Cardiac involvement in acute thrombotic thrombocytopenic purpura: association with troponin T and IgG antibodies to ADAMTS 13

Summary.  Introduction: Evidence for cardiac involvement in thrombotic thrombocytopenic purpura (TTP) is uncommonly described. Methodology: We retrospectively reviewed 41 patients assessing troponin T as a marker for cardiac involvement in acute TTP with clinical symptoms, electrocardiograms (ECG) and echocardiograms. A histopathological review of five patients who died of acute TTP was also undertaken. Results: In 54% (22/41) of patients, troponin T was ≥ 0.05μg L⁻¹ (normal range 0–0.01 μg L⁻¹). Half (12/22) had cardiac symptoms and 8/22 with a raised troponin T reported chest pain. ECG changes were present in 62% of patients with a raised troponin T. Median anti-ADAMTS 13 IgG antibody was significantly higher ( P  = 0.018) in patients with troponin T ≥ 0.05 μg L⁻¹ (58.5% (range 17–162%), compared with patients with troponin T < 0.05 μg L⁻¹ (35%, range 9–134%). Patients who died had higher troponin T levels (median 0.305 μg L⁻¹) and raised anti-ADAMTS 13 IgG (median 66.5%). On admission, there were no deaths in those with troponin T ≤ 0.04μg L⁻¹. Histology confirmed widespread myocardial microvascular thrombi. Conclusion: Clinical symptoms, ECG changes and echocardiograms are poor predictors of cardiac disease in acute TTP. Troponin T is specific for cardiac muscle and a sensitive marker of myocardial damage. In TTP patients, raised levels (≥ 0.05 μg L⁻¹) signify myocardial necrosis associated with microvascular thrombi. Mortality and acute morbidity was associated with higher admission troponin T and raised IgG antibody (> 67%) to ADAMTS 13.     

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. Objective: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. Methods and Results: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. Conclusions: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.     

Screening and characterization of specific anti-lipopolysaccharide antibodies in Belgian blood donors by enzyme-linked immunosorbent assays

The goal of this project was to find and collect high concentrations of endotoxin-specific antibodies for therapeutic IgG- or IgM-enriched preparations. Various enzyme-linked immunosorbent assays (ELISAs) were developed to perform longitudinal studies of the serological response to a large panel of smooth and rough purified lipopolysaccharide (LPS) extracts in a population of healthy blood donors. To accomplish this, 1612 human serum samples from volunteer blood donors collected by seven different blood banks in Belgium were screened and specific IgM and IgG activities were measured. Approximately 17% of the donors had anti-LPS concentrations higher than 40 mg L⁻¹. Of these, 10.9% had anti-smooth LPS antibodies, 3.7% had anti-rough LPS antibodies and 2.8% were found to be positive towards both types of LPS. The mean anti-LPS antibody concentration was 8 mg L⁻¹ for rough LPS and 14 mg L⁻¹ for smooth LPS. Age- and sex-related distributions of the activities indicated that the greatest prevalence of high anti-LPS concentration was in women aged 40–49 years and in men older than 60 years. Differential absorption experiments showed that the pooled serum of selected blood donors contained a mixture of specific and cross-reacting antibodies. We detected predominantly anti-LPS activities due to the IgG₁ and IgG₂ subclasses. The range of specificities to different LPS was increased by the pooling of selected sera. It was concluded that pools of naturally occurring specific anti-LPS immunoglobulin antibodies may be obtained in Belgium by screening blood donors using ELISAs that we have developed.     

Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura

BACKGROUND: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. METHODS: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. RESULTS: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. CONCLUSION: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.     

The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission

Summary.  Background:  Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. Objective:  To study ADAMTS13 and VWF-related features in TTP patients in remission. Methods:  ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibα-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. Results:  ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. Conclusion : ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.     

