C.L. Eckhardt

Intensive peri‐operative use of factor VIII and the Arg593→Cys mutation are risk factors for inhibitor development in mild/moderate hemophilia A

Summary.u2002 Background:u2002A severe and challenging complication in the treatment of hemophilia A is the development of inhibiting antibodies (inhibitors) directed towards factor VIII (FVIII). Inhibitors aggravate bleeding complications, disabilities and costs. The etiology of inhibitor development is incompletely understood. Objectives:u2002In a large cohort study in patients with mild/moderate hemophilia A we evaluated the role of genotype and intensive FVIII exposure in inhibitor development. Patients/methods:u2002Longitudinal clinical data from 138 mild/moderate hemophilia A patients were retrospectively collected from 1 January 1980 to 1 January 2008 and analyzed by multivariate analysis using Poisson regression. Results:u2002Genotyping demonstrated the Arg593Cys missense mutation in 52 (38%) patients; the remaining 86 patients had 26 other missense mutations. Sixty‐three (46%) patients received intensive FVIII concentrate administration, 41 of them for surgery. Ten patients (7%) developed inhibitors, eight of them carrying the Arg593Cys mutation. Compared with the other patients, those with the Arg593Cys mutation had a 10‐fold increased risk of developing inhibitors (RR 10; 95% CI, 0.9–119).The other two inhibitor patients had the newly detected mutations Pro1761Gln and Glu2228Asp. In both these patients and in five patients with genotype Arg593Cys, inhibitors developed after intensive peri‐operative use of FVIII concentrate (RR 186; 95% CI, 25–1403). In five of the 10 inhibitor patients FVIII was administered by continuous infusion during surgery (RR 13; 95% CI, 1.9–86). Conclusion:u2002The Arg593Cys genotype and intensive peri‐operative use of FVIII, especially when administered by continuous infusion, are associated with an increased risk for inhibitor development in mild/moderate hemophilia A.

Surgery and inhibitor development in hemophilia A: A systematic review

Summary.u2002 Background: Although the association between intensive treatment and the formation of inhibiting antibodies towards factor VIII (FVIII) in hemophilia A has been demonstrated, the contributing effect of surgery is presently unclear. The release of immunological danger signals resulting from tissue damage during surgery in the presence of a high FVIII antigen load may elicit the formation of FVIII antibodies. The aim of this systematic review was to investigate the role of surgery in the inhibitor risk associated with intensive treatment as compared with treatment for bleeding and prophylactic administration of FVIII. Methods: A comprehensive literature search was performed that identified four cohort studies and three case control studies, comprising 342 inhibitor patients among a total of 957 hemophilia A patients. Results: Intensive treatment increased the inhibitor risk, most pronounced with intensive treatment of ≥u20035 exposure days (EDs) compared with <u20033 EDs (OR, 4.1; 95% confidence interval, 2.6–6.5). Pooled odds ratio for inhibitor development in severe hemophilia patients that received intensive treatment for surgery at first exposure was 4.1 (95% confidence interval, 2.0–8.4) compared with treatment for bleeding or prophylaxis. Information on continuous infusion, previously treated patients and non‐severe hemophilia A was insufficient for valid meta‐analyses. Conclusions: Intensive FVIII treatment for surgery at first exposure leads to a higher inhibitor risk in hemophilia A patients compared with intensive treatment for bleeding.

Inhibitor development and mortality in non-severe hemophilia A

The life expectancy of non‐severe hemophilia A (HA) patients equals the life expectancy of the non‐hemophilic population. However, data on the effect of inhibitor development on mortality and on hemophilia‐related causes of death are scarce. The development of neutralizing factor VIII antibodies in non‐severe HA patients may dramatically change their clinical outcome due to severe bleeding complications.

The Fc gamma receptor IIa R131H polymorphism is associated with inhibitor development in severe hemophilia A.

The development of factor (F) VIII neutralizing alloantibodies (inhibitors) is a major complication of treatment with FVIII concentrates in hemophilia A and the etiology is still poorly understood. The low‐affinity Fc gamma receptors (FcγR), which are expressed on immune cells, provide an important link between cellular and humoral immunity by interacting with IgG subtypes. Genetic variations of the genes encoding FcγRs (FCGR genes) have been associated with susceptibility to infectious and autoimmune diseases.

