Handbook of Clinical Neurology, Vol. 120 (3rd series) Neurologic Aspects of Systemic Disease Part II Jose Biller and Jose M. Ferro, Editors © 2014 Elsevier B.V. All rights reserved

Chapter 71

Thrombophilic states IDA MARTINELLI*, SERENA MARIA PASSAMONTI, AND PAOLO BUCCIARELLI A. Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine and Medical Specialties, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, University of Milan, Milan, Italy

THROMBOPHILIC STATES Definition Thrombophilia is defined as a hypercoagulable state leading to a thrombotic tendency. Thrombophilic abnormalities can be inherited, acquired, or “mixed” (both congenital and acquired) and the risk of thrombosis is different according to each abnormality (Lane et al., 1996) (Table 71.1). Inherited thrombophilias include deficiencies of the natural anticoagulant proteins antithrombin (AT), protein C (PC), and protein S (PS), as well as the gain-of-function mutations in the factor V gene (factor V Leiden (FVL)) and prothrombin gene (PT G20210A). The anticoagulant effect of AT, PC, and PS on various procoagulant factors is shown in Figure 71.1. Acquired thrombophilia is mainly represented by the presence of antiphospholipid antibodies; the most frequently investigated mixed abnormality is mild to moderate hyperhomocysteinemia.

Inherited thrombophilia Deficiencies of the natural anticoagulant proteins AT, PC, and PS are rare but strong risk factors for venous thromboembolism (VTE) and are generally found in young patients with positive family history and frequent recurrences (De Stefano et al., 1994; Martinelli et al., 1998). AT is a glycoprotein synthesized by the liver and is the major inhibitor of the coagulation serine proteases, in particular thrombin and activated factor X (Xa), other than factors IXa, XIa and XIIa; its inhibition is amplified by the presence of heparin and the endogenous sulphated glycosaminoglycans that lie on the endothelial surface. Two functional domains are recognized: the reactive site

domain (which interacts with the serine residue of the protease) and the heparin-binding domain. The antithrombin gene (SERPIN1), located at chromosome 1q 23-25, has been cloned and its entire nucleotide sequence determined; currently, more than 80 different mutations have been identified (Lane and Caso, 1989). AT deficiency has a dominant autosomal transmission and was firstly identified in 1964 in a Norwegian family (Egeberg, 1965). Its prevalence is 1:3000 in the general population and 3–5% in nonselected and selected patients with VTE, respectively (Tait et al., 1994; De Stefano et al., 1996). The risk of VTE in carriers of AT deficiency is 50-fold higher than in noncarriers (Rosendaal, 1999). On the basis of functional and immunologic assays, AT deficiency is classified into types I and II. Type I is due to a wide variety of heterogeneous DNA mutations and is a quantitative defect characterized by reduced functional and antigen AT levels; type II is due to missense mutations (leading to single amino acid substitutions) and is a qualitative defect in which antigenic level are normal with an impaired plasma activity due to the production of a variant protein (Lane and Caso, 1989). Clinically, type I is associated with recurrent juvenile VTE, whereas in type II the risk of thrombosis is closely related to the position of the mutation within the protein. Thus, heterozygous mutations within the heparin binding domain of AT have a relatively low risk of thrombosis compared to those with mutations at or close to the reactive site of the molecule. PC, described for the first time in 1979 (Kisiel, 1979), is a vitamin K-dependent glycoprotein synthesized in the liver in an inactive form. It is activated by thrombin (after its binding to thrombomodulin on endothelial cell surfaces) through the binding with the endothelial cell protein C receptor), a type I transmembrane protein highly expressed on the endothelium of large blood

*Correspondence to: Ida Martinelli, M.D., Ph.D., Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, University of Milan, Via Pace, 9, 20122 Milan, Italy. Tel: þ39-02-55-03-54-68, Fax: þ39-02-55-0344-39, E-mail: [email protected]

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Table 71.1 Prevalence of thrombophilia abnormalities and relative risk of venous thromboembolism

Thrombophilia abnormality

Prevalence in the general population (%)

Prevalence in patients with VTE (%)

Relative risk of VTE

Antithrombin deficiency Protein C deficiency Protein S deficiency Factor V Leiden* Prothrombin G20210A* Antiphospholipid antibodies Hyperhomocysteinemia

0.02–0.17 0.14–0.5 ? 2–10 1–4 1–2 5

1.1 3.2 2.2 20–50 6–18 5–15 10–15

50 15 6–10 7 3–4 11 1.5

VTE, venous thromboembolism. *Heterozygous.

