Antithrombin is a protease inhibitor. Based on kinetic rates of inhibition, its primary targets are thrombin and factor Xa, and hence antithrombin both regulates generation of thrombin and inhibits thrombin that has been generated. Inhibition of target proteases is increased approximately 1,000-fold by glycosaminoglycan activation of antithrombin, which is the mechanism by which heparin acts as a pharmacological anticoagulant. The activation process involves an induced conformational change in the structure of antithrombin which enables formation of an irreversible covalent complex with the target protease [2]. The complex undergoes a further dramatic conformational change involving both the inhibitor and the inhibited protein which alters the properties of each, resulting in rapid clearance from the circulation.

Two laboratory (intermediate) phenotypes of heritable antithrombin deficiency are recognized. Type I is characterized by a quantitative reduction of antithrombin with a parallel reduction in function (measured as inhibitory activity against factor Xa or thrombin) and the level of protein in the plasma (measured immunologically as the antigenic level). Type 2 deficiency is due to the production of a qualitatively abnormal antithrombin protein characterized by disturbance of the complex inhibitory mechanism of protease inhibition as a result of a mutation in the SERPINC1 gene. The functional activity is discrepantly low compared to the antigenic level. Type 2 deficiency is subclassified according to the nature of the functional deficit:

Approximately 100 point mutations (missense, nonsense, or insertions or deletions causing frameshifts) and several whole or partial gene deletions have been identified as causes of type 1 deficiency. Numerous point mutations causing qualitative type 2 deficiency have been identified. Homozygous type 1 deficiency and type 2RS mutations are incompatible with life. Type 2 HBS and some PE mutations are associated with a lower risk of thrombosis; homozygosity, and compound heterozygosity, involving these mutations is compatible with life.

Functional activity assays typically use a chromogenic substrate and factor Xa as the target protease. The total amount of antithrombin protein can be measured immunologically with antibodies, for example, by enzyme-linked immunosorbent assay (ELISA). As antithrombin antigen levels may be normal or near normal in type 2 deficiency, immunological assays may fail to identify patients with these variants and so a functional assay should be used as the initial assay.

Although there is little reported variation in the plasma concentration of antithrombin both during healthy pregnancy and following delivery (as stated in Chap. 1), antithrombin levels may be slightly reduced in pregnancy and are reduced in women taking estrogen preparations, as well as in other situations. Consequently, the clinical significance of a low antithrombin level must be interpreted by an experienced clinician who is aware of all the relevant factors that may have influenced the test result in a specific patient.

Protein C is the zymogen precursor of activated protein C (APC). Protein C is activated to APC by thrombin bound to thrombomodulin on the endothelial surface. APC inactivates the activated cofactors (VIIIa and Va) and so inhibits thrombin generation. Factors VIII and V are activated by small amounts of thrombin during initiation of coagulation to nonenzymatic cofactors required for assembly of macromolecular complexes that are required for the full thrombin explosion. The enzymatic components of these complexes are factors IXa and Xa, and so inactivation of VIIIa and Va by APC leads to disassembly of the enzymatic complexes, thus attenuating thrombin generation.

The laboratory diagnosis of protein C deficiency is based on a functional assay. As protein C antigen levels may be normal or near normal in type 2 deficiency, immunological assays may fail to identify patients with these variants, so a functional assay should be used as the initial assay. Most commercially available functional assays use a snake venom to activate protein C and a chromogenic substrate to quantify APC activity. A chromogenic assay will detect type 1 and most type 2 defects. The diagnosis of type 1 protein C deficiency is problematic because of the wide overlap in protein C activity between heterozygous carriers and unaffected individuals. The diagnosis of type 2 defects is problematic because a chromogenic assay will only detect defects affecting the enzymatic site.

Protein C levels are not affected by pregnancy or estrogen exposure. Acquired low levels of protein C occur during anticoagulant therapy with oral vitamin K antagonists, vitamin K deficiency, disseminated intravascular coagulation (DIC), and liver disease. Consequently, the clinical significance of a low protein C level must be interpreted by an experienced clinician who is aware of all the relevant factors that may have influenced the test result in a specific patient.

Protein S is a vitamin K-dependent glycoprotein produced in the liver, endothelial cells, and megakaryocytes. Protein S is a nonenzymatic cofactor for APC-mediated inactivation of factors VIIIa and Va and additionally is involved with tissue factor pathway inhibitor-dependent natural anticoagulation. Approximately 60 % of protein S circulates bound to C4b-binding protein and is inactive. The remaining 40 %, designated free protein S, is uncomplexed and is the active form. Free protein S increases the affinity of activated protein C for negatively charged phospholipid surfaces on platelets or the endothelium and increases complex formation of APC with the activated forms of factors VIII and V (VIIIa & Va). However, the degree of C4b binding has not yet been shown to be a determinant of thrombosis risk. In addition to APC cofactor activity, protein S has an independent anticoagulant activity as a cofactor for TFPI (tissue factor pathway inhibitor).

Protein S is usually quantified immunologically rather than measured functionally. Nowadays, monoclonal antibodies that detect only free protein are used to quantify free protein S. Functional protein S assays are imprecise and are not used in the majority of coagulation laboratories.

Protein S levels are significantly lower in females, so much so that different normal reference ranges are required for males and females. There is a significant risk of a false-positive diagnosis of protein S deficiency in women. Protein S levels are reduced by estrogens and fall progressively during normal pregnancy. Acquired low levels of protein S occur during anticoagulant therapy with oral vitamin K antagonists, vitamin K deficiency, DIC, and liver disease.