Inhibitor incidence after intensive FVIII replacement for surgery in mild and moderate haemophilia A: a prospective national study in the Netherlands

Inhibitor development is currently the most severe complication in mild/moderate haemophilia A patients, causing increased bleeding tendency, hospitalization and mortality. It has been suggested that receiving high doses of factor VIII (FVIII) concentrates for surgical procedures is an important risk factor for inhibitor development in these patients. The current multicentre study aimed to determine prospectively the incidence of inhibitor development after intensive FVIII replacement therapy for surgical procedures in patients with mild/moderate haemophilia A. All consecutive patients with mild/moderate haemophilia A were included when they required at least 10 000 iu of FVIII concentrates (or 250 iu/kg) for 5 or more days for a surgical procedure. Potential clinical risk factors for inhibitor development and results of inhibitor tests were collected. Forty‐six patients with a median age of 54 years (interquartile range, 40–59 years) were included in the study. F8 genotyping revealed 20 different missense mutations. Patients received either recombinant (65%) or plasma‐derived FVIII concentrates (35%) by intermittent bolus injections (41%) or continuous infusion (57%). Two patients developed a low titre inhibitor post‐operatively. The incidence of inhibitor development following intensive treatment for surgery in this unselected prospective cohort of mild/moderate haemophilia A patients was 4% (95% confidence interval, 0·5–14·8).

Variation in baseline factor VIII concentration in a retrospective cohort of mild/moderate hemophilia A patients carrying identical F8 mutations

Essentials Factor VIII levels vary in mild and moderate hemophilia A (MHA) patients with the same mutation. We aimed to estimate the variation and determinants of factor VIII levels among MHA patients. Age and genotype explain 59% of the observed inter‐individual variation in factor VIII levels. Intra‐individual variation accounted for 45% of the variation in the three largest mutation groups.

Intensity of factor VIII treatment and the development of inhibitors in non-severe hemophilia A patients: results of the INSIGHT case–control study

Essentials Research suggests that intensive treatment episodes may increase the risk to develop inhibitors. We performed an international nested case‐control study with 298 non‐severe hemophilia A patients. Surgery and a high dose of factor VIII concentrate were associated with increased inhibitor risk. Physicians need to review arguments for factor VIII dose and elective surgery extra critically.

Inhibitor development in mild and moderate hemophilia A seems to be associated with an increased mortality risk

