The inhibition of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies isolated from patients with antiphospholipid syndrome by chromatography methods

Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis; however, the mechanism remains unknown. Recent studies have focused on the impediment of protein C anticoagulant activity by anti-β₂-glycoprotein I (β₂GPI) antibodies (aβ₂GPI Ab). We purified IgG fractions containing a high concentration of aβ₂GPI Ab from patients with antiphospholipid syndrome (APS) and then investigated the effect of purified aβ₂GPI Ab on the activity of activated protein C (APC). Using a three-step chromatography method (DEAE-sepharose column, phosphatidylserine polyacrylamide gel column dependent on the presence of β₂GPI, and protein G column chromatography), we successfully isolated anti-β₂GPI IgG from nine patients with APS. Seven of nine samples inhibited APC activity in a concentration-dependent manner only in the presence of β₂GPI, as observed by a chromogenic assay that was able to determine thrombin activity even in the presence of APC. The extent of APC inhibition by these fractions appeared to be related to aβ₂GPI Ab titers of the purified IgG. However, the inhibitory effect of IgG from patients was not detected in the absence of β₂GPI. IgG purified from three normal subjects did not affect APC activity. Herein, we show a useful method for the isolation of IgG containing a high concentration of aβ₂GPI Ab. Moreover, the present findings indicate that inhibition by aβ₂GPI Ab on APC anticoagulant activity could explain one of the mechanisms for the thrombotic state in APS. Received: April 9, 2001 / Accepted: July 10, 2001     

Interpretation and recommended testing for antiphospholipid antibodies

The antiphospholipid syndrome (APS) is defined by the association of arterial and/or venous thrombosis and/or pregnancy complications with the presence of at least one of the main laboratory-detected antiphospholipid antibodies (aPL) (i.e., lupus anticoagulants [LA], IgG and/or IgM anticardiolipin antibodies [aCL], and IgG and/or IgM anti-β2-glycoprotein I antibodies [aβ2GPI]). During the last decade efforts have been made to improve the harmonization and reproducibility of laboratory detection of aPL and guidelines have been published. The prognostic significance of aPL is being clarified through the fine elucidation of their antigenic targets and pathogenic mechanisms. Several clinical studies have consistently reported that LA is a stronger risk factor for both arterial and venous thrombosis compared with aCL and aβ2GPI. In particular, LA activity dependent on the first domain of β2-glycoprotein I and triple aPL positivity are prognosticators of the thrombotic and obstetric risks. Hopefully, this increasing knowledge will help improve diagnostic and treatment strategies for APS.     

The epidemiology of the antiphospholipid syndrome: who is at risk?

Arterial and venous thrombosis are the most common and clinically relevant events of the so-called antiphospholipid syndrome; they are reported in approximately one third of patients with the antiphospholipid (aPL) antibodies. APL antibodies are part of a wide family of immunoglobulins directed against proteins complexed with negatively charged phospholipids. They include lupus anticoagulants (LA), anticardiolipin (aCL) antibodies, and the most recently recognized anti-beta-2-glycoprotein I (β2-GPI) and antiprothrombin (aPT) antibodies. Previous thrombotic events and the presence of LA, particularly if identified by the dilute Russell viper venom test, appear to be the strongest risk factors for vascular complications. High-titer aCL antibodies have been reported to be associated with an increased thrombotic tendency, but this was not confirmed in all studies. The data only partially support the concept that anti-β2-GPI and aPT antibodies may be considered as independent risk factors for thrombosis. Further prospective studies are required.     