Endothelial cell surface

Chininogen Precallicrein

Callicrein

XII

(VIIa)

TF Xa, IXa

VIIa

XIIa Ca2+

VIIa

XIa

XI

IIa Ca2+

IX

VIIa

VII

IXa

Protein S

VIII

VIIIa

APC

TM+IIa

Protein C

Ca2+ Xa

X

Va

IIa

APC inhibitor

V

Antithrombin

Ca2+

XIIIa

II

IIa

XIII

IIa Fibrinogen

Fibrin monomer

Ca2+

Fibrin clot

Fig. 71.1. Simplified scheme of the coagulation system. “a” means activated factor. On the left side are the procoagulant factors leading to thrombin activation and fibrin production, essential in venous thrombi. On the right side are the anticoagulant mechanisms of antithrombin, protein C, and its cofactor protein S. Dotted lines indicate inhibition. TF, tissue factor; APC, activated protein C.

vessels that enhances protein C activation more than 10fold (Fukudome et al., 1996). Upon activation, activated PC has an important natural anticoagulant activity and, together with its cofactor PS, suppresses thrombin generation by inhibition of coagulation factors Va and VIIIa. PC gene (PROC) lies on 2q13-q14 chromosome; numerous loss-of-function mutations can cause the deficiency. PC deficiency has a dominant autosomal transmission and is very rare in the general population (around 0.2%), while its frequency in nonselected and selected patients with VTE is 3% and 8%, respectively (Tait et al., 1995; De Stefano et al., 1996). The risk of VTE is approximately 15-fold higher in carriers than in noncarriers (Rosendaal, 1999). PC deficiency is defined on the basis of PC plasma level activity and antigen measurements. As for AT deficiency, there are two types of

PC deficiency: type I is a quantitative defect (plasma reduction of antigen and function) and type II is a qualitative defect (reduction of activity with normal plasma antigen) (Griffin et al., 1981). Type I deficiency is the most common PC defect, and it is due to a reduced synthesis or stability of PC. PS is the PC cofactor and derives its name from Seattle, the town where it was discovered in 1984 (Comp and Esmon, 1984). Like PC, it is a vitamin K-dependent glycoprotein of liver synthesis that circulates in a free form (40%) and in a form bound with the acute phase C4bbinding protein (60%), the latter having no anticoagulant activity. Together with PC cofactor activity, free PS can inhibit the prothrombinase and tenase complex independently (Dahlba¨ck, 1991). Its gene (PROS1) lies on chromosome 3q11.2 and more than 130 loss-of-function

THROMBOPHILIC STATES genetic mutations have been described. The deficiency has a dominant autosomal transmission and its frequency in the general population is difficult to establish, particularly because of the absence of laboratory standardized criteria. The prevalence in patients with VTE is similar to that of PC deficiency, ranging from 2% to 7% (De Stefano et al., 1996). The risk of VTE is 6–10-fold higher in carriers than in noncarriers (Rosendaal, 1999). Three types of PS defects have been described: type I is a quantitative deficiency with decreased plasma levels of functional and antigenic total and free PS; type II is a qualitative deficiency with decreased cofactor activity but normal total and free PS levels; type III is a quantitative deficiency with reduced functional activity and free PS antigen levels and normal total PS levels. The two most common genetic risk factors for VTE are FVL (a G to A substitution at position 1691 in exon 10 of the factor V gene) and PT G20210A (a G to A substitution in the 30 -untranslated region of the prothrombin (factor II) gene). In 1993 a poor anticoagulant response to activated PC was associated with an increased risk of VTE (Dahlba¨ck et al., 1993; Koster et al., 1993). The so-called activated PC resistance is mainly caused by the G1691A mutation (chromosome 1q23) in the Arg506 cleavage site of FVa, described first in Leiden in 1994 (Bertina et al., 1994; Voorberg et al., 1994). FVL is inactivated by activated PC more slowly than wild type factor Va, thus promoting a hypercoagulable state and an increased susceptibility to VTE. The abnormality has a dominant autosomal transmission and, in its heterozygous form, is the most common prothrombotic mutation in the Caucasian population, with a prevalence of about 3%, ranging from 2% to 10% from Southern to Northern Europe (Rees et al., 1995) that rises to 20% and 50% in nonselected and selected patients with VTE, respectively. FVL is very rare in Africans and in Asian populations. The risk of VTE is increased approximately sevenfold in heterozygous and 80-fold in homozygous carriers compared to noncarriers (Rosendaal et al., 1995; Rosendaal, 1999). PT G20210A was discovered in 1996 by the Leiden group, and is associated with an approximately 20% increase of prothrombin plasma levels (Poort et al., 1996). As for FVL, this mutation has a dominant autosomal transmission and it is the second most common coagulation abnormality, with a prevalence of the heterozygous form in populations of Caucasian descent ranging from 1% to 4% from Northern to Southern Europe. PT G20210A is found in 6% of unselected and 18% of selected patients with VTE (Rosendaal et al., 1998). The risk of VTE is three- to fourfold higher in heterozygous carriers than in noncarriers. Homozygous PT G20210A is extremely rare in the general population and therefore the risk of VTE associated with this abnormality remains unknown.

