Arteriosclerosis obliterans associated with anti-cardiolipin antibody/beta2-glycoprotein I antibodies as a strong risk factor for ischaemic heart disease in patients with systemic lupus erythematosus.

OBJECTIVE: The main objective of this study was to clarify the role of aPLs in the pathogenesis of arteriosclerosis obliterans (ASO), ischaemic heart disease (IHD) and cerebral vascular disorder (CVD) in patients with SLE. METHODS: We evaluated 155 patients with SLE by using objective tests for diagnosing ASO, IHD and CVD and laboratory tests including ELISA for aCL/beta2-glycoprotein I antibodies (aCL/beta2-GPI) and anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT). RESULTS: Twenty-five (16.1%) of the 155 SLE patients were diagnosed with ASO. Both aCL/beta2-GPI and anti-PS/PT levels were significantly higher in SLE patients with ASO (mean +/- S.E., 104.3 +/- 38.8 U/ml for aCL/beta2-GPI, P < 0.01; 72.6 +/- 48.9 U/ml for anti-PS/PT, P < 0.05) than in SLE patients without ASO (22.8 +/- 9.9 U/ml for aCL/beta2-GPI; 18.3 +/- 4.4 U/ml for anti-PS/PT). Multivariate logistic analysis including aCL/beta2-GPI, anti-PS/PT and traditional risk factors (hypercholesterolaemia, hypertension and diabetes mellitus) confirmed that the presence of aCL/beta2-GPI was the most significant risk factor for ASO in SLE patients [odds ratio (OR) 3.45; 95% CI 1.40, 8.56; P < 0.01]. Furthermore, the prevalence of ASO was associated strongly with IHD (OR 11.8; 95% CI 3.45, 40.1; P < 0.0001) but not CVD (OR 1.84; 95% CI 0.65, 5.21; P = 0.25). CONCLUSIONS: The presence of aCL/beta2-GPI contributes to the risk of development of ASO, which may represent an important mechanism for the pathogenesis of IHD in patients with SLE.

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus.

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P < 0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).

Measurement of a newly developed thrombomodulin addition activated partial thromboplastin time assay in patients with deep venous thrombosis.

We developed a simple assay using rabbit thrombomodulin (TM) based on an activated partial thromboplastin time method, which detected the response to TM in plasma coagulation. We call it thrombomodulin addition clotting time (TACT). The anticoagulant response to TM was calculated by dividing the clotting time with TM by the clotting time with buffer solution. Results were expressed as TACT ratio, which indicates the degree of inhibition of plasma clotting by TM. Using this assay, we measured the TACT ratio in 80 patients with deep-vein thrombosis (DVT) and in 126 controls matched to the patients according to age and sex. A significant difference in the TACT ratio was observed between patients with DVT (mean 1.874) and controls (mean 1.956) (p < 0.001). Twenty- three patients (29%) had TACT ratios below the 10th percentile (1.757) of distribution of control subjects (odds ratio: 3.5; 95% confidence interval (CI): 1.7-7.2). After excluding subjects with a deficiency of protein C, protein S and antithrombin III, we found an odds ratio for DVT of 3.4 (95% CI: 1.6-7.2). These data suggest that natural anticoagulant deficiencies do not influence the TACT ratio, and our case-control study may show that the plasma of patients with DVT has a low response to TM.

Association between the prevalence of antibodies to beta(2)-glycoprotein I, prothrombin, protein C, protein S, and annexin V in patients with systemic lupus erythematosus and thrombotic and thrombocytopenic complications.