Atherosclerosis in autoimmune diseases

Lipid peroxidation occurs frequently in patients with systemic autoimmune diseases and contributes to autoimmune vascular inflammation. Oxidized low-density lipoprotein (oxLDL) interacts with β2-glycoprotein I (β2GPI), forming oxLDL/β2GPI complexes. Circulating oxLDL/β2GPI complexes and autoantibodies to these complexes have been demonstrated in patients with systemic lupus erythematosus and antiphospholipid syndrome. These findings suggest an immunogenic nature of the complexes and an active proatherogenic role in autoimmunity. Biochemical characterization of the complexes and immunohistochemical studies of atherosclerotic lesions suggest that most of the complexes originate in the arterial wall and are released into circulation. The in vitro macrophage uptake of oxLDL/β2GPI complexes increased significantly in the presence of antiphospholipid antibodies (anti-β2GPI), suggesting that macrophage Fcγ receptors are involved in the lipid intracellular inflthat leads to foam cell formation. These findings provide an immunologic explanation for the accelerated development of atherosclerosis seen in systemic lupus erythematosus and antiphospholipid syndrome.     

Thrombolysis in antiphospholipid syndrome: Current hematologic perspectives

Antiphospholipd antibodies (aPL) can cause thromboembolic events, but the reason for the thrombogenesis has not been fully elucidated. Studies show that the true pathogenic targets of aPL are plasma proteins involved in hemostasis (eg, β₂-glycoprotein I and prothrombin). These plasma proteins in turn bind to phospholipids, leading to the misclassification as “antiphospholipid” antibodies. The hemostatic system has abundant checks and balances to avoid excess hemorrhage and thrombosis. Thus, thrombosis requires more than interrupting one protein in the complex system. This review examines host genetic factors important in predisposition to thrombosis associated with aPL and concentrates on how antibodies in antiphospholipid syndrome interact with our natural anticoagulants and lead to thrombosis.     

Antibodies to Serine Proteases in the Antiphospholipid Syndrome

It is generally accepted that the major autoantigen for antiphospholipid antibodies (aPL) in the antiphospholipid syndrome (APS) is β₂-glycoprotein I (β₂GPI). However, a recent study has revealed that some aPL bind to certain conformational epitope(s) on β₂GPI shared by the homologous enzymatic domains of several serine proteases involved in hemostasis and fibrinolysis. Importantly, some serine protease–reactive aPL correspondingly hinder anticoagulant regulation and resolution of clots. These results extend several early findings of aPL binding to other coagulation factors and provide a new perspective about some aPL in terms of binding specificities and related functional properties in promoting thrombosis. Moreover, a recent immunological and pathological study of a panel of human IgG monoclonal aPL showed that aPL with strong binding to thrombin promote in vivo venous thrombosis and leukocyte adherence, suggesting that aPL reactivity with thrombin may be a good predictor for pathogenic potentials of aPL.     

Anti-beta 2-glycoprotein I antibodies: a useful marker for the antiphospholipid syndrome

We studied anti-beta 2-glycoprotein I antibodies (a beta 2GPI) in autoimmune disease patients to evaluate their relationship to clinical findings. Seventy-nine systemic lupus erythematosus (SLE) patients [44 with antiphospholipid antibodies (aPL)], 21 with primary antiphospholipid syndrome (APS), eight asymptomatic individuals with aPL and 60 controls were studied. Sixteen SLE patients (14 with aPL and two without aPL) and six with primary APS had a beta 2GPI. A significant relationship was found between a beta 2GPI and aPL (P < 0.01). In SLE, a significant correlation was found between previous thrombosis or thrombocytopenia and a beta 2GPI or a beta 2GPI + aPL, but not between fetal losses and a beta 2GPI. These data suggest that a beta 2GPI may be useful in the study of APS.      