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Acquired thrombophilia Antiphospholipid antibodies are a strong acquired risk factor for thrombosis (Ginsburg et al., 1992; Runchey et al., 2002; De Groot et al., 2005; Naess et al., 2006). The corresponding syndrome is characterized by the presence of circulating antiphospholipid antibodies in plasma and either arterial and/or venous thrombosis, or pregnancy complications, particularly fetal loss (Wilson et al., 1999; Miyakis et al., 2006). The clinically relevant antiphospolipid antibodies include lupus anticoagulant, anticardiolipin, and anti-b2-glycoprotein I antibodies. The term “antiphospholipid antibodies” derives from the previous opinion that they acted against negatively charged endothelial and cellular membrane phospholipids. Now we know that these autoantibodies are directed towards a wide variety of protein cofactors that exert their role on phospholipid membrane surfaces (b2-glycoprotein I, prothrombin, PC, PS, annexin V, coagulation factor XII, and others) (Galli et al., 2003). The resulting complexes interact with several cell types, including endothelial cells, monocytes and platelets, all of which play an important role in hemostasis and thrombogenesis. The indirect activation of these cells results in the release of prothrombotic and proinflammatory mediators (e.g., tissue factor-bearing microparticles, interleukin6, proteins of the complement system), leading to the activation of platelet and coagulation pathways (Urbanus et al., 2008). Recently, it has been shown that antiphospholipid antibodies interact directly with the vessel wall and cause alterations of plasma lipoprotein (i.e., high-density lipoprotein) function increasing the atherotrombotic risk (Charakida et al., 2009). The antiphospholipid antibodies can be isolated, or drug-related, or associated with autoimmune (e.g., systemic lupus erythematosus or rheumatoid arthritis), lynphoproliferative or inflammatory diseases (Harris et al., 1985). The prevalence of thrombosis in the presence of antiphospholipid antibodies is around 15% in the general population, with an incidence of 7.5 patientyears (Galli and Barbui, 2003). The relative risk of thrombosis varies depending on the type of antibody and is higher in the presence of lupus anticoagulant with or without anticardiolipin antibodies (OR 11.0 (95%CI 3.81–32.3)) and lower if only anticardiolipin antibodies are present at a clinically significant titration (>40 IU) (OR 3.21 (95%CI 1.11–9.28)), or at a titer< 40 IU (OR 1.56 (95%CI 1.10–2.24)) (Wahl et al., 1998).

Mixed thrombophilia Mild to moderate hyperhomocysteinemia (HHcy) is a weak risk factor for both venous and arterial thrombosis (Falcon et al., 1994; den Heijer et al., 1996; Ortel, 1999). Homocysteine is an intermediate degradation product in

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the metabolic pathway in which the aminoacid methionine is converted into cysteine. An impairment of this metabolic pathway leads to increased plasma homocysteine levels. The possible mechanisms by which HHcy promotes thrombosis are different and still under investigation; they include a toxic effect on endothelial cells, smooth muscle cell proliferation and intimal thickening, impaired generation of nitric oxide and prostacyclin, increased platelet adhesion, activation of factor V, interference with PC activation and thrombomodulin expression, induction of tissue factor activity and inhibition of tissue-type plasminogen activator (D’Angelo and Selhub, 1997). Both genetic (e.g., mutations in methylenetetrahydrofolate reductase (MTHFR) and cystathionine-b-synthase genes) and acquired factors (e.g., deficiencies of folate, vitamin B12 and vitamin B6, renal impairment, the use of antifolic drugs, and older age) interact to determine plasma homocysteine concentrations. Hence, HHcy is considered a “mixed” risk factor for thrombosis (Cattaneo, 1999).

