift of the hemostatic balance
towards the enhancement of procoagulant forces. When the hypercoagulability surpasses a certain threshold, thrombus formation will occur (1 6).

The body possesses natural antithrombotic protective mechanisms to counteract the initial thrombotic processes. One of them
is inactivation of the coagulation esterases by physiologic inhibitors such as antithrombin III, protein C, and protein S. Individuals with reduced activities of these inhibitors have a propensity for developing thrombosis. Disorders of these homeostatic
mechanisms can lead to an imbalance of coagulation (prothrombotic) over anticoagulation (antithrombotic) activities.
In some of the hypercoagulable patients, a definite molecular abnormality can be identified in the coagulation laboratory.
1st Department of Internal Medicine, Faculty of Medicine, Comenius
University, Bratislava, Slovakia
Address for correspondence: A. Remkova, MD, PhD, DSc, 1st Dept
of Internal Medicine, Faculty of Medicine, Comenius University,
Mickiewiczova 13, SK-813 69 Bratislava 1, Slovakia.
Phone: +421.2.57290249
Acknowledgement. This work was supported by Slovak Ministry of
Education grant VEGA No 1/2290/05.

Remkova A. Diagnostic approach to hypercoagulable states

Tab. 1. Classification of hypercoagulable states.
Inherited (Primary) Hypercoagulable States
Antithrombin III deficiency
Protein C deficiency
Protein S deficiency
Activated protein C resistance due to factor V gene mutation
Prothrombin gene mutation
Dysfibrinogenemia (rare)
Acquired (Secondary) Hypercoagulable States
Antiphospholipide syndrome
In association with physiologic or other thrombogenic stimuli
(e.g., pregnancy and the post-partum period, estrogen use, advancing age, immobilization, injury, postoperative state etc.)
In association with some clinical disorders (e.g., malignancy,
nephrotic syndrome, congestive heart failure etc.)
Mixed Hypercoagulation States
Hyperhomocysteinemia

Disorders of these homeostatic mechanisms can lead to an imbalance of coagulation (prothrombotic) over anticoagulation
(antithrombotic) activities (14).
Multiple genetic and environmental factors contribute to the
development of thrombosis. According to this concept, thrombosis is either a congenital disorder of the natural anticoagulant
pathways or an acquired disorder, which triggers the mechanism
of coagulation and/or reduces the natural anticoagulant activity
(Tab. 1). Two types of molecular risk factors for thrombosis can
be recognized: genetic factors and abnormal laboratory phenotypes (4).
Primary hypercoagulable states
The primary hypercoagulable states are inherited thrombotic
disorders, resulting from mutations in genes encoding a plasma
protein component of one of the coagulation mechanisms (3).
The term of inherited thrombophilia is applied to individuals with
atendency to thrombosis due to predisposing genetic defects.
The inherited thrombotic disorders are almost exclusively asso-

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ciated with venous thrombosis. It is currently possible to define

several genetic risk factors. The anticoagulant pathways most
frequently involved include antithrombin III (AT), protein C (PC),
and protein S (PS) deficiencies and activated PC (APC) resistance. APC-resistance is a defect in the protein C anticoagulant
pathway, resulting in the poor anticoagulant response of plasma
to activated protein C. Around 80 % of all individuals with APCresistance carry a mutation of the factor V gene (factor V Leiden).
Factor V (FV) Leiden is due to replacement of arginine-506 to
glutamine-506 (Arg506Gln) in one of the cleavage sites for APC.
The mutation in the gene of FV delays inactivation of the activated FV. Abnormalities in procoagulant pathways, which result
in enhanced fibrin formation, mostly will concern elevated levels and /or function of procoagulant factors. These can be genetically determined or be the result of an interaction with the
environment. Elevated levels of factor II, factor VIII, and fibrinogen are associated with an increased risk of thrombosis. Elevation in plasma prothrombin (FII) levels is associated with a FII
gene mutation, due to a G A transition in nucleotide 20210 in
the 3' untranslated region. Hyperhomocysteinemia, as a common
risk factor for both venous and arterial thrombosis, is determined
by genetic as well as by dietary factors. A common genetic cause
is the variant of the methylene tetrahydrofolate reductase
(MTHFR) gene that leads to a thermolabile variant of the enzyme and mildly elevated homocysteine levels. Genetic risk factors for thrombosis comprise all gene mutations that results in a
loss or gain function of the encoded protein. Loss of function is
responsible for hereditary deficiencies, like AT, PC, and PS deficiency. Such mutations prevent the synthesis of a normal protein
(type I deficiency) or result in the synthesis of an abnormal, functionally defective protein (type II deficiency). Laboratory diagnosis relies on the measurement of protein concentrations and/
or function in plasma. Mutations that result in a gain of function
include FV Leiden and FII gene mutation. DNA analysis seems
to be the method of choice for the determination of these molecular risk markers (4).
Examples of abnormal laboratory phenotypes as risk factors
for thrombosis include APC-resistance, mild hyperhomocystei-

