Blood coagulation.pdf

HAEMATOLOGY Haematology Blood coagulation Björn Dahlbäck Under normal circumstances, the coagulation system is balanced in favour of anticoagulation. Thrombin is the key effector enzy...

HAEMATOLOGY Haematology Blood coagulation Björn Dahlbäck Under normal circumstances, the coagulation system is balanced in favour of anticoagulation. Thrombin is the key effector enzyme of the clotting cascade. Antagonists of vitamin K inhibit a vitamin-K-dependent post-translational modification of several coagulation proteins, which is required for these proteins to attain a phospholipid-binding conformation. Heparin stimulates the activity of antithrombin, a serine-protease inhibitor. Analysis of knock-out mice has shown the relative importance of the coagulation factors in vivo. Gene therapy may soon be a therapeutic option for inherited deficiencies of factors VIII and IX. Blood coagulation and platelet-mediated primary Prothrombin is activated to thrombin by the haemostasis have evolved as important defence prothrombinase complex, which consists of the mechanisms against bleeding. The coagulation system is phospholipid-bound complex between the enzyme, triggered in response to rupture of endothelium, which factor Xa, and its cofactor, activated factor V (Va). The allows exposure of blood to the extravascular tissue. The substances that activate factor V are factor Xa (on the responses of the coagulation system are coordinated with phospholipid surface) and thrombin (both in solution and the formation of the platelet plug that initially occludes on the surface). Thrombin feedback amplifies the system the vascular lesion. Anticoagulant mechanisms ensure by activating not only factor V, but also factors VIII and careful control of coagulation and, under normal XI. Factor VIII circulates bound to von Willebrand factor, conditions, they prevail over the procoagulant forces. which is an adhesive protein important for the generation Disturbances of the natural balance between the of the initial platelet plug.8 After activation, factor VIIIa procoagulant and anticoagulant systems due to genetic or dissociates from von Willebrand factor and forms a acquired factors may result in bleeding or thrombotic complex on the platelet surface with factor IXa; this diseases. complex (denoted the tenase complex) then activates factor X. Activation of factor XI by thrombin is another Coagulation pathway amplification loop, resulting in the generation of Thrombin is the key effector enzyme of the coagulation additional factor IXa, which in turn activates factor X.9 system, having many biologically important functions The initiation of coagulation via the exposure of tissue such as the activation of platelets, conversion of fibrinogen factor (tissue-factor pathway) as described above, the to a fibrin network, and feedback amplification of extrinsic pathway, is the mechanism by which coagulation coagulation. The precise and balanced generation of is initiated in vivo in response to trauma. The intrinsic thrombin at sites of vascular injury is the result of an pathway is an alternative mechanism by which the ordered series of reactions collectively referred to as blood coagulation system can be initiated. It involves factor XII, coagulation.1–3 The system is triggered on the surface of high-molecular-weight kininogen, prekallikrein, and factor extravascular cells by the exposure of tissue factor to XI; it results in the activation of factor XI. The blood (figure). physiological role of this pathway is not fully understood, Tissue factor is a membrane protein abundantly present because it is not important in trauma-initiated in cells surrounding the vascular bed. It binds both coagulation. Thus, inherited deficiencies of proteins of zymogen and activated forms of factor VII (factor VIIa). A this system are not associated with bleeding problems, fraction of factor VII in blood circulates as active enzyme except for deficiency of factor XI, which leads to a and the binding of this form to tissue factor triggers moderately severe bleeding disorder. coagulation by converting factors IX and X to their active Maximum thrombin generation occurs after the forms (IXa and Xa).4,5 Feedback amplification is achieved formation of the fibrin clot.5 This thrombin is important when factor VII bound to tissue factor is activated by for additional fibrin generation as well as for activation of factors VIIa, IXa, and Xa. Factors IXa and Xa may factor XIII and the thrombin-activatable fibrinolysis remain associated with the tissue-factor-bearing cell or inhibitor. Activated factor XIII (XIIIa) is a diffuse into the blood and bind