M. Martinuzzo

A prospective study of antibodies to β2‐glycoprotein I and prothrombin, and risk of thrombosis

Summary.  Antiphospholipid syndrome (APS) is a clinical autoimmune disorder characterized by thrombosis/pregnancy morbidity associated with the persistence of lupus anticoagulant (LA) and/or anticardiolipin (aCL) antibodies. We assessed the contribution of antibodies to β2‐glycoprotein I (anti‐β2GPI) and prothrombin (anti‐PT) to the thrombotic risk in a cohort of 194 consecutive patients with persistent LA and/or aCL. Median follow‐up was 45 months. A total of 39 patients (20.1%) had one documented episode of thrombosis during follow‐up. Eleven of these patients had no previous thrombosis before enrollment in the study and 28 had recurrences of thrombosis. There were 21 venous and 18 arterial thrombotic events and the overall incidence of thrombosis was 5.6% per patient‐year. After multivariate analysis, the male sex (P = 0.025), a previous thrombosis (P < 0.01), the presence of anti‐β2GPI (P = 0.001), and the presence of anti‐PT (P = 0.03) remained as independent risk factors for recurrent thrombosis. Only IgG anti‐β2GPI and anti‐PT were associated with an increased risk of thrombosis (P < 0.01 and P = 0.005). Patients testing positive for anti‐β2GPI had a higher rate of thrombosis than did antiphospholipid patients without anti‐β2GPI (8.0% vs. 3.1% per patient‐year). Similarly, a higher rate of thrombosis was found in patients with positive anti‐PT compared with patients without anti‐PT (8.6% vs. 3.5% per patient‐year). Considering only the group of 142 LA positive patients, the highest incidence of thrombosis was found in LA patients positive for both anti‐β2GPI and anti‐PT (8.4% per patient‐year). In conclusion, the presence of IgG anti‐β2GPI and anti‐PT in patients with LA and/or aCL and mainly in those with LA predicts a higher risk of thromboembolic events.

Circulating levels of tissue factor and proinflammatory cytokines in patients with primary antiphospholipid syndrome or leprosy related antiphospholipid antibodies

The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) in patients with thromboembolic complications. In APS, most aPL are autoantibodies to β2-glycoprotein I and prothrombin, which play a major role in the APS pathogenesis. Nevertheless, antibodies with the same antigen specificity are also found in aPL patients with leprosy, in whom thromboembolic complications are uncommon. The in vivo upregulation of the tissue factor (TF) pathway and the imbalance of cytokines have been proposed as potential mechanisms of thrombosis in the APS. We measured the circulating levels of TF, interleukin 6 (IL-6), IL-6 receptor (sIL-6R), tumor necrosis factor (TNF-a) and interferon g (IFN-g) in 83 patients with autoimmune aPL (42 with and 41 without clinical features of definite primary APS), 48 leprosy patients (33 with aPL) and 48 normal controls. There was a trend (P = 0.06) to higher median sTF in patients with autoimmune aPL (139 pg/mL) compared with leprosy patients (103.5 pg/mL) and controls (123 pg/mL). In addition, the frequency of raised sTF levels (.187 pg/mL) was significantly higher in the group with autoimmune aPL [22.9% (APS 21.4%, non-APS 24.4%)] but not in leprosy (10.4%) compared with controls (4.2%). Elevated levels of IL-6 and TNF-a and a trend to lower IFN-g were found in patients with definite APS. Leprosy patients with aPL, however, had increased TNF-a and IFN-g but normal IL-6 levels. Levels of sIL-6R did not differ between controls and either patients with autoimmune aPL or leprosy. The different cytokine profiles as well as differences in circulating levels of TF might contribute to the high thrombotic risk found in patients with autoimmune aPL but not in leprosy related aPL patients.

Autoimmune antiphospholipid antibodies impair the inhibition of activated factor X by protein Z/protein Z‐dependent protease inhibitor

Summary.  The hemostatic process is tightly regulated by several antithrombotic mechanisms. Among them, protein Z (PZ)‐dependent protease inhibitor (ZPI) potently inhibits factor (F)Xa in a manner dependent on calcium ions, phospholipids and PZ. Autoimmune antiphospholipid antibodies (aPL) are mainly directed against phospholipid‐binding plasma proteins such as β2‐glycoprotein I (β2GPI) and prothrombin, and are known to interfere with phospholipid‐dependent hemostatic pathways. In this study, we investigated whether purified aPL are able to interfere with inhibition of FXa by PZ/ZPI. β2GPI modestly delayed the FXa inactivation by PZ/ZPI and most isolated aPL‐IgGs were found to further increase the inhibitory potential of β2GPI on PZ/ZPI activity. Without β2GPI, the PZ/ZPI activity was unaffected by the addition of aPL‐IgG. As PZ deficiency is hypothesized to lead to a prothrombotic state, we performed a case‐control study to measure plasma levels of PZ and ZPI in 66 patients with autoimmune aPL and 152 normal controls. The prevalence of low PZ levels (below the 5th percentile of controls) was significantly greater in the 37 patients with definite antiphospholipid syndrome (APS) (24.3%) but not in the 29 aPL patients not fulfilling the criteria for APS (10.3%) compared with the normal group (4.6%, P < 0.001 vs. APS). ZPI antigen levels were similar in patients with aPL and normal controls. Concomitant PZ deficiency increased by approximately sevenfold the risk of arterial thrombosis in aPL patients. Taken together, these data suggest that the PZ/ZPI system is commonly impaired in aPL patients thus probably increasing the thrombotic risk.

