Raymond Camilleri

Thrombotic thrombocytopenic purpura and pregnancy: presentation, management, and subsequent pregnancy outcomes

Pregnancy can precipitate thrombotic thrombocytopenic purpura (TTP). We present a prospective study of TTP cases from the United Kingdom Thrombotic Thrombocytopenic Purpura (UK TTP) Registry with clinical and laboratory data from the largest cohort of pregnancy-associated TTP and describe management through pregnancy, averting fetal loss and maternal complications. Thirty-five women presented with a first TTP episode during pregnancy: 23/47 with their first congenital TTP (cTTP) episode and 12/47 with acute acquired TTP in pregnancy. TTP presented primarily in the third trimester/postpartum, but fetal loss was highest in the second trimester. Fetal loss occurred in 16/38 pregnancies before cTTP was diagnosed, but in none of the 15 subsequent managed pregnancies. Seventeen of 23 congenital cases had a missense mutation, C3178T, within exon 24 (R1060W). There were 8 novel mutations. In acquired TTP presentations, fetal loss occurred in 5/18 pregnancies and 2 terminations because of disease. We also present data on 12 women with a history of nonpregnancy-associated TTP: 18 subsequent pregnancies have been successfully managed, guided by ADAMTS13 levels. cTTP presents more frequently than acquired TTP during pregnancy and must be differentiated by ADAMTS13 analysis. Careful diagnosis, monitoring, and treatment in congenital and acquired TTP have assisted in excellent pregnancy outcomes.

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.

The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies

Summary.  The importance of testing for anticardiolipin antibodies (aCL) in the diagnosis of antiphospholipid syndrome (APS) in patients with thrombosis has recently been challenged (ISTH SSC meeting, Boston 2002). We have analyzed the antiphospholipid serology of 123 patients with persistent antiphospholipid antibodies (aPL) attending our hematology department. The cohort was tested for anti‐β2‐glycoprotein I (β2‐GPI) antibodies and aCL of IgG and IgM class and for lupus anticoagulant (LA). Ninety‐six of these patients fulfilled Sapporo clinical criteria for APS and 70 of these patients had venous and/or arterial thrombosis. Patients with LA plus anti‐β2‐GPI antibodies had significantly higher levels of IgG aCL and anti‐β2‐GPI antibodies than those exhibiting positivity for only LA or anti‐β2‐GPI antibodies (P < 0.05). Patients with aCL IgG levels over 60 GPLU were found in all cases to be positive for LA and anti‐β2‐GPI antibodies; 25.2% (31/123) of all patients and 26.04% (25/96) of patients fulfilling Sapporo clinical criteria for APS were positive for aCL only. The mean IgG aCL level in the Sapporo clinical criteria positive patients who had aCL only was 11.5 GPLU (normal < 5 GPLU). These data indicate that omission of aCL testing from the clinical investigation of APS could lead to a failure to diagnose the syndrome in a proportion of patients.

A phenotype–genotype correlation of ADAMTS13 mutations in congenital thrombotic thrombocytopenic purpura patients treated in the United Kingdom

Summary.  Background:  ADAMTS13 mutations play a role in thrombotic thrombocytopenic purpura (TTP) pathogenesis.

The expression of prion protein by endothelial cells: a source of the plasma form of prion protein?

Summary. The neuronal prion protein (PrPC) is also expressed within peripheral tissues including human blood. The majority of blood PrPC is found within the plasma fraction. We hypothesized that the vascular endothelium could be a source of this PrPC. Reverse transcription polymerase chain reaction demonstrated that both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC‐1) expressed PrPC mRNA. Flow cytometry confirmed PrPC expression on HMEC‐1s and HUVECs (120 900 ± 15 058 and 58 327 ± 4577 molecules PrPC/cell respectively), with no upregulation following cellular activation. Confocal immunofluorescence microscopy confirmed that HMEC‐1s and HUVECs were positive for PrPC on the plasma membrane. Time‐resolved dissociation‐enhanced fluoroimmunoassay (DELFIA®) analysis of cell culture medium demonstrated a slow constitutive release of soluble PrPC not associated with activation. In contrast to von Willebrand factor antigen, PrPC plasma levels in vivo decrease following desmopressin therapy in patients with von Willebrand disease. Measurement of PrPC plasma levels in patients with varying blood counts demonstrated no association between cell count and PrPC concentration. However, there was a higher level of PrPC in plasma from patients with end‐stage renal failure. In conclusion, endothelial cells of both macrovascular and microvascular origin expressed high levels of PrPC which can be constitutively released into the cell culture medium.