to the surface of nearby transglutaminase that stabilises the clot by covalent cross- activated platelets, which have formed the primary platelet linking of fibrin.3 Thrombin-activatable fibrinolysis plug.6 Activation of platelets is associated with the inhibitor is a carboxypeptidase that releases carboxy- exposure of negatively charged phospholipids, which have terminal lysines from fibrin. Because these lysines are high potential to bind coagulation factors and assemble important for the binding of fibrinolytic enzymes to fibrin, enzyme-cofactor complexes that are crucially important activation of the inhibitor prevents further fibrinolytic for efficient propagation of the system.7 attack.10 Phosphatidylserine is a negatively charged phospholipid Lancet 2000; 355: 1627–32 required for the assembly of the tenase and Department of Clinical Chemistry, Lund University, University prothrombinase complexes.1,7,11 Under normal conditions, Hospital, Malmö, S-20502 Malmö, Sweden (Prof B Dahlback MD) phosphatidylserine is present in the inner-layer leaflet of (e-mail: [email protected]) the plasma cell membrane. During platelet activation, it is THE LANCET Vol 355 May 6, 2000 1627 HAEMATOLOGY Activation and propagation of coagulation Thrombin Tissue VIIa VIIIa Va factor IXa Tissue IX/X Xa Prothrombin Xa IXa X factor Activation of protein C and inhibition of coagulation Protein S APC Thrombomodulin Protein C V VIIIi Va Vi VIIIa T APC Protein S APC Schematic models illustrating some of the phospholipid-bound reactions that are involved in the activation and regulation of coagulation Factor VIIa binds to tissue factor and activates factors IX and X. Factors IXa and Xa together with factors VIIIa and Va, respectively, form the tenase and prothrombinase complexes that activate factor X and prothrombin, respectively. Thrombin-mediated activation of factor XI, factor V, and factor VIII, which gives positive feedback amplification of the system, is not shown. Thrombomodulin is present on endothelial cells. Thrombin generated in the vicinity of intact endothelial cells binds to thrombomodulin and efficiently activates protein C. Activated protein C (APC) and protein S form a complex on the plasma membrane of endothelial cells and possibly also on other cells. This complex inactivates factors Va and VIIIa, which results in down-regulation of the coagulation system. The degradation of factor VIIIa by APC is stimulated by protein S and by factor V, which in this context functions as an anticoagulant protein. translocated from the inner layer of the membrane to the phospholipid surfaces increases the local concentration of outer layer.7 In the tenase and prothrombinase complexes, the coagulation components and counteracts regulation all participating proteins (ie, the enzymes factors IXa and by anticoagulant mechanisms. Xa, the cofactors VIIIa and Va, and the substrates factor The relative importance of the various coagulation X and prothrombin), have affinity for the negatively factors in vivo has been elucidated by knock-out mice charged phospholipid surface. The enzymes and the technology.12 The crucial importance of the tissue-factor substrates are vitamin-K-dependent proteins that interact pathway is shown by the finding that mice lacking tissue with the phospholipid membrane via their amino-terminal factor die as embryos.13–15 Mice deficient in factor VII domains, which contain ␥-carboxyglutamic acid residues. develop normally in utero but die shortly after birth from This post-translationally modified glutamic acid residue is severe bleeding.16 The difference in severity between present only in the vitamin-K-dependent proteins. The deficiency of tissue factor and deficiency of factor VII residues are involved in calcium binding, important for suggests a role for tissue factor during embryogenesis the correct folding of the ␥-carboxyglutamic acid domain. beyond fibrin formation. Deficiencies of factor V and Inhibition of the ␥-carboxylation reaction by antagonists prothrombin are both associated with fatal haemorrhage of vitamin K results in defective calcium binding of the ␥- and partial embryonic lethality,17–19 whereas animals carboxyglutamic acid domains and loss of ability to deficient in factor IX or VIII develop normally during fetal interact with the phospholipid membrane.2 This is the life, but show haemophilia-like disease after birth.20,21 As molecular basis for anticoagulant therapy with vitamin K in human beings, fibrinogen deficiency in mice is antagonists. associated with normal fetal development and a moderate The concentrations of the various coagulation proteins to severe bleeding phenotype,22 