Acute thrombocytopenia in patients treated with amiodarone is caused by antibodies specific for platelet membrane glycoproteins

Amiodarone has been implicated as a cause of thrombocytopenia but the responsible mechanism is unknown. We performed studies in three patients to characterize the pathogenesis of this complication. No amiodarone‐dependent, platelet‐reactive antibodies were identified using conventional serological techniques. However, water‐insoluble amiodarone solubilized in methanol and diluted to 1·0 mg/ml in aqueous buffer reproducibly promoted binding of IgG antibodies in patient serum to platelets. Solid phase assays identified drug‐dependent antibodies specific for platelet glycoproteins (GP)Ia/IIa (integrin α₂β₁) in each patient and a second antibody specific for GPIIb/IIIa (α_IIbβ₃ integrin) in one patient. When studied by ion mobility analysis and transmission electron microscopy, the serologically active amiodarone preparation, a milky suspension, was found to consist of particles 2–30 nm in diameter, typical of a coacervate, a state characteristic of amiodarone in aqueous medium. The findings provide evidence that thrombocytopenia in the three patients studied was caused by drug‐dependent antibodies specific for platelet glycoproteins GPIa/IIa and/or GPIIb/IIIa. We postulate that, in vivo, amiodarone may become incorporated into occult lipophilic domains in platelet glycoproteins, producing structural modifications that are immunogenic in some individuals, and that the resulting antibodies can cause platelet destruction in a person taking this drug.     

Rifampicin‐dependent antibodies target glycoprotein IIb/IIIa and cause clearance of human platelets in NOD/SCID mice

Dysregulation of MYC is the genetic hallmark of Burkitt lymphoma (BL) but it is encountered in other aggressive mature B‐cell lymphomas. MYC dysregulation needs other cooperating events for BL development. We aimed to characterize these events and assess the differences between adult and paediatric BLs that may explain the different outcomes in these two populations. We analysed patterns of genetic aberrations in a series of 24 BLs: 11 adults and 13 children. We looked for genomic imbalances (copy number variations), copy‐neutral loss of heterozygosity (CN‐LOH) and mutations in TP53, CDKN2A, ID3 (exon 1), TCF3 (exon17) and CCND3 (exon 6). Young patients displayed more frequent 13q31.3q32.1 amplification, 7q32q36 gain and 5q23.3 CN‐LOH, while 17p13 and 18q21.3 CN‐LOH were only detected in adult BLs. ID3 mutations were present in all adult samples, but only in 42% of childhood cases. CCND3 and ID3 double‐hit mutations, as well as 18q21 CN‐LOH, seemed to be associated with poorer outcome. For the first time, we report different genetic anomalies between adult and paediatric BLs, suggesting age‐related heterogeneity in Burkitt lymphomagenesis. This may explain the poorer prognosis of adult BLs. Additional studies are needed to confirm these results in the setting of clinical trials.     

Warfarin‐induced skin necrosis associated with Factor V Leiden and protein S deficiency

Thrombotic events are rare complications during anticoagulation therapy. The thrombosis varies from localized cutaneous involvement to catastrophic thromboembolism and is usually associated with an underlying thrombophilia. We describe a patient who developed skin necrosis during warfarin treatment for a pulmonary thromboembolism. The management was complicated by the development of heparin‐induced thrombocytopenia and further thrombotic events. Thrombophilia screen demonstrated the presence of protein S deficiency and Factor V Leiden as the prothrombotic factors, together with the demonstration of antiplatelet factor 4 antibodies, which confirms the diagnosis of heparin‐induced thrombocytopenia (type II). Reinstitution of warfarin at a low loading dose was successful without the recurrence of skin lesions nor any further thrombosis.     

