John C. Giddings

C-Reactive Protein–Induced In Vitro Endothelial Cell Activation Is an Artefact Caused by Azide and Lipopolysaccharide

Objective—C-reactive protein (CRP) has been proposed to be an independent risk factor for cardiovascular disease. In vitro studies investigating the mechanism behind this have used purified commercial CRP (cCRP) and endothelial cells. We investigated the role of contaminants in cCRP preparations. Methods and Results—Human umbilical vein endothelial cells and the human endothelial cell line EA.hy926 were incubated with Escherichia coli–derived cCRP, in-house–generated azide-free recombinant, and ascites-purified CRP, azide, or lipopolysaccharide (LPS) equivalent to the concentration present in cCRP preparations. Cells were investigated for change in cell proliferation, morphology, apoptosis, and expression of endothelial NO synthase and intercellular adhesion molecule-1. Cell supernatants were assessed for monocyte chemoattractant protein-1 (MCP-1), interleukin-8, von Willebrand factor secretion, and pH change. Only cCRP was able to induce all activation events analyzed; however, this ability was lost on extensive dialysis, suggesting that low molecular weight contaminants were responsible for these events. Indeed, the effects of cCRP were mirrored by azide or LPS. Conclusions—We investigated a wide range of effects on endothelial cells ascribed to CRP; however, azide and LPS, but never CRP itself, were responsible for the cell activation events. We conclude that CRP, per se, does not activate endothelial cells.

A population based, unselected, consecutive cohort of patients with acquired haemophilia A

Previous studies in acquired haemophilia A have reported on cohorts of patients referred to specialist centres or were retrospective surveys of specialist centre experience. This may have resulted in the literature representing a more severe group of patients than seen in routine haematological practice. We report on a consecutive, unselected cohort of all patients in south and west Wales who presented with acquired haemophilia A between 1996 and 2002. There were 18 patients, an incidence of 1·34/million/year. Compared with previously reported cohorts our patients were older, with a median age of 70 years, and less likely to have an underlying diagnosis (27%). The bleeding phenotype was less severe, with only 27% having life or limb threatening bleeds and 41% required no haemostatic treatment. One patient died of bleeding, but three died of complications related to immunosuppression. Response to immunosuppression was high compared with other series, with 88% of treated patients attaining an undetectable inhibitor and normal factor VIII level. These data suggest that previously reported cohorts may represent more severely affected patients and, whilst guidelines for treatment based on these assumptions may be valid for severely affected patients, they may not be universally applicable.

Protective Effects of Ticlopidine and Aspirin, Administered Alone and in Combination, on Thrombus Formation in Rat Cerebral Vessels

The protective effects of ticlopidine and d,l-lysine acetylsalicylate (L-ASA), used alone and in combination, on the pathogenesis of thrombosis in cerebral blood vessels were investigated in a rat animal model using a He-Ne laser method. Ticlopidine and L-ASA, given orally at a concentration from 100 mg/kg, inhibited thrombus formation in a dose-dependent manner. Ticlopidine (300 mg/kg p.o.) inhibited thrombosis in arterioles and venules for 3 days after administration. The inhibitory activity of L-ASA (300 mg/kg p.o.) was less prolonged than that of ticlopidine and was observed for only approximately 24 h. Combined administration of ticlopidine and L-ASA significantly enhanced and prolonged the antithrombotic effects of either drug given alone. The results demonstrate that ticlopidine and L-ASA have potent antithrombotic properties in rat cerebral blood vessels in vivo.

Unresponsiveness to factor VIII inhibitor bypassing agents during haemostatic treatment for life-threatening massive bleeding in a patient with haemophilia A and a high responding inhibitor.

Summary.   We report a case of haemophilia A with a high responding inhibitor of factor VIII (FVIII) who had a serious retroperitoneal haematoma caused by penetration of a duodenal ulcer. Inhibitor‐bypassing therapy was commenced immediately on admission. On the 17th day of treatment with activated prothrombin complex concentrate (APCC; FEIBA®), re‐bleeding occurred and thrombelastography (TEG) demonstrated resistance to therapy. Treatment was changed to recombinant activated factor VII (rFVIIa; NovoSeven®) and resulted in clinical improvement together with an improvement in TEG parameters. On the 10th day of continuous infusion with NovoSeven®, however, TEG again showed resistance to therapy. FEIBA® infusions were re‐introduced and TEG results remained satisfactory for 7 days. On day 34, however, further retroperitoneal bleeding was evident and a decline in the haemostatic efficiency of FEIBA® was recorded by TEG. NovoSeven® was again successfully administered for 7 days. There were no laboratory findings to indicate disseminated intravascular coagulation (DIC), hypercoagulability or abnormal fibrinolysis. The plasma‐based clotting tests did not show any additional prolongation on the occasions when the TEG demonstrated unresponsiveness to FEIBA® or NovoSeven®. These findings suggested that some component of whole blood, other than plasma might have governed the TEG data. The long‐term use of APCC such as FEIBA® or rFVIIa, requires careful monitoring in terms of FVIII inhibitor bypassing activity as well as the tendency to DIC.

The characterization and synthesis of antigens related to factor VIII in vascular endothelium

Abstract Umbilical cord endothelial cells were obtained by collagenase digestion and maintained in culture. Release of factor VIII related antigen (FVIIIRAG) and ristocetin co-factor activity were demonstrated by specific assays. Synthesis of FVIIIRAG was confirmed by incorporation of 35S L-methionine into specific immunoprecipitate and demonstrated by two dimensional crossed immunoelectrophoresis (2DCIE) and autoradiography. FVIIIRAG synthesised by endothelial cells had increased anodal electrophoretic mobility compared to that in normal adult and cord blood. Release of procoagulant factor VIII (FVIIIC) by cultured endothelial cells was not demonstrated by coagulation assay. A rabbit antiserum to FVIIIRAG/FVIIIC was rendered non precipitating and apparently monospecific for FVIIIC by absorption with immobilised dissociated FVIIIRAG. Using this antiserum antigenic determinants of FVIIIC were not detected by immunohistological means in cultured endothelial cells or vascular intima. The results suggest that the endothelial cells released a modified, incomplete or precursor form of factor VIII.