Protein S defects are divided into three types:

This complicated classification reflects the complexity of the biology of protein S but has no mechanistic reference to disturbance of natural anticoagulant activity. Given these limitations and the imprecision of laboratory methodology, the diagnosis of heritable protein S deficiency is less precise and the clinical implication of a low protein S level in an individual is more uncertain than it is for antithrombin or protein C.

Factor V is a cofactor required for thrombin generation. Factor V has no cofactor activity until cleaved by thrombin or factor Xa. Activated factor V (Va) is inactivated by APC (see Sect. 3.2.2 above). Resistance to activated protein C (APC resistance) is a laboratory phenomenon in which there is a suboptimal anticoagulant response to addition of APC to a patient’s plasma. In 95 % of cases of familial APC resistance, this is due to the same point mutation in the gene for FV, a guanine to adenine transition at nucleotide position 1691 in exon 10 (F5G1691A), resulting in a mutant protein FVR506Q. The mutation is known as the factor V Leiden mutation and the mutant factor Va has normal procoagulant activity, but substitution of glutamine for arginine at position 506 (which is an APC cleavage site) results in slower inactivation by APC. Nowadays, the mutation is frequently detected by direct DNA analysis (rather than by a clotting assay) to detect the presence of the mutant protein.

The mutation is present in around 4 % of the Caucasian population and around 15 % of unselected consecutive Caucasian patients with a first venous thrombosis. The prevalence is highest in Northern Europeans. The mutation is infrequent in other populations. The high prevalence and founder effect suggest positive selection, and this may relate to a favorable effect on embryo implantation and hence reproduction [3] rather than a lower risk of fatal hemorrhage in females during childbirth, as originally thought.

Acquired APC resistance is common, in part often due to increased FVIII levels, and is observed in pregnancy and in association with estrogen exposure.

A number of other anticoagulant proteins have been investigated as potential factors causing thrombophilia, but a relationship between venous thrombosis and low protein levels or associated gene mutations has not been established.

A causal relationship between levels of specific individual proteins involved in regulating fibrinolysis and venous thrombosis has not been established. However, in a case–control study using a global measure of fibrinolytic potential, there was an approximately doubled risk of venous thrombosis in patients with clot lysis times above the 90th percentile of controls [4]. Further analysis of a larger study confirmed this finding and demonstrated that hypofibrinolysis in combination with established acquired and genetic risk factors, such as F5G1691A, had a synergistic effect on venous thrombosis risk [5]. The genetic basis for hypofibrinolysis in these patients was not investigated.

Hyperhomocysteinemia may be caused by genetic abnormalities but only the severe inherited abnormalities of homocysteine metabolism (homozygous cystathionine beta-synthase deficiency and homozygous deficiency of methylenetetrahydrofolate reductase) result in congenital homocystinuria associated with an increased risk of both arterial and venous thrombosis, as well as premature atherosclerosis and mental retardation, epilepsy, and skeletal and eye problems. Fifty percent of patients present with venous or arterial thrombosis before the age of 30 years. The thermolabile variant of methylenetetrahydrofolate reductase (MTHFR), due to a common genetic polymorphism (C677T), is not a risk factor for venous thrombosis [6, 7].

The antiphospholipid syndrome (APS) is the most common acquired form of thrombophilia. APS is diagnosed when a patient with arterial or venous thrombosis (or pregnancy morbidity in women) is found to have antiphospholipid antibodies (anticardiolipin, aCL; and/or lupus anticoagulant, LA; and/or anti-beta-2-glycoprotein I, aß2-GPI). The updated international consensus (revised Sapporo) classification criteria for definite antiphospholipid syndrome [8] require the presence of a LA and/or IgG or IgM aCL present in medium or high titer (i.e., >40 GPL or MPL or > the 99th percentile) and/or aß₂GPI (IgG and/or IgM) >99th percentile. These aPL should be persistent, defined as being present on two or more consecutive occasions at least 12 weeks apart. The international consensus criteria were originally designed for scientific clinical studies, and there remains a need for firm diagnostic criteria for routine clinical use which may differ from these. APS has conventionally been divided into primary and secondary forms, the latter being associated with systemic lupus erythematosus (SLE) or a related rheumatological condition. However, this distinction was abandoned in the revised Sapporo classification [8] on the basis that it is unknown whether APS and SLE are two diseases coinciding in an individual, underlying SLE offers a setting for the development of APS, or APS and SLE represent two elements of the same process.

Laboratory test results are subject to considerable pre-analytical variation. In addition, transiently abnormal results may be found in normal healthy individuals. For these reasons, for a patient to be considered to have antiphospholipid activity (aCL, LA, or aß2-GPI), test results must be positive on two separate occasions. The probability of misdiagnosing APS has been reduced by stricter criteria for antibody titers (>40 GPL or MPL for aCL or >90th percentile for aCL or aß2-GPI) and demonstration of persistence of antibodies (present on at least 2 consecutive occasions at least 12 weeks apart) [9]. Positivity in all 3 assays (aCL, LA, aß2-GPI) is associated most strongly with thrombosis and pregnancy complications. Recent evidence suggests that the antibodies most strongly associated with thrombosis and pregnancy morbidity are against domain I of β2-GPI; these antibodies are responsible for lupus anticoagulant activity specifically associated with clinical events and are responsible for positive aCL results. While the criteria for diagnosis of APS are unlikely to change again soon, it is possible that the laboratory identification of clinically relevant antibodies to domain I β2-GPI will eventually simplify the diagnosis and improve the clinical utility of laboratory tests.