High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome

Summary.  As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant TFPI (anti‐TFPI) using an optimized ELISA. Anti‐TFPI (>18 U mL−1 for IgG and/or > 15 U mL−1 for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P < 0.0001). Among aPL patients, the prevalence of positive anti‐TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL−1) anti‐TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P= 0.017). Most aPL recognized full‐length TFPI, but not a truncated form of TFPI lacking the C‐terminus of the molecule. Isolated IgGs from subjects with anti‐TFPI impaired the dose‐dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti‐TFPI limit TFPI action and are associated with the APS.

High prevalence of antiphospholipid antibodies in leprosy: evaluation of antigen reactivity.

Antiphospholipid antibodies (aPL) have been reported not only in autoimmune disorders but also in various infectious diseases. Accumulating evidence indicates that b2 glycoprotein I (b2GPI) and prothrombin are the main proteins to which autoimmune aPL bind. The aim of this study was to evaluate the prevalence of different aPL in patients with leprosy. We included 51 outpatients (42 lepromatous and 9 borderline leprosy) without any clinical feature of the antiphospholipid syndrome (APS). 35 had lupus anticoagulant and 31 had anticardiolipin antibodies (aCL). Anti-β2GPI antibodies were highly positive in 29=51 and anti-prothrombin antibodies (anti-II) were detected in 23=51. Almost all aCL and anti-b2GPI were of IgM isotype, while IgG isotype was more frequent among anti-II. No statistical difference was found when aPL were evaluated in patients grouped according to their bacteriological status. Furthermore, patients under treatment (n ‘ 33) had a similar frequency of positive aPL compared to patients in vigilance (n ‘ 14). Assessing the specificity of antibody binding to CL and b2GPI in ELISA by means of inhibition studies with cardiolipin-b2GPI liposomes, leprosy and APS sera showed a similar behaviour. Comparable results were also found in both groups of patients when inhibition experiments with lysate of Mycobacterium leprae were carried out. In summary, leprosy-related aPL resemble those found in patients with APS but the immunoglobulin isotype is different, with IgM much more prevalent in leprosy patients.

Major and Potential Prothrombotic Genotypes in a Cohort of Patients With Venous Thromboembolism

Factor V Leiden (FVL) and the prothrombin 20210A (PT-20210A) variant are well-known risk factors for venous thromboembolism (VT). The thermolabile variant (TT) of the methylenetetrahydrofolate reductase (MTHFR) gene, and homozygosity for the 4G allele of the promoter region of the plasminogen activator inhibitor-1 (PAI-1) are potential genetic polymorphisms that have not been consistently associated with increased risk of VT. A case-control study was performed on 192 consecutive unrelated patients referred for evaluation of thrombophilia because of VT and 200 healthy controls. FVL was found in 10.4% of patients compared to 3.0% of controls, while 6.3% of patients were carriers of the PT-20210A allele compared to 2.0% of controls. The adjusted odds ratios (OR) were 5.92 and 4.03 for FVL (P=.001) and the PT-20210A (P=.033), respectively. The prevalence of homozygotes for MTHFR (TT) and PAI-1 (4G/4G) among patients and controls were 13.7% versus 13.0% and 21.6% versus 23.5%, respectively (P=ns). A total of 121 patients underwent a complete screening for FVL, the PT-20210A, protein C (PC), protein S (PS), antithrombin III (ATIII), levels of factor VIII, and antiphospholipid antibodies (aPL). In 59 patients (48.8%) at least one defect was found, being a single defect in 55 and combined defects in 4 patients. Plasma levels of homocysteine (Hcy) were measured in 138 patients and 144 controls. Subjects from both groups carrying the MTHFR-TT variant had higher Hcy levels than those with the normal genotype. Hyperhomocysteinemia (HHcy) by itself is a risk factor for VT (OR 4.92, P<.0001). We conclude that FVL and the PT-20210A are risk factors for VT as well as Hcy levels, but the MTHFR and PAI-1 polymorphisms do not appear to be associated with VT in our country.