Primary immune thrombocytopenia (ITP) is characterized by isolated thrombocytopenia. The mechanisms leading to a low platelet count include antibody-mediated destruction of platelets and suppression of megakaryocyte and platelet development. The causes of loss of tolerance and autoantibody production are unknown and it is likely that both genetic and environmental factors are involved. Platelet-specific autoantibodies are directed against a restricted number of ‘dominant’ epitopes of GPIIbIIIa, less frequently of GPIbIX or other platelet glycoproteins. There is evidence to suggest that these autoantibodies are produced from a limited number of clonal B cells by antigen-driven selection. Abnormalities of T cells certainly play a role in causing or perpetuating ITP. Chronic ITP is characterized by a Th1 profile and an impaired T-regulatory cell function, both of which are reversed upon successful treatment. Furthermore, cytotoxic T cells have been shown to cause platelet destruction in vitro and can probably suppress megakaryopoiesis. Chronic infections such as HIV, HCV and Helicobacter pylorican present with isolated thrombocytopenia and therefore mimic ITP. Antibodies cross-reacting with platelet antigens have been identified in thrombocytopenia associated with these infections, supporting a role for molecular mimicry in the development of this disorder. Disclosure of Interest: Honararia for participation on advisory boards and as a speaker at medical education events supported by GlaxoSmithKline, Amgen and RocheIntroduction: Hemophilia A patients have a 50% lower cardiovascular mortality than the general population. Whether this is caused by less atherosclerosis due to hypocoagulability is unclear. We assessed whether hemophilia A patients with obesity, a major atherosclerotic risk factor, have a lower prevalence of subclinical atherosclerosis than obese controls. Methods: Fifty obese (body mass index [BMI] > 30 kg/m2) and 48 normal-weight (BMI 1.3 mm, P = 0.11), irrespective of the severity of hemophilia. (Figure presented) Conclusion: Hemophilia A patients with obesity have the same degree of subclinical atherosclerosis as obese control subjects, which suggests that the lower cardiovascular mortality is likely caused by a decreased risk of arterial thrombosis.The antiphospholipid syndrome (APS) is an autoimmune disease associated with the presence of antiphospholipid antibodies (APL) and the occurrence of thrombosis and pregnancy complications. One of the assays to detect APL is based on the prolongation of phospho-lipid dependent coagulation assays caused by these antibodies; lupus anticoagulant (LAC). To prevent thrombotic complications, APS patients use long-term anticoagulant treatment that could interfere with LAC determination. Rivaroxaban is a new direct factor Xa inhibitor. In this study we assessed whether rivaroxaban interferes with the detection of LAC. We tested normal pooled plasma (NPP), LAC-positive plasma of SLE patients and LAC-negative plasma from SLE patients. These plasmas were spiked with rivaroxaban. LAC ratios were determined by measuring the LAC-dependent and -independent coagulation times (dRVVT screen/dRVVT confirm, aPTT low and high phospho-lipids, taipan venom and ecarin times). Taipan venom and ecarin are snake venoms that directly activate prothrombin. The taipan venom time is LAC sensitive, whereas the ecarin time is not. Rivaroxaban added to plasma of healthy individuals prolonged the dRVVT LAC ratio, leading to a false positive lupus anticoagulant signal. Rivaroxaban had no influence on the aPTT LAC ratio of normal plasma, but slightly increased the ratio in plasma of SLE patients. For some plasmas negative for LAC, the presence of rivar-oxaban lead to a false positive LAC signal in the aPTT. We observed that the ratio of the taipan venom time over ecarin time remained uninfluenced by the presence of rivaroxaban, both in the absence or presence of antiphospholipid antibodies. This study shows that rivaroxaban can interfere with conventional LAC assays. The taipan venom time/ecarin time ratio may be a good alternative for the detection of LAC in patients using rivaroxaban.Introduction: Cancer greatly increases the risk of venous thromboem-bolism (VTE). Here, we investigated the contribution of microparti-cle-dependent procoagulant activity to the prothrombotic state in these patients. Methods: In 43 cancer patients without VTE at entry and 22 healthy volunteers, markers of in vivo and microparticle-dependent coagulation were measured and patients were prospectively followed for 6 months for the development of VTE. Procoagulant activity of microparticles (MPs) was measured using a phospholipid dependent test (STA Procoag PLL), a factor Xa-generation assay (Xa-assay) with and without anti-tissue factor (TF), and a fibrin generation test (FGT) with and without anti-factor VII(a). Results: Markers of in vivo coagulation activation and total number of circulating MPs were elevated in cancer patients compared to controls (F1+2 246 vs. 156 pM, thrombin-antithrombin complexes 4.1 vs. 3.0 mg/L, D-dimer 0.76 vs. 0.22 mg/L and 5.53 x 106 vs. 3.37 x 106 microparticles per mL; all P <0.001). Five cancer patients (12%) developed VTE during follow-up. Patients with VTE had comparable levels of coagulation activation markers and phos-pholipid dependent MP procoagulant activity. However, TF-mediated Xa-generation (0.82 vs. 0.21 pg/mL, P = 0.016) and the VIIa-dependent FGT (13% vs. 0%, P = 0.036) were higher in the VTE group compared with the non-VTE group.

Dissecting intensive treatment as risk factor for inhibitor development in haemophilia.

The treatment of hemophilia A can be severely complicated by the development of neutralizing antibodies towards factor VIII (inhibitors). Intensive treatment with factor VIII concentrates has been previously indicated as an important risk factor for inhibitor development. As intensive treatment is administered for major bleeding or surgery, the association between intensive treatment and inhibitor development may originate from two different triggers of the immune system: a.) a high dose of factor VIII administered within a short time span and b.) immunological “danger signals” arising from tissue damage and inflammation. Before changing our treatment strategies in order to lower the inhibitor risk, the contribution of these factors has to be dissected in order to establish their causal role. In the present paper we discuss the methodological issues that are required for observational studies in order to distinguish the separate effects of high dose of factor VIII administration and immunological danger signs on inhibitor development. TXC 20140714 De Groot.indd 200 4-8-2014 16:30:15 201 Dissecting intensive treatment as risk factor

Sevenfold risk for inhibitors in mild/moderate hemophilia A with factor VIII missense mutations in two distinct regions