which shows that in circulating blood relate to their specific roles in the thrombin generation is more important than fibrin pathway.2,3 The predominant clotting factor is fibrinogen deposition. This fact may be related to the multiple (10 ␮mol/L); this concentration is about 50 000 times functions of thrombin, including its ability to activate higher than that of factor VIII (0·2 nmol/L). The high platelets—a reaction that is crucial for the haemostasis fibrinogen concentration is required for the formation of response to vascular injury. the fibrin network, whereas the low amount of factor VIII is more than sufficient to support factor IXa in the activation of factor X. There are also variations in the Regulation of blood coagulation by concentrations of the vitamin-K-dependent proteins: anticoagulant pathways factor VII (10 nmol/L) is the least abundant, factors IX Regulation of coagulation is exerted at each level of the and X are present in intermediate concentrations (100 pathway, either by enzyme inhibition or by modulation of nmol/L), and prothrombin circulates at the highest the activity of the cofactors. The tissue-factor-pathway concentration (2 ␮mol/L). Thus, early components of the inhibitor inhibits the reactions involving tissue factor and pathway circulate at lower concentrations than the factors factor VIIa.23 This inhibitor is mostly bound to LDL in that act at later stages, which is consistent with the plasma or to heparan sulphate when associated with principal organisation of the system with multiple endothelial cells. The lack of tissue-factor-pathway reactions and amplification potential. The assembly of inhibitor may not be compatible with life, since no enzyme-cofactor complexes on negatively charged deficiency states have been described in human beings. 1628 THE LANCET Vol 355 May 6, 2000  HAEMATOLOGY This idea is supported by the lethal phenotype found in disease and die during embryogenesis, even before tissue-factor-pathway-inhibitor knock-out mice.24 These development of a functional cardiovascular system.30 animals show uncontrolled activation of coagulation with consumption of coagulation factors. Inherited and acquired coagulation disorders. Most of the enzymes generated during activation of Inherited deficiencies of factor VIII (haemophilia A) and coagulation are inhibited by the serine-protease inhibitor factor IX (haemophilia B) are rare inherited bleeding antithrombin, previously called antithrombin III. disorders with prevalence of about one in 10 000. The Antithrombin preferentially inhibits free enzymes, whereas genes for both factors are on the X chromosome, which is enzymes that are part of the tenase or prothrombinase why only males are affected, whereas females are carriers complexes are less accessible for inhibition. The of the disease. Almost half of the disease-causing physiological role of antithrombin is to limit the mutations are novel, and many boys with haemophilia are coagulation process to sites of vascular injury and to born in families with no previous history of the disease. protect the circulation from liberated enzymes. Severe haemophilia is associated with less than 1% of the Antithrombin is, in itself, an inefficient serine-protease normal plasma concentration of either factor IX or factor inhibitor, but heparin and the heparin-like molecules that VIII, whereas moderate and mild forms have plasma are present on the surface of endothelial cells stimulate its concentrations of 1–5% and 5–20% of normal, activity.25 This mechanism is the molecular basis for the respectively. The normal plasma concentrations of these use of heparin as a therapeutic anticoagulant. components seem therefore to be higher than required for The protein C anticoagulant system regulates a normal physiological response, which is noteworthy coagulation by modulation of the activity of the two considering the very low normal concentrations of factor cofactors, factors VIIIa and Va.26 Protein C, the key VIII in particular. The classic symptoms of haemophilia component of the system, is a vitamin-K-dependent are bleeding episodes affecting joints, muscles, internal zymogen to an anticoagulant protease. It is activated on organs, and the brain. Joint bleeding (haemarthrosis) is the surface of intact endothelial cells by thrombin that has the most characteristic feature of severe haemophilia. bound to the membrane protein thrombomodulin Repeated bleeds result in chronic arthropathy with loss of (figure). Thus, thrombin has the capacity to express both joint movement, fixed flexion contracture, and severe procoagulant and anticoagulant functions depending on muscle