Inherited thrombophilia: memorandum from a joint WHO/International Society on Thrombosis and Haemostasis meeting.

Memoranda I Memorandums Inherited thrombophilia: Memorandum from a joint WHO/International Society on Thrombosis and Haemostasis meeting* Inherited thrombophilias are common disorders with a worldwide distribution, including antithrombin, protein C, and protein S deficiencies as well as resistance to activated protein C. Increased understanding of these disorders suggests that thrombophilia can arise from interaction between defective genes and environmental factors. WHO and the Intemational Society on Thrombosis and Haemostasis (ISTH) discussed the problems of inherited thrombophilia at a joint meeting held in Geneva on 6-8 November 1995. The present article reports on the various possibilities for controlling the disorder and makes a series of recommendations for diagnosis, treatment, and research into the condition. Introduction Before 1990 the results of studies to identify isolated hereditary deficiencies or defects were consistent with two principle endogenous anticoagulant path- ways (Fig. 1): the antithrombin-heparan sulfate pathway and the protein C/protein S pathway. How- ever, until that time, only three single gene disorders had been identified that were associated with a signifi- cant increase in the risk for venous thromboembol- ism in families with a symptomatic deficient patient: antithrombin, protein C and protein S deficiencies. These deficiencies occurred only in about 15% of * Based on the report of a Joint WHO/International Society on Thrombosis and Haemostasis (ISTH) meeting on Inherited Thrombophilia, held in Geneva, 6-8 November 1995 (unpublished document WHO/HGN/ISTH/WG/95.5 available upon request from Division of Noncommunicable Diseases, World Health Organiza- tion, 1211 Geneva 27, Switzerland. The full report has been pub- lished in Thrombosis and haemostasis, 1996, 76 (5): 651-652; and 1996, 76 (6): 824-834. Requests for reprints should be sent to Dr V. Boulyjenkov, Division of Noncommunicable Diseases, World Health Organization, 1211 Geneva 27, Switzerland. The participants at the meeting were as follows: Dr K.A. Bauer, Boston, MA, USA; Dr R.M. Bertina, Leiden, Netherlands; Dr N.P. Bochkov, Moscow, Russian Federation; Dr M. Chandy, Vellore, Tamilnadu, India, and Al-Khod, Oman; Dr B. Dahlback, Malmo, Sweden; Dr E.K. Ginter, Moscow, Russian Federation; Dr D. Lane, London, England (Rapporteur); DrJ.P. Miletich, St. Louis, MO, USA; Dr F.R. Rosendaal, Leiden, Netherlands; Dr U. Seligsohn, Tel Hashomer, Israel. ISTH Secretariat: Dr P.M. Mannucci (Chairman and Co-Rapporteur). WHO Secretariat: Dr N.P. Napalkov, Dr D.E. Barmes, Dr V. Boulyjenkov (Secretary) and Dr. C.-C. Heuck. Reprint No. 5768 families with familial thrombosis and only in a small proportion of all patients with venous thrombosis (1). The awareness that for 85% of families predis- posed to thrombosis no explanation could be found stimulated the application of genetic and epide- miological approaches to the problem. As a result, a major breakthrough in the study of familial throm- bosis has been achieved over the past 3 years. First, activated protein C resistance (APC-R) was discov- ered, and, second, a mutation in the factor V gene (1691 G -* A in exon 10, leading to 506Arg -+ Gln substitution) was identified as the molecular basis for the phenotype of APC-R in the large majority of affected individuals (2,3). This mutation, which is associated with a significant increase in thrombotic risk (3-5), has been found in about 50% of selected families with thrombophilia and in 20% of consecu- tive patients with thrombosis. A consequence of this advance has been a conceptual change in how thrombophilia is viewed, which has implications for its diagnosis and treatment. Pathogenesis and genetic basis of thrombophilia Thrombophilia's predisposing defects do not neces- sarily cause continuous clinical impairment; they only weaken the ability to cope with fluctuations induced by interactions with the environment. A list of potential genetic risk factors that have been asso- ciated with thrombosis is shown in Table 1. The term inherited thrombophilia reflects the presence of an inherited factor that, per se, predis- Bulletin of the World Health Organization, 1997, 75 (3): 177-189 © World Health Organization 1997 177 Memorandum Fig. 1. Schematic representation of the two principle anticoagulant pathways that are important in the regulation of coagulation proteinase activity. a) Coagulation regulation by antithrombin; b) Coagulation regulation by protein C/protein S. To the left of each diagram is a simplified view of the coagulation cascade, illustrating the positive "procoagulant" feedback loops through which thrombin activates factors V and VIII. To the right, are the "anticoagulant" pathways which prevent excessive activation of coagulation. These pathways involve antithrombin (which directly inhibits the coagulation proteinases such as factor Xa and thrombin), and protein C/protein S (which inactivate factor Va and factor Villa). Protein S normally forms a complex with C4bBP and it is only the free form of protein S that acts as a cofactor for protein C. a) -, factor IXa ~~~+ factor VIIIo- factor Vllla factor X factor Xa factor V - . factor Va Prothrombin Thron Anticoagulant Antithrombin + Heparin or EC heparan sulfate Anticoagulant nbin Procoagulant / Fibrinogen Fibrin b) -i factor IXa - factor VIII - factor Villa - factor VIII factor X factor Xa factor V . . > factor Va > factor VI Protein Thrombin-Antithrombin complex Protein S Ca -+-- Protein C Prothrombin Thrombin A 0) Procoagulant Anticoagulant Fibrinogen Fibrin Thrombomodulin factor Xa-Antithrombin complex v WHO Bulletin OMS. Vol 75 1997178 Inherited thrombophilia Table 1: Causes of inherited thrombophiliaa Acquired Antithrombin deficiency Protein C deficiency Protein S deficiency APC-R/factor V 506 Arg - Gln Dysfibrinogenaemia Thrombomodulin Acquired/inheriteda Hyperhomocysteinaemia Elevated factor VilI Elevated fibrinogen Potentially inheritedb