Lack of association of beta2-glycoprotein I polymorphisms Val247Leu and Trp316Ser with antiphospholipid antibodies in patients with thrombosis and pregnancy complications

Beta2-glycoprotein I (beta2GPI) is an important target antigen for antiphospholipid antibodies (aPL) and thus beta2GPI polymorphisms may influence aPL production and the development of antiphospholipid syndrome. We have studied the relationship between the Val247Leu and Trp316Ser beta2GPI polymorphisms and the aPL status of 230 patients referred for aPL screening. Sixty-one (26.5%) had persistent aPL [anticardiolipin antibodies (IgG and/or IgM), lupus anticoagulants and/or IgG anti-beta2GPI antibodies]. A comparison of the genotypic and allelic frequencies of these two polymorphisms between the Caucasian patient population and an ethnic-matched normal control group (n = 308) showed no significant differences between aPL-positive patients, aPL-negative patients and the normal control group. This suggests that the Val or Leu allele at position 247 and the Trp or Ser allele at position 316 of beta2GPI do not play a role in the production of aPL. There was a significantly decreased prevalence of the Ser316 allele in aPL-negative women (n = 98) when compared with female normal control subjects (n = 249) [0.020 [95% confidence interval (CI) 0.00-0.04]vs 0.060 (95% CI 0.04-0.08), P = 0.0286]. Subgroup analysis showed no significant difference between female patients with thrombosis and female normal control subjects. Thus, the Ser316 allele may protect women from developing pregnancy complications by influencing an anticoagulant function of beta2GPI via a mechanism distinct from aPL production.     

Clinical significance of antiphospholipid antibodies in Indian scleroderma patients

In patients with systemic sclerosis (SSc), antiphospholipid antibodies (aPL) have been reported to be associated with more severe manifestations including digital infarct, gangrene and pulmonary hypertension. But these findings are not consistent in all studies; moreover, there are no data available from Indian subcontinent. The objective of this study is to assess the prevalence of antiphospholipid antibodies in Indian SSc patients and correlate them with clinical and immunological features. Seventy-two patients were recruited prospectively from rheumatology clinic from 2002 to 2006. Their medical records were reviewed. Anticardiolipin antibodies (IgG, IgM) by ELISA and lupus anticoagulant (LA) were tested in standardized pattern and repeated after 6 weeks. Anti-β2 glycoprotein-I antibodies were done in patients who had aPL antibodies. Nineteen patients had diffuse cutaneous SSc and 53 had limited disease. Seven patients (9.7%) were positive for aPL antibodies in their sera. Only one patient had clinical features of antiphospholipid antibody syndrome and manifested with recurrent abortions and deep vein thrombosis. She was positive for aCL, LA and anti-β2 glycoprotein-I antibodies. Four patients were only aCL (IgG) positive in moderate titers and one each had only aCL (IgM) and LAC positivity. None of the clinical parameters showed an association with aPL antibody.     

Val/Leu<Superscript>247</Superscript> and Trp/Ser<Superscript>316</Superscript> polymorphisms in β<Subscript>2</Subscript> glycoprotein I and their association with thrombosis in unselected Chilean patients

It is known that polymorphisms of β ₂-glycoprotein I (β ₂GPI) in exon 7 affect interaction between the phospholipid binding site and the antibodies, and that other polymorphisms in exon 8 increase the generation of antibodies. In this study, we analyzed genetic polymorphisms of β ₂GPI in unselected Chilean patients to determine the prevalence of β ₂GPI polymorphisms in the phospholipid domain in patients with venous and arterial thrombosis and the clinical correlation with thromboembolic complications. This study comprised 149 patients with venous and arterial thrombosis (62 with venous thrombosis and 87 with arterial thrombosis) and 160 healthy controls with no previous history of thrombosis. Polymorphisms of exons 7 and 8 of β ₂GPI, which encode for its fifth domain, were determined by PCR-RFLP. The presence of aPL or anti-β ₂GPI in the patients was detected by ELISA. Anti-β ₂GPI were present in 8/149 patients (5.4%); of these, five had aCL antibodies of low titer. The allele containing Val/Leu²⁴⁷ and Trp/Ser³¹⁶ was significantly more frequent in patients with thrombosis than in the control group (OR=3.1, CI 1.6–6.0, p=0.0003; OR=2.9, CI 1.1–8.6, p=0.027, respectively). These polymorphisms did not correlate with aPL or anti-β ₂GPI but significant differences were observed with venous thrombosis (p=<0.0001) and arterial thrombosis (p=0.026). In conclusion, the β ₂GPI polymorphisms Val/Leu²⁴⁷ and Trp/Ser³¹⁶ are not related to the presence of anti-β ₂GPI antibodies in unselected Chilean patients with venous and arterial thrombosis, but they are significantly associated with venous and arterial thrombosis.     

