Kengo Gohchi

Distinguishing β2‐glycoprotein I dependent (systemic lupus erythematosus type) and independent (syphilis type) anticardiolipin antibody with Tween 20

Summary We investigated whether or not the use of Tween 20 could help to distinguish β2‐glycoprotein I (GPI) independent anticardiolipin antibody (aCL) (syphilis‐type aCL) from GPI‐dependent aCL (SLE‐type aCL) in a GPI‐dependent/ independent aCL ELISA. aCL was positive in all 16 SLB patients arid all 15 syphilis patients, who were positive for aCL in the standard ELISA. in the GPI‐independent ELISA with Tween 20. GPI‐dependent aCL was detected in 12/16 SLE patients by the GPI‐dependent ELISA with Tween 20. aCL was not detected in any of the syphilis patients by GPI‐dependent ELISAs. On the basis of these results, we recommend that Tween 20 should be used in ELISAs to distinguish GPI‐dependent aCL from GPI‐independent aCL.

Effect of Total-Body Cold Exposure on Plasma Concentrations of von Willebrand Factor, Endothelin-1 and Thrombomodulin in Systemic Lupus erythematosus Patients with or without Raynaud’s Phenomenon

The effect of total-body cold exposure on plasma concentrations of von Willebrand factor (vWF), endothelin-1 (ET) and thrombomodulin (TM), all of which are considered to be generated from the endothelium, was studied in systemic lupus erythematosus (SLE) patients with and without Raynauds phenomenon. The plasma levels of vWF, ET and TM in SLE patients, irrespective of the presence of Raynauds phenomenon, were significantly higher than in normal controls even before the cold provocation test. After the cold provocation test, plasma levels of vWF and ET were significantly higher in SLE patients with Raynauds phenomenon than in those without and in normal controls. No significant increase in TM was observed in either the SLE patients or the controls. These results suggest that SLE patients, regardless of the presence of Raynauds phenomenon, are in a hypercoagulable state and that this state may be further intensified by cold exposure. Hence, it is concluded that we should consider antithrombotic therapy for SLE patients, especially those with Raynauds phenomenon, to prevent unwanted activation of the coagulation system and possible endothelial damage.

Resistance to activated protein C activity of an anti‐/2 ‐glycoprotein I antibody in the presence of β‐glycoprotein I

Some researchers claim that lupus anticoagulant‐positive plasma may cause a false‐positive reaction in the test for activated protein C (APC) resistance, a hereditary thrombophilic state characterized by abnormal factor V, which frequently causes venous thrombosis, We investigated whether anti‐/32 ‐glycoprotein I antibody (aGPI), which has recently come to be regarded as an anti‐cardiolipin antibody (aCL) itself, might have an effect on the APC resistance test.

β2-glycoprotein I -dependent and -independent anticardiolipin antibody in patients with end-stage renal disease

Abstract We investigated whether anticardiolipin antibody (aCL), which is common in patients with end-stage renal disease (ESRD), was dependent on β2-glycoprotein I (GPI), a cofactor of aCL. Lupus anticoagulant (LA) was identified in 13/39 hemodialysis (HD) patients. GPI-independent aCL was positive in 12/39 of this group, five of whom had both GPI -independent aCL and LA. Three of the 20 patients on symptomatic and supportive treatment other than HD had LA, but none of them were aCL positive. When GPI was added to the assay system to allow for GPI-dependent aCL measurement, the optical density (OD) readings of 10/12 HD patients decreased, thus proving that they were negative for aCL. In contrast, the OD readings of 6 control patients with systemic lupus erythematosus (SLE) increased markedly (3/6) or decreased slightly to moderately (3/6), however, within the limits of positive range, indicating GPI-dependent cCL positivity. These results suggested that LA and GPI-independent aCL were produced in ESRD patients, especially those on HD, possibly through mechanisms related to the hemodialysis membrane. However, the significance of GPI-independent aCL in this clinical setting, which may differ from the GPI-dependent aCL detected in SLE patients, as well as the possible participation of serum GPI in the production of aCL remain to be clarified.