Lack of association of β2-glycoprotein I polymorphisms Val247Leu and Trp316Ser with antiphospholipid antibodies in patients with thrombosis and pregnancy complications

Summary. Beta2‐glycoprotein I (β2GPI) is an important target antigen for antiphospholipid antibodies (aPL) and thus β2GPI polymorphisms may influence aPL production and the development of antiphospholipid syndrome. We have studied the relationship between the Val247Leu and Trp316Ser β2GPI polymorphisms and the aPL status of 230 patients referred for aPL screening. Sixty‐one (26·5%) had persistent aPL [anticardiolipin antibodies (IgG and/or IgM), lupus anticoagulants and/or IgG anti‐β2GPI antibodies]. A comparison of the genotypic and allelic frequencies of these two polymorphisms between the Caucasian patient population and an ethnic‐matched normal control group (n = 308) showed no significant differences between aPL‐positive patients, aPL‐negative patients and the normal control group. This suggests that the Val or Leu allele at position 247 and the Trp or Ser allele at position 316 of β2GPI do not play a role in the production of aPL. There was a significantly decreased prevalence of the Ser316 allele in aPL‐negative women (n = 98) when compared with female normal control subjects (n = 249) {0·020 [95% confidence interval (CI) 0·00–0·04]vs 0·060 (95% CI 0·04–0·08), P = 0·0286}. Subgroup analysis showed no significant difference between female patients with thrombosis and female normal control subjects. Thus, the Ser316 allele may protect women from developing pregnancy complications by influencing an anticoagulant function of β2GPI via a mechanism distinct from aPL production.

-455G/A β-fibrinogen gene polymorphism, factor V Leiden, prothrombin G20210A mutation and MTHFR C677T, and placental vascular complications

Hyperfibrinogenaemia is associated with systemic arterial and venous thromboembolism and therefore may contribute to placental vascular disease associated with obstetric complications. The fibrinogen-raising −455G/A β-fibrinogen gene polymorphism may enhance the physiological increase in fibrinogen levels during pregnancy and thereby predispose to obstetric complications. This retrospective case–control study looked at the association between the β-fibrinogen gene polymorphism −455G/A, the hereditary thrombophilic markers factor V Leiden, prothrombin G20210A mutation (PGM) and C677T methylene tetrahydrofolate reductase (MTHFR), and obstetric complications associated with placental vascular disease. The study group (n = 247) comprised 147 women (90 Caucasian) who met the clinical criteria and a control group of 100 parous women (90 Caucasian) with no history of obstetric or medical complications. No significant differences were observed in the −455A allelic frequencies of the patient and normal control groups, with (allelic frequencies, 0.156 and 0.178, respectively; P = 0.5716, χ2 test, odds ratio = 1.17, 95% confidence interval = 0.65–2.13) or without (allelic frequencies, 0.129 and 0.170, respectively; P = 0.2077, χ2 test, odds ratio = 1.38, 95% confidence interval = 0.81–2.35) the exclusion of non-Caucasian women. There was an increased prevalence of factor V Leiden among Caucasian patients compared with normal controls (allelic frequencies, 0.056 and 0.017, respectively; P = 0.048, χ2 test, odds ratio = 0.29, 95% confidence interval = 0.05–1.15) but there were no differences in the prevalences of PGM or MTHFR. These data suggest that factor V Leiden is associated with an increased risk of obstetric complications, but that the −455A allele of β-fibrinogen, PGM and MTHFR do not appear to be implicated.

No association between pulmonary embolism or deep vein thrombosis and the –455G/A β-fibrinogen gene polymorphism

Hyperfibrinogenaemia has been reported to be associated with deep vein thrombosis (DVT). However, whether or not the ‘fibrinogen-raising’ –455G/A polymorphism of the β-fibrinogen gene is associated with DVT is uncertain and there are no data on whether this polymorphism is associated with pulmonary embolism (PE). We have studied relationships between the –455G/A β-fibrinogen gene polymorphism and the occurrence of PE and/or DVT (n = 339) (PE only, n = 76; DVT only, n = 216; PE and DVT, n = 47). There was no difference between the –455A allelic frequencies for the control (n = 190) and patient groups — PE, 0.187 and 0.171, respectively [P = 0.6087, χ2 test; odds ratio (OR), 1.12; 95% confidence interval (CI), 0.72–1.74]; DVT, 0.187 and 0.171, respectively (P = 0.5408, χ2 test; OR, 1.11; 95% CI, 0.78–1.59). This also applied when only Caucasian individuals were considered — PE allelic frequencies, 0.192 and 0.193, respectively (P = 0.9764, χ2 test; OR, 0.99; 95% CI, 0.62–1.60); DVT allelic frequencies, 0.192 and 0.186, respectively (P = 0.8404, χ2 test; OR, 1.04; 95% CI, 0.71–1.51). While the results should be interpreted with caution as the frequency of the –455A allele is rare, the –455A allele of the β-fibrinogen gene does not appear to be associated with an increased risk of PE or DVT.

Paradoxical association between the 316 Trp to Ser beta 2-glycoprotein I (Beta2GPI) polymorphism and anti-Beta2GPI antibodies.

Summary. We report a woman with an obstetric history ofa stillbirth at 28 weeks, associated with hypertension and severe intrauterine growth restriction and a miscarriage at 9 weeks. She was persistently positive for immunolgobulin G (IgG) anticardiolipin antibodies and IgG anti‐Beta‐2‐glycoprotein I (anti‐Beta2GPI) antibodies. She has delivered three healthy babies when managed antenatally with aspirin and low‐molecular‐weight heparin prophylaxis. Genotyping revealed that she was homozygous for the 316 Trp to Ser Beta2GPI polymorphism. Studies examining the binding of her plasma Beta2GPI to purified cardiolipin showed markedly reduced binding in comparison with Beta2GPI in pooled normal plasma.