Some antiphospholipid antibodies recognize conformational epitopes shared by beta2-glycoprotein I and the homologous catalytic domains of several serine proteases

OBJECTIVE: To test the hypothesis that some antiphospholipid antibodies (aPL) in patients with the antiphospholipid syndrome (APS) recognize a conformational epitope shared by beta2-glycoprotein I (beta2GPI; the major autoantigen for the antiphospholipid antibodies) and the homologous catalytic domains of several serine proteases (such as thrombin, activated protein C [APC], and plasmin) involved in hemostasis. METHODS: We generated 4 new IgG monoclonal aPL (2 screened against beta2GPI, 1 against thrombin, and 1 against protein C) from 2 APS patients. The monoclonal antibodies (mAb) were analyzed for binding to beta2GPI, thrombin, APC, and plasmin, as well as for anticardiolipin antibody (aCL) activity. To demonstrate a shared epitope between beta2GPI and a serine protease, 1 mAb was studied by cross-inhibition analysis. RESULTS: Both of the IgG anti-beta2GPI mAb bound to thrombin, APC, and plasmin. On the other hand, the 1 anti-thrombin mAb and the 1 anti-protein C mAb also bound to beta2GPI. Moreover, the binding of 1 cross-reactive mAb to beta2GPI was inhibited by alpha-thrombin (which contains only the catalytic domain of thrombin). All 4 mAb displayed aCL activity. CONCLUSION: Taken together with the findings that some aCL bind to several serine proteases that participate in hemostasis and share homologous catalytic domains, these data demonstrate that some aCL in APS patients recognize one or more conformational epitopes shared by beta2GPI and the catalytic domains of disease-relevant serine proteases.     

Detection of acquired resistance to activated protein C associated with antiphospholipid antibodies using a novel clotting assay.

Antiphospholipid antibodies (aPA) frequently interfere with the protein C pathway. This manifests as acquired activated protein C (APC) resistance in the absence of factor V Leiden and has been proposed as a putative mechanism for the pathogenesis of the antiphospholipid syndrome (APS). We have developed a Russell's viper venom test, performed with and without activation of endogenous protein C, which is sensitive to aPA-associated APC resistance. Results were reported as the endogenous APC ratio (EAPCr); the ratio of the two clotting times normalized against pooled normal plasma. Forty-four patients with aPA, anticardiolipin and/or lupus anticoagulant, including 34 with a history of thrombosis or pregnancy morbidity; a control group of aPA-negative patients; and 26 healthy normals were studied. EAPCr (mean, SD) was significantly higher in APS patients (1.94, 0.58) than normals (0.98, 0.12) or controls (1.14, 0.19; P < 0.00001). Elevated EAPCr (> 1.22) occurred in 91% of aPA-positive patients, predominantly due to resistance to APC (87%) rather than prolonged basal clotting times alone (15%). Significant correlation was observed between the EAPCr value and dilute Russell's viper venom time (rs = 0.44, P = 0.003), IgG anticardiolipin (rs = 0.54, P = 0.002), protein S (r = -0.46, P = 0.01) and activated partial thromboplastin time-based APC resistance (r = -0.61, P = 0.001). There was no significant relationship between EAPCr and protein C concentration, anti-beta2-glycoprotein-I (anti-beta2GPI) or IgM anticardiolipin. Purified aPA IgG caused a dose-dependent increase in APC resistance when added to normal plasma. We conclude that aPA-associated acquired APC resistance is a common feature of APS and may be independent of anti-beta2GPI.     