ADAMTS‐13 activity and autoantibodies classes and subclasses as prognostic predictors in acquired thrombotic thrombocytopenic purpura

Summary. Background: Thrombotic thrombocytopenic purpura (TTP) is a rare life‐threatening disease. Of surviving patients, 45% develops an exacerbation or a late recurrence. Severe ADAMTS‐13 deficiency, both during the acute episode and remission, is a well‐established predictor of recurrence. The predictive value of anti‐ADAMTS‐13 antibodies, their inhibitory activity and Ig class subtype for disease recurrence is still to be established. Objectives: To analyze ADAMTS‐13‐related biomarkers (ADAMTS‐13 and anti‐ADAMTS‐13 immunoglobulins, classes and subclasses) and their potential relationship with prognosis. Patients/Methods: In 115 patients with TTP, we assessed the association between levels of these biomarkers and the severity of acute episodes; we analysed also the hazard ratio (HR) and 95% confidence interval (CI) of recurrence in association with biomarkers levels retrieved at the previous acute episode or during remission, using Cox regression models. Results: During the acute phase, higher IgA, IgG1 and IgG3 titers showed the strongest association with acute episode severity. In the survival analyzes, the only biomarker significantly associated with a high hazard of recurrence after an acute episode was the presence of IgG. Conversly, low ADAMTS‐13 activity or antigen levels (< 10%), the presence of ADAMTS‐13 inhibitor or IgG during remission were all significantly associated with a higher hazard of recurrence. Conclusions: Both the Ig class and subclass are of predictive value for acute episode severity in patients with TTP. Although markers that could predict the risk of recurrence in the acute phase are limited, a thorough assessment of ADAMTS‐13‐related parameters during remission is warranted.     

Early atherosclerosis and IgG2 to bacteria are associated with FcγRIIa genotype in non-smokers

Background  Involvement of low density lipoprotein (LDL) immune complexes (ICs) in atherogenesis has been proposed. Human FcγRIIa receptor (CD32) plays a crucial role in the phagocytosis of IgG₂ ICs and a functional point mutation 131His/Arg diminishes IgG₂ binding to the receptor.
Study design  We examined FcγRIIa-131His/Arg polymorphism, IgG₂ antibody titres to oxidized low-density lipoprotein (OxLDL) and Streptococcus pneumoniae cell wall polysaccharide (CWPS) and subclinical atherosclerosis in a large cohort of Finnish subjects ( n  = 1041).
Results  Non-smoking subjects with homozygous 131His/His genotype had more premature atherosclerosis ( P  =   0·004) and higher IgG₂ to bacterial CWPS ( P  =   0·002) compared with other genotypes. Smoking subjects had significantly higher intima-media thickness (IMT) than that of non-smokers ( P  <   0·001) and genotype-dependent associations were indistinct. There was no association between FcγRIIa genotype and antibody titres to OxLDL.
Conclusions  Our data demonstrate that FcγRIIa 131His/Arg polymorphism is associated with subclinical atherosclerosis in non-smoking subjects. Furthermore, FcγRIIa genotype is associated with IgG₂ titres to bacterial CWPS, but not to OxLDL. These data propose possible involvement of FcγRIIa receptor in atherogenesis.     