Anti-endothelial cell antibodies to the endothelial hybridoma cell line (EAhy926) in systemic lupus erythematosus patients with antiphospholipid antibodies

The endothelial hybridoma (EAhy926) cell line was employed to clarify whether antiphospholipid antibodies (aPA) [lupus anticoagulant (LA), antiprothrombin antibody (aPT) and/or anticardiolipin antibody (aCL)] and anti‐endothelial cell antibodies (AECA) are identical, and establish whether β2‐glycoprotein I (β2‐GPI) is needed for reactivity of aPA to endothelial cells. Ig‐G AECA was positive in 9/30 SLE patients with aPA (30.0%) and 10/22 SLE patients negative for aPA (45.5%). Ig‐M AECA was positive in one SLE patient with aPA and one SLE patient without aPA. AECA‐positivity was not significantly different among unfixed, TNF‐stimulated and fixed EAhy926. The influence of β2‐GPI on the reactivity of serum to EAhy926 was minimal, and absorption experiments of serum with cardiolipin‐liposome/β2‐GPI or phosphatidylserine‐liposome/prothrombin gave little evidence of cross‐reactivity of aPA and AECA. The results of our study suggest that aPA and AECA may have partially cross‐reacted, but were different antibodies. However, further study is needed to clarify the clinico‐pathological significance of AECA.

Clinical Significance of the Venous Occlusion Test on Systemic Lupus erythematosus Patients with a Focus on Changes in Blood Levels of Tissue Plasminogen Activator, von Willebrand Factor Antigen, and Thrombomodulin

We investigated the clinical significance of the venous occlusion (VO) test on patients with systemic lupus erythematosus (SLE) with or without circulating lupus anticoagulant (LA) concerning whether changes in the blood coagulation and fibrinolysis system in vivo subsequent to VO reflect mechanical stimulation of the endothelium or presence/development of endothelial damage. The tissue plasminogen activator antigen (tPA:Ag) before VO was much lower in the LA-positive patients than in the LA-negative ones (p < 0.01) and the von Willebrand factor antigen (VWF:Ag) pre-VO was significantly higher in the patient group, regardless of LA status, than in the control group (p < 0.01). But the mean increment in tPA:Ag and VWF:Ag post-VO, when expressed as the percentage of the baseline level, showed no appreciable difference between LA-positive and -negative groups. Thrombomodulin (TM) basically, on the other hand, was higher in the patients of either LA status than in the controls (p < 0.01) with a significant post-VO increase in the SLE group, which was more marked in the LA-positive patients, against no substantial change in the controls (p < 0.01). It is known that tPA and VWF:Ag are released simply as a result of endothelial stimulation and that the release of TM is preceded by endothelial damage. Based on the present results, we may well conclude that (1) the endothelium is functionally intact in SLE patients, (2) an injury of the endothelium, possibly as a consequence of vasculitis, preexists in LA-positive patients, and thus to measure the TM response to VO would offer a helpful tool in diagnosing the preexisting endothelial damage in these clinical settings.

Resistance to activated protein C in systemic lupus erythematosus patients with antiphospholipid antibodies

To the Editor: Dahlback et al. (1) have recently reported three families who are thrombophilic due to an as yet uniuentified “cofactor” abnormality to activated protein C (APC), namely resistance to APC. Some researchers (2) confirmed that its prevalence rate, especially among thrombophilic patients, was higher than any other known hereditary causative abnormalities for venous thrombosis and suggested that the poor response to APC might be the most important hereditary cause of venous thrombosis. Antiphospholipid antibodies (aPL) (3), sometimes detected in patients with systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome, are also known as causative autoantibodies to arterio-venous thrombosis; however, its true mechanism of action is still remained to be clarified. It seems highly possible that a risk of thrombotic event may be further increased in patients positive for both aPL and resistance to APC. However, to our knowledge, there is no report concerning this point. This fact prompted us to investigate a prevalence rate of resistance to APC in SLE patients with aPL. The studied subjects were 50 SLE patients who fulfilled the ARA diagnostic criteria for SLE. They have been maintained on < 10 mg/day of prednisolone and were not taking any drugs to influence this study for at least 7 days. Fifteen of them had previous history of arterio-venous thrombosis, habitual abortion/fetal loss, thrombocytopenia and/or biological false positive reaction to syphilis (BFP). aPL (anticardiolipin-, antiphosphatidyl serine-, antiphosphatidyl inositol-, antiphosphatidic acid-antibody) was measured previously reported ELISA method (4). Resistance to APC in plasma was measured using test kit (Coatest APC Resistance, Chromogenix AB, Sweden). Resistance to APC was judged positive when the ratio of clotting time of the sample/ control was < 2 according to the instruction of the manufacturer. Twenty-five of 50 SLE patients ( 5 0 2 ) ) were positive for at least one of aPL. Twelve of 15 SLE patients with previous history of aPL related complications were positive for aPL. Resistance to APC was initially positive in 3 of 50 SLE patients. Two patients (patient A, female, 42 years old, patient B, female, 49 years old) were repeatedly positive for resistance to APC at different test occasions, and thus 2 patients were finally diagnosed as definitely positive cases for resistance to APC. They were also positive for aPL (negative for lupus anticoagulant), but neither of them had a previous history of thrombosis other than habitual abortion (patient A) and thrombocytopenia (patient B). Thus we have confirmed the existence of resistance to APC in 2 SLE patients with aPL. But since they had no previous history of thrombosis, we could not confirm our hypothesis that the co-existence of APC-resistance in patients with aPL was an additional risk factor for thrombotic event. Svensson et al. (5) reported that the probability of a person positive for resistance to APC in families with thrombophilia being free of thrombosis at the age of 45 was approximately 59%. Provided this holds true, our 2 SLE patients could be said to be still at risk for thrombosis; hence, we need further follow-up studies to record whether they develop venous thrombosis in the near future. Since not all patients with aPL necessarily develop thrombosis, additional facto(s), such as resistance to APC may play an important role along with aPL for developing thrombosis in this clinical setting.