BACKGROUND: Anti-phospholipid (aPL) antibodies (Abs) frequently found in the plasma of patients with systemic lupus erythematosus (SLE) have been associated with thrombotic complications. Our aim was to clarify the roles in thrombosis of aPL Abs that react with complexes of phospholipids and plasma proteins such as beta(2)-glycoprotein I (beta(2)-GPI), prothrombin, protein C, protein S, and annexin V. METHODS: We determined the prevalence of aPL Abs to various phospholipid-binding plasma proteins in SLE patients with arterial thrombosis (30 cases), venous thrombosis (19 cases), thrombocytopenia (14 cases), fetal loss (14 cases), and patients without complications (91 cases). The aPL Abs were measured by an ELISA system in which human plasma proteins (beta(2)-GPI, prothrombin, protein C, protein S, and annexin V) were immobilized on gamma-irradiated or plain polystyrene plates. RESULTS: All types of aPL Abs were frequently observed in the patients with SLE when gamma-irradiated polystyrene plates were used (51 of 168 cases positive for anti-beta(2)-GPI, 94 of 168 cases positive for anti-prothrombin, 36 of 168 cases positive for anti-protein C, 47 of 168 cases positive for anti-protein S, and 50 of 168 cases positive for anti-annexin V), whereas no Abs to these plasma proteins were detected when plain polystyrene plates were used. Multivariate analysis confirmed that both anti-beta(2)-GPI and anti-prothrombin Abs were significant risk factors for arterial thrombosis [odds ratios (ORs), 8.8 and 14.5, respectively; 95% confidence intervals (CIs), 3.2-25 and 1.8-116, respectively] but not for venous thrombosis. The presence of anti-protein S Abs was a significant risk factor for venous thrombosis (OR, 30.4; CI, 3.3-281) but not for arterial thrombosis. The only significant risk factor for fetal loss was the presence of anti-annexin V Abs (OR, 5.9; CI, 1.4-14.8). CONCLUSIONS: Patients with SLE frequently have some aPL Abs to beta(2)-GPI, prothrombin, protein C, protein S, and annexin V. Thrombotic complications in SLE may depend on the antigenic specificities of these Abs, alone or in combination.

Frequency of natural coagulation inhibitor (antithrombin III, protein C and protein S) deficiencies in Japanese patients with spontaneous deep vein thrombosis.

One hundred and thirteen consecutive Japanese patients with deep venous thrombosis (DVT) were studied for the incidences of antithrombin III (AT-III), protein C (PC) and protein S (PS) deficiencies, and the results were compared with those of normal subjects. Ten of the 392 normal Japanese subjects were found with PS deficiency (n = 8, 2.02%) or PC deficiency (n = 2, 0.5%). PS deficiencies comprised type I (1/8, 12.5%), type 11 (4/8, 50%), and type III (3/8, 37.5%). All PC deficiencies were type I. Among patients with DVT, 32 (28.3%) were deficient in AT-III, PC and PS. These patients consisted of two AT-III deficiency (1.77%), nine PC deficiency (7.96%), 20 PS deficiency (17.7%), and one combined deficiency of PC and PS (0.88%). Both of the patients with AT-III deficiency were classified as type II, all those with PC deficiency as type I, and those with PS deficiency as type I in 25% (5/20), type II in 55% (11/20) and type III in 20% (4/20). The frequency of PC and PS deficiencies in patients with DVT were 15.6 and 7.38 times the control population frequency, respectively, and this difference was statistically significant (P < 0.05). These data suggest that the Japanese population has a high frequency of PC and PS deficiencies. We recommend that PS activity should be measured for screening of thrombosis since type II deficiency accounted for approximately 50% of PS deficiency cases in both patients and the normal group in the Japanese.

[Informed consent for the use of the remaining portion of laboratory specimens for education, research, and quality control of laboratory tests].

The remaining portion of a laboratory specimen is usually used for education, research, and quality control of laboratory tests in hospitals, but informed consent has not been obtained because of the high volume of patients who undergo laboratory tests. However, patients must be informed in some manner. Therefore, we decided to inform patients that any remaining specimen would be used for various purposes by placing such a notice on walls in the central clinical laboratory and hospital lobby. We then obtained a signature on a dissent document, instead of a consent document, from any patient who dissented from such use. This indirect process for obtaining informed consent was approved by the ethics committee of Osaka University Medical School. The number of dissent documents sent in to the director was 54 of about 400,000 patients who underwent laboratory tests over the last 3 years, and there was no complaint against this "informed consent process".

[Measurement of reticulated platelet using whole blood: methodology and clinical application].