wasting. The first bleeding manifestations appear the context under which it is generated. At sites of in early childhood, but not in the neonatal period. vascular disruption, the procoagulant effects of thrombin Bleeding from the mouth caused by the eruption of the are fully expressed. In contrast, in an intact vascular child’s teeth is a common early manifestation of system, thrombin has anticoagulant function since it binds haemophilia. Haemorrhages in muscles and joints after to thrombomodulin and activates protein C. Activated minor twists or knocks are associated with the early protein C (APC) can cleave the phospholipid-membrane- crawling and walking efforts. Since primary haemostasis is bound cofactors factors Va and VIIIa, which results in unaffected, the bleeding time is normal and the patients inhibition of the coagulation system. A vitamin-K- do not experience major problems with bleeding from dependent cofactor protein, protein S, supports the mucous membranes and minor skin lesions. anticoagulant activity of APC. In human plasma, about von Willebrand’s disease is caused by quantitative or 30% of protein S is free, the remainder being bound to the qualitative defects in von Willebrand factor. This results in complement regulatory protein C4b-binding protein.26 a primary haemostasis defect, which is caused by deficient APC and free protein S form a membrane-bound adhesion of platelets to exposed subendothelial collagen. complex, which can cleave factors VIIIa and Va, even Bleeding from skin and mucous membranes is common, when they are part of fully assembled tenase and and the bleeding symptoms begin soon after birth in many prothrombinase complexes. cases. This disorder occurs in both male and female In vivo, APC does not cleave intact factor VIII because children. The disease is clinically heterogeneous, and the the binding of factor VIII to von Willebrand factor severity of the symptoms depends not only on the nature prevents it from interacting with the phospholipid of the disease-causing mutation, but also on whether or membranes. In contrast, factor V binds phospholipids as not both alleles are affected. Three major categories of von well as factor Va does, and APC is able to cleave the intact Willebrand’s disease are distinguished. Type I refers to form of factor V. The consequence of APC-mediated partial deficiency (heterozygous) and is inherited as an cleavage of factor V is the generation of anticoagulant autosomal dominant trait. Type II (several subtypes factor V that functions in synergy with protein S as an distinguished) is associated with a qualitative defect in von APC cofactor in the degradation of factor VIIIa. Thus, Willebrand factor, which commonly affects the multimeric factor V can function as a procoagulant and an structure of the protein. Type III refers to total deficiency anticoagulant cofactor, procoagulant factor Va being (homozygous or compound heterozygous) and is inherited formed after limited proteolysis by thrombin or factor Xa, as an autosomal recessive disease. Severe forms of von whereas the anticoagulant factor V activity is expressed by Willebrand’s disease have a secondary deficiency of factor factor V that has been proteolytically cleaved by APC.27,28 VIII, since von Willebrand factor is a carrier of factor VIII The anticoagulant potential of factor V may be in blood. particularly important in the regulation of the tenase Acquired bleeding disease may be caused by an complex by APC and protein S. The physiological autoantibody directed against a coagulation factor, the importance of the protein C system is shown by the severe most common being antibodies directed against factor thromboembolic disease that is associated with VIII or V. Unlike inherited haemophilia, the acquired homozygous deficiency of protein C in both human beings bleeding disorders mainly affect elderly people. The and mice.29 In both cases, the severe lethal thrombotic molecular mechanisms involved in the generation of the disease manifests shortly after birth. Mice lacking a autoantibodies are not known. The associated bleeding functional thrombomodulin gene have even more severe tendency may be severe and occasionally life-threatening. THE LANCET Vol 355 May 6, 2000 1629 HAEMATOLOGY Another type of acquired bleeding disorder is that related low. High concentrations of fibrin degradation products, to the requirement for vitamin K in the biosynthesis of including D-dimers, result from activation of the many of the coagulation proteins. Conditions associated fibrinolytic system. with malabsorption of vitamin K can lead to deficient ␥- carboxylation of the vitamin-K-dependent