Association of antiphospholipid antibodies with valvulopathy in systemic lupus erythematosus: a systematic review

Over the last few years, several studies in patients with systemic lupus erythematosus (SLE) have demonstrated an association between valvulopathy, including Libman–Sacks endocarditis (LS) and the presence of antiphospholipid antibodies (aPL), suggesting a role of these antibodies in the pathogenesis of the lesions. On the other hand, other studies found no such association. The aim of the present study is to analyze, by means of a systematic review, the existent evidences about the association between aPL and valvular lesions, including LS endocarditis, in patients with SLE. The information was obtained through searches for articles in the MEDLINE (1966 to 2010), SciELO, and LILACS databases, using the following key words: “Libman–Sacks endocarditis”, “antiphospholipid and Libman–Sacks”, “antiphospholipid and valvular heart disease”, “antiphospholipid and verrucous endocarditis”, “antiphospholipid and heart”, “antiphospholipid and valvular vegetations”, “anticardiolipin antibodies” and the corresponding translations in Portuguese. Twenty articles were found, which evaluated the association between the presence of aPL and valvulopathy. Thirteen of these studies evaluated the association of aPL with the LS lesion. Of the 20 articles, 15 demonstrated a positive association between aPL and valvular lesions, whereas 5 articles demonstrated that there was no association. Evidence of an association between the presence of aPL and valvular lesion was demonstrated in the majority of studies. Nevertheless, the possible role of these antibodies in the etiopathogenesis of the lesion has not yet been proved.     

ANTIBODIES TO ENDOTHELIAL CELLS AND TO {beta}2-GLYCOPROTEIN I IN THE ANTIPHOSPHOLIPID SYNDROME: PREVALENCE AND ISOTYPE DISTRIBUTION

The aim of this study was to analyse the prevalence and isotypedistribution of antibodies to endothelial cells (aEC and toß2-glycoprotein I (aß2GPI) in the antiphospholipidsyndrome (APS). Fifteen patients with an APS [nine associatedwith systemic lupus erythematosus (SLE) and six ‘primary’]and 15 with SLE without an APS were prospectively studied. TheaEC were determined by an enzyme-linked immunosorbent assay(ELISA) using endothelial cells derived from human umbilicalvein and the aß2GPI by ELISA using highly purifiedß2GPI. A positive titre of aEC was detected in 20out of 30 patients (67%), but in none of the control group.Ten patients had both IgG and IgM isotypes, five had IgG onlyand five had only IgM. Thirteen patients with the APS (87%)were found to have a positive titre of aEC, while only sevenwith SLE but without a history of APS (47%) had aEC (P <0.05). Nine patients with the APS (60%) had a positive titreof aß2GPI (four had both IgG and IgM isotypes, onehad IgG only and four had only IgM), while none of the patientswithout an APS (0%) had these antibodies (P < 0.001). A significantassociation was also found between the presence of aPL and aEC(P < 0.05), as well as between aPL and aß2GPI (P< 0.001). Both aEC and aß2GPI can be found in theAPS. This reinforces the theory that APS represents a complexautoimmune disorder in which several autoantibodies co-existwith aPL. KEY WORDS: Antiphospholipid antibodies, Anticardiolipin antibodies, Lupus anticoagulant, Endothelial cells, ß2-glycoprotein I     

Antibody specificity and related clinical features in antiphospholipid syndrome

The concept of antiphospholipid syndrome (APS) has been widely accepted. Antiphospholipid antibodies originally included anticardiolipin antibodies and lupus anticoagulants. However, recent advances have shown that most pathogenic antiphospholipid antibodies are directed to phospholipid binding proteins such as β₂-glycoprotein I (β₂-GPI) and prothrombin. Other candidates for antigens of so-called antiphospholipid antibodies are protein C, protein S, factor V, factor X, annexin V, high and low molecular weight kininogens, and complex with β₂-GPI and oxidized low-density lipoprotein (LDL). These autoantibodies directed to different phospholipid binding proteins are present in the blood stream, and contribute to triggering procoagulant effects on endothelial cells and platelets, leading to thrombosis. The heterogeneity of these antiphospholipid antibodies appears to explain various clinical manifestations in patients with APS. The preliminary classification criteria for definite APS and a general management policy have been proposed, although successful treatment of patients with antiphospholipid antibodies have only been shown by retrospective studies. Further prospective investigations are required to confirm the diagnostic and therapeutic criteria for patients with APS.     