We established simple and rapid method to measure reticulated platelets using whole blood. After whole blood was fixed with paraformaldehyde, sample was stained with thiazole orange and analyzed using flow cytometer. %RP was significantly higher in patients with idiopathic thrombocytopenic purpura(ITP) (27.7 +/- 14.3%) compared with that of normal subjects(11.9 +/- 3.1%), and elevated in 73% of ITP patients. Furthermore, %RP reflected the status of disease very well in a patient with ITP undergoing therapy. Our method can be effectively applied for differential diagnosis and assessment of the status of thrombocytopenic disorders.

Frequency of protein S deficiency in general Japanese population.

We measured protein S (PS) activity in a large group of Japanese subjects to study the frequency of PS deficiency. The study group comprised 213 men, ages 18-58 years, and 179 women, ages 18-60 years. PS activity in the total 392 subjects was 58-135% (mean +/- 2 SD), 65-135% in the men and 54-120% in the women. The men showed significantly higher levels of PS activity than the women (p<0.001). We identified 8 subjects (4 men and 4 women) in whom PS activity was lower than the mean-2SD for men and women, respectively. Moreover, we examined the classifications of PS deficiency. The frequency of PS deficiency in this study was 2.04% (Type I: 0.51%, Type II: 1.02%, Type III: 0.51%). Based on our findings, it would appear that the frequency of Type II PS deficiency in the Japanese population is approximately 1%. The screening of PS decrease should be judged by activity.

Platelet activation induced by combined effects of anticardiolipin and lupus anticoagulant IgG antibodies in patients with systemic lupus erythematosus--possible association with thrombotic and thrombocytopenic complications.

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p<0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+ x LA+ plasma only, but was significantly augmented by aCL+ x LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+ x LA-, aCL- x LA+ and aCL- x LA- plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+ x LA+ plasma (aCL+ x LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+ x LA--IgG and aCL- x LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.

Three novel missense mutations in unrelated Japanese patients with type I and type II protein S deficiency and venous thrombosis.

A molecular analysis of protein S deficiency in three unrelated Japanese patients was performed. An approximately 50% reduction in both functional and immunologic levels of protein S was detected in the plasmas from two unrelated patients, designated protein S Osaka 1 and protein S Osaka 2. An approximately 50% reduction in the functional level, but a normal immunologic level of protein S, was detected in plasma from a third patient, designated protein S Osaka 3. All of the exons and exon/intron junctions of the protein S gene were studied using a strategy combining polymerase chain reaction amplification and rapid non-radioactive single-strand conformational polymorphism analysis. We identified a G-to-A change in exon X of the protein S gene in protein S Osaka 1. This mutation resulted in the substitution of Gly for Ser at position 295 in the sex hormone-binding globulin-like region. In protein S Osaka 2, a G-to-C change at the position of the 3' end of exon III was identified, leading to the amino acid substitution of Val46 by Leu in the aromatic stack region. In protein S Osaka 3, an A-to-G change in exon II was identified, leading to the substitution of Lys9 by Glu in the Gla domain. It was concluded that the Gly295-to-Ser mutation and Val46-to-Leu mutation cause type I protein S deficiency and that the Lys9-to-Glu mutation causes type II deficiency.

High prevalence of thrombocytopenia in SLE patients with a high level of anticardiolipin antibodies combined with lupus anticoagulant.

The relationship between thrombocytopenia and the level of anticardiolipin antibodies (aCL) and/or the existence of lupus anticoagulant (LA) ware studied in 146 patients with systemic lupus erythematosus (SLE). These patients were divided into six groups: A, those LA positive with a high level of aCL (>10 U/ml) (10 cases); B, those LA positive with a low level of aCL (3-10 U/ml) (15 cases); C, those LA positive but aCL negative (<3 U/ml) (12 cases); D, LA negatives with a high level of aCL (12 cases); E, LA negatives with a low level of aCL (16 cases); and F, aCL and LA double negatives (81 cases). The prevalence of thrombocytopenia (platelet count < or = 100 x 10(9)L) was by far the highest in group A (9/10 cases, 90.0%, P < 0.005, Fisher's exact probability test) as compared with group B (4/15 cases, 26.7%), group C (4/12 cases, 33.3%), group D (1/12 cases, 8.3%), group E (4/16 cases, 25.5%), and group F (9/81 cases, 11.1%). When the relationship between moderate thrombocytopenia and arterial or venous thrombosis was studied in these patients with SLE, thrombocytopenia was detected in 10 (83.3%, P < 0.005, Fisher's exact probability test) of 12 patients with arterial thrombosis; however, it was present in only 4 (23.5%) of 17 patients with venous thrombosis and in 14 (12.3%) of 114 patients without thrombosis. These findings suggest that a high aCL activity combined with LA positively reflects a high risk for both thrombocytopenia and arterial thrombosis.

