Evaluation of the hypercoagulable state

Whom to screen, how to test and treat

Amy P. Barger, MD; Randy Hurley, MD

VOL 108 / NO 4 / SEPTEMBER 15, 2000 / POSTGRADUATE MEDICINE

CME learning objectives

• To recognize the most common inherited thrombophilic disorders
• To identify the acquired risk factors for thrombosis
• To understand what constitutes an appropriate laboratory evaluation for hypercoagulability

The authors disclose no financial interests in this article.

This is the third of four articles on thromboembolism

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Preview: As new inherited and acquired prothrombotic abnormalities have been discovered, our knowledge about the mechanisms of venous thrombosis has advanced. However, questions about whom to screen and how to manage remain. Dr Barger and Dr Hurley discuss the inherited and acquired causes of thrombosis, describe an approach to risk assessment and laboratory evaluation, and present general guidelines for management.
Barger AP, Hurley R. Evaluation of the hypercoagulable state: whom to screen, how to test and treat. Postgrad Med 2000;108(4):59-66

Hypercoagulable conditions are a group of inherited and acquired disorders that predispose to venous thromboembolism. Recently, heightened interest in these conditions has been sparked by the discovery of several common inherited abnormalities, including factor V Leiden, the prothrombin 20210 mutation, and hyperhomocystinemia. Laboratory-based testing has made it possible to identify a predisposing genetic cause in up to 50% of patients with venous thromboembolism (1-4).

The ongoing discovery of new inherited and acquired prothrombotic abnormalities and coexistent abnormalities in individual patients has complicated the diagnostic approach. Paradoxically, the technology to diagnose a multitude of hypercoagulable conditions has outpaced the practical application of this new information to treatment. Short-term treatment of venous thromboembolism varies little. Moreover, convincing evidence regarding the intensity or duration of anticoagulation is still lacking, since much of the information on underlying biologic defects is new and evolving.

This article reviews the major inherited abnormalities associated with venous thromboembolism. (The mechanisms of arterial thrombosis differ and are primarily associated with atherosclerosis and embolic disease, except in the antiphospholipid syndrome and hyperhomocystinemia, which uniquely cause both arterial and venous thrombosis.) We also discuss an approach to clinical risk assessment, review appropriate laboratory testing, and provide basic guidelines for management and prophylaxis.

Coagulation and major anticoagulant mechanisms

Under normal hemostatic conditions, the relationship between the coagulation cascade (figure 1: not shown) and the mechanisms designed to regulate or limit coagulation is complex. Coagulation is a result of the sequential activation of several serine proteases that ultimately generate thrombin at the site of vascular injury. Circulating anticoagulants that function to limit thrombus formation include antithrombin, activated protein C (APC), and plasmin. Antithrombin, a serine protease inhibitor, binds heparin and inactivates a number of serine proteases. Thrombomodulin, an endothelial cell surface protein, binds thrombin and alters its substrate specificity, thus allowing it to convert protein C to APC. APC exerts its anticoagulant effect by proteolytically cleaving and inactivating the activated forms of factors V and VIII (Va and VIIIa, respectively). Protein S is a cofactor required for protein C activity. Although 60% of protein S circulates bound to a carrier protein, only free protein S has cofactor activity. Plasmin arises from plasminogen and acts as the major fibrinolytic enzyme.

Our understanding of these mechanisms continues to evolve as new genetic abnormalities are defined and new anticoagulant pathways are discovered.

Classification of hypercoagulable conditions

Hypercoagulable conditions can be classified as either primary (inherited) or secondary (acquired). The common primary conditions are deficiencies of antithrombin, protein C, and protein S; factor V Leiden (which results in resistance to APC), the prothrombin 20210 mutation, hyperhomocystinemia, and elevated levels of factor VIII (table 1). Secondary hypercoagulable conditions are a heterogeneous group of disorders ranging from well-known clinical conditions that predispose to venous thromboembolism, such as pregnancy and the postoperative state, to medical conditions such as the antiphospholipid syndrome and cancer (table 1).