影响获得性血栓性血小板减少性紫癜首次缓解期内复发的相关指标研究

目的 探讨血管性血友病因子裂解蛋白酶(ADAMTS) 13活性和抗ADAMTS13抗体表达水平,与获得性血栓性血小板减少性紫癜(TTP)于首次缓解期内复发的关系.方法 选择2008年3月至2014年6月于陕西延安大学附属医院和陕西省渭南市富平县医院诊治的37例获得性TTP患者为研究对象,按照其在首次缓解随访期内是否复发,分为研究组(n=15)和对照组(n=22).分别采用残余胶原结合试验、ELISA、免疫印迹等方法,检测两组患者的ADAMTS13活性,ADAMTS13抗原水平,抗ADAMTS13抗体,ADAMTS13抑制物,血管性血友病因子(vWF)抗原和超大分子量vWF(ULVWF)多聚体等指标,并且进行统计学分析;采用多因素非条件logistic回归分析法,评估获得性TTP患者于首次缓解期内复发的独立影响因素.本研究遵循的程序符合病例收集医院人体试验委员会所制定的伦理学标准,得到该伦理会批准,分组征得受试对象本人的知情同意,并与之签订临床研究知情同意书.结果 ①研究组患者的中位ADAMTS13活性为11％(7％～124％),低于对照组的53％(7％～151％),差异有统计学意义(u=4.018,P＜0.05).研究组与对照组患者的ADAMTS13活性显著降低率分别为53.3％(8/15)和22.7％(5/22),二者比较,差异有统计学意义(P=0.049).②37例获得性TTP患者的血浆ADAMTS13活性和ADAMTS13抗原水平呈正相关关系(rs=0.810,P=0.001).研究组患者的中位ADAMTS13抗原水平为33％(3％～99％),低于对照组的59％(3％～128％),差异有统计学意义(u=4.121,P＜0.05).研究组与对照组ADAMTS13抗原水平显著降低率分别为13.3％(2/15)和9.1％(2/22),二者比较,差异有统计学意义(P=0.008).③研究组患者的抗ADAMTS13抗体检出率为66.7％(10/15),高于对照组的36.4％(8/22),差异有统计学意义(P=0.007).④研究组患者中抗ADAMTS13抑制物检出率为46.7％ (7/15),高于对照组患者的18.2％ (4/22),差异有统计学意义(P=0.011).⑤研究组与对照组患者ULVWF多聚体检出率分别为20.0％(3/15)和13.6％(3/22),二者比较,差异有统计学意义(P=0.042).⑥多因素非条件logistic回归分析结果显示,获得性TTP患者于首次缓解期内复发的独立危险因素包括ADAMTS13活性显著降低(OR=2.95,95％ CI:1.13～6.96,P＜0.05),检出抗ADAMTS13抗体(OR=3.31,95％CI:1.08～8.19,P＜0.05),检出抗ADAMTS13抑制物(OR=3.24,95 ％CI:1.24～9.03,P＜0.05).结论 获得性TTP患者在首次缓解期内,ADAMTS13活性水平显著降低,存在抗ADAMTS13抗体及抗ADAMTS13抑制物,会显著增加疾病复发风险,可以考虑将其作为预测获得性TTP患者于首次缓解期内复发的重要指标.     

Post-transfusion purpura: a survey of 12 Danish cases with special reference to immunoglobulin G subclasses of the platelet antibodies

SUMMARY. The clinical and immunological data of 12 Danish cases of post-transfusion purpura (PTP) are summarized. All patients but one were women. All except one had thrombocytopenic purpura which occurred 4–11 days after transfusion, usually with a nadir of the platelet count below 10times10⁹ 1^-1. The typical haemorrhagic symptoms were cutaneous bleedings, melena and haematuria lasting from 3 to 12 days. The time until normalization of platelet count varied from 3 to 50 days after onset. One patient had recurrence of PTP and one patient died due to intracranial haemorrhage.
Ten of the HPA-la negative patients (83%) had platelet-specific HPA-la antibodies and two HPA-lapositive individuals had anti-HPA-lbantibodies. In 10 patients, HLA antibodies were also detectable and in one patient a delayed haemolytic transfusion reaction due to anti-E was seen.
All of seven patients investigated had anti-HPA antibodies of both IgGl and IgG3 subclasses during the thrombocytopenic period while all of 10 patients had anti-HPA of only the IgGl subclass after recovery from PTP Thus, the destruction of autologous platelets in PTP seems to be associated with the presence of anti-HPA of the IgG3 subclass which may be of importance in the pathogenesis of PTP.     