Significance of quantitative measurement of heparin‐induced platelet antibodies

Background: Heparin‐induced thrombocytopenia (HIT) is a prothrombotic immune‐mediated adverse drug reaction. Antigen and platelet activation assays are used for detection of antibodies. Quantitative results from platelet factor 4 (PF4)‐dependent immunoassays may lead to inter‐laboratory standardization of measurements. Objectives: The aim was to modify a PF4‐dependent immunoassay to measure PF4/heparin antibodies quantitatively. Methods: Over five consecutive years, 1070 samples from thrombocytopenic, heparin‐treated patients were analyzed by a PF4/heparin ELISA and the heparin‐induced platelet activation assay (HIPA). Results of ELISA assay were expressed as arbitrary units per liter (AU/L). Results: Precision of ELISA at the concentration of 50 AU/L was 3.6%. Of 1070 samples, 117 were positive for antibodies by ELISA and/or HIPA assay. The higher the antibody concentration was, the higher was the proportion of HIPA positive cases (>140 AU/L, 100%, n = 26; 100–140 AU/L, 55%, n = 20; 50–99 AU/L, 38%, n = 29; 30–49 AU/L, 17%, n = 36). Conclusions: The measurement of anti‐PF4/heparin antibody concentration is a new parameter that may improve the diagnosis of HIT. All samples with extremely strong antibody concentration were positive also by HIPA. For accuracy, antibody concentrations must be in the linear range of the assay and an international standard is needed.     

Time course of hepatitis E‐specific antibodies in adults

Hepatitis E virus (HEV) is highly endemic in industrialized countries, but there is a lack of knowledge on individual and overall antibody concentration dynamics. The aim of this study was to characterize longitudinal concentration changes of anti‐HEV immunoglobulin G (anti‐HEV IgG) by enzyme immunoassay (EIA). In total, 199 serum samples collected from 45 subjects over 18 years were analysed. A wide range of anti‐HEV IgG levels was found. Overall, anti‐HEV IgG significantly decreased after an observation period of at least 5 years. One negative seroconversion was observed. Four individual profiles suggested single and even multiple HEV reinfections despite pre‐existing HEV antibodies.     

A prospective study of the prevalence of heparin-induced antibodies and other associated thromboembolic risk factors in pediatric patients undergoing hemodialysis

Heparin, which is used at high doses in hemodialysis patients, may induce antibodies favoring thromboembolic complications. We prospectively investigated the prevalence of heparin-induced platelet-reactive antibodies in a cohort of 38 pediatric hemodialysis patients, by means of heparin/platelet factor 4 (H/PF4) ELISA and heparin-induced platelet activation assay (HIPA). We also assessed other acquired and congenital hypercoagulable states. Heparin-induced antibodies were detected in 13 and 21% of patients with HIPA and ELISA, respectively. Anti-H/PF4 antibodies were negatively correlated with the number of hemodialysis sessions. These antibodies disappeared after a median time of 6 months despite continuing heparin treatment. The prevalence of antiphospholipid antibodies was 21% (anticardiolipin 10.5%, anti-beta2GPI 13%, and lupus anticoagulant 5%). Blood levels of homocysteine, factor VIII, and fibrinogen were significantly higher and factor II levels were significantly lower in hemodialysis patients than in controls, whereas factor VII, factor IX, and natural coagulation inhibitor levels were similar in patients and controls. Overall, 26 of 38 patients had at least one biomarker of hypercoagulability, but only 1 patient, without anti-H/PF4 antibodies, presented with thrombosis. In conclusion, heparin induces the transient production of anti-H/PF4 antibodies in children undergoing hemodialysis, but other abnormalities probably contribute to hypercoagulability. These findings may help to improve the diagnosis and management of thrombotic events in hemodialysis patients.     

Novel nonsense mutation in the platelet glycoprotein Ibbeta gene associated with Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder caused by quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. This complex is composed of four subunits, GPIbalpha, GPIbbeta, GPIX, and GPV, and the coordinated assembly of GPIbalpha, GPIbbeta, and GPIX is required for the efficient surface expression of a functional complex. We report here a novel nonsense mutation of the GPIbbeta gene associated with BSS. Flow cytometric analysis of the patient's platelets showed markedly reduced GPIbalpha and absent GPIX surface expression. Immunoblot analysis of solubilized platelets showed that a small amount of GPIbalpha was detected; however, GPIbbeta and GPIX were undetectable. DNA sequencing analysis revealed a novel nonsense mutation of the GPIbbeta gene that converts Trp (TGG) to a stop codon (TAG) at residue 123. Transient transfection studies revealed that the mutant GPIbbeta polypeptide was not detected in the transfected 293T cells, suggesting that null expression of the mutant GPIbbeta impairs expression of the GPIbalpha and GPIX subunits and results in a BSS phenotype in the patient.     