Table 1. Classification of hypercoagulable conditions

Primary (inherited) Antithrombin deficiency Protein C deficiency Protein S deficiency Factor V Leiden (resulting in APC resistance) Prothrombin 20210 mutation Hyperhomocystinemia Elevated factor VIII levels Dysfibrinogenemia Factor XII deficiency Disorders of plasmin generation Secondary (acquired) Pregnancy Immobility Trauma Postoperative state Use of oral contraceptives, estrogen, tamoxifen (Nolvadex) Antiphospholipid syndrome Hyperhomocystinemia Other disease states Malignancy Nephrotic syndrome Myeloproliferative disorders Congestive heart failure Heparin-induced thrombocytopenia with thrombosis Paroxysmal nocturnal hemoglobinuria Behçet's disease APC, activated protein C.

Although this classification system is useful for categorizing the causes of thrombosis and directing testing, it is an oversimplification. Venous thromboembolism is now thought to be a disease with multiple causes. Thrombosis is usually a consequence of the interplay of acquired and inherited defects (5-7); for example, venous thromboembolism occurring postoperatively in a patient with factor V Leiden. Also, since several of the thrombophilic disorders are relatively prevalent (table 2), one person can have multiple defects, leading to thrombosis without an obvious external stimulus. Occasionally, the site of thrombosis may provide a clue to the cause; for example, paroxysmal nocturnal hemoglobinuria, Behçet's syndrome, and myeloproliferative disorders are associated with visceral thrombosis.

Table 2. Estimated prevalence of various thrombophilic defects
Defect	General population	Venous thromboembolism population
Antithrombin	0.3%	3%
Protein C	0.3%	3%
Protein S	0.3%	3%
Factor V Leiden	5%	10%-20%
Hyperhomocystinemia	5%	10%-20%
Prothrombin 20210	2%-3%	6%-18%
Antiphospholipid	2%	14%
Factor VIII	6%-8%	10%-15%
Adapted from Lensing et al (3) and Ginsberg et al (22).

Antithrombin, protein C, and protein S
Deficiencies of antithrombin, protein C, and protein S are inherited in an autosomal dominant pattern. All three generally present with a first episode of thrombosis between the ages of 10 and 50 years. Homozygous antithrombin deficiency is extremely rare. The heterozygous form carries a significant lifetime risk of thrombosis ranging from 17% to 100% (8,9), which is generally higher than the risk associated with either protein C or protein S deficiency. Acquired antithrombin deficiency is seen with liver disease, oral contraceptive use, the nephrotic syndrome, pregnancy, and disseminated intravascular coagulation.

Homozygous forms of protein C and protein S deficiency do exist and are associated with purpura fulminans in newborns. Adults with heterozygous protein C or protein S deficiency may experience skin necrosis shortly after starting warfarin therapy without concomitant heparin therapy. Both protein C and protein S are vitamin K-dependent cofactors whose levels may drop precipitously after initiation of warfarin, leading to transient hypercoagulation. As with antithrombin deficiency, several acquired conditions, including liver disease and disseminated intravascular coagulation, can lead to decreased activity of protein C and protein S. A decreased protein S level has also been specifically noted to occur with pregnancy, oral contraceptive use, and the nephrotic syndrome.

Screening for a deficiency of antithrombin, protein C, or protein S is accomplished by assessing the activity level. A decreased activity level may be due to a reduced antigenic level of normal protein or to a dysfunctional protein. Assessing the activity level of protein S is more complicated, since protein S activity can fluctuate with the level of its C4 binding protein, an acute phase reactant. Therefore, both free and total levels of protein S should be determined.

Factor V Leiden
First described in 1993, APC resistance is most commonly due to a point mutation in the gene encoding factor V. Replacement of arginine with glutamine at position 506, termed factor V Leiden, renders factor V resistant to inactivation by protein C. Factor V Leiden is the single most common inherited thrombophilic defect (4,10). The estimated prevalence among people of European ancestry is 5%, but the defect is less common in other ethnic groups (5,6). Heterozygosity for factor V Leiden mutation imparts a sevenfold increased lifetime risk of venous thromboembolism, whereas homozygous expression confers an 80-fold increased risk (5,11).