Prevalence of the ADAMTS-13 missense mutation R1060W in late onset adult thrombotic thrombocytopenic purpura

Summary.  Background:  Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. Objectives:  We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. Patients/Methods/Results:  We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls ( n  = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS-13 activity levels <5% at presentation (normal: 66–126%). Antibodies/inhibitors to ADAMTS-13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first-degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS-13 activity. Conclusion:  The high prevalence of R1060W ADAMTS-13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.     

Significance of IgG-subclass antibody determinations in bullous pemphigoid

The presence of circulating basement membrane zone (BMZ) antibodies is characteristic of patients with bullous pemphigoid (BP) and are routinely employed in making the diagnosis. The positive tests, however, occur in 50–70% of patients with BP, thus necessitating consideration of other tests in a significant number of patients. The purpose of this study was to examine the efficacy of indirect immunofluorescence (IF) tests specific for various IgG subclasses antibodies to BMZ in BP, especially in patients who are seronegative on routine indirect IF tests with fluorescein-conjugated antibodies to IgG. BMZ antibodies primarily are of IgG4 subclass and are present in all BMZ antibody positive BP cases. Of BP patients negative for BMZ antibodies, 72% were found positive when tested for IgG4 subclass antibodies. In conclusion, testing for IgG4 subclass BMZ antibodies enhances the sensitivity of serum tests from 68.5% to 91%. This may be due in part to the inherent increased sensitivity of the assay and for detecting subclasses of IgG and in part, due to the subclass distribution of the BP antibodies. © 1996 Wiley-Liss, Inc.     

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl₄) in rats. Six groups were analysed: (1) CCl₄ (0.4 g kg⁻¹, i.p., three times a week) for 13 weeks; (2) CCl₄ for 8 weeks followed by colchiceine (60 μg kg⁻¹) + CCl₄ for 5 weeks; (3) CCl₄ for 8 weeks and thereafter UDCA (25 mg kg⁻¹) + CCl₄ for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na⁺,K⁺- and Ca²⁺-ATPase activities and the cholesterol–phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.     

Inhibitory autoantibodies against ADAMTS-13 in patients with thrombotic thrombocytopenic purpura bind ADAMTS-13 protease and may accelerate its clearance in vivo

BACKGROUND: Many patients with acquired thrombotic thrombocytopenic purpura (TTP) harbor autoantibodies that may bind and/or inhibit ADAMTS-13 proteolytic activity and accelerate its clearance in vivo. METHODS: To test this hypothesis, we determined ADAMTS-13 activity and antigen levels in parallel plasma samples from patients clinically diagnosed with TTP. Collagen binding, GST-VWF73 and FRETS-VWF73 assays were used to determine ADAMTS-13 activity and to detect inhibitory autoantibodies. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation plus Western blotting (IP/WB) were used to detect total anti-ADAMTS-13 IgG (inhibitory and non-inhibitory). RESULTS: Among 40 patients with TTP (21 idiopathic and 19 non-idiopathic), inhibitory autoantibodies were detected (by FRETS-VWF73) in 52% of idiopathic and 0% of non-idiopathic TTP patients. In contrast, non-inhibitory IgG autoantibodies were detected in 29% of idiopathic and 50% of non-idiopathic TTP patients. The concentration of inhibitory IgG autoantibody in idiopathic TTP patients was significantly higher than that of non-inhibitory IgG in either idiopathic or non-idiopathic TTP patients. Idiopathic TTP patients demonstrated significantly reduced ADAMTS-13 activity compared with non-idiopathic patients, but only slightly lower ADAMTS-13 antigen levels. Interestingly, patients with inhibitory autoantibodies exhibited significantly lower ADAMTS-13 antigen levels than those with only non-inhibitory IgG autoantibodies or no autoantibody. Serial plasma exchanges increased levels of ADAMTS-13 activity and antigen concurrently in patients with inhibitory autoantibodies. CONCLUSION: The identification of severe ADAMTS-13 deficiency and autoantibodies or inhibitors appears to be assay-dependent; the inhibitory IgG autoantibodies, in addition to binding and inhibiting ADAMTS-13 proteolytic activity, may accelerate ADAMTS-13 clearance in vivo.     