Lipoprotein(a) Since the homology between apoliproprotein(a) (apo(a)) and plasminogen was described in 1987, the role of lipoprotein(a) (Lp(a)) as an inhibitor of normal fibrinolytic activity of plasminogen was hypothesized. It has recently been reported that the inhibition of plasminogen activation by apo(a) results from the interaction of apo(a) with the ternary complex that includes tissue-type plasminogen activator, plasminogen and fibrin, rather than competition of apo(a) and plasminogen for binding site of fibrin, and that Lp(a) types containing smaller apo(a) isoforms bind more avidly to fibrin and are better inhibitors of plasminogen activation (Angle´s-Cano et al., 2001). Recent clinical studies showed that Lp(a), either alone or in synergy with other thrombophilic risk factors (Nowak-G€ ottl et al., 2008), significantly increases the risk of arterial (Danesh et al., 2000; Smolders et al., 2007) and venous thrombosis, the latter particularly in childhood (Nowak-G€ ottl et al., 1999). Individuals with smaller apo (a) isoforms have an approximately twofold higher risk of coronary heart disease or ischemic stroke than those with larger proteins (Erqou et al., 2010). To date, whether or not smaller apo(a) isoforms are independent from Lp (a) concentration and other risk factors is not clarified.

THROMBOPHILIC STATES AND CEREBRAL VENOUS SINUS THROMBOSIS Epidemiology and clinical symptoms The incidence of cerebral venous sinus thrombosis (CVST) is uncertain, because of the absence of epidemiologic studies. At variance with arterial stroke, CVST

affects mainly young adults and children, with an estimated annual incidence of three to four cases per million adults and seven cases per million neonates and children (deVeber et al., 2001; Stam, 2005). Symptoms are varied and related to the involved venous structure. When thrombosis involves the cortical veins, localized edema and parenchymal infarction generally develop (Stam, 2005). Intracranial hemorrhage complicates 14–39% of CVST (Ferro et al., 2004; Wasay et al., 2008). The most common symptoms and signs are headache and papilledema due to intracranial hypertension, seizures, focal neurologic deficits, and altered consciousness. Headache occurs in 90% or more of patients and papilledema (30% of patients) may cause visual loss and, if the sixth cranial nerve is compressed, diplopia. Seizures (focal or generalized) develop in up to 40% of patients, as well as motor deficits, whereas symptoms such as dysarthria and aphasia are uncommon. The onset of symptoms is subacute, developing from 2 days to 1 month in 50– 80% of patients, and can be even longer in the 10–20% of patients with isolated intracranial hypertension. Prognosis of CVST is favorable in more than 80% of cases (Dentali et al., 2006a) and recurrence occurs in only 2.2% of patients (Ferro et al., 2004). Death is mainly caused by cerebral herniation in the acute phase and to underlying illnesses, e.g., cancer, during follow-up.

Inherited thrombophilia Inherited thrombophilia is an established risk factor for CVST (Table 71.2). Due to the rarity of the disease, only few studies with a relatively small sample size are Table 71.2 Prevalence of thrombophilia abnormalities and relative risk of cerebral venous sinus thrombosis*

Thrombophilia abnormality Antithrombin deficiency Protein C deficiency Protein S deficiency Factor V Leiden{ Prothrombin G20210A{ Antiphospholipid antibodies Hyperhomocysteinemia

Prevalence in patients with CVST (%)

Relative risk of CVST

1–7 3–6 3–8 3–12 11–21 4–17

3 11 12 4 9 9{

4–29

4

CVST, cerebral venous sinus thrombosis. *Percentage ranges derive from single studies (Martinelli et al., 2003; Ferro et al., 2004; deVeber et al., 2001; Stam, 2005) and risks from a revision paper (Dentali et al., 2006a). { Heterozygous. { From Christopher et al. (1999).

THROMBOPHILIC STATES available in the literature. The largest study investigating the risk of a first episode of CVST associated with thrombophilia included 121 patients and 242 controls and showed that by far the most common thrombophilia abnormality was PT G20210A, present in 22% of patients and 2% of controls, for an 11-fold increased risk of the disease (Martinelli et al., 2003). These data have been consistently reported in the literature, as well as the strong association of CVST with the use of oral contraceptives. Indeed, CVST affects predominantly women of childbearing age (Stam, 2005) because of the use of oral contraceptives. A meta-analysis of eight casecontrol studies showed a sixfold increased risk of CVST in oral contraceptive users and a ninefold increased risk in heterozygous carriers of PT G20210A (Dentali et al., 2006b). Both PT G20210A and oral contraceptive use are independent risk factors for CVST, and their combined presence increases the risk of CVST in multiplicative terms, up to 79-fold (Martinelli et al., 2003; Gadelha et al., 2005). The same is true for heterozygous FVL, which increases the risk of a first CVST fivefold when present alone and up to 30-fold when associated with oral contraceptive use. The relationship between the risk of CVST and the lack-of-function deficiencies of AT, PC, and PS is less established because of the relatively small number of patients investigated so far and the low prevalence of these coagulation abnormalities in patients and controls. Taken together, AT, PC, and PS deficiencies increase sixfold the risk of a first CVST (Martinelli et al., 2003). When inherited thrombophilia is investigated in relation to the risk of recurrent venous thrombosis after a first CVST, it appears that only “severe” abnormalities (AT, PC, PS deficiencies, antiphopsholipid antibodies, or combined abnormalities) are associated with an increased risk of VTE, mainly deep vein thrombosis of the lower limbs (hazard ratio 4.71 (95%CI 1.34–16.5)), and not of CVST, whereas PT G20210A and FVL are not. Therefore, only patients with severe thrombophilia have an indication for anticoagulant therapy of indefinite duration (Martinelli et al., 2010).