Tab. 2. Differential diagnosis of hypercoagulable states.

Clinical characteristics

Laboratory tests

Personal history of proven venous thromboembolism

The hemostatic profile of AT, PC, PS, FV Leiden and FII gene mutation
should be investigated. The measurement of antiphospholipid antibodies
should also be performed.

Age at the first thromboembolic episode

A first episode before 40 45 years of age is frequent in patients with AT, PC,
PS deficiency. A first episode at an older age (over 60 years) without any
systemic disease (e.g. cancer) may be associated with FV Leiden.

Recurrence of thromboembolic episodes

In about 50 % of patients with thrombophilia due to AT, PC, PS deficiency.

Family history of venous thromboembolism

The hemostatic profile of AT, PC, PS, FV Leiden and FII gene mutation
should be investigated.

Personal or family history of fetal loss

In AT deficiency and in combined congenital coagulation abnormalities as

well as in FV Leiden. An antiphospholipid syndrome should be suspected in
subject with recurrent fetal loss.

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Bratisl Lek Listy 2006; 107 (8): 292 295

Tab. 3. Screening laboratory evaluation for hypercoagulable states.

Clotting assay for resistance to activated protein C
Genetic test for factor V Leiden
Genetic test for prothrombin gene mutation (G20210A)
Functional assay of antithrombin III
Functional assay of protein C
Functional assay of protein S
Immunological assays of total and free protein S. Screen for dysfibrinogenemias (immunologic and functional assays of fibrinogen,
thrombin time)
Measurement of total plasma homocysteine
Clotting assay for lupus anticoagulant
Serologic tests for antiphospholipid antibodies

nemia, and elevated factor VIII levels. The molecular basis for
some of these plasma phenotypes is incompletely known.
While hereditary deficiencies of AT, PC, or PS will be found
in fewer than 5 % of unselected patients presenting with venous
thromboembolism, the likelihood of identifying these defects is
increased several-fold by screening patients with the initial thrombosis occurring prior to age of 50 years, a family history of venous
thromboembolism, and recurrent venous thrombosis. FV Leiden,
FII gene mutation (FII G20210A), and hyperhomocysteinemia
are more prevalent defects, and can be found in significant numbers of patients with first episodes of idiopathic venous thrombosis (i.e., no apparent precipitating factor) after age 50 years in
the absence of a positive family history. Thrombotic episodes
can be triggered by various stimuli (e.g., pregnancy, estrogen
use, surgery) in perhaps 50 % of individuals with hereditary defects (6 9). Selection of patients for the laboratory investigation
of molecular risk markers is indicated in Table 2.
A diagnostic approach to patients suspected of having a biologic defect predisposing to thrombosis is based on screening
laboratory evaluation (Tab. 3).
The laboratory evaluation for a natural anticoagulant deficiency is performed by functional and immunological assays.
Functional tests are based either on amidolytic or clotting methods. They are the best screening tests for deficiencies of AT, PC,
and PS because they can detect both quantitative and qualitative
defects. Immunologic (antigenic) assays detect only quantitative
deficiencies of these proteins. They are recquired when functional tests are abnormal for classification in types I (quantitative) or type II (qualitative) deficiencies. The FV Leiden and FII
gene mutation can be performed by DNA analysis. Coagulation
assays for the FV Leiden mutation are based on the resistance of
the activated mutant FV molecule to inactivation by APC. To
screen for a dysfibrinogenemia, the thrombin time is recommended along with measurements of plasma fibrinogen by clotting and immunologic assay. Testing for hyperhomocysteinemia
should be done after an overnight fast and on normal diet.
For the laboratory diagnosis of primary hypercoagulable states
careful interpretation of the results aims toward the exclusion of
disseminated intravascular coagulation (DIC), or elimination of
another acquired hypercoagulable state or acquired natural anticoagulants deficiencies. The existence of underlying conditions