[Association between antiphospholipid antibodies and arterial or venous thrombosis/thrombocytopenia].

The relationship between thrombotic or thrombocytopenic complications and the existence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA) was studied in 146 patients with systemic lupus erythematosus (SLE). The prevalence of arterial thrombosis was obviously higher in patients who had both aCL and LA than in patients with either aCL or LA alone or in those with neither. Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia, there is a possibility that aCL and LA might enhance platelet activation and aggregation. To test this hypothesis, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb). The IgG fraction purified from aCL+.LA+ plasma apparently enhanced platelet activation induced by adenosine diphosphate (ADP) at a low concentration, but IgG fractions from aCL+.LA- or aCL-.LA+ plasma did not cause enhancement of platelet activation. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis in patients with SLE.

Hypercholesterolemia as a risk factor for deep-vein thrombosis.

Our retrospective study has shown that hyperlipidemia is a novel etiologic factor in deep-vein thrombosis (DVT) and that most of the idiopathic DVT patients were hyperlipidemic (Thrombosis Research 79, 147-151, 1995). The aim of our current study is to analyze the interrelationship between hyperlipidemia and DVT by means of a case-control study. A series of lipid parameters were analyzed using serum from 109 patients with deep vein thrombosis (DVT). One hundred nine age- and sex-matched subjects served as controls. Diagnosis of hyperlipidemia was made if the serum cholesterol level was above 220 mg/dL or if the triglyceride level was above 150 mg/dL. Among several types of hyperlipidemia examined, the risk factor associated with the highest estimated odds ratio was carriage of hypercholesterolemia associated with hypertriglyceridemia (odds ratio 5.1) followed in order by hypercholesterolemia without hypertriglyceridemia (odds ratio 2.6) and hypertriglyceridemia without hypercholesterolemia (odds ratio 0.9). These findings support the hypothesis that hypercholesterolemia plays an important role in the pathogenesis of DVT.

Risk of arterial thrombosis in patients with anticardiolipin antibodies and lupus anticoagulant.

The relationship between arterial or venous thrombosis and the levels of anticardiolipin antibodies (aCL) and/or existence of lupus anticoagulant (LA) was studied. The 141 patients with systemic lupus erythematosus (SLE) were divided into four groups: aCL single positive (25 cases), LA single positive (11 cases), aCL and LA double positive (25 cases), aCL and LA double negative (80 cases). The prevalence of thrombosis was higher in aCL and LA double positive patients (21/25 cases, 84.0%, P <0.01) than that in aCL single positive patients (4/25 cases, 16.0%), LA single positive patients (1/11 cases, 9.1%) and double negative patients (3/80 cases, 3.8%). Furthermore, in these double positive patients, all patients (10/10 cases) with a high positive level of aCL (> 10 units/ml) had arterial thrombosis, whereas only 2/15 patients (13.3%) with a low positive level of aCL (3-10 units/ml) were affected. Venous thrombosis was frequently found in the low positive group (9/15 cases, 60.0%). On the contrary, none of 105 LA negative patients had arterial thrombosis and only seven (6.7%) had venous thrombosis. These findings indicate that a high aCL activity combined with a LA positive result might be a risk factor for arterial thrombosis.

Involvement of dysplasminogenemia in occurrence of deep vein thrombosis.