Acquired thrombophilia Antiphopsholipid antibodies are associated with an increased risk of both arterial and venous thrombosis. Among the latter, the association is demonstrated with the most common manifestation by far, namely, deep vein thrombosis of the lower limbs. The magnitude of the risk of CVST associated with antiphospholipid antibodies is not well established due to both the rarity of the disease (leading to studies of small sample sizes) and the rarity of acquired thrombophilia among controls. A case-control study on 31 patients and the same number

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of controls showed an approximately ninefold increased risk of CVST associated with anticardiolipin antibodies, with wide confidence intervals that skimmed statistical significance (Christopher et al., 1999). A larger casecontrol study showed the presence of antiphospholipid antibodies in 4% of patients but in no controls, thus rendering it unfeasible to estimate the relative risk of the disease (Martinelli et al., 2003). As for the more common thrombotic manifestations, venous or arterial, the presence of antiphospholipid antibodies is an indication for the maintenance of anticoagulant therapy.

Mixed thrombophilia The first observation of mild to moderate HHcy as a risk factor for deep vein thrombosis of the lower limbs was done in 1994 (Falcon et al., 1994). Some years later, the same group reported a fourfold (OR 4.2 (95%CI 2.3– 7.6)) increased risk of CVST associated with fasting or postmethionine load homocysteine plasma levels (Martinelli et al., 2003), which was confirmed by other smaller subsequent studies of fewer than 50 patients (Boncoraglio et al., 2004; Cantu et al., 2004; Ventura et al., 2004). The concomitant use of oral contraceptives, as for FVL and PT G20210A, leads to a synergistic interaction with HHcy relating to the risk of CVST, which rises to almost 20-fold (OR 18.3 (95%CI 4.9–68.1)) (Martinelli et al., 2003).

Cerebral venous sinus thrombosis in children In children and neonates the main risk factors for CVST are gestational or perinatal complications (24% of cases), dehydration (25%), head infections (18%), and thrombophilia (32%). Concerning the latter, a recent meta-analysis showed a statistically significant association between CVST and AT (OR 18.4 (95%CI 3.3–104.3)), PC (OR 6.3 (95%CI 1.6–25.4)), PS (OR 5.3 (95%CI 1.5–18.3)) deficiency and FVL (OR 2.7 (95%CI 1.7– 4.3)), and a trend toward an association with PT G20210A (OR 2.0 (95%CI 0.9–4.1)) and combined inherited thrombophilic abnormalities (OR 6.1 (95%CI 0.9– 43.1)) (Kenet et al., 2010). A previous multicenter study by the same authors as the meta-analysis showed a role for thrombophilia in idiopathic CVST and not in CVST associated with comorbid conditions (such as head/neck trauma, systemic illnesses, and infections) (Kenet et al., 2004). It has to be noted that laboratory investigation of inherited thrombophilia based on plasma measurements, such as AT, PC, and PS testing, is difficult to interpret in neonates and children because of the lack of specific normal ranges. Common concomitant liver or kidney diseases or sepsis are often responsible for an acquired deficiency of one or more of these proteins. Whether or not elevated Lp(a), that is associated with an increased