Plasminogen deficiency Heparin cofactor 11 deficiency Elevated histidine-rich glycoprotein Plasminogen activator deficiency? Elevated plasminogen activator inhibitor? a Precise relative contributions currently uncertain. b To date, there is no firm evidence that these conditions are linked to inherited thrombophilia. The development of thrombosis is often believed to be caused by interaction between genetic and acquired factors (the best recognized of the latter being advancing age, immobilization, major surgery, orthopaedic surgery, neurosurgery, pregnancy, puerperium, use of estrogen-containing hormones, malignancies, and antiphospholipid syndrome). poses towards thrombosis, but which, because of the episodic nature of thrombosis, requires interaction with other factors (inherited or acquired) before on- set of the clinical disorder. The concept of inherited thrombophilia is operational, the definition of which has undergone continuous refinement. It was origi- nally based on early presentation of thrombosis, usu- ally coupled with inherited phenotypic abnormality of one of the inhibitory proteins - antithrombin, protein C or protein S. Progress in understanding the molecular basis of thrombosis has enabled a more genetically based definition to be formulated: in- herited thrombophilia is a genetically determined tendency to venous thromboembolism. Severe ab- normalities or combinations of less severe defects may be clinically apparent from an early age of on- set, frequent recurrence, or family history. Milder traits may be discovered only by laboratory investi- gation and all the genetic influences and their inter- actions are not yet understood. The nature and heterogeneity of the molecular defects associated with the established genetic risk factors for venous thrombosis, including anti- thrombin deficiency, protein C deficiency, protein S deficiency and factor V Leiden, are discussed below. Antithrombin deficiency Antithrombin is a single-chain plasma glycoprotein (58kDa) that is synthesized in the liver and which belongs to the superfamily of serine protease in- hibitors (serpins); its concentration in plasma is 2.5[imol/l. It is the primary inhibitor of most of the activated serine proteinases involved in blood coagulation (thrombin, factor Xa, factor IXa, factor XIa, factor XIIa, kallikrein) and therefore is one of the most important physiological regulators of fibrin formation. Antithrombin deficiency is a heterogenous dis- order. The subclassification of antithrombin defi- ciency was originally based mainly on the results of functional and immunological assays of plasma. Sub- sequently, as understanding of the mutations in the antithrombin gene increased, the nomenclature was modified (6, 7). Currently, two types of antithrombin deficiency are recognized: type I (identified by a con- cordant reduction of both functional and immuno- logical antithrombin); and type II (identified by a variant antithrombin molecule, which has a defect in the reactive site (II RS), a defect affecting the heparin binding site (II HBS), or multiple functional defects (pleiotropic effect) (II PE)). Clinically anti- thrombin deficiency is heterogeneous, with muta- tions causing type II HBS deficiency being of much less risk than those causing the other subtypes (7, 8). Recurrent mutations occur in all types of antithrombin deficiency, but especially in type II HBS; of the 21 distinct repeat mutations, nine in- volve a CpG dinucleotide (a "hotspot" for muta- tion). Only preliminary data are currently available that throw light on whether these recurrent muta- tions are the result of independent mutations or a founder effect (identity by descent) (9). Protein C deficiency Protein C is a vitamin-K-dependent plasma glyco- protein that is the precursor of the serine-proteinase- activated protein C (APC). Protein C is synthesized in the liver as a single chain (62kDa), which is con- verted into two chains by removal of a dipeptide (Argl57-Thr158) probably in the Golgi complex. In plasma most protein C occurs in the double chain form (41 kDa heavy chain and 21 kDa light chain); the concentration of protein C in plasma is normally 65 nmol/I but is reduced during treatment with oral anticoagulants. Protein C deficiency is a heterogeneous disorder (10, 11). A phenotypic subclassification has been proposed, based on the results of functional and im- munological assays of protein C. In type I protein C deficiency there is a concordant reduction in pro- tein C activity and in the level of protein C antigen, while in the type II deficiency there is evidence for the presence of an abnormal protein C molecule (reduced protein C activity, normal protein C anti- WHO Bulletin OMS. Vol 75 1997 179 Memorandum gen). A further classification of the type II protein C deficiency can be made by comparing the results of different functional tests (clotting test vs chromo- genic test). In total, 160 different mutations have been re- ported to cause type I or type II protein C deficiency. Surprisingly, ca. 60% of the mutations causing the type I deficiency are missense mutations. Probably these amino acid substitutions lead to changes in the interactions with other residues and thus interfere with protein folding, a condition associated with rapid intracellular degradation of the protein. Protein S deficiency Protein S is a vitamin-K-dependent plasma glyco- protein (70kDa) that is synthesized in the liver, but also in endothelial cells, megakaryocytes and Leydig cells in the testis. Its concentration in plasma is 25,tg/ml, but is lower during treatment with oral anticoagulants. Consistent with the subclassifications used for other hereditary deficiencies, type I deficiencies/ defects result in a reduction of total protein S antigen (and of free protein S antigen and protein S activity). Type II deficiency defines the presence of a function- ally abnormal protein S molecule (normal levels of total protein S antigen and of free protein S antigen but reduced protein S activity). Type III protein S deficiency is defined by normal total