coagulation Treatment of haemophilia and von Willebrand’s proteins. In severe cases, this process results in an disease increased bleeding tendency. However, a more common Concentrates of factor IX or VIII, derived either from disorder is deficiency of vitamin K due to excessive intake plasma or produced by recombinant techniques, are used of antagonists, for example warfarin, used as anticoagulant in the treatment of haemophilic patients. Treatment can therapy. Severe liver disease may be associated with be given on demand or as prophylaxis. Drugs that inhibit bleeding tendency due to deficient synthesis of platelet function, such as aspirin, should be avoided. coagulation factors. The acquired bleeding tendency Intramuscular injections can trigger severe bleeding associated with disseminated intravascular coagulation is episodes and should therefore not be used. In most cases, due to consumption of platelets and coagulation factors, the administration of factor concentrates is uneventful but which is the result of widespread pathological proteolysis. may be complicated by virus transfer (plasma-derived In this disorder, many proteolytic enzyme systems are products) or the induction of antibody formation by the activated, including both coagulation and fibrinolysis, administered products. The management of haemophilic which result in microvascular thrombosis and major patients who have inhibitory antibodies is difficult and disturbances of the capillary circulation. Disseminated may include the use of high doses of factor VIIa or efforts intravascular coagulation is commonly caused by severe to induce immune tolerance.31,32 Gene therapy has not so infections with septicaemia or malignant disease, but it far been established as a therapeutic modality, although can also complicate traumatic injury, surgery, or pregnancy. research in this area is very intense. In animals, gene therapy of factor IX in dogs has shown promising results. Bleeding episodes in patients with severe forms of von Laboratory investigation of bleeding disorders Willebrand’s disease are treated with concentrates of The initial investigation of patients with bleeding von Willebrand factor. DDAVP (D-amino-D-arginine symptoms includes measurements of platelet counts, vasopressin) increases the release of von Willebrand factor bleeding time, and global clotting tests such as the from endothelial cells. It is therefore useful in the activated partial thromboplastin time (intrinsic pathway) treatment of type I von Willebrand’s disease. However, and the prothrombin time (extrinsic or tissue-factor because an efficient response to DDAVP requires that the pathway). Patients with primary haemostatic disorders von Willebrand factor can be synthesised, it is not effective who have normal results of clotting tests but long bleeding in type III von Willebrand’s disease. Type II shows times are further investigated with functional platelet tests, variation in the response to DDAVP, which should and immunological and functional assays for von therefore only be used after individual testing. Willebrand factor. When a coagulation disorder is suspected, specific functional and immunological testing of coagulation factors is possible. Severe haemophilia Inherited and acquired thrombotic disorders shows long clotting times in laboratory tests that are Venous thrombosis is common, each year affecting one in sensitive to the tenase complex (the activated partial 1000 individuals, with higher rates among elderly people thromboplastin time but not the prothrombin time, which than in the young. Pulmonary embolism or post- is insensitive to the tenase complex owing to the high thrombotic syndrome may complicate the disease, but in tissue-factor concentrations used in the assay). Specific most cases the recovery is uneventful. Inherited and functional assays for factors VIII and IX are used to acquired risk factors are involved in the pathogenesis of confirm the diagnosis. Identification of causative thrombosis. The inherited risk factors are life-long, mutations has so far been carried out only in research whereas most acquired risk factors are of short duration laboratories and has generated large databases with many (eg, pregnancy, surgery, and immobilisation). An acquired different disease-generating mutations. Moderate and risk factor may seem to be the cause of a thrombotic mild forms of haemophilia have either normal or only episode that is in fact due to a combination of genetic and slightly longer than normal activated partial acquired risk factors.33 Most inherited risk factors for thromboplastin times, and the diagnosis relies on the thrombosis