1066 I. MARTINELLI ET AL. risk for VTE in children, is also a risk factor for The possible association between AIS and the two CVST has not yet been properly investigated gain-of-function mutations FVL and PT G20210A has (Young et al., 2008). been addressed by several studies reviewed in metaanalyses. One of these, published as part of the CopenaTHROMBOPHILIC STATES AND ghen City Heart Study, included eight studies for a total ISCHEMIC STROKE of 1270 adult patients with AIS and 2269 healthy controls, and did not find any significant association Inherited thrombophilia between FVL and AIS (OR 1.1 (95%CI 0.7–1.5)) (Juul In the pathogenesis of arterial ischemic stroke (AIS) and et al., 2002). A small but not statistically significant assoof arterial thrombosis in general, a major role is played ciation between AIS and FVL was found in a second by such well-established risk factors as diabetes, high meta-analysis of 15 case-control studies for a total of blood pressure, dyslipidemia, obesity, smoking, and 3039 patients and 12 200 controls (OR 1.3 (95%CI family history. Inherited thrombophilia has long been 0.9–1.9)) (Kim and Becker, 2003). Ten case-control studrecognized as contributing to VTE, but its association ies from the same meta-analysis (1625 patients and 5050 with arterial thrombosis is controversial (de Moerloose controls) also investigated the impact of PT G20210A on and Boehlen, 2007). A few cases of arterial thrombosis, AIS, finding a similar weak association (OR 1.3 (95%CI including cerebral thromboembolic events, associated 0.9–1.9)). Combining AIS and myocardial infarction, with a deficiency of AT (Vomberg et al., 1987; and considering individuals younger than 55 years, the Ueyama et al., 1989; Johnson et al., 1990), PC or PS association was significantly stronger for PT G20210A (Israels and Seshia, 1987; Girolami et al., 1989; Sacco (OR 1.7 (95%CI 1.1–2.5)) than for FVL (OR 1.4 (95%CI et al., 1989; Camerlingo et al., 1991; Green et al., 1992) 1.0–2.0)). A third meta-analysis of 26 case-control studhave been reported, mainly in children, but the associaies for a total of 4588 patients and 13 798 controls (Casas tion between these inherited coagulation abnormalities et al., 2004) found a moderate and statistically signifiand AIS is very uncommon. Few studies have specificant association between FVL and AIS (OR 1.3 (95%CI cally addressed such a relationship in large populations, 1.1–1.6)) which disappeared when one study (mainly and prospective studies have given conflicting results. responsible of the high heterogeneity observed) was An early prospective study (Finazzi and Barbui, 1994) excluded (OR 1.2 (95%CI 1.0–1.4)). In this meta-analysis, with more than 160 patient-years of follow-up found 19 case-control studies (3028 patients and 7131 controls) no cases of AIS among nine patients with AT deficiency, evaluated the contribution of PT G20201A on the risk of 36 patients with PC deficiency, and 36 patients with PS AIS, finding a statistically significant association (OR deficiency. Two other prospective studies found that 1.44 (95%CI 1.11–1.86)) without any significant interlow PC (but not AT) levels had a borderline significant study heterogeneity. After these three meta-analyses, a association with the risk of AIS over a 6–9-year few studies have been published focusing on the effect follow-up, and were also associated with cerebral infarcof the two gain-of-function mutations on the risk of AIS. tions as identified by MRI imaging (Folsom et al., 1999; In a study of 49 young patients with cryptogenic stroke, Knuiman et al., 2001). However, baseline MRI was not Aznar et al. (2004) found a significant association with performed for comparison, and no distinction was made PT G20210A (OR 3.8 (95%CI 1.1–13.3)) but not with FVL between inherited and acquired PC and AT deficiencies. (OR 2.6 (95%CI 0.5–14.0)), perhaps because the study A case-control study of 219 patients with a first episode was underpowered. The same study highlighted the of AIS and 205 healthy controls failed to find an associeffect of inherited thrombophilia in enhancing the risk ation between AIS (all pathogenic subtypes) and AT, PC, of AIS in oral contraceptive users (OR 14.3 (95%CI or PS deficiency (Hankey et al., 2001). A retrospective 0.7–31.0) for carriers of both risk factors). In another cohort family study of 150 families with inherited thromcase-control study (294 patients and 286 controls) carbophilia found that 0% of AT-, 1.6% of PC-, and 4.8% of ried out in a population from Sardinia, an Italian island PS-deficient relatives experienced an AIS, compared to with a well-known elevated genetic homogeneity, no 0.6% of relatives without deficiencies, but the overall association between AIS and either FVL or PT rate of arterial thrombotic events (either AIS or myocarG20210A was observed (Rubattu et al., 2005). dial infarction) was not significantly different between Other coagulation abnormalities or genetic variants in deficient and nondeficient relatives (Martinelli et al., genes coding for proteins involved in primary hemosta1998). Finally, Brouwer et al. (2005) investigated the sis, coagulation, and fibrinolysis have been recently risk of venous and arterial thrombosis in 156 and 268 associated with the risk of AIS, but their role as potential first-degree relatives from type I- and type III-deficient risk factors is still under investigation. These include: PS probands, finding no association with arterial high plasma levels of factor VIII and von Willebrand facthrombosis. tor, dysfibrinogenemia, b-fibrinogen G/A -455 and 448