Frequency of anti-heparin-platelet factor 4 antibodies in hemodialysis patients and correlation with recurrent vascular access thrombosis

Heparin-induced thrombocytopenia (HIT), characterized by the formation of antibodies to a complex of platelet factor 4 (PF4) and heparin, is a well-recognized risk factor for thromboembolic complications. The frequency of antibody development varies among patient populations. Hemodialysis patients have repeated heparin exposure and should be at risk of developing HIT. This might, contribute to the development of vascular access thrombosis. We prospectively evaluated 88 hemodialysis patients for the presence of anti-PF4/heparin antibodies. Eighteen patients (20%) had a prior history of 1 or more prior access thrombosis. One patient (1.14%), without a history of graft thrombosis, tested positive for anti-PF4/heparin antibodies. In our study, the presence of anti-PF4/heparin antibodies was rare and was not increased in patients with a history of vascular access thrombosis.     

Drug‐induced methaemoglobinaemia presenting with angina following the use of dapsone

Summary Anaemia may result in tissue hypoxia which may induce or exacerbate symptoms of ischaemia. Tissue hypoxia may however also result from the presence of haemoglobin with altered oxygen‐binding characteristics. Drug‐induced methaemoglobinaemia in which oxygen is irreversibly bound to haemoglobin may complicate the use of some common drugs. This condition may result in severe tissue hypoxia, which is rapidly and cheaply reversed by methylene blue.     

Novel heterozygous missense mutation in the platelet glycoprotein Ib beta gene associated with isolated giant platelet disorder.

The glycoprotein (GP) Ib/IX/V complex plays an important role in primary hemostasis, serving as the platelet receptor for von Willebrand factor (vWF). Recent studies have shown that the phenotype caused by mutations in the subunits of the GPIb/IX complex spans a wide spectrum; from the normal phenotype, to isolated giant platelet disorders (GPD), and to the full-blown bleeding disorder, the Bernard-Soulier syndrome (BSS). We characterize here a novel missense mutation of the GPIb beta gene associated with isolated GPD. In the patient's platelets, the expression level of the GPIb/IX complex was moderately reduced compared with that of the GPIIb/IIIa complex, whereas the latter was expressed at higher levels than in a normal control. Immunoblot analysis showed normal electrophoretic mobility of GPIb alpha, GPIb beta, and GPIX. However, the amount of GPIb beta was approximately 66% of the normal value. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIb beta gene that converts Arg (CGC) to Cys (TGC) at residue 17. Transient transfection studies demonstrated that mutant GPIb beta protein was not detected in transfected 293T cells. These findings indicated that null expression of the abnormal GPIb beta causes decreased expression of the complex and results in the GPD phenotype in the patient, and suggested that homozygosity of the mutation may lead to a BSS phenotype in vivo.     

Use of systemic bivalirudin with catheter‐directed thrombolysis in a patient with heparin‐induced thrombocytopenia: A case report

In patients with submassive pulmonary embolism, the use of catheter‐directed thrombolysis (CDT), using low‐dose alteplase is associated with improvement in overall hemodynamics. The data for use of CDT in patients with heparin‐induced thrombocytopenia are limited. We report a case of CDT in a patient with HIT using bivalirudin anticoagulation. Data of the use of bivalirudin and argatroban for systemic anticoagulation with CDT are limited.     

Catheter-directed thrombolysis of acute lower extremity arterial thrombosis in a patient with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an underdiagnosed problem, and the optimal treatment of arterial thrombosis in patients with HIT remains controversial. There are many angiographic procedures which require heparin as an adjunctive agent; however, some of the heparin-related complications and their management remains unclear. We are presenting a 77-year-old male patient with HIT, who developed acute lower extremity limb threatening ischemia due to arterial thrombosis. In our case, the patient has been successfully treated with percutaneous catheter-directed thrombolysis with tissue plasminogen activator and a direct thrombin inhibitor argatroban.