affect the natural balance between specific tests for the respective factor. Diagnosis of procoagulant and anticoagulant forces, and most are acquired bleeding disorders caused by autoantibodies is found in the protein C system.26 The most common based on inhibition in clotting tests by the antibodies. inherited risk factor is a single point mutation in the gene Identification of which specific factor is recognised by the for factor V (G1691A), which results in phenotype called antibody involves specific coagulation or immunological APC resistance, found in 20–40% of patients with tests (eg, western blotting, which analyses the reactivity of thrombosis.34–36 The factor V mutation predicts the the antibodies with various coagulation proteins). The replacement of arginine 506 with a glutamine residue, laboratory diagnosis of vitamin K deficiency is based on which results in the loss of one of the APC cleavage sites the use of clotting tests examining the tissue-factor in factor V/Va.37 Mutant factor V (VR506Q, V:Q506, or pathway (prothrombin time). The activated partial factor V Leiden) has full procoagulant capacity. Dual thromboplastin time is normal in most patients with this mechanisms cause the hypercoagulable condition that disorder. Disseminated intravascular coagulation is characterises APC resistance.27 The factor V mutation is characterised by consumption of both platelets and associated with impaired degradation of factor Va by coagulation factors. Thus, platelet counts are low, the APC, since the arginine 506 site is one of three APC activated partial thromboplastin time long, and the cleavage sites in factor Va. In addition, the factor V concentrations of fibrinogen, factor V, and factor VIII mutation affects factor VIIIa degradation, because the 1630 THE LANCET Vol 355 May 6, 2000  HAEMATOLOGY anticoagulant activity of factor V is stimulated by the the blood are APC resistant, even though DNA analysis arginine 506 cleavage. The factor V mutation is found suggests heterozygosity. Diagnosis of the prothrombin predominantly in populations of caucasian origin, which is mutation (G20210A) relies on DNA testing, whereas explained by a single mutational event that took place deficiencies of protein C, protein S, and antithrombin are around 30 000 years ago and a subsequent founder best diagnosed by functional or immunological assays. For effect.38 The prevalence of the factor V mutation varies protein S, assays for the free form of the protein are between different countries of Europe and, with some preferable to those measuring the total protein S, since exceptions, a north-south gradient is apparent with they have higher predictive value for deficiency. Global highest prevalence (10–15%) in the north and lowest in clotting tests, such as the activated partial thromboplastin the south (about 2%). In populations with mixed ethnic time, are useful for the initial identification of patients backgrounds, such as the USA, the prevalence is about with antiphospholipid syndrome: in many cases of this 5%. APC resistance due to the factor V mutation is disorder the clotting times in all tests are long because the associated with a slightly increased risk of thrombosis (five antibodies disturb the interaction between the coagulation to ten fold) in its heterozygous state and a greatly factors and the phospholipids. Further investigation of a increased risk in the homozygous state (50–100 fold).33 On lupus anticoagulant includes the use of other clotting the other hand, APC-resistant women have a reduced tests, such as the dilute Russell’s viper venom time, a test bleeding tendency after delivery, which, during evolution, in which a prothrombinase complex is generated due to may have provided a survival advantage explaining the activation of factors X and V by the venom. high prevalence of the mutation.39 Demonstration of inhibitory activity in mixtures between The second most common genetic risk factor for normal plasma and the patient’s plasma is used to assess thrombosis (found in 6–8% of patients with thrombosis) is the potency of the lupus anticoagulants. The laboratory a single mutation (G20210A) in the 3⬘ untranslated region investigation may also include neutralisation tests with of the prothrombin gene.40 The mutation does not affect excess phospholipids and immunological tests that prothrombin function but is associated with slightly measure the binding of the patient’s antibodies to increased concentrations of prothrombin in plasma. It is immobilised phospholipids.43 found in around 2% of white people and is associated with slightly increased risk of thrombosis (three to five fold). Treatment