Although antithrombin, protein S, and protein C deficiencies usually present with thrombosis relatively early in life, the risk of thrombosis due to factor V Leiden increases with age. Coinheritance of other thrombophilic mutations, such as protein C deficiency, prothrombin 20210, or hyperhomocystinemia, further increases thrombotic risk (11,12). The APC ratio, a clotting-based assay, can be used to screen for APC resistance. Molecular-based assays to determine the characteristic point mutation can confirm the presence of factor V Leiden.

Prothrombin 20210
In 1996, prothrombin 20210, an autosomal dominant inherited defect, was discovered. A guanine-to-adenine base substitution at position 20210 of the 3'-untranslated region of the prothrombin gene results in elevated prothrombin levels. This mutation is slightly less prevalent in the general population than factor V Leiden and may follow the same European ethnic pattern. Prothrombin 20210 confers a weaker thrombotic risk (about threefold) than factor V Leiden (8,12). Coinheritance of both factor V Leiden and prothrombin 20210 compounds the risk of venous thromboembolism, pregnancy-associated venous thromboembolism, and recurrent venous thromboembolism (5,6,12,13).

Hyperhomocystinemia
Elevated levels of homocysteine, an intermediary in methionine metabolism, have been associated with both arterial and venous thrombosis. Hyperhomo-cystinemia, defined as fasting plasma levels greater than 15 micromole/L, is relatively common in the general population and can result from inherited enzyme deficiencies or acquired disorders (14). Dietary deficiencies of folate and vitamins B₆ and B₁₂, chronic renal failure, pernicious anemia, and hypothyroidism have all been associated with elevated homocysteine levels.

Hyperhomocystinemia is determined by testing for the known enzyme deficiencies (eg, cystathionine beta-synthase, the thermolabile variant of tetrahydrofolate reductase) or by measuring fasting homocysteine levels. Evaluation of homocysteine levels following a standard methionine loading dose may increase the sensitivity of testing.

Treatment consists of nutritional replacement of folate and vitamins B₆ and B₁₂. Although nutritional supplementation can decrease homocysteine levels, it is still unclear whether the lowering of homocysteine levels will actually decrease the incidence of thrombosis or the other conditions associated with early atherosclerosis and cerebrovascular disease.

Antiphospholipid syndrome
The antiphospholipid syndrome is caused by a heterogeneous group of antibodies to various proteins complexed with negatively charged phospholipids. It can be a primary or secondary condition. In the latter case, it can result from autoimmune diseases (eg, systemic lupus erythematosus, rheumatic arthritis), use of certain drugs (eg, antibiotics, procainamide hydrochloride [Pronestyl, Procanbid], hydralazine hydrochloride [Apresoline]), viral infections (eg, HIV, syphilis, hepatitis C), or lymphoma. Clinical manifestations include arterial and venous thrombosis, recurrent fetal loss, thrombocytopenia, livedo reticularis, and neurologic symptoms.

Diagnosis is confirmed by the occurrence of one or more clinical manifestations in the presence of positive antibody studies on two occasions more than 3 months apart (8). Antiphospholipid antibodies can be demonstrated by a lupus anticoagulant assay, such as the dilute Russell's viper venom time test, or by an enzyme-linked immunosorbent assay for anticardiolipin antibodies.

One retrospective analysis (15) suggests that patients with the antiphospholipid syndrome are best treated with a higher-intensity anticoagulant therapy (target international normalized ratio of 3.0 to 3.5, compared with usual target of 2.0 to 3.0) because of the substantial risk for recurrent thrombosis.

Cancer and clotting
The link between malignancy and thrombosis, first described by Trousseau, has been recognized since the 1800s. Although often associated with adenocarcinoma, thrombosis has been described with many types of cancer and likely occurs through multiple mechanisms. Risk of a subsequent diagnosis of cancer in a patient with idiopathic venous thromboembolism has been estimated at about 7% to 8%; risk increases to 17% if the patient has recurrent idiopathic venous thromboembolism (16). Despite this risk, the questions of which patients with venous thromboembolism should undergo evaluation for cancer and how such an evaluation could be performed in a cost-effective way have not been conclusively answered. One prospective cohort study (17) suggests that an appropriate clinical evaluation to detect potential malignancy in such patients would include extensive history taking and physical examination, routine laboratory studies, and a chest radiograph.