protein S anti- gen but reduced free protein S antigen and activity. Although this phenotype seems to be rather preva- lent, it is not yet clear whether it is caused by a hereditary defect and if so, whether it is linked to the protein S locus. Genetic analysis of the protein S genes of symp- tomatic protein S deficient probands has been ham- pered by the structural complexity of the protein S gene and by the existence of the highly homologous pseudogene. In three separate studies mutations were only found in 50-60% of the patients, although all coding and flanking regions had been amplified and sequenced (12-14). The majority of genetic lesions that cause a type I deficiency are single nucleotide substitutions, inser- tions, and deletions; so far, at least 33 unique events have been reported. Four different mutations have been reported to cause a type II protein S deficiency (15,16); two in the propeptide, one in the first EGF domain and one in the second EGF domain. Factor V Arg5O6 -* Gin mutation Factor V is a single-chain plasma glycoprotein pro- cofactor that is synthesized in the liver and in megakaryocytes. Its concentration in human plasma is 20nmol/l and in platelets, 4 ig per 109 platelets. During blood coagulation factor V is converted into factor Va by meizothrombin and/or factor Xa. More recently it has been reported that factor V is not only a pro-cofactor in the prothrombinase re- action but also a cofactor in the inactivation of factor Vllla by APC (17). A recent review provides more information on the structure and function of human factor V (18). In 1994, the single-point mutation in the factor V gene was identified as the genetic defect that caused the phenotype of APC-R in the vast majority of affected individuals (3, 5, 19). This mutation in- volves a G -* A transition of nucleotide 1691 in exon 10, which predicts the synthesis of a variant factor V molecule (factor V 506 Arg -* Gln or factor V Leiden). The mechanism through which the muta- tion leads to the APC-R phenotype is still the subject of detailed biochemical studies. So far, the factor V 506Arg -* Gln mutation is the only genetic defect that has seen identified in APC-R families. Its frequency is relatively high in Caucasian populations (up to 6%) but is much lower in African and Asian populations (down to 0%) (20). Evidence for a founder effect in the spread of this disorder was obtained from the results of haplotype analysis of 53 Dutch carriers of the muta- tion (3). Other candidates? There are a number of other genetic defects or iso- lated deficiencies that have been implicated in contributing to the risk of thrombosis in families with thrombophilia. In most cases these are based on observations of case families. Sometimes gene- tic defects have been identified but no data on genotype-phenotype relationships are currently available (21). Hereditary dysfibrinogenaemia is characterized by a prolonged plasma thrombin time. Clinical symp- toms vary from none, to mild bleeding, to venous or arterial thrombosis. The phenotype may follow recessive or dominant inheritance. Recently the evi- dence for a causal relationship between an isolated dysfibrinogenaemia and venous thrombosis has been critically reviewed and discussed (22). Mild hyperhomocysteinaemia has recently been found in 19% of patients with juvenile venous thrombosis, and family studies showed that in most cases this phenotype was inherited (23). Thrombomodulin (TM), a further component of the protein C anticoagulant pathway (24), is a transmembrane protein synthesized by endothelial cells and acts as a receptor for thrombin and as cofactor of thrombin in the activation of protein C. WHO Bulletin OMS. Vol 75 1997ISO Inherited thrombophilia By analogy with protein C and protein S deficiencies, it might be expected that deficiencies or defects in thrombomodulin may be associated with an in- creased risk of thrombosis. However, to date, infor- mation is still very limited on the co-segregation of these mutations with thrombophilia in the proband families. Plasminogen deficiency and dysplasminogenae- mia, have frequently been reported to be associated with thrombophilia. However, studies reveal that in most families with a type I plasminogen deficiency (parallel reduction of plasminogen activity and anti- gen) only the proband suffers from thrombotic dis- ease (25), which can be taken as evidence against its causative role in thrombosis. The frequency of plasminogen deficiency among the general popula- tion seems to be slighter lower (0.4%) than among cohorts of patients with thrombosis (1-3%) (26,27). Since reduced plasminogen levels may cause thrombophilia, it seems reasonable to propose that an inherited elevated level of histidine-rich glycoprotein (HRG) in plasma is also a risk factor for thrombosis. HRG (a non-enzymatic protein) forms a 1:1 complex with plasminogen in plasma (via its lysin-binding sites) and thus reduces the free plasminogen concentration to around 50% (28). Complex formation with HRG interferes with the binding of plasminogen to fibrin. Although several families with thrombophilia and high HRG levels have been reported (29, 30), there is still no formal evidence for its association with thrombosis. A further potential risk factor of thrombosis tissue factor pathway inhibitor (TFPI) deficiency - has been investigated, but no mutation has been found in the TFPI genes of 30 symptomatic probands of families with thrombophilia (P.H. Reitsma & R. M. Bertina, unpublished observations). Finally, a phenotype has recently been identi- fied as a risk factor for thrombosis in large patient- control studies: elevated factor VIII levels (31). The hereditability of this phenotype and the possible underlying molecular defects have not yet been reported. Epidemiology of inherited thrombophilia Venous thrombosis has an overall annual incidence of 1 per 1000 population. It is rare among the young, and becomes more frequent with advancing age. The true prevalence of hereditary thrombophilia has not yet been determined. All the genetic abnormalities