A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70.

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease.

Gene-based continuous expression of FVIIa for the treatment of hemophilia.

Qualitative or quantitative defects in the genes for coagulation factors VIII (FVIII) or IX (FIX) result in a life-threatening, bleeding phenotype (hemophilia A (HA) or B (HB), respectively). Although hemophilia treatment by clotting factor replacement is effective, a proportion of patients develop neutralizing antibodies (inhibitors) to the infused factor that complicate the disease management. For inhibitor patients, recombinant human activated coagulation Factor VII (rhFVIIa), when administered at therapeutic doses, has been shown to bypass the deficiency in FVIII or FIX and result in hemostasis. As an alternative to this protein infusion therapy, a gene-based approach for the treatment of hemophilia with inhibitors has been developed, using continuous expression of a transgene coding for FVIIa following viral-mediated delivery. This approach was validated in hemophilic mice and, notably, in dogs as a model that closely resembles the human disease. In particular, liver-directed FVIIa gene delivery in hemophilic dogs resulted in multi-year transgene expression that ameliorated the bleeding phenotype, without thrombotic complications. These data support the gene-based FVIIa expression as a novel bypass therapy for hemophilia with inhibitors.

Thrombin activatable fibrinolysis inhibitor activation and bleeding in haemophilia A.

Individuals with haemophilia A exhibit bleeding tendencies that are not always predicted by their factor (F)VIII level. It has been suggested that bleeding in haemophilia is due not only to defective prothrombin activation but also aberrant fibrinolysis. Thrombin activatable fibrinolysis inhibitor (TAFI) activation was measured in tissue factor (TF)-initiated blood coagulation in blood samples of 28 haemophiliacs and five controls. Reactions were quenched over time with FPRck and citrate and assayed for TAFIa and thrombin-antithrombin (TAT). The TAFIa potential (TP), TAFI activation rate and the TAFIa level at 20 min (TAFIa(20 min)) was extracted from the TAFI activation progress curve. In general, the time course of TAFI activation follows thrombin generation regardless of FVIII activity and as expected the rate of TAFI activation and TP decreases as FVIII decreases. The magnitude of TP was similar among the control subjects and subjects with <11% FVIII. In severe subjects with <1% FVIII at the time of blood collection, the TAFIa(20 min) was inversely and significantly correlated with haemarthrosis (-0.77, P = 0.03) and total bleeds (-0.75, P = 0.03). In all cases, TAFIa(20 min) was more strongly correlated with bleeding than TAT levels at 20 min. Overall, this study shows that TAFI activation in whole blood can be quantified and related to the clinical bleeding phenotype. Measuring TAFIa along with thrombin generation can potentially be useful to evaluate the differential bleeding phenotype in haemophilia A.

The changing face of hepatitis in boys with haemophilia associated with increased prevalence of obesity.

Hepatitis in children with haemophilia was historically most often associated with transfusion-transmitted infections. However, with the use of recombinant clotting factor concentrates, acquisition of such infections has now become rare. We studied the profile of hepatitis in North-American children with haemophilia in the modern era of safe blood products and excess childhood obesity. A total of 173 boys (<18 years) registered in the Pediatric Comprehensive Care Haemophilia Program were included in this retrospective study. Hospital records were reviewed for baseline data, serial height and weight measurements and serial alanine aminotransferase (ALT) levels. A body mass index (BMI) ranking was available for 170 boys, of whom 25 (14.7%, 95% CI 9.7-20.9%) were obese. The rate of obesity was higher in severe haemophilic boys. Compared with the general childhood population, the rate of obesity trended towards being higher in young haemophilic boys (2-5 years), but was similar in other age groups. A persistently high ALT (≥80 U L(-1) ) was documented in 5 boys and was associated with obesity. Three boys had clinical and imaging studies compatible with non-alcoholic fatty liver disease (NAFLD). Overweight and obesity are common among haemophilic boys, especially those who are younger and with severe disease. In this large group of haemophilic boys, chronic viral hepatitis was rare and NAFLD was a more common cause of liver disease. Overweight and obese haemophilic boys should be evaluated for NAFLD and interventional programmes should be designed to reduce the potential complications associated with obesity.

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Evaluation of the efficacy and safety of etoricoxib in the treatment of hemophilic arthropathy.