Although various hematological disorders have been considered to be etiologic factors in deep vein thrombosis (DVT), the involvement of dysplasminogenemia (DPG) in DVT has not been studied in detail. In 72 consecutive DVT patients, the presence of DVT was suspected based on a history of lower limb swelling and tenderness with acute onset, and was confirmed by duplex scanning, radioisotope venography, or contrast venography. DPG was identified by the observation of dissociation between the activity and antigenicity of plasminogen. Of the 72 patients, 9 (12.5%) were diagnosed as having DPG, and several antithrombin-III deficiency and protein C and S deficiency were identified. The mean age of the genetically normal and DPG patients was 52 +/- 15 and 40 +/- 15 years, respectively (p < 0.05). Thus, these findings suggest that DPG is deeply related to the development of DVT, and that abnormality of the fibrinolytic system is one of the major etiologic factors in DVT.

Study on an antibody against F1F2 fragment of human factor V in a patient with Hashimoto's disease and bullous pemphigoid.

We found a 81-year-old man with Hashimoto's disease and bullous pemphigoid whose activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged. Mixing studies with normal plasma failed to correct APTT and PT, suggesting the presence of circulating inhibitor. The patient's factor V activity was 11% of normal control and was not corrected by mixing with normal plasma, indicating the presence of an inhibitor against factor V. This inhibitory activity was observed up to 32 times dilution of the patient's plasma. The inhibitor activity against factor V was not detected in the fraction of the patient's plasma that passed through Protein A Sepharose, but detected in the elution with glycine-HCl buffer. Furthermore, using SDS-PAGE and immunoblotting method, purified IgG from the patient's plasma reacted with a protein with molecular weight (74KD) equivalent to F1F2 of factor Va which was activated by thrombin. The inhibitory activity was associated with IgG4 subclass. These data indicate that the inhibitor was IgG antibody which inhibit F1F2 of factor V. Autoimmune mechanism was strongly suggested for the development of the antibody.

Portal vein calcification: a clinical review of the last 50 years and report of a case associated with dysplasminogenemia.

We report herein a case of a 68-year-old Japanese woman in whom calcification of the portal vein was recognized by plain abdominal X-ray radiograph and computed tomography (CT) scan when she presented with repeated thrombosis of the portal system. Following emergency small bowel resection for intestinal necrosis caused by superior mesenteric vein thrombosis, hematological studies revealed the association of dysplasminogenemia. A review of 21 cases of portal vein calcification reported between 1940 and 1990 revealed the average age to be 53.7 +/- 10.2 years and the male/female ratio 17:4. Although the majority of cases suffered from portal hypertension (81%), only 38% had any evidence of liver cirrhosis, while 52% had normal liver function, being comparable to idiopathic portal hypertension. The calcified lesions were located in the portal vein in 100% of cases, the splenic vein in 62%, the superior mesenteric vein in 33%, and the inferior mesenteric vein in 0%. The precise etiology of the calcification was not elucidated in any of the reviewed cases. The patient reported herein is the first reported case of portal vein calcification due to repeated thrombosis of the portal system caused by dysplasminogenemia, which could be accounted as a cause of idiopathic portal hypertension.

[Negative interference with peroxidase-coupled enzymatic serum creatinine assay by a hemostatic drug containing a hydroquinone structure].

We report a discrepancy between serum creatinine levels obtained by the method based on alkaline picric acid (Jaffé reaction) and that by automated analyzer employing enzymatic assay based on peroxidase-coupled reaction in a patient with renal dysfunction taking Ethamsylate, a capillary vessel stabilizer. Serum creatinine level obtained by the Jaffé reaction was 83 mg/l, and that by the enzymatic method was 22 mg/l. Recovery test of creatinine using the patient's serum showed 101 to 102% recovery by the Jaffé reaction and 77 to 89% by the enzymatic method. The same results were observed both in an in vitro test and in serum obtained from a healthy volunteer who had been given Ethamsylate previously. As the chemical structure of Ethamsylate includes a hydroquinone unit, it was thought that the compound may consume hydrogen peroxide produced by the reaction of peroxidase as the hydrogen donor and compete with the substrate used as the hydrogen donor for the color development. Thus evaluation of test results obtained by peroxidase coupled methods should be carefully interpreted in a patient who is consuming drugs which hydrogen peroxide such as hydroquinone.