Factor VIII
Recently, elevated levels of factor VIII have been implicated as an independent risk factor for venous thromboembolism. One study (7) reported that levels greater than 150% of normal conferred a 4.8-fold increased risk of thrombosis compared with normal controls. The mechanism of elevated factor VIII and the cause (whether inherited or acquired) are unknown.

Clinical assessment of risk

When a patient presents with a venous thromboembolism, the question clinicians often ask is, Should this patient undergo evaluation for a hypercoagulable state? Several factors must be considered in determining which patients should have such an evaluation. First, a thorough history should be obtained to document any previous thrombotic events and identify any acquired prothrombotic conditions. Next, the presence of any of the clinical indicators of a hypercoagulable state (table 3) should be ascertained. If one of these factors or any findings on history taking are suggestive of a hypercoagulable state, appropriate laboratory testing should be performed.

Table 3. Clinical indicators of a hypercoagulable state

Family history of thrombosis Recurrent thrombosis Thrombosis at a young age (generally <50 yr) Idiopathic thrombosis (ie, no precipitating factor identified) Thrombosis at unusual sites (eg, cerebral, mesenteric, portal, hepatic veins)

The issue of screening for thrombophilic defects is controversial. The laboratory evaluation is expensive, and the short-term treatment of venous thromboembolism is the same in all patients, regardless of cause; therefore, it is unclear which patients warrant screening. In addition, there is little evidence to show that testing would influence the intensity or duration of long-term anticoagulation, except in the antiphospholipid syndrome.

Venous thromboembolism with a clear precipitating cause (such as trauma) should be treated with anticoagulation for 3 months, while the first episode of idiopathic venous thromboembolism (in which a precipitating cause is not identified) should be treated for at least 6 months. Evidence is accumulating that idiopathic venous thromboembolism should be considered a chronic disease, since risk of cumulative recurrence is high at 25% to 30% after 4 years (18-20).

Perhaps, then, patients with idiopathic venous thromboembolism should be considered for long-term anticoagulation, regardless of the presence of an underlying inherited defect. When warfarin therapy is continued indefinitely, however, the risk of major hemorrhage is significant--about 5% to 7% per year (21). Possibly, more intense monitoring in special anticoagulation monitoring clinics would decrease this risk. Regardless, a recommendation for lifelong anticoagulation requires serious consideration and a global assessment of the risks and benefits for the individual patient.

In contrast, there are many compelling arguments to test appropriate patients for inherited thrombophilia. Testing advances the knowledge base of the pathophysiology of venous thromboembolism. Although data on specific recommendations for length and intensity of anticoagulant therapy are lacking, this information will likely become available as more research data accumulate.

In certain situations, treatment does vary, notably the use of antithrombin concentrates in antithrombin deficiency and the antiphospholipid syndrome, which requires more intense anticoagulation. More important, identifying patients at risk for thrombosis carries significant implications in family counseling and high-risk situations. The affected family members of individuals with an identified hypercoagulable defect are also at increased risk of thrombosis.

Prophylactic therapy in high-risk situations such as surgery, immobilization, pregnancy, or the puerperium is beneficial for persons with a thrombotic tendency. Oral contraceptive use may pose risks in some instances. Risk of thrombosis conferred by oral contraceptive use alone is fourfold, but this risk increases to 35-fold if factor V Leiden is present (11). Because the absolute risk of thrombosis is small in women using oral contraceptives (largely owing to young age), screening for factor V Leiden is not indicated (11). However, when venous thromboembolism occurs in a woman taking oral contraceptives, testing may be warranted in order to provide adequate counseling about continued oral contraceptive use and the risks of thromboembolism in pregnancy. Similarly, testing for factor V Leiden may be warranted when venous thromboembolism occurs in breast cancer patients taking tamoxifen (Nolvadex), because of an increased risk of thrombosis in this group.

Screening laboratory evaluation

Laboratory evaluation of a patient presenting with venous thromboembolism begins with baseline hematologic and coagulation studies. A complete blood cell count may detect a myeloproliferative disorder such as polycythemia or thrombocytosis. A prolonged partial thromboplastin time, unrelated to heparin use, may be a clue to a lupus anticoagulant. If baseline tests yield no clues, the next steps are to obtain a detailed patient and family history and to ascertain the clinical circumstances related to the venous thromboembolism. If no precipitating cause of thrombosis can be identified or no high-risk clinical indicators are present (table 3), initial thrombophilic screening tests should be done (table 4).