Primary immune thrombocytopenia (ITP) is characterized by isolated thrombocytopenia. The mechanisms leading to a low platelet count include antibody-mediated destruction of platelets and suppression of megakaryocyte and platelet development. The causes of loss of tolerance and autoantibody production are unknown and it is likely that both genetic and environmental factors are involved. Platelet-specific autoantibodies are directed against a restricted number of ‘dominant’ epitopes of GPIIbIIIa, less frequently of GPIbIX or other platelet glycoproteins. There is evidence to suggest that these autoantibodies are produced from a limited number of clonal B cells by antigen-driven selection. Abnormalities of T cells certainly play a role in causing or perpetuating ITP. Chronic ITP is characterized by a Th1 profile and an impaired T-regulatory cell function, both of which are reversed upon successful treatment. Furthermore, cytotoxic T cells have been shown to cause platelet destruction in vitro and can probably suppress megakaryopoiesis. Chronic infections such as HIV, HCV and Helicobacter pylorican present with isolated thrombocytopenia and therefore mimic ITP. Antibodies cross-reacting with platelet antigens have been identified in thrombocytopenia associated with these infections, supporting a role for molecular mimicry in the development of this disorder. Disclosure of Interest: Honararia for participation on advisory boards and as a speaker at medical education events supported by GlaxoSmithKline, Amgen and RocheIntroduction: Hemophilia A patients have a 50% lower cardiovascular mortality than the general population. Whether this is caused by less atherosclerosis due to hypocoagulability is unclear. We assessed whether hemophilia A patients with obesity, a major atherosclerotic risk factor, have a lower prevalence of subclinical atherosclerosis than obese controls. Methods: Fifty obese (body mass index [BMI] > 30 kg/m2) and 48 normal-weight (BMI 1.3 mm, P = 0.11), irrespective of the severity of hemophilia. (Figure presented) Conclusion: Hemophilia A patients with obesity have the same degree of subclinical atherosclerosis as obese control subjects, which suggests that the lower cardiovascular mortality is likely caused by a decreased risk of arterial thrombosis.The antiphospholipid syndrome (APS) is an autoimmune disease associated with the presence of antiphospholipid antibodies (APL) and the occurrence of thrombosis and pregnancy complications. One of the assays to detect APL is based on the prolongation of phospho-lipid dependent coagulation assays caused by these antibodies; lupus anticoagulant (LAC). To prevent thrombotic complications, APS patients use long-term anticoagulant treatment that could interfere with LAC determination. Rivaroxaban is a new direct factor Xa inhibitor. In this study we assessed whether rivaroxaban interferes with the detection of LAC. We tested normal pooled plasma (NPP), LAC-positive plasma of SLE patients and LAC-negative plasma from SLE patients. These plasmas were spiked with rivaroxaban. LAC ratios were determined by measuring the LAC-dependent and -independent coagulation times (dRVVT screen/dRVVT confirm, aPTT low and high phospho-lipids, taipan venom and ecarin times). Taipan venom and ecarin are snake venoms that directly activate prothrombin. The taipan venom time is LAC sensitive, whereas the ecarin time is not. Rivaroxaban added to plasma of healthy individuals prolonged the dRVVT LAC ratio, leading to a false positive lupus anticoagulant signal. Rivaroxaban had no influence on the aPTT LAC ratio of normal plasma, but slightly increased the ratio in plasma of SLE patients. For some plasmas negative for LAC, the presence of rivar-oxaban lead to a false positive LAC signal in the aPTT. We observed that the ratio of the taipan venom time over ecarin time remained uninfluenced by the presence of rivaroxaban, both in the absence or presence of antiphospholipid antibodies. This study shows that rivaroxaban can interfere with conventional LAC assays. The taipan venom time/ecarin time ratio may be a good alternative for the detection of LAC in patients using rivaroxaban.Introduction: Cancer greatly increases the risk of venous thromboem-bolism (VTE). Here, we investigated the contribution of microparti-cle-dependent procoagulant activity to the prothrombotic state in these patients. Methods: In 43 cancer patients without VTE at entry and 22 healthy volunteers, markers of in vivo and microparticle-dependent coagulation were measured and patients were prospectively followed for 6 months for the development of VTE. Procoagulant activity of microparticles (MPs) was measured using a phospholipid dependent test (STA Procoag PLL), a factor Xa-generation assay (Xa-assay) with and without anti-tissue factor (TF), and a fibrin generation test (FGT) with and without anti-factor VII(a). Results: Markers of in vivo coagulation activation and total number of circulating MPs were elevated in cancer patients compared to controls (F1+2 246 vs. 156 pM, thrombin-antithrombin complexes 4.1 vs. 3.0 mg/L, D-dimer 0.76 vs. 0.22 mg/L and 5.53 x 106 vs. 3.37 x 106 microparticles per mL; all P <0.001). Five cancer patients (12%) developed VTE during follow-up. Patients with VTE had comparable levels of coagulation activation markers and phos-pholipid dependent MP procoagulant activity. However, TF-mediated Xa-generation (0.82 vs. 0.21 pg/mL, P = 0.016) and the VIIa-dependent FGT (13% vs. 0%, P = 0.036) were higher in the VTE group compared with the non-VTE group.