that cause a tendency to venous thrombosis are clearly not known, since only in about half the pa- tients from families selected on the basis of a high number of unexplained thromboses is an underlying defect found (32). Hence, the prevalence of heredi- tary thrombophilia in the general population will be higher, possibly twice as high as in estimates from large prevalence studies on known genetic defects, and the high prevalence of hereditary thrombophilia will make it an important factor in the overall inci- dence of thrombosis. The prevalence of protein C and antithrombin deficiences has been investigated in a very large study of almost 10000 blood donors (33, 34) (Table 2). Repeated testing of the levels of these proteins, coupled with family studies and DNA analysis, led to prevalence estimates of 1 in 500 for protein C defi- ciency and 1 in 5000 for type I antithrombin defi- ciency. These values lie in the same range found in a previous study of over 5000 blood donors, where 1 in 250 were considered to be protein C deficient (35). If the approximate prevalence of protein C deficiency is taken to be 1: 350, the prevalence of severe (homozygous or compound heterozygous) defi- ciency is 1:700 x 1:700 = 2:106. For APC-R, the groups that have been studied are smaller than those used in the blood donor studies. However, since the prevalence of this abnormality is ten times greater than that of the other inhibitor deficiencies, the estimates for its prevalence are as reliable. Such estimates for Caucasians lie in the range 3-7% (4, 36, 37), which, since they are based on self-selected individ- uals without a history of cardiovascular disease or venous thrombosis, are under- rather than over- estimates. The prevalence of the homozygous fac- tor V 506 Gln mutation has been estimated to be approximately 1:5000 (4). Approximately 5% of consecutive patients with objectively confirmed deep-vein thrombosis are ac- counted for by protein C, protein S, and antithrom- bin deficiences combined, while APC-R is present in 20% of consecutive patients with deep-vein throm- bosis (4, 38). Among selected patient groups, the prevalences of protein C, protein S, and antithrombin deficiences are mostly in the range 5-10%, much higher than the levels found in population studies, and also somewhat higher than those among consecutive unselected patients. The higher prevalences among patients with thrombosis than among healthy indi- viduals, and among thrombophilic individuals than among unselected patients also indicate that these deficiencies indeed lead to venous thrombosis and venous thrombophilia. APC-R appears to account for half of all cases of hereditary thrombophilia, and as shown in Table 2 clearly emerges as the most important cause of hereditary thrombosis and per- haps of venous thrombosis in general. WHO Bulletin OMS. Vol 75 1997 181 Memorandum Table 2: Prevalence of the major thrombophilic cloning abnormalities % prevalence of: Protein C Protein S Antithrombin deficiency deficiency deficiency APC-R Healthy individuals Tait et al. (n = 9669) (ref. 33, 34) 0.2a - 0.02a.b Miletich et al. (n = 5422) (ref. 35) 0.4 - Svensson & Dahlback (n = 130) (ref. 36) - - - 7 Rosendaal et al. (n = 474) (ref. 4) - - - 3a Ridker et al. (n = 704) (ref. 37) - - -6a Consecutive patients with first deep venous thrombosis Heijboer et al. (n = 277) (ref. 64) 3 2 1 Koster et al. (n = 474) (ref. 65) 3 1 1 Rosendaal et al. (n = 471) (ref. 4) - - - 20a Thrombophilic patients Bribteteal. (n = 113) (ref. 66) 8 13 4 Scharrer et al. (n = 158) (ref. 67) 9 6 5 Ben Tal et al. (n = 107) (ref. 68) 6 3 7 Tabernero et al. (n = 204) (ref. 69) 1 1 0.5 - Griffin et al. (n = 25) (ref. 32) - - - 52 a DNA confirmed. bType I antithrombin deficiency. Clinical manifestations of inherited thrombosis The commonest clinical manifestation of inherited thrombosis is deep vein thrombosis of the lower limbs, with or without pulmonary embolism, which accounts for approximately 90% of all the throm- botic episodes (Table 3). Unusual sites of venous thrombosis, such as the mesenteric or cerebral veins, account for less than 5% of the total episodes in patients with antithrombin, protein C or protein S deficiencies; in patients with APC-R, thrombosis seems to occur less frequently at such sites. A total of 50-60% of individuals from families with antithrombin, protein C, and protein S deficien- cies have a history of thrombosis at diagnosis with a 50% recurrence rate; in approximately 80% of pa- tients the first thrombotic episode occurs before 40 years of age. For antithrombin deficiency, the overall risk of venous thrombosis is considered to be greater than with protein C or protein S deficiency (39), but contradictory results have been obtained (40). In patients with antithrombin, protein C and protein S deficiencies, 32-50% of the venous thrombotic episodes occur when other risk factors are concomitantly present (surgery, pregnancy, im- mobilization) (40-43). In individuals with APC-R, the need for the presence of such risk factors to trigger thrombotic episodes appears to be greater (62%) than for the other thrombophilic syndromes (44). The following risk factors are often associ- ated with the occurrence of thrombosis: pregnancy, puerperium, and surgery. Among women with antithrombin deficiency, the frequency of thrombo- sis during pregnancy and puerperium is 37-44%; in instances of protein C or protein S deficiency, 12-19% (45); in APC-R, 28% (46). Thrombotic epi- sodes occur most frequently during puerperium, accounting for 60-75% of all such episodes that complicate pregnancy. Retrospective analysis of a large number of antithrombin, protein C or protein S deficient individuals produced an overall frequency of venous thrombosis complicating surgery of 22%, with no significant differences arising because of the Table 3: Clinical features of patients with inherited thrombophilia arising from defects in anticoagulant pathways Venous thromboembolism (>90% of cases) Deep vein thrombosis of lower limbs (common) Pulmonary embolism (common) Superficial thrombophlebitis