Comparison of IgG subclasses and complement binding activity of autoantibodies from patients with bullous pemphigoid and pemphigus

Bullous pemphigoid and pemphigus are autoimmune disorders of skin of unknown etiology and are characterized by the presence of immunoreactants in the skin and circulating autoantibodies to skin components. The distribution of IgG subclass antibodies to intercellular substance (ICS) of pemphigus and basement membrane zone substance (BMZ) of bullous pemphigoid was analyzed by using monoclonal antibodies to human IgG subclasses. IgG4 type anti-BMZ antibody was found in the majority of patients with bullous pemphigoid (88% in skin and 96% in serum). One third to one half of bullous pemphigoid patients had IgG1 and IgG2 anti-BMZ antibodies. The majority of bullous pemphigoid skin (92%) had complement in skin, however only one third of their sera had complement binding activity in vitro. IgG1 anti-ICS antibody was the predominant one in patients with pemphigus (86% in skin and 80% in circulation). IgG4 anti-ICS antibody was seen in two thirds of specimens from pemphigus patients. IgG3 subclass antibody was more frequently seen in pemphigus than in bullous pemphigoid patients. Two-thirds of pemphigus sera were capable of activating complement in vitro. The complement binding activity was directly associated with IgG1 and/or IgG3 subclass antibodies. The possible mechanisms for the restricted IgG4 subclass antibodies in bullous pemphigoid and pemphigus are discussed.     

Treatment of autoimmune thrombotic thrombocytopenic purpura in the more severe forms

Daily therapeutic plasma exchange (TPE) transformed the historically fatal prognosis of acquired, anti-ADAMTS13 antibody-mediated thrombotic thrombocytopenic purpura (TTP), leading to the current overall survival rates of >80%. However, relapses occur in up to 40% of patients and refractory disease with fatal outcomes still occurs. In this context, the introduction of rituximab has probably been the second major breakthrough in TTP management. Rituximab is now routinely recommended during the acute phase, typically in patients with a suboptimal response to treatment, or even as frontline therapy, with high response rates. In more severe patients, salvage strategies may include twice daily TPE, pulses of cyclophosphamide, vincristine, as well as splenectomy in the more desperate cases. In this life-threatening disease, relapses can be efficiently prevented in patients with a severe acquired ADAMTS13 deficiency and otherwise in remission with the use of rituximab. In the coming years, the TTP therapeutic landscape should be enriched by original strategies stemming from clinical experience and new agents that are currently being evaluated in large, ideally international, clinical trials. Promising agents under evaluation include N-acetylcysteine, bortezomib, recombinant ADAMTS13 and caplacizumab, an inhibitor of the glycoprotein-Ib/IX-von Willebrand factor axis.     

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model

Summary.  Objective: The pathogenicity of anti-human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 n m , followed by injection of human B domain-deleted FVIII at 180 U kg⁻¹, producing peak plasma concentrations of ∼2 n m . At 2 h, blood loss following a 4-mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti-A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti-C2 inhibitors, (iii) 2–77 and B45, non-classical type II anti-C2 inhibitors, and (iv) 2–117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg⁻¹ overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL⁻¹ and 3 U mL⁻¹ FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose FVIII rather than bypassing agents may be warranted.     