that influence normal hemostasis (such as pregnancy, oral contraception, hormonal replacement therapy, cancer, chemotherapy, liver
disease, oral anticoagulation should be taken in consideration.
Secondary hypercoagulable states
The acquired or secondary hypercoagulable states consist of
a heterogenous group of disorders with an increased risk for developing thrombotic complications (2, 5, 6). Hyperhomocysteinemia is a common abnormality that results in an increased risk
of venous as well as arterial thrombosis. Plasma homocysteine
levels are determined by genetic as well as by environmental
factors, the latter including dietary intake and absorption of folic
acid and vitamins B12 and B6. Many underlying conditions (e.g.,
malignancies) may induce changes in the coagulation system and
lead to a hypercoagulable state. Cancer cells may have direct
procoagulant effects by the production of tissue factor and of
cancer procoagulant. Certain acquired conditions (e.g., elevated
factor VIII levels, lupus anticoagulant, pregnancy, oral contraceptive use) could result in the laboratory phenotype of APCresistance. This type of APC-resistance is not associated with
FV Leiden. It seems to be also a risk factor for thrombosis, although less strong. The origin and molecular basis of APC-resistance in the absence of FV Leiden is not well known and is
likely to be of mixed genetic and acquired origin.
The risk of thrombosis is increased in individuals with antiphospholipid antibodies (APA). This abnormal plasma phenotype is found in about one-half of patients with systemic lupus
erythematosus, but also in the course of infections, neoplastic
diseases, administration of certain drugs, and sometimes in apparently normal subjects (10). The APA are directed against a variety
of phospholipid (PL) binding-proteins of which 2-glycoprotein I
(2GPI) is considered to be the major antigen. Some of these antibodies prolong PL-dependent clotting reactions and are termed
lupus anticoagulants (LA). Autoimmune APA which are bound
through 2GPI to cardiolipin are called anticardiolipin antibodies
(ACA). The origin of APA is unknown. Some arise as a response
to infections, other may occur as a result of cellular injury or
apoptosis. Among patients with venous thrombosis, a LA has been
reported in 5 % to 15 %. It can lead to a nine-fold increased risk of
thrombosis. It seems that LA is a stronger risk factor for thrombosis than ACA. The thrombosis recurrence rate in patients with APA
is exceedingly high. Thrombogenic mechanisms remain unclear.
More than one mechanism seems likely since APA are associated
with such a wide spectrum of venous, arterial and microvascular
thromboses. Antiphospholipid syndrome (APS) is an autoimmune
disorder, defined as the association of APA with arterial or venous
thrombosis, recurrent fetal loss or thrombocytopenia (10) (Tab.
4). Pregnancy loss is most likely a result of disruption of placental
blood flow due to vascular thrombosis. About 515 % of women
with recurrent pregnancy loss are found to have evidence of APA.
The APA are clearly linked to myocardial infarction and thrombotic or embolic stroke, particularly in young people.
Two general types of assays are used to identify APA in clinical practice (10). Enzyme-linked immunosorbent assays (ELISA)