This 2-part, double-blind, placebo-controlled study was conducted to determine the safety and efficacy of etoricoxib, a COX-2 selective inhibitor, for the treatment of hemophilic arthropathy. In part 1 (6 weeks), 102 patients (> or = 12 years old) with hemophilic arthropathy were randomized to receive 90 mg etoricoxib once daily or placebo (1:1 ratio). In part 2 (6 months), 51 patients taking placebo in part 1 were randomized to receive 90 mg etoricoxib or 25 mg rofecoxib once daily; patients taking etoricoxib in part 1 continued the same treatment. Efficacy end points included Patient Assessment of Arthropathy Pain, Patient Global Assessment of Arthropathy Disease Status, and Investigator Global Assessment of Arthropathy Disease Status. Safety was evaluated at each study visit. Etoricoxib provided significant improvement in all end points versus placebo (P < .001). Fewer patients taking etoricoxib discontinued due to a lack of efficacy versus placebo (P = .048). During part 2, efficacy was maintained; etoricoxib and rofecoxib demonstrated similar results. The most common adverse experiences were upper respiratory infection and headache. The incidence of joint bleeding during part 1 was similar between etoricoxib (66.7%) and placebo (72.6%) and during part 2 between etoricoxib (77.0%) and rofecoxib (78.9%). We conclude that etoricoxib provided superior efficacy versus placebo for the treatment of hemophilic arthropathy and was generally safe and well tolerated.

HIV and HCV coinfection in haemophilia.

A substantial number of haemophilic patients are infected with both human immunodeficiency virus (HIV) and hepatitis C (HCV). HIV has been shown to accelerate the course of HCV chronic liver disease and there is evidence that HCV infection may worsen the prognosis of HIV. As many HIV infected patients are stable on highly active antiretroviral therapy (HAART) HCV should be actively managed in coinfected individuals. Pegylated interferon (Peg-IFN)/ribavirin combination therapy is the treatment of choice for HCV infection and should be considered in patients with stable HIV on or off HAART with CD4 counts >200 x 10(6)/l. Results of on-going trials of combination therapy in coinfected individuals are awaited. For coinfected patients with end stage liver disease who are stable on HAART liver transplantation should be considered.

Does an enzyme other than thrombin contribute to unexpected changes in the levels of the different forms of thrombin activatable fibrinolysis inhibitor in patients with hemophilia A, hemophilia B and von Willebrand disease?

Pro-thrombin activatable fibrinolysis inhibitor (pro-TAFI), also called plasma procarboxypeptidase B or U, is one of the modulators of fibrinolysis in blood. Pro-TAFI is activated by thrombin/thrombomodulin complex or by plasmin to a carboxypeptidase B-like enzyme (TAFI) of 35.8 kD molecular weight. TAFI spontaneously becomes inactive as a result of a temperature-dependent conformational change in the protein (TAFIi). In this study, pro-TAFI, total TAFI antigen and TAFI-TAFIi antigen levels were measured in 32 patients with hemophilia A, 4 patients with hemophilia B, 21 patients with von Willebrand disease (VWD) and 13 healthy controls. A statistically significant decrease in pro-TAFI was found in all groups (10.72+/-4.57 mg/L (p<0.001); 8.00+/-2.35 mg/L (p<0.01) and 8.98+/-2.33 mg/L (p <0.001) for hemophilia A, hemophilia B and VWD, respectively) compared to controls (17.85+4.61 mg/L). A statistically significant increase in TAFI-TAFIi antigen was found in hemophilia A (1.05+/-1.01 mg/L) (p<0.05) and in VWD patients (0.96+/-1.01 mg/L) (p<0.05) compared to controls (0.55+/-0.36 mg/L). There was no difference in total TAFI antigen levels between any group of patients and the controls. Neither did pro-TAFI nor TAFI-TAFIi levels differ within the group of hemophilia A patients in relation to severity (mild, moderate and severe) or among the VWD patients in relation to subtype (type 1, type 2A and type 3). These findings indicate an increased conversion of pro-TAFI to TAFI and/or TAFIi in patients with bleeding disorders. As thrombin generation is seriously impaired in these patients and almost absent in hemophilia A and B and in type 3 VWD, it is possible that plasmin mediates pro-TAFI activation in these patients. Enhanced fibrinolysis via generation of plasmin has previously been reported in hemophilia and VWD. Activation of pro-TAFI by plasmin may be a feedback mechanism that counterbalances increased fibrinolysis in patients with bleeding disorders. The relationship between the TAFI activation pathway and bleeding complications associated