Homocysteine levels are associated with the severity of peripheral arterial disease in Type 2 diabetic patients

Summary.  Background : Homocysteine levels are positively associated with the risk of cardiovascular disease. They might be determined by both MTHFR677C→T polymorphisms and folate or B-vitamin status. Objectives : To investigate the possible association between plasma homocysteine levels and its genetic or environmental determinants and either the presence or the severity of peripheral arterial disease (PAD), in Type 2 diabetic patients. Methods : From a cohort of 944 patients with Type 2 diabetes, 135 patients with PAD were selected, and frequency-matched for age and sex with 219 Type 2 diabetic control patients without macrovascular complications. According to the increasing severity of the disease, patients were divided into PAD₁ (only diffuse calcifications of the arteries without any stenosis or occlusion), PAD₂ (one or two stenosis or occlusions) and PAD₃ (three or more). Results : Homocysteine levels were similar in control and case patients (10.3 µmol L⁻¹ vs. 10.7 µmol L⁻¹, P  = 0.53); however, a significant increase was found in PAD₃ patients: odds ratio = 2.77 (95% confidence interval 1.14, 6.72) for patients with homocysteine levels above the median vs. those under the median in multivariate analysis. Although all significantly associated with homocysteine levels, neither MTHFR genotype nor folic acid or vitamin B₁₂ levels were associated with severity of PAD. A significant interaction ( P  < 0.05) was found between folic acid and MTHFR polymorphism in determining the levels of homocysteine. Conclusions : In Type 2 diabetes, homocysteine was associated with the angiographic severity of PAD, but neither the genotypes nor vitamin levels contributed to this association.     

IgG heavy-chain subclass restriction of thyrotropin-binding inhibitory immunoglobulins in Graves'' disease

The IgG subclass composition of antibodies is an important determinant of their function. Thyrotropin receptor antibodies cause the hyperthyroidism of Graves' disease but their subclass distribution has been incompletely investigated. We have therefore purified IgG subclasses from Graves' sera by passage over affinity columns designed to deplete all but a single subclass, and then assayed those pure subclass fractions for their ability to displace radiolabelled thyrotropin from its solubilized receptor as a measure of thyrotropin receptor antibody activity. Sufficient activity was recovered for analysis in nine of 10 Graves' patients, in five of whom activity was almost completely (97-100%) restricted to the IgG1 subclass; in the remaining four patients the response was predominantly IgG1 and IgG4 with marked under-representation of the IgG2 subclass. This contrasts with the unrestricted subclass response, in the same fractions, for autoantibodies against thyroglobulin and microsomes. These results suggest that there may be a primary defect at the B-cell level in Graves' disease.     

Insulin inhibits tissue factor expression in monocytes

Summary.  Objectives:  Platelets from healthy subjects are inhibited by insulin but type 2 diabetes mellitus (T2DM) platelets have become insulin-resistant, which might explain their hyperactivity. In the present study we investigated whether monocytes are responsive to insulin. Methods and results:  LPS-induced tissue factor (TF) upregulation was measured in human monocytes and monocytic THP-1 cells in a factor Xa generation assay. Insulin (0.1–100 nmol L⁻¹) induced a dose-dependent inhibition in both cell types and in monocytes 100 nmol L⁻¹ insulin inhibited cytosolic, membrane-bound and microparticle TF by 32 ± 2, 27 ± 3 and 52 ± 4% ( n  = 3). Insulin induced Tyr phosphorylation of the insulin receptor (INS-R) and formation of an INS-R – G_iα₂ complex, suggesting interference with LPS-induced cAMP control. Indeed, insulin interfered with LPS-induced cAMP decrease and TF upregulation in a manner similar to an inhibitor of G_i (pertussis toxin) and agents that raise cAMP (iloprost, forskolin, IBMX) reduced TF upregulation. Although LPS failed to raise cytosolic Ca²⁺, quenching of Ca²⁺ increases (BAPTA-AM) reduced and induction of Ca²⁺ entry (ionophore, P2X7 activation) enhanced upregulation of TF mRNA and procoagulant activity. Insulin interfered with MCP-1-induced Ca²⁺ mobilization but not with ATP-induced Ca²⁺ rises. Conclusions:  Insulin inhibits TF expression in monocytes and monocyte-derived microparticles through interference with G_iα₂-mediated cAMP suppression, which attenuates Ca²⁺-mediated TF synthesis.