of patients with thrombosis Heterozygous deficiency of protein C, protein S, or Venous thrombosis is initially treated with a combination antithrombin also increases the risk of thrombosis (found of heparin and vitamin K antagonists. Either in 1–3% of thrombosis patients), but these deficiencies are unfractionated or low-molecular-weight heparin can be uncommon in the general population (protein C and used; the latter is prepared from unfractionated heparin by protein S deficiency in about one in 300 and antithrombin chemical or enzymic cleavage methods. Low-molecular- deficiency in one in 2000).41,42 Many different mutations in weight heparin has better pharmacokinetic properties than each of these genes cause the deficiency states. The risk of unfractionated heparin, and adequate haemostatic control thrombosis in deficiency of protein C or protein S is is achieved with a single daily subcutaneous injection. In similar to that in APC resistance, whereas antithrombin addition, laboratory monitoring is not needed with low- deficiency is a somewhat stronger risk factor. Since the molecular-weight heparin. After a few days of the risk of thrombosis associated with the inherited disorders combined treatment, the concentrations of functional is low, most individuals with a single genetic risk factor vitamin-K-dependent coagulation proteins fall into the will not have thrombosis. Individuals with more than one therapeutic range, and heparin is discontinued. Treatment risk factor, either genetic or acquired, have a higher risk. with vitamin K antagonists, which should be regularly Venous thromboembolism is now considered to be a monitored by prothrombin time (international normalised typical multigenetic/multifactorial disease. ratio), is generally continued for 3–6 months. The risk of The antiphospholipid syndrome (lupus anticoagulant) bleeding complications must always be weighed against is an acquired risk factor for thrombosis, which can be of the benefits of the anticoagulation effect, especially if an long duration and can cause both arterial and venous oral anticoagulant is used for periods exceeding 3–6 thrombosis. The antibodies in the plasma of patients with months, when the risk of thrombotic recurrence probably this syndrome are directed against a protein-lipid declines. Whether the presence of a genetic risk factor is complex. In most cases, the protein is ␤2-glycoprotein 1, associated with an increased risk of recurrence is not known, though several studies of APC resistance suggest but antibodies against prothrombin have also been this association.44 Patients with combined genetic defects, identified. The antiphospholipid syndrome is associated and probably also patients with single gene defects, may with increased risk of pregnancy-related complications be at increased risk of recurrence, and long-term including miscarriages.43 anticoagulation beyond 6 months can be considered, even after an isolated thromboembolic event. Prophylactic Laboratory investigation of risk factors for treatment with low-molecular-weight heparin or oral thrombosis anticoagulants is recommended for individuals with Simple functional clotting-based APC resistance tests are multiple genetic defects (including homozygous APC available with close to 100% sensitivity and specificity for resistance) in situations known to be associated with a the factor V mutation. Positive results are generally high risk of thromboembolic complications. This confirmed by DNA-based tests, which distinguish recommendation holds even if the patient neither has heterozygous from homozygous forms. In rare cases, the experienced thrombosis nor has any family history of such APC resistance test suggests a more severe phenotype complications. In symptom-free carriers of single genetic than the DNA test, which may be due to risk factors lacking a family history of thrombosis, short- pseudohomozygosity with one mutant factor V allele and term prophylaxis may be considered in high-risk one null allele. In this condition, all factor V molecules in situations. THE LANCET Vol 355 May 6, 2000 1631 HAEMATOLOGY References inhibitor gene disruption produces intrauterine lethality in mice. Blood 1 Mann KG, Lorand L. Introduction: blood coagulation. Methods 1997; 90: 944–51. Enzymol 1993; 222: 1–10. 25 Lindahl U, Kjellen L. Heparin or heparan sulfate—what is the 2 Furie B, Furie BC. Molecular and cellular biology of blood difference? Thromb Haemost 1991; 66: 44–48. coagulation. N Engl J Med 1992; 326: 800–06. 26 Dahlback B. The protein C anticoagulant system: inherited defects as 3 Davie EW. 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