Summary. Vascular occlusion has a central role in the pathophysiology of sickle cell disease (SCD) and, although there is little evidence that thrombosis alone is responsible, patients with sickle cell disease are known to have an illdefined but increased thrombotic risk. The most serious complication of this in childhood is stroke which occurs in 7-10% of children and a further 14% have asymptomatic cerebrovascular disease (CVD) on imaging. We have performed a comprehensive profile of coagulation inhibitors and markers of thrombin generation in 96 children (83 nontransfused [NTx] and 13 transfused [Tx]) with steady-state SCD and 18 healthy sibling controls. The levels of protein S (free and total) and heparin cofactor II were reduced in both the NTx and Tx groups compared to controls and protein C and APC resistance ratios were reduced in the NTx group only. Antithrombin levels were not different from controls. Thrombin-antithrombin complexes and prothrombin fragment F1þ2 were increased in both patient groups. In the NTx subgroups with or without CVD there were no differences for any of the parameters measured except for lower haemoglobin levels and higher white cell counts in those with asymptomatic CVD. We conclude that children with SCD have a reduction in levels of the majority of the coagulation inhibitors and increased thrombin generation in the steady-state and these are only partially reversed by transfusion. However, these abnormalities do not appear to play a primary role in the development of cerebrovascular disease.

Summary. The association between antiphospholipid antibodies and an increased risk of thrombosis in antiphospholipid syndrome (aPS) patients is probably caused by numerous mechanisms, including the effects of antibodies to phospholipid-binding proteins such as b 2 -glycoprotein I and prothrombin. In this study, we investigated the inhibition of tissue factor pathway inhibitor (TFPI) in 33 patients with primary antiphospholipid syndrome (PAPS). TFPI was measured in PAPS patients using an amidolytic assay, dependent on the generation of activated factor X (Fxa), and this was compared with 55 healthy subjects. Functional levels of TFPI (mean^SD) were significantly lower in PAPS patients (0´89^0´37 U/ml) than the control group (1´05^0´15 U/ml) (P 0´02). The difference was caused by a subset of five patients who had TFPI levels below the lower 99% confidence interval of the normal reference range, representing increased FXa generation in the assay system. IgG fractions were isolated from these five patients and five control subjects, then incorporated into normal plasma to measure FXa generation in the TFPI assay system. FXa generation was increased when polyclonal rabbit anti-human TFPI IgG (P , 0´0001) or PAPS IgG (P 0´0001) were added to normal plasma, demonstrating inhibition of TFPI. The apparent anti-TFPI activity demonstrated in the five subjects with PAPS in this study may represent a significant new mechanism for thrombosis in patients with aPS, as it implies that increased tissue factor FVIIa-mediated thrombin generation might occur.

Anti‐phospholipid antibodies (aPL) are associated with an increased risk of thrombosis and recurrent fetal loss. Antibodies to prothrombin (aPT) have been associated with the anti‐phospholipid syndrome (aPS). We assessed variations in aPT assay methodology to optimize an aPT method that was used to screen patients with aPS (n = 66). Detection of aPT using enzyme‐linked immunosorbent assay was influenced by the concentration of the capture antigen, the microtitre plate type and the buffer system. The combination of γ‐irradiated plates, a phosphate‐buffered saline buffer and coating antigen of 10 μg/ml prothrombin was the most sensitive. Both serum and citrate samples are suitable for the detection of aPT. Under these conditions aPT IgM but not IgG were found to be associated with thrombosis and/or fetal loss.

SUMMARYIt is known that antiphospholipid antibodies (aPL) hamper the anticoagulant activity of the protein C system, but the mechanism is still obscure. In this study, we demonstrate that anticardiolipin antibodies (not anti-protein C autoantibodies) can bind protein C via b 2 -GPI, which bears their binding epitope, in a fashion dependent on negatively charged phospholipids. We studied the binding of IgG from aPL to protein C in the presence of b 2 -GPI by ELISA (anti-'protein C' antibody ELISA), and compared their binding with those obtained in the absence of b 2 -GPI. In the anti-'protein C' antibody ELISA system, 47% of 78 aPL þ patients had a positive titre in the presence of cardiolipin (CL) and b 2 -GPI, but binding was not found in the absence of b 2 -GPI. Highly significant correlations were found between the titre of anti-'protein C' antibody in the presence of b 2 -GPI and that of anti-b 2 -GPI antibody (r ¼ 0·802, P ¼ 0·0001). We further analysed the interaction between protein C, phospholipids, b 2 -GPI and human aCL MoAbs established from patients with antiphospholipid syndrome. In a first set of experiments, the binding of b 2 -GPI to protein C and its phospholipid dependency were investigated. b 2 -GPI bound to protein C in the presence of CL or phosphatidylserine, but not in the presence of phosphatidylcholine or phosphatidylethanolamine. In a second group of experiments, the binding of three human monoclonal aCL recognizing the cryptic epitope of b 2 -GPI (virtually anti-b 2 -GPI antibodies) was evaluated in the presence of cardiolipin and b 2 -GPI. All three human monoclonal aCL bound to protein C in the presence of CL and b 2 -GPI, whereas they did not in the absence of either b 2 -GPI or CL. These data suggest that protein C could be a target of aCL by making a complex with CL and b 2 -GPI, leading to protein C dysfunction.

Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis and recurrent miscarriage. We assessed levels of coagulation activation markers and aPL during normal pregnancy and in women with the antiphospholipid syndrome (aPS). Fluctuations in aPL levels were observed in all patients with aPS. No particular pattern of antibody positivity, or fluctuation in aPL level, was associated with poor pregnancy outcome. A significant increase was observed in levels of factor Xlla (FXIIa; P < 0.001), factor VIIa (FVIIa, P < 0.001), thrombin antithrombin complexes (TAT; P < 0.001), prothrombin fragment F1.2 (F1.2; P < 0.001) and D-dimer (DD; P < 0.05) during normal pregnancy. Factor VIIa, TAT, F1.2 and DD increased significantly before 20 weeks gestation, while a statistically significant increase in FXIIa levels was first detected between weeks 20 and 30 of gestation. In pregnant women with aPS, increases in FXIIa were similar to those in normal pregnancy, but increased FVIIa levels were not observed until after 30 weeks gestation. Similar to normal pregnancy, increased levels of TAT and F1.2 were detected in aPS pregnancies before 20 weeks gestation, but increased DD were not observed until after week 20. Surprisingly, women with aPS receiving low molecular weight heparin prophylaxis had significantly higher (P = 0.02) levels of TAT (median 8.6; interquartile range (IQR) 6.5-20.8) between weeks 20 and 30 of gestation compared to the normal pregnant population (median 5.9; IQR 4.7-7.9), thus indicating increased thrombin generation in women with aPS in mid-pregnancy.