THROMBOPHILIC STATES gene polymorphisms (Ortel, 1999), platelet glycoprotein Ib-a Thr!Met and plasminogen activator inhibitor 1 (PAI1) 4G/5G polymorphism (Casas et al., 2004), two haplotypes in factor VIII and one haplotype in factor XIIIA1 carrying minor alleles for V35L and P565L variants (Smith et al., 2008), the factor XIIIA1 Tyr204Phe variant (Pruissen et al., 2008). The presence of inherited thrombophilia does not change the normal attitude towards therapy after an episode of AIS. The standard therapy is represented by antiplatelet agents (either aspirin 100 mg per day or clopidogrel 75 mg per day), unless a cardioembolic origin of the stroke is demonstrated (atrial fibrillation or patent foramen ovale with paradoxical embolism), that requires an anticoagulant therapy with vitamin K antagonists.

Acquired thrombophilia A number of studies demonstrated that antiphospholipid antibodies are an independent risk factor for first and possibly recurrent AIS in young adults (Brey, 2005). Among these are one cohort and four case-control studies (Brey et al., 1990; Nencini et al., 1992; Toschi et al., 1998; Blohorn et al., 2002; Brey et al., 2002). All but one showed an increased risk of incident AIS in young people. The negative study tested only anticardiolipin antibodies, whereas the others tested both lupus anticoagulant and anticardiolipin antibodies. Lupus anticoagulant is associated with a higher risk of AIS than isolated anticardiolipin antibodies. In the elderly the contribution of antiphospholipid antibodies to the risk of AIS is less evident, likely because other major systemic risk factors (such as diabetes, hypertension, dyslipidemia) play a major role in the pathogenesis of the disease. The best therapeutic strategy for preventing antiphospholipid antibody-related recurrent stroke in patients with a first episode of AIS is not completely clear. A randomized double-blind controlled trial comparing the risk of recurrent stroke and other thromboembolic diseases over a 2 year period in patients with a first episode of AIS who were randomized to either aspirin therapy (325 mg per day) or warfarin therapy (target INR 2.0) showed that in patients with antiphospholipid antibodies at the time of the first AIS and without atrial fibrillation or high-grade carotid stenosis, aspirin and warfarin therapy (at a mean INR target of 2.0) were equally effective (APASS Investigators, 2004). The WAPS study showed that high-intensity warfarin therapy (INR range 3.0–4.5) was not clearly superior to standard therapy (warfarin with INR range of 2.0–3.0 or aspirin 100 mg per day) in preventing recurrent thrombosis in patients with antiphospholipid antibody syndrome, and was associated with an increased rate of minor hemorrhagic events (Finazzi et al., 2005).

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Mixed thrombophilia HHcy is associated with a mild increased risk of arterial thrombosis (den Heijer et al., 1996; Ortel, 1999). Although the risk factor is represented by HHcy independently of its genetic or acquired origin, the effect of the mutated homozygous MTHFR 677TT polymorphism on AIS was investigated in two meta-analyses (Kim and Becker, 2003; Casas et al., 2004) that showed a mild increased risk (OR 1.2 (95%CI 1.1–1.4)) (Casas et al., 2004). Patients with HHCy can be treated with folic acid, cobalamin, and pyridoxine in order to lower plasma homocysteine levels and thereby potentially decreasing the risk of thrombosis. Previous randomized clinical trials and meta-analyses of these trials on the efficacy of vitamin supplementation either in primary or in secondary prevention of arterial thrombosis gave conflicting results. It is likely that the possible benefit of reducing the smaller effect of HHcy on the risk of arterial thrombosis was masked by the effect of such stronger risk factors as arterial hypertension, diabetes, smoking, and dyslipidemia. A recent meta-analysis showed that folic acid supplementation was not effective in secondary prevention of AIS, but there might be a mild benefit in primary prevention, especially in males and in combination with B vitamins (Lee et al., 2010).