Preventive Effects of Hesperidin, Glucosyl Hesperidin and Naringin on Hypertension and Cerebral Thrombosis in Stroke‐prone Spontaneously Hypertensive Rats

The effects of hesperidin, glucosyl hesperidin (G‐hesperidin), a water‐soluble derivative of hesperidin, and naringin on blood pressure and cerebral thrombosis were investigated using stroke‐prone spontaneously hypertensive rats (SHRSP). Hesperidin, G‐hesperidin and naringin were mixed with diet and fed to the animals for 4 weeks. No effect was evident on body weight, but the supplements significantly suppressed the age related increase in blood pressure. Thrombotic tendency, as assessed using a He‐Ne laser technique in the cerebral blood vessels, was significantly decreased in the treated animals compared with the control animals. Measurements of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) demonstrated that the supplements had strong antioxidant activity. Furthermore, these supplements significantly increased the production of nitric oxide (NO) metabolites in urine measured with Griess reagent. Vasodilation induced by acetylcholine‐mediated NO production in the endothelium was assessed using thoracic aortic ring preparations and indicated that endothelial function was significantly improved by the administration of these supplements.

Effects of Ginkgo biloba extract (EGb 761) on cerebral thrombosis and blood pressure in stroke‐prone spontaneously hypertensive rats

1. An extract of Ginkgo biloba (EGb 761) has been reported to alleviate cerebrovascular problems. In the present study, we investigated the antithrombotic effects of EGb 761 in cerebral blood vessels of stroke‐prone spontaneously hypertensive rats (SHRSP/Izm).

Immunological characterization of factor VIII autoantibodies in patients with acquired hemophilia A in the presence or absence of underlying disease

The development of a factor VIII autoantibody results in a severe hemorrhagic diathesis known as acquired hemophilia A. Underlying pathologies, such as autoimmune disease or chronic inflammatory disease, are observed in about half of the patients. We have investigated a total of 16 cases with acquired hemophilia A and divided the patients into two groups according to the presence or absence of other clinical conditions. Group A comprised nine cases with no detectable associated pathology. Group B consisted of seven cases with other clinical diagnoses. Significant levels of factor VIII activity (FVIII:C) and factor VIII antigen (FVIII:Ag) were detected in Group A and the pattern of FVIII:C inactivation was characteristic of Type 2 inhibitors. In contrast, no FVIII:C was detected in Group B and, in five of seven cases, the inhibitory pattern was Type 1. IgG(4) antibody subclass specificity was dominant in both groups. IgG1 antibody reactivity was higher in Group B than in Group A. Our results suggested a close relationship between the presence of underlying disease and immunological and coagulation characteristics in acquired hemophilia A.

Segregation of a pure form of spastic paraplegia and NOR insertion into Xq11.2

A 3-year-old boy, his 7-year-old brother, and a maternal uncle had a pure form of spastic paraplegia and a variant X chromosome with a faintly stained gap at Xq11.2. The mother of the propositus also had the variant X chromosome but was clinically unaffected. Three other unaffected females in the family did not have the variant X chromosome. The gaps in the variant X chromosome from the affected members and the mother were Ag-NOR staining positive, C-banding negative, rDNA FISH analysis positive, and alpha-satellite FISH analysis negative. The gap, therefore, represented an insertion of the nucleolus organizer region (NOR) derived from the short arm of an acrocentric chromosome. The variant X chromosome of the mother was randomly inactivated, as evidenced by BrdU replication analysis of her Epstein-Barr virus-transformed lymphoblastoid cells. Because mutations of the proteolipid protein gene at Xq21 have been responsible for a pure form of spastic paraplegia, this was also investigated but found to be negative in all affected relatives. Summing up these findings, it is proposed that the NOR insertion in the affected members of the family disrupted a hitherto unknown gene for a pure form of spastic paraplegia, situated at Xq11.2, and caused the disorder.

MEASUREMENT OF ANTI-FACTOR IX IGG SUBCLASSES IN HAEMOPHILIA B PATIENTS WHO DEVELOPED INHIBITORS WITH EPISODES OF ALLERGIC REACTIONS TO FACTOR IX CONCENTRATES

We have established a simple enzyme-linked immunosorbent assay (ELISA) for the detection of anti-factor IX IgG subclasses in haemophilia B patients with inhibitors. The assay was performed using immobilized purified factor IX. Specific IgG subclasses were detected by peroxidase-conjugated anti-human IgG1,2,3 and 4. Ten plasma samples from 6 haemophilia B patients with inhibitors ranging from 1.0 to 253 Bethesda Units/ml were analyzed. All samples were positive for IgG4. Six out of 10 samples were positive only for IgG4. Three samples were positive for IgG2. Five of the 6 patients had previously had allergic reactions to factor IX concentrates. Three patients had allergic episodes within the past month. Three samples from these latter patients taken on the day when the allergy had occurred showed positive also for IgG1. In later samples, however, taken at 4 days and 4 weeks respectively from two of these same patients. IgG1 was not detected. In two of the five patients in whom allergic reactions had occurred more than one month previously IgG1 was not detected. The results suggested that allergic reactions in patients with haemophilia B treated with factor IX concentrates were associated with the development of the specific IgG1 subclass of antibody to factor IX.