Table 4. Thrombophilic screening tests

Initial APC resistance Clotting-based assay Molecular-based assay for factor V Leiden Antiphospholipid antibodies Lupus anticoagulant assay (RVVT) ELISA for anticardiolipin antibody Prothrombin 20210 mutation Fasting homocysteine level Factor VIII level Remote from acute event Antithrombin activity Protein C activity Protein S activity, free and total levels APC, activated protein C; ELISA, enzyme-linked immunosorbent assay; RVVT, Russell's viper venom time.

Although certain tests can be performed at the time of the initial event, heparin interferes with clotting-based assays for APC resistance, the lupus anticoagulant, and factor VIII levels. Protein C, protein S, and antithrombin functional and antigenic tests should be performed only in strongly thrombophilic patients: those with a venous thromboembolism prior to age 50, with recurrent venous thromboembolism, or with an extensive family history of thrombus. In addition, testing for protein C, protein S, and antithrombin cannot be reliably performed during an acute event or while the patient is taking anticoagulants, because the levels fluctuate during active thrombosis and are suppressed by warfarin therapy. If indicated, testing for antithrombin, protein C, and protein S can be performed 3 weeks after anticoagulant therapy has been discontinued.

Management guidelines

Risk data do not exist for every possible combination of gene-gene defect or gene-environment defect, much less for the risk of recurrent thrombosis associated with all of the possible scenarios. However, general conclusions have been drawn on the basis of existing data--namely, multiple genetic defects, spontaneous thromboses, and specific secondary risk factors all increase the risk of thrombosis. The following guidelines are based on these general risk assessments (7).

Long-term warfarin therapy is recommended for patients at high risk of thrombosis, which include:

• Those who have had two or more spontaneous events
• Those who have had one spontaneous life-threatening event
• Those with the antiphospholipid syndrome, antithrombin deficiency, or more than one genetic defect who have had one spontaneous event

Prophylaxis is recommended for patients at moderate risk, which include:

• Those who have had one provoked event
• Those who are asymptomatic and have not had a previous event

Summary

Venous thromboembolism is a common disease that causes significant morbidity and mortality. In recent years, the ability to diagnose inherited genetic defects and common acquired conditions predisposing to thrombosis has greatly increased. Venous thromboembolism is now understood to be a complex interaction of genetic and environmental factors leading to thrombosis. Integrating the various factors to individually assess thrombotic risk still poses a challenging clinical problem that will likely become easier as more data accumulate. As the ability to accurately assess risk increases, the data can then be translated into tailored treatment regimens. Until then, only general guidelines regarding evaluation and management are available. In the future, it is likely that other prothrombotic conditions will be elucidated, adding to the pool of data.

References

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Dr Barger is assistant clinical professor of medicine, University of Minne-sota Medical School--Minneapolis, and a hospitalist, department of internal medicine, Regions Hospital, St Paul. Dr Hurley is assistant clinical professor of medicine, division of hematology/oncology, University of Minnesota Medical School--Minneapolis, and staff member, department of hematology/oncology, Regions Hospital. Correspondence: Amy P. Barger, MD, Department of Internal Medicine, Regions Hospital, 640 Jackson St, St Paul, MN 55101. E-mail: amy.p.barger@healthpartners.com.

Symposium Index

• VENOUS THROMBOEMBOLISM: Introduction to a four-article symposium by Russell L. Holman, MD
• IMPROVING DETECTION OF VENOUS THROMBOEMBOLISM: New technology holds promise for early, precise diagnosis by Paramjit Gill, MD, Avi Nahum, MD, PhD
• ANTICOAGULATION FOR VENOUS THROMBOEMBOLISM: What are the current options? by Thomas Yacovella, MD, Michael Alter, MD
• EVALUATION OF THE HYPERCOAGULABLE STATE: Whom to screen, how to test and treat by Amy P. Barger, MD, Randy Hurley, MD
• THROMBOEMBOLISM DURING PREGNANCY: Risks, challenges, and recommendations by Jonathan S. Sellman, MD, Russell L. Holman, MD

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