Prevalence of antiphospholipid antibodies is not different in Chilean diabetic patients and normal individuals

Diabetes mellitus (DM) is complicated by vascular and neurological events. Antiphospholipid (aPL) antibodies have already been associated with many clinical conditions, including venous and arterial thrombosis, as well as recurrent fetal loss. However, a significant association between aPL antibodies and DM has not been widely reported yet. In the present study, we investigated the prevalence of aPL antibodies in diabetic patients. This study included 100 Chilean diabetic patients (67 of them with some complications and 33 without complications; 28 with Type 1 and 72 with Type 2 DM) and 100 healthy blood donor controls. Each sample was analyzed for IgG, IgM and IgA anticardiolipin (aCL), anti-beta(2) glycoprotein I (anti-beta(2)GPI), antiprothrombin (aPT) antibodies, and lupus anticoagulant (LA). Fourteen out of 100 (14%) diabetic patients presented some type of aPL antibodies. Four patients were positive for aCL antibodies, two for anti-beta(2)GPI antibodies, and nine for aPT antibodies. All patients were LA negative. The incidence of different isotypes was similar in each of the aPL antibodies studied, and their activities were low. No significant correlation was observed between aPL antibodies and vascular complications.     

Low levels of activated protein C in patients with systemic lupus erythematosus do not relate to lupus anticoagulants but to low levels of factor II

The presence of lupus anticoagulants (LAC) in plasma is a major risk factor for thrombosis. An attractive hypothesis to explain a LAC-mediated thrombotic tendency is that LAC interfere with activation of protein C, a natural antithrombotic in plasma. We investigated the relationship between LAC and protein C activation in vivo. We selected 20 patients with systemic lupus erythematosus (SLE) with LAC (and not using oral anticoagulants), 36 patients with SLE without LAC and 25 healthy volunteers. In these, we measured circulating levels of activated protein C (APC), prothrombin (FII), free protein S, C4BP, protein C, and antibodies to protein C, protein S, FII and beta2-glycoprotein I (beta2GPI). In SLE patients (n = 56), mean levels of APC, FII and free protein S were significantly (P < 0.001) lower than those in healthy volunteers (respectively 13%, 17% and 14%). Mean protein C levels and C4BP levels were similar for SLE patients and healthy volunteers. In contrast to the above hypothesis, the decreased levels of APC could not be attributed to the presence of LAC. Levels of APC were correlated with both FII levels and protein C levels. Decreased levels of APC, FII, protein C and free protein S were related to the presence of anti-FII antibodies. None of the patients had antibodies against protein C or protein S. In conclusion, although the mean levels of APC, FII and free protein S were significantly decreased in SLE patients, no correlation with LAC was found. However, anti-FII antibodies were related to decreased levels of APC, FII, protein C, free protein S and C4BP. As FII levels, and not protein C levels, were decreased in SLE patients and correlated with APC levels, we conclude that the decreased FII levels are responsible for the low levels of APC.     