Mesenteric vein thrombosis (rare but characteristic) Cerebral vein thrombosis (rare but characteristic) Family history of thrombosisa First thrombosis usually at <45 years of agea Frequent recurrencesa Neonatal purpura fulminans (homozygous protein C and protein S deficiency) a Less evident in patients with APC-R, who appear to be less severely affected clinically. WHO Bulletin OMS. Vol 75 1997182 Inherited thrombophilia type of deficiency or surgical procedure. Intake of oral contraceptives is associated with an increased thrombotic risk, particularly for women with anti- thrombin deficiency and APC-R (46). Homozygous antithrombin deficiency is ex- tremely rare and almost exclusively reported in patients with HBS defects; such individuals have a severe thrombotic history of early onset, often affecting their arteries (47). Homozygous type I anti- thrombin deficiency has a poor prognosis: in one report, two brothers with this defect died within 3 weeks of birth (48). Homozygous protein C defi- ciency is associated with unusual phenotypic and clinical expressions (reviewed in 49). Among pa- tients whose protein C levels are too low to be mea- sured, purpura fulminans, caused by thrombosis of small vessels with cutaneous and subcutaneous ischaemic necrosis, may occur soon after birth or during the first year of life. Among those patients with very low but measurable protein C levels (5- 20%), clinical manifestations are milder and gener- ally similar to those for heterozygous deficiency. Homozygous protein S deficiency has rarely been reported, but is also associated with neonatal purpura fulminans (50). In view of the high fre- quency of the mutant factor V among the general population, homozygous APC-R is relatively fre- quent (ca 1:5000) (4). Management of inherited thrombophilia Acute events The management of acute venous thrombosis or pul- monary embolism involving patients with inherited thrombophilia is generally not different from that of other patients. Thrombolytic therapy can be used for patients with massive acute venous thrombosis or pulmonary embolism. An intravenous bolus of 5000 units of heparin should be initiated followed by an infusion of 1400 units per hour (51), or if a weight- adjusted regimen is used, a bolus of 80 units per kg body weight followed by an infusion of 18 units per kg per hour (52). The infusion of 50 units of antithrombin con- centrate per kg body weight (one unit is the amount of antithrombin present in 1 ml of pooled normal human plasma) will usually raise the plasma anti- thrombin level to approximately 120% in a congeni- tally deficient individual whose baseline level is 50% (53). Plasma levels should be monitored to ensure that they remain above 80%; administration of 60% of the initial dose at 24-hour intervals is recom- mended to maintain inhibitor levels in the normal range (53). After an episode of venous thrombosis or pul- monary embolism, patients are usually given oral anticoagulants for 3-6 months. Recent data indicate that the risk of recurrence is greater for patients with permanent rather than temporary risk factors for thrombosis (54, 55) and it is therefore appropriate that patients with inherited thrombophilia be given warfarin for at least 6 months at an international normalized ratio (INR) of 2.0-3.0. Following 6 months of anticoagulant therapy for an acute thrombotic event, the relative benefit con- ferred by such long-term therapy in preventing future thromboembolic complications versus the potential side-effects, cost, and inconvenience for the patient should be assessed. Unfortunately there are a paucity of reliable data on the magnitude of the thrombotic risk or the benefit of anticoagulant therapy for patients with antithrombin, protein C, or protein S deficiencies since these are relatively un- common disorders. Because of the high frequency of APC-R among patients presenting with a first epi- sode of venous thrombosis, reliable data are just emerging on the risk of recurrence (37). Here how- ever, we give only general guidelines for managing patients with the various hereditary defects that predispose to thrombosis rather than provide rigid recommendations. Inherited thrombotic disorders When a heterozygous patient with one of the heredi- tary thrombotic disorders is identified, family studies should be conducted since approximately half the first-degree relatives will be affected. Affected asym- ptomatic individuals should be counselled about the implications of the diagnosis and given advice about those symptoms that require immediate medical at- tention. Among women of child-bearing age, oral contraceptives are generally contraindicated be- cause of the increased thrombotic risk associated with their use, although individual circumstances need to be considered. The replacement dose of estrogens administered to postmenopausal women is much lower than the contraceptive dose and has not been shown to increase the risk of venous throm- bosis among the general population (56). Since no data are available that indicate that postmenopausal estrogen replacement therapy increases the risk of thrombosis among patients with a hereditary throm- botic disorder, such therapy is not absolutely con- traindicated. All individuals with inherited thrombotic disor- ders should be carefully evaluated prior to surgical, medical, or obstetric procedures that carry an in- WHO Bulletin OMS. Vol 75 1997 183 Memorandum creased thrombotic risk and should then receive ap- propriate prophylactic anticoagulation regimens. If specific concentrates are available for the patient's deficiency state, under certain circumstances these might also be administered to raise the plasma levels of the protein to the normal range during the peri- operative period. Among patients with an inherited thrombotic disorder, the occurrence of two or more spontaneous thromboembolic episodes often leads to the continu- ation of oral anticoagulants for life, even though some workers hold that the risks of bleeding could exceed those of recurrence of thrombosis. Management of pregnancy The management of pregnancies among women with hereditary thrombotic disorders poses special problems. The incidence of thrombotic complica- tions during pregnancy and the postpartum period appears to be greater for women with antithrombin deficiency than protein C or protein S deficiencies (57). Recent data indicate that 60% of women who develop a first episode of venous thrombosis during pregnancy have APC-R. During pregnancy, adjusted-dose heparin administered subcutane- ously is the anticoagulant of choice because its efficacy and safety for the fetus are established (58). Centres with greater experience in using low molecular weight heparins, however, might find that they are advantageous since laboratory monitoring may not be required. Patients with a history of thrombotic episodes should receive treat- ment throughout pregnancy, while women with antithrombin deficiency but who have not yet experi- enced thrombosis should probably be treated. Treat- ment of asymptomatic women with other hereditary thrombotic disorders should be considered on an individual basis. Both the dose and duration of heparin therapy in pregnancy are uncertain since appropriately de- signed clinical trials have not been performed. Pa- tients considered to be at high risk should receive full-dose heparin subcutaneously every 12 hours for the duration of pregnancy. The dose of heparin should be adjusted to ensure that the 6-hour post- injection activated partial thromboplastin (APTT) level is 1.5 times the control value. For women con- sidered to be at intermediate risk, lower doses of heparin can be used (5000-10000 units subcutane- ously every 12 hours) and therapy can be started during the second or third trimester and continued for approximately 6 weeks into the postpartum period. Low-risk patients can be observed closely throughout pregnancy with duplex ultrasound imaging of their leg veins at regular intervals. Coumarin-induced skin necrosis and neonatal purpura fulminans A clear association has been established between coumarin-induced skin necrosis and hereditary pro- tein C deficiency, with about a third of patients with such necrosis having hereditary protein C deficiency (59). This complication has also been described in a patient with homozygous protein S deficiency (60). Since coumarin-induced skin necrosis is a rare com- plication, therapy has been guided primarily by un- derstanding of its pathogenesis; such therapy should consist of immediate discontinuation of warfarin, administration of vitamin K, and infusion of heparin at therapeutic doses. Fresh frozen plasma has been used, but improved results can be expected with the administration of a highly purified protein C concen- trate, which facilitates the rapid and complete nor- malization of plasma protein C levels (61). Management of neonatal purpura fulminans in association with homozygous or doubly heterozy- gous protein C deficiency is more complicated and heparin therapy as well as antiplatelet agents are not effective (62). Administration of a source of protein C appears to be critical in the initial treatment of such patients, and fresh frozen plasma has been used with success to treat infants. However, the half-life of protein C in the circulation is only about 6-12 hours (63), and administration of plasma on a frequent basis is limited by the development of hyperpro- teinaemia, hypertension, loss of venous access, and the potential for exposure to infectious viral agents. Warfarin has been administered to infants without the redevelopment of skin necrosis during the phased withdrawal of fresh frozen plasma infusions (62), and has been used chronically to control throm- botic diathesis. General overview Currently, mutations in four genes are clearly linked to increased risk for venous thromboembolism. Many discrete mutations cause antithrombin, pro- tein C, and protein S deficiencies that diminish the capacity to balance procoagulant activity. One spe- cific mutation in factor V (506Arg -* Gln) has a similar impact by rendering this procoagulant factor resistant to proteolytic degradation. Roughly 50% of cases of familial thrombophilias can be explained by these four established risk factors for thrombosis. Apparently a number of other genetic risk factors have escaped detection, so far; it is, however, un- likely that these will include plasminogen, heparin co-factor II, tissue factor pathways inhibitor, or t2- glycoprotein-1 deficiencies. Other risk factors will WHO Bulletin OMS. Vol 75 1997184 Inherited thrombophilia need further evaluation (e.g. dysfibrinogenaemia, thrombomodulin defects and inherited hyperhomo- cysteinaemia). Increasingly it is becoming apparent that coinheritance of more than one relatively mild thrombophilic risk factor results in more severe clini- cal expression. Greater attempts are being made to quantify genetic and acquired risk factors. Risk estimates de- pend heavily on how study subjects are selected and do not necessarily apply to individuals chosen differ- ently. In particular, results from studies of families with a marked tendency to thrombophilia probably overestimate the risk for individuals who have had a single thrombotic event. Finally, when gene-gene and gene-environment interactions are required to bring about thrombosis, within-family and between- family differences may well be comparable. Laboratory evaluation of thrombophilia should involve the use of assays with the highest sensitivity and specificity for the genetic defect being detected. Such assays can be immunological or functional - the former may not detect cases with truly functional defective proteins. A practical approach should be taken and the selection of analytical procedures should be governed by the aim of the investigation as well as of locally determined factors such as prevalence of the genetic defects to be detected and availability of technical support. Based on avail- able scientific information, the laboratory evalu- ation should include the determination of the lev- els of protein C, total and free protein S, and anti- thrombin, as well as use of a functional APC-R test that is sensitive and specific for the presence of the factor V 506Gln allele. For protein C, assays that are based on its activation by the protein C activator Protac and determination of the active enzyme with synthetic substrate fulfil the required quality criteria. At present, no functional protein S assays can be recommended for general screening of thrombophilic patients. Immunological assays of to- tal as well as of free protein S are, however, recom- mended. Recently published results indicate that free protein S in the best marker for genetically determined protein S deficiency, but further studies are required before a recommendation can be made only to determine free protein S. Functional assays for antithrombin that are based on heparin- stimulated inhibition of factor Xa are recommended for screening thrombophilic patients. For initial screening of APC-R, functional tests are recom- mended; such tests can be improved by diluting the patient plasma in factor-V-deficient plasma. Since assays for protein C and protein S have distinctly lower sensitivity and specificity for detecting the presence of inherited deficiency during the acute thrombotic episode and oral anticoagulation, at present it is recommended that the laboratory inves- tigation for these components be performed after discontinuation of therapy. The clinical manifestations of the defects of naturally occurring anticoagulant systems (anti- thrombin, protein S, and protein C deficiencies; and APC-R) are similar. In heterozygotes, the typical manifestations are venous thromboembolism (e.g. deep-vein thrombosis of the legs, pulmonary embo- lism, and superficial thrombophlebitis). Visceral and cerebral vein thrombosis are rarer but quite typical for inherited thrombophilia. Patients with homozy- gous defects usually have more severe clinical manifestations with an earlier age of onset. Some manifestations are quite typical, e.g. skin necrosis and widespread neonatal thrombosis in cases of protein C and protein S deficiencies. Preliminary data suggest that some homozygous defects (anti- thrombin type II HBS deficiency) may be also asso- ciated with an increased risk for arterial thrombosis in the young, but more data on this and other homozygous deficiencies are warranted to establish any relationship with arterial disease. When a symptomatic patient with inherited thrombophilia due to a known genetic defect is identified, family studies should be conducted since approximately half the first-degree relatives will be affected. Asymptomatic individuals who carry the genetic defect should be counselled on the implica- tions of the diagnosis and on symptoms that require medical attention. In general, management of symp- tomatic individuals with the genetic defect is similar to that for symptomatic patients without an identifi- able genetic defect. An exception is provided by pa- tients with neonatal purpura fulminans in association with homozygous or doubly heterozygous protein C deficiency, for whom administration of a source of protein C is critical during initial treatment. Since future thrombotic events in patients with inherited thrombophilia cannot be accurately predicted and there is a risk of bleeding associated with anticoagu- lant therapy, recommendations on long-term treat- ment are best carried out individually at the present time. Once an individual is defined as having heredi- tary thrombophilia, as many family members as pos- sible need to be examined for the particular defect detected in the proband and a pedigree constructed. Family members who are affected should be coun- selled about the risk of thrombosis. Evaluation of the potential risk of giving birth to severely affected neonates is usually carried out for families between which intermarriage is practised, and consequently counselling, extensive carrier detection, and prenatal diagnosis are carried out. Targets for antenatal diagnosis of hereditary thrombophilias are those WHO Bulletin OMS. Vol 75 1997 185 Memorandum families into which infants have seen born with severe thrombosis caused by homozygosity or com- pound heterozygosity for protein C, protein S, or antithrombin deficiency, as well as the consan- guineous families mentioned above. For such target families attempts need to be made to detect the responsible mutation(s), and to devise a simple method for their detection, e.g., polymerase chain reaction and restriction analysis or Southern analy- sis. This is followed by an extensive search of family members of child-bearing age for carriers, who are then counselled. Antenatal diagnosis is based on analysis of DNA obtained by chorionic villus sam- pling or amniocentesis. Deep vein thrombosis and pulmonary embolism have a lower incidence in developing than in devel- oped countries, possibly because of a combination of racial and environmental factors. A few studies with complete laboratory evaluation on inherited thrombophilia have, however, been carried out in developing countries; the results indicate that there is a higher chance of finding an underlying gene- tic defect (protein C, protein S, and antithrombin deficiency) in patients with thrombosis in such countries. Preliminary data on APC-R suggest that this defect is rare among Asians, Africans, and Chinese. Conclusions and recommendations * Recent reports support the hypothesis that famil- ial thrombophilia is a multiple gene disorder and that its penetrance is higher among carriers of multiple gene defects. Prophylactic and therapeutic measures therefore need to be adjusted to the number of inde- pendent risk factors present in an individual. Hence, efforts should be intensified towards identifying those genetic risk factors that so far have escaped detection, so that they can be included in diagnostic screening procedures. * Guidelines need to be developed for the use of specific laboratory tests in screening procedures aim- ing at identifying individuals who carry a genetic risk factor for venous thrombosis. Collaborative interna- tional investigations with standardized recruitment protocols should be encouraged. * More specific recommendations need to be devel- oped for the classification of hereditary protein S deficiency. In this respect, it needs to be established whether type III protein S deficiency is an independ- ent risk factor for venous thrombosis or a different phenotype of type I protein S deficiency. * At present, general screening of the population for the protein C, protein S, and antithrombin genetic defects is not recommended because of their low prevalence in populations and because of the low predictive value of a positive test. Studies should be carried out to determine whether general screening for APC-R (factor V:506Gln allele), e.g. before surgery or hospitalization and use of oral contra- ceptives, is beneficial for decision-making on therapeutic and prophylactic regimens. * Heterozygous protein C, protein S, or anti- thrombin deficiencies should not be the target of antenatal diagnosis. In view of the very low expected frequency of severe cases of such homozygous or compound heterozygous defects in the general popu- lation, it is not recommended that population screen- ing for carriers be carried out. * Recommendations for screening for hereditary thrombophilia at the national level in health care services can be made only after adequate data on the epidemiology, risk of thrombosis, and results of therapeutic intervention are available. * In developing countries, where thrombotic disor- ders in general appear to have a low prevalence, all patients with venous thromboembolism should be screened in order to determine the proportion with hereditary thrombophilia. Family studies should be carried out on all patients for whom a genetic defect is documented. * Since facilities for screening may be available only in reference centres, treatment should be initiated without delay, where appropriate, and tests per- formed in the reference centre after use of antico- agulants has been discontinued. * Accurate data are needed on the frequency and impact of thrombophilia in developing countries. For this purpose, individual laboratories should be identified in different regions and these should de- velop the necessary expertise to screen for and docu- ment the genetic defect responsible, in association with WHO Collaborating Centres. * Data on the thrombotic risk in patients and asymptomatic family members with thrombophilia should be collected to determine whether the risk profile is different in developed and developing countries. Careful documentation of the risk of haemorrhage when anticoagulants are taken is necessary to determine the risk: benefit ratio for therapeutic intervention involving patients with thrombophilia. * In order to increase awareness about inherited thrombophilia in developed and developing WHO Bulletin OMS. Vol 75 1997186 Inherited thrombophilia countries, a WHO Collaborating Centre should be designated to improve diagnosis, clinical recognition, and treatment of related thrombosis. Such a centre should serve as a reference centre for an appropriate WHO region and improve education of both health professionals and the general public. Acknowledgements The participants at the meeting express their sincere thanks to the following individuals for their support and ex- pertise: Dr V. Kalinine, Institut fur Molekularbiologische Diagnostik, Hamburg, Germany; Dr P. de Moerloose, Divi- sion of Angiology and Haemostasis, University Cantonal Hospital, Geneva, Switzerland; and, Dr F. Peyvandi, Narmak, Tehran, Islamic Republic of Iran. Resume La thrombophilie h6reditaire A l'occasion d'une reunion conjointe qui s'est tenue a Geneve du 6 au 8 novembre 1995, I'OMS et la Soci6te internationale de thrombose et d'hmo- stase (ISTH) ont examine les problemes poses par la thrombophilie h6reditaire, fait 6tat des diverses possibilites de lutte contre la maladie, et formul6 une serie de recommandations pour le diagnostic, le traitement et la recherche concernant cette affec- tion. L'hypothese que la thrombophilie familiale est une maladie multigenique et que sa penetrance est plus grande chez les porteurs d'anomalies multigeniques est confortee par des observations recentes. On peut donc s'attendre a ce que les mesures prophylactiques et therapeutiques re- fletent par n6cessit6 le nombre de facteurs de risque independants presents chez un individu donne. 11 est par suite souhaitable de faire porter les efforts sur l'identification des facteurs de risque genetiques qui n'ont pas encore ete identifies, de maniere a pouvoir en tenir compte dans la d6mar- che diagnostique. La predisposition a la thrombose ne s'explique pas chez 85% des familles atteintes. Ce vide ex- plicatif a favorise l'utilisation des methodes gen6- tiques et 6pidemiologiques et il en est r6sulte des decouvertes majeures dans l'tude de la thrombose familiale au cours des trois dernieres annees. C'est tout d'abord la resistance a la proteine C activ6e (APC-R) qui a ete identifiee, puis une mutation du gbne du facteur V (1 691G-A dans l'exon 10, con- duisant au remplacement 506Arg-*Gln) consid6r6e comme la base mol6culaire du phenotype APC-R chez la plupart des individus atteints. Cette muta- tion, identifiee a une augmentation considerable du risque thrombotique, est pr6sente chez 50% des families selectionnees atteintes de thrombophilie, et chez 20% des cas consecutifs de thrombose. Cette d6couverte a eu pour consequence de modi- fier la perception de la thrombophilie, et donc des repercussions sur son diagnostic et son traitement. 11 serait bon de recueillir des donnees sur le risque thrombotique, a la fois chez des patients et chez les membres asymptomatiques des familles atteintes, pour determiner si le profil de risque est different dans les pays developpes et en developpement. 11 est necessaire de documenter soigneusement le risque d'hemorragie chez les sujets sous anticoagulants pour 6valuer le rapport risque/avantage de l'intervention therapeutique en cas de thrombophilie. 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