Remkova A. Diagnostic approach to hypercoagulable states

Tab. 4. Classification criteria for the antiphospholipid syndrome.
Antiphospholipid syndrome (APS) is established if at least one clinical
and one laboratory criterion is present. Persistent positivity of laboratory tests is important.
Clinical criteria
1. Vascular thrombosis
One or more clinical episodes of arterial, venous, or small vessel
thrombosis, in any tissue or organ.
2. Pregnancy morbidity
One or more unexplained deaths of a morphologically normal fetus, or premature births of a morphologically normal neonate because of eclampsia or severe preeclampsia, or recognized features
of placental insufficiency, or three or more unexplained consecutive spontaneous abortions.
Laboratory criteria
1. Lupus anticoagulant in plasma, on 2 or more occasions at least 12
weeks apart.
2. Anti-cardiolipin antibody IgG and/or IgM in serum or plasma,
present in medium or high titer, on 2 or more occasions, at least 12
weeks apart, measured by a standardized ELISA.
3. Anti-2 glycoprotein-I antibody IgG and/or IgM in serum or plasma,
present on 2 or more occasions, at least 12 weeks apart, measured
by a standardized ELISA.

are used for APA against specific proteins: ACA and anti-2-GPI
antibodies. Lupus anticoagulants (LA) are detected by using coagulation tests. LA are heterogeneous and their reactivity depends on the nature of the assay, the origin and amount of PL in
the coagulation reagents, and their target antigens. More than
one test must be used, because a single test will identify only
60 70 % of patients with lupus inhibitors. Both activated partial
thromboplastin time (APTT)-based assays and dilute Russells
viper venom time (dRVVT) is suitable for LA determination.
Molecular markers of coagulation activation in assessment
of prethrombotic state
Increased plasma levels of coagulation activation markers
indicate an activation of the coagulation system that is also termed
as hypercoagulable state. Under certain conditions, this may
be a prodromal state of thrombosis. Activation of the coagulation system may take place locally at a site of vascular injury, or
systemically, within the entire blood volume. Systemic coagulation activation can be triggered by a variety of mechanisms, leading to the DIC syndrome.
Certain assays reflect the coagulation reactions leading to a
formation of fibrin thrombus. Such tests can reflect the generation of thrombin, which is responsible for the transformation of
fibrinogen into fibrin. During the process of prothrombin to
thrombin activation, measurable prothrombin fragments 1+2
(F1+2) are released. The thrombin-antithrombin (TAT) complexes
measure the interaction between thrombin and its inhibitor. Other
tests reflect the action of thrombin on fibrinogen with fibrin formation. After the release of fibrinopeptides A (FPA) from fibrinogen, the resulting fibrin monomer molecules display an in-

295

tense tendency to polymerize. Soluble fibrin detected in plasma

samples includes a variety of complexes consisting mainly of
fibrin monomer units. The advantage of measuring soluble fibrin over FPA is the considerably longer half-life of soluble fibrin in the circulation. It can be detected by a variety of methods, including paracoagulation and precipitation assays. Finally,
some tests reflect the action of plasmin on fibrinogen and fibrin.
Perhaps the best known of these tests involve fibrinogen/fibrin
degradation products. Tests that are more specific for fibrin digestion may have more potential when evaluating patients with
thrombosis. Markers of this category include the D-dimer, which
is digestion product of cross-linked fibrin. D-dimer is a marker
of fibrin turnover. Plasma measurements of D-dimer are used
routinely in diagnosis of DIC and of venous thromboembolism.
Increased fibrin turnover represents a prothrombotic state, which
might favor not only thrombogenesis but also atherogenesis.
Plasma fibrinogen is a strong and consistent predictor of clinical
ischemic events, most of which are caused by arterial thrombosis.
In general, recognition of hypercoagulable state and coagulation activation using special laboratory methods is useful information for a targeted prevention and /or therapy of thrombosis and thromboembolic complications.
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Received June 14, 2006.
Accepted June 29, 2006.