Tissue factor in antiphospholipid syndrome: shifting the focus from coagulation to endothelium

Thrombosis, in both the arterial and the venous circulation,is one of the major manifestations of antiphospholipid syndrome(APS), a prothrombotic disorder associated with recurrent thromboticevents and pregnancy morbidity in the presence of antiphospholipidantibodies (aPL) [1]. In the past decade, many studies have investigated the pathophysiologyof thrombosis in APS and considerable interest has focused onthe role of aPL as a clue to the mechanism of thrombosis. Resultsof intensive research have significantly advanced our understandingof the mechanisms by which these antibodies may play a directrole in clot formation. It is now recognized that many of theautoantibodies associated with APS are directed against phospholipid-bindingplasma proteins, such as ß₂-glycoprotein I (ß2GPI)and prothrombin, or phospholipid–protein complexes, expressedon or bound to the surface     

Annexin A2: biology and relevance to the antiphospholipid syndrome

Antiphospholipid antibodies (aPL), the majority of which are directed against beta(2)-glycoprotein I (beta(2)GPI), are associated with an increased incidence of venous and arterial thrombosis. The pathogenesis of antiphospholipid/anti-beta(2)GPI-associated thrombosis has not been defined, and is likely multifactorial. However, accumulating evidence suggests an important role for endothelial cell activation with the acquisition of a procoagulant phenotype by the activated endothelial cell. Previous work demonstrated that endothelial activation by antiphospholipid/anti-beta(2)GPI antibodies is beta(2)GPI-dependent. We extended these observations by defining annexin A2 as an endothelial beta(2)GPI binding site. We also observed that annexin A2 plays a critical role in endothelial cell activation induced by anti-beta(2)GPI antibodies, and others have described direct endothelial activation by anti-annexin A2 antibodies in patients with aPL . Similar findings have been reported using human monocytes, which also express annexin A2. Because annexin A2 is not a transmembrane protein, how binding of beta(2)GPI/anti-beta(2)GPI antibodies, or anti-annexin A2 antibodies, to endothelial annexin A2 causes cellular activation is unknown. Recent studies, however, suggest an important role for the Toll-like receptor family, particularly TLR4. In this article, we review the role of these interactions in the activation of endothelial cells by aPL . The influence of these antibodies on the ability of annexin A2 to enhance t-PA-mediated plasminogen activation is also discussed.     

Prevalence and isotype distribution of antiphospholipid antibodies in unselected Chilean patients with venous and arterial thrombosis

Antiphospholipid antibodies (aPL) are a heterogeneous family of antibodies associated with thrombotic events and other complications. The objective of this study was to investigate the prevalence of aPL in a group of Chilean patients with thrombosis. Two hundred and twenty-six patients with venous and arterial thrombosis and 95 healthy controls were studied. Anticardiolipin (aCL), anti-₂ glycoprotein I (anti-₂GPI), and antiprothrombin (aPT) antibodies were determined. Eighty-eight out of 226 (38.9%) patients with thrombosis had some type of aPL. Fifty-seven patients (25.2%) were positive for aCL, 31 (13.7%) for aPT, and 14 (6.2%) for anti-₂GPI antibodies. Twelve patients (5.3%) were positive for more than one aPL. IgG, IgM and IgA isotypes were observed in aCL, anti-₂GPI, and aPT antibodies. Twenty-six out of 92 (28.3%) patients with venous thrombosis and 31/134 (23.1%) patients with arterial thrombosis were positive for aCL antibodies. With regard to the control group (4/95=4.2%), the odd ratios (OR) were 5.2 (1.3–19.8; p0.01) and 5.7 (1.6–22.3; p0.01), respectively. Additionally, we observed statistically significant OR with aPT and anti-₂GPI antibodies; in the first, with venous and arterial thrombosis, and in the second, only with arterial thrombosis. Our results show a significant prevalence of aPL, predominantly aCL and aPT antibodies, in patients with thrombosis. Additionally, aCL and aPT antibodies appear to be a risk factor for venous and arterial thrombosis, and anti-₂GPI antibodies appear to be a risk factor for arterial thrombosis.Abbreviations aCL Anticardiolipin antibodies - aPL Antiphospholipid antibodies - AMI Acute myocardial infarction - BSA Bovine serum albumin - DVT Deep venous thrombosis - FBS Fetal bovine serum - IS Ischemic stroke - LA Lupus anticoagulant - RT Room temperature - RVT Retinal vein thrombosis - SLE Systemic lupus erythematosus