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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

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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 assoTab. 2. Differential diagnosis of hypercoagulable states. Clinical characteristics Personal history of proven venous thromboembolism

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-

Laboratory tests 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. 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. In about 50 % of patients with thrombophilia due to AT, PC, PS deficiency. The hemostatic profile of AT, PC, PS, FV Leiden and FII gene mutation should be investigated. 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.

Age at the first thromboembolic episode

Recurrence of thromboembolic episodes Family history of venous thromboembolism Personal or family history of fetal loss

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

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

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.

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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. References
1. Bauer KA, Goodnight SH, Ridker PM. Hypercoagulable states translation of risk factors to clinical practice. 255273. In: McArthur JR (Ed). Hematology. Washington, Amer Soc Hematol, 1998. 2. Ens GE. Disorders leading to thrombosis. 675-681. In: Stiene-Martin EA, Lotspeich-Steininger ChA, Koepke JA (Eds). Clinical Hematology: principles, procedures, correlations. Philadelphia, Lippincott-Raven Publishers, 1998. 3. Mannucci PM. Laboratory detection of inherited thrombophilia: a historical perspective. Semin Thromb Hemost 2005; 31 (1): 510. 4. Favaloro EJ. Diagnostic issues in thrombophilia: a laboratory scientists view. Semin Thromb Hemost 2005; 31 (1): 1116. 5. Johnson CM, Mureebe L, Silver D. Hypercoagulable states: a review. Vasc Endovascular Surg 2005; 39 (2): 123133. 6. Remkov A. Trombofiln stavy ako rizikov faktor trombz. Intern Med 2004; 4 (3): 183185. 7. Remkov A, Androv A, Bulas J, Kiov S. Intrakardilna trombza pri zpalovom ochoren reva komplikovanom sepsou. Intern Med 2002; 2 (2): 113115. 8. Remkov A, Reptov A, Holom K, imko J. Trombza jugulrnych l ako komplikcia gravidity po umelom oplodnen. Intern Med 2003; 3 (78): 458460. 9. Remkov A. Trombza aprotrombotick stavy vgynekolgii aprodnctve 1. as: Rizikov stavy vzniku trombz vgynekolgii aprodnctve. Intern Med 2005; 5 (10): 529533. 10. Miyakis S, Lockshin MD, Atsumi T et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4 (2): 295306. Received June 14, 2006. Accepted June 29, 2006.

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-