Antiplatelet Factor 4—Heparin Antibodies in Patients with Antiphospholipid Antibodies

Antibodies directed against platelet factor 4-heparin are present in patients with heparin-induced thrombocytopenia (HIT). Additionally, it has been suggested that heparin can be an antigenic target of antiphospholipid antibodies (aPL). We investigated the presence of heparin-platelet factor 4-induced antibodies (HPIA) in 33 patients with aPL. There were 30 patients with lupus anticoagulant, 25 with anticardiolipin antibodies, 21 with anti-beta2 glycoprotein I, and 18 with antiprothrombin antibodies. 20 patients had a history of thrombosis and 19 had received heparin during the last 60 months. We found 7 (21.2%) who had HPIA; 5 of them also had anti-beta2 glycoprotein I antibodies. Four patients had severe thrombocytopenia and suspicion of HIT. Among them, two presented high positive HPIA results, one of them with positive platelet aggregation test. The third patient showed grey zone HPIA and borderline aggregation test and the fourth one had negative results. Among patients without a history of HIT, 2 who had never received heparin presented high positive, one a moderate positive, and one a grey zone HPIA result; all of them with negative aggregation tests. Five positive sera samples were incubated with cardiolipin liposomes in the presence of beta2 glycoprotein I, and whereas an inhibition greater than 50% was achieved in anticardiolipin and anti-beta2 glycoprotein I activities, HPIA results did not change. We demonstrate that HPIA could be frequently found in patients with aPL. They are responsible for HIT in some cases but can also be found in patients who have not received heparin. Whether they predispose patients with aPL to HIT is not known; nevertheless, a close follow-up of heparin treatment in these patients seems to be mandatory.

Increased lipid peroxidation correlates with platelet activation but not with markers of endothelial cell and blood coagulation activation in patients with antiphospholipid antibodies

Recent studies have shown that patients with antiphospholipid antibodies (aPL) have increased lipid peroxidation. We evaluated the urinary excretion of 11‐dehydro thromboxane B2 (11‐DH‐TXB2) and isoprostane F2αIII (IPF2αIII), reflecting platelet activation and lipid peroxidation in vivo, and plasma soluble markers of endothelial cell, platelet and blood coagulation activation: soluble vascular cell adhesion molecule‐1 (sVCAM‐1), P‐ and E‐selectin (sPsel and sEsel), F1 + 2 fragment of prothrombin (F1 + 2), thrombin–antithrombin complexes (TAT) and D‐Dimer (DD). We studied 79 patients with aPL (47 with previous thrombosis), 45 healthy volunteers (normal controls, NC), 12 patients with systemic lupus erythematosus (SLE) without aPL and a thrombosis control group (TCG) without thrombophilia (n = 16). Urinary levels (mean, range) of eicosanoids and isoeicosanoids were significantly increased in 39 patients with aPL compared with 25 NC, 11‐DH‐TXB2 164·0 ng/mmol creatinine (9·5–1162·8) versus 43·4 ng/mmol creatinine (4·2–87·6), P < 0·001; IPF2αIII 56·9 pg/mg creatinine (5·5–388·7) versus 27·0 pg/mg creatinine (4·6–87·6), P = 0·03. Both metabolites were significantly correlated (ρ= 0·49, P = 0·014), but none correlated with any clinical manifestation or antibody profile. The aPL group presented increased levels of sPsel, sEsel, sVCAM‐1, TAT, F1 + 2 and DD, but any soluble marker correlated with IPF2αIII. Urinary 11‐DH‐TXB2 correlated with sPsel (ρ= 0·39, P = 0·04). Compared with SLE controls, the SLE group with aPL had higher levels of F1 + 2. Plasma levels of F1 + 2 and DD were significantly increased and a trend to higher sPsel was found in aPL patients with thrombosis compared with the TCG. Platelet activation, lipid peroxidation and blood coagulation activation seem to be important in the pathophysiology of antiphospholipid syndrome.

Antiphospholipid antibodies and antibodies to tissue factor pathway inhibitor in women with implantation failures or early and late pregnancy losses