Circulating intercellular adhesion molecule‐1 and soluble interleukin 2‐receptor in patients with systemic lupus erythematosus

To the Editor: It is well known that clinical complications, such as thrombosis and fetal loss, are sometimes seen in patients with systemic lupus erythematosus (SLE), especially in those found to be positive for antiphospholipid antibody (aPL) (1). Intercellular adhesion molecule1 (ICAM1) serves in intercellular adhesion by binding to its ligand, leukocyte integrins lymphocyte functionassociated antigen 1 (LFA-1) and complement receptor type 3, which allows participation in many immunological functions. ICAM1 is expressed primarily in endothelial cells, and its surface expression may be augmented in association with cell activation and/or the production of various cytokines. Thus, it seems possible that ICAM-I is a marker reflecting endothelial activation and/or injury (2). We measured serum ICAM-1 level in patients with SLE to investigate a possible correlation with aPL positivity. Furthermore, we measured serum soluble interleukin-2 receptor (sIL2-R), known as lymphocyte activation marker (3), to clarify whether or not there was any correlation between the levels of ICAM-1 and sIL2-R in SLE patients. The subjects were 48 patients with SLE who ful-

Detection of Beta-2-Glycoprotein-l-Dependent Antiphospholipid Antibodies and Anti-Beta-2-Glyco-protein-l Antibody in Patients with Systemic Lupus Erythematosus and in Patients with Syphilis

We investigated whether or not antiphospholipid antibodies (aPLs; antiphosphadidylserine antibody, aPS; antiphosphatididylinositol antibody, aPI; antiphosphatidic acid antibody, aPA, and antiphosphadidylethanolamine antibody, aPE) were beta 2-glycoprotein-I (GPI)-dependent antibodies like anticardiolipin antibody (aCL) in patients with systemic lupus erythematosus (SLE). None of the patients with syphilis or healthy controls was positive for any GPI-dependent aPL. By contrast, GPI-dependent aCL (40%), aPS (20%), aPI (18%), aPA (12%) and aPE (8%) were detected in patients with SLE. Among these, 4 patients were negative for aCL, but positive for aPS. Those who were positive for more than 2 types of aPL, along with lupus anticoagulant, had a high incidence of arteriovenous thrombosis, fetal loss, thrombocytopenia and biological false-positive reaction to syphilis. From these findings we conclude that GPI-dependent aPLs, other than aCL, are present in patients with SLE, and we should examine more than 2 types of aPL, such as a combination of aCL and aPS, to avoid overlooking aPL. Furthermore, we confirmed that GPI-independent aPL was not rare in SLE patients, but the clinical significance of this type of aPL in this clinical setting is unclear.

Plasma concentrations of total/free and functional protein S are not decreased in systemic lupus erythematosus patients with lupus anticoagulant and/or antiphospholipid antibodies

SummaryWe conducted an investigation to clarify whether or not the levels of total, free, and functional protein S and C4-binding protein (C4bp) in plasma are decreased in systemic lupus erythematosus (SLE) patients, especially those with antiphospholipid antibody (aPL), which is known to be a causative factor of such complications as habitual abortion and arteriovenous thrombosis. Fifty patients with SLE were recruited as subjects of the study. Serum aPL (anticardiolipin, antiphosphatidyl serine, antiphosphatidyl inositol, and antiphosphatidic acid antibodies) were measured by ELISA. Lupus anticoagulant was determined by aPTT, KCT, and diluted RVVT. Furthermore, plasma concentrations of total, free, and functional protein S and C4bp were measured. There were no significant differences in the mean levels of total, free, or functional protein S and C4bp between aPL-positive, aPL-negative SLE patients, and the healthy controls. From these results, we concluded that the protein S level is not the sole factor causing complications, and that other factor(s) may be involved in the induction of such complications in this clinical setting.