Stroke in children The meta-analysis by Juul et al. (2002) included seven studies on infants and children with AIS (453 patients and 1180 controls), and the pooled analysis demonstrated a fivefold increased risk of AIS in carriers of FVL (OR 4.8 (95%CI 3.3–7.0)). A systematic review of 18 casecontrol studies (3235 patients and 9019 controls) assessed the risk of first AIS associated with thrombophilia in children, finding a pooled OR of 1.2 (95%CI 0.8–1.9) for FVL, 1.1 (95%CI 0.5–2.3) for PT G20210A, 1.0 (95%CI 0.3–3.7) for AT, 6.5 (95%CI 3.0–14.3) for PC, and 1.1 (95%CI 0.3–3.8) for PS deficiency (Haywood et al., 2005). A 5 year prospective followup study of 301 children with AIS and an objectively demonstrated inherited thrombophilia abnormality confirmed the association between the risk of recurrent AIS and PC deficiency (relative risk 3.5 (95%CI 1.1–10.9)), but not AT or PS deficiency, FVL or PT G20210A, and found that also Lp(a) was an independent risk factor for stroke (relative risk 4.4 (95%CI 19–10.5)) (Strater et al., 2002). A recent meta-analysis of 22 observational studies (1526 patients with AIS and 2799 controls) showed a significant association between a first AIS and PC deficiency (OR 11.0 (95%CI 5.1–23.6)), FVL (OR 3.7 (95% CI 2.8–4.9)), PT G20210A (OR 2.6 (95%CI 1.7–4.1)),

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combined genetic defects (OR 18.8 (95%CI 6.5–54.1)), antiphospholipid antibodies (OR 7.0 (95%CI 3.7–13.1)), Lp(a) (OR 6.5 (95%CI 4.5–9.6)), and, to a lesser extent, MTHFR 677 TT (OR 1.6 (95%CI 1.2–2.1)), whereas no statistically significant association was found for AT (OR 3.3 (95%CI 0.7–15.5)) and PS deficiencies (OR 1.5 (95%CI 0.3–6.9)) (Kenet et al., 2010). Concerning neonates, a case-control study of 91 neonates found an association between AIS and FVL (OR 4.0 (95%CI 1.7–9.0)) or Lp(a) (OR 4.8 (95%CI 2.2– 10.9)) (Gunther et al., 2000). A high prevalence of FVL (21%) has also been found in a case series of 24 neonates with cerebral infarction (Mercuri et al., 2001). The follow-up of these patients showed that all five patients with FVL had hemiplegia (100%), compared to only 21% of patients without FVL.

Stroke related to paradoxical embolism Studies investigating the role of thrombophilia in AIS secondary to paradoxical embolism (i.e., in the presence of patent foramen ovale) gave conflicting results. Some of them showed that either FVL or PT G20210A (Karttunen et al., 2003; Pezzini et al., 2003) was significantly and independently associated with the occurrence of AIS in patients with patent foramen ovale. The Young Adult Myocardial Infarction and Ischemic Stroke (YAMIS) study investigated the frequency of venousto-arterial circulation shunts, usually caused by patent foramen ovale, and thrombophilia (AT, PC, and PS deficiency, FVL, PT G20210A, antiphospholipid antibodies) in young adults with AIS and in matched healthy controls (Sastry et al., 2006), finding a higher prevalence of venous-to-arterial shunts in the former but no association between thrombophilia and AIS. Anticoagulant therapy with vitamin K antagonists is the best choice when the cardioembolic origin of AIS is demonstrated.

CONCLUSIONS Thrombophilia abnormalities are well recognized risk factors for CVST, especially in those cases not related to such strong risk factors as tumors in the brain or other sites, cerebral infections, or traumas. In most patients, thrombophilia interacts with environmental risk factors, the most frequent being oral contraceptive use and pregnancy/puerperium, leading to CVST. This explains, at least in part, the higher prevalence of women than men with a first CVST. Thrombophilia seems to be also a risk factor for CVST recurrence, but only in its severe form (AT, PC, PS deficiencies, antiphospholipid antibodies, or combined abnormalities). A complete thrombophilia screening (AT, PC, PS, FVL, PT G20210A, antiphospholipid antibodies, FVIII, homocysteine) should be considered for all patients in which CVST is

not related to cancer, brain infections, or traumas. Testing for thrombophilia is rarely urgent, so careful consideration is required concerning the timing as well as the need for assessment. The role of thrombophilia in the pathogenesis of AIS and, in general, of arterial thrombosis, is less important than its role in VTE, and it is still debated. The two thrombophilic defects recognized as risk factors for both VTE and arterial thrombosis are antiphospholipid antibodies and HHcy, while the risk associated with the two common mutations, FVL and PT G20210A, is of little value, and limited to particular subsets of patients (e.g., young patients with AIS in the absence of classic risk factors, such as arterial hypertension, diabetes, smoking, dyslipidemia, or patients with cryptogenic AIS). Deficiencies of naturally occurring inhibitors AT, PC, and PS do not play a significant role in the etiopathogenesis of AIS. Hence, thrombophilia screening should be reserved for patients with AIS at a young age (i.e.