The association of antiphospholipid antibodies (aPL) with early recurrent spontaneous abortions and fetal deaths is well established [1,2]. A role for aPL as a possible cause of failure to achieve pregnancy after in vitro fertilization (IVF), however, is controversial. An association between the hypercoagulable state because of the presence of aPL and unsuccessful embryo implantation has been observed in some [3], but not in other studies [4–6]. Pregnancy complications constitute one of the two major clinical criteria of the antiphospholipid syndrome (APS) [7] and adverse pregnancy outcomes may result from poor placental perfusion because of localized thrombosis. Recently, we found that high titers of antibodies to tissue factor pathway inhibitor (TFPI) (anti-TFPI) in women with autoimmune aPL seem to increase the risk of aPL-related reproductive failures [8]. TFPI is aKunitz-type protease inhibitor that tightly regulates tissue factor-mediated coagulation. During normal pregnancy, some of the hemostatic changes occurring in the placenta are characterized by increased tissue factor expression and low expression of TFPI. Both tissue factor and TFPI seem to be essential for the maintenance of hemostasis in the placenta [9]. The objective of the present study was to evaluate the presence of aPL [lupus anticoagulant (LA) andmoderate or high titers of anticardiolipin antibodies (aCL)] and anti-TFPI in women with a history of pregnancy loss, as well as in women with recurrent IVF failures. The study included patients referred to our institution in Argentina for evaluation of aPL because of a history of two or more consecutive spontaneous abortions (early pregnancy loss before 10 weeks of gestation), at least one fetal death (late pregnancy loss at or beyond 10 weeks of gestation) or recurrent (two ormore) IVF failures despite good visual quality embryos. Patients were referred after excluding other common etiologies of pregnancy failures (infections and hormonal, metabolic, uterine anatomic or genetic abnormalities). There were 243 women (median age 32 years old, range 21–37) with early and/ or late pregnancy losses (98 with early, 116 with late, and 29 with both), and 48 (median age 33 years old, range 23–39) with IVF failures. Themedian number of pregnancy losses was three (range 2–7) in the early group and two (range 1–5) in the late group, and the median number of IVF procedures was three (range 2–6) in the IVF failure group. A group of 80 normal control women (median age 35 years old, range 25–42) was also evaluated. They had had only successful pregnancies. None of the patients or control women had a history of thrombosis or systemic lupus erythematosus. In all cases, blood collection took place at least 3 months after pregnancy complications and if aPL were positive a second blood sample was taken 3 or 6 months later. LA activity was identified through at least three different screening tests, mixing studies and confirmatory procedures according to the ISTHguidelines. aCL (IgG and IgM isotypes) were measured by using a standardized in-house ELISA and titers above 20 GPL or MPL units were considered to be a positive result. The in-house ELISAs for IgG and IgM antibodies to b2 glycoprotein I (antib2GPI) and to prothrombin (anti-PT) were performed as previously reported using electron beam(100 kGy) and c-irradiated microtiter plates, respectively. The cut-off values (10 arbitrary units for IgG or IgM) were previously determined as the 99th percentiles of 95 normal sera [10]. Antibodies to TFPI (IgG and IgM isotypes) were detected in patients sera as recently described using recombinant full-length TFPI coated on c-irradiated microtiter plates [8]. Results were expressed as U mL, referred to internal standards arbitrarily fixed at 100 U mL. The 99th percentiles in 70 healthy controls were previously chosen as the cut-off points (18 U mL and 15 U mL for IgG and IgM anti-TFPI, respectively). Of the 80 control women, positive aCL was found in only one subject (1.2%). Similarly, only two of 48 (4.2%) patients with IVF failures had aCL. None of them had either LA or anti-b2GPI and/or anti-PT. On the other hand, 43 of the 243 (17.7%) women with pregnancy loss had persistent classical aPL. LAwas positive in 32; aCL in 37 and 34 women had antib2GPI and/or anti-PT. All women with positive anti-b2GPI and/or anti-PT had LA and/or aCL. The prevalence of aPL Correspondence: Ricardo R. Forastiero, Hematologia, Universidad Favaloro, Solis 453, (C1078AAI) Buenos Aires, Argentina. Tel.: +54 11 4378 1145; fax: +54 11 4378 1311; e-mail: forastiero@favaloro.edu.ar

Thrombophilia in Human Immunodeficiency Virus—Infected Patients with Osteonecrosis: Is There a Real Connection? The First Case-Control Study

Several reports have described an increased incidence of osteonecrosis in human immunodeficiency virus—infected patients (HIV+), but the cause has not been established. The association between thrombophilia and osteonecrosis in HIV+ was studied. A case-control study in HIV+, 19 cases and 38 controls, was designed. Magnetic resonance imaging was made in both groups to confirm or exclude hip osteonecrosis. The extensive tests of thrombophilia were measured, and the clinical data were recorded, nadir of CD4+ cell count and well-known risk factors for osteonecrosis. Thrombophilia has been frequently found both in patients with and without osteonecrosis (thrombophilia, 68.4% vs 60.5%), but no specific thrombophilia tests were significantly associated with osteonecrosis. A low nadir of CD4+ (<60 cells/μL) and corticoid use were significantly (P < .05) associated with osteonecrosis. In multivariate analysis, only nadir of CD4+ <60 cells/μL remained a predictor of osteonecrosis (odds ratio = 7.33; 95% confidence interval, 1.80-29.82, P = .005). Thrombophilia might have a limited role in the development of osteonecrosis in HIV+. Nadir of CD4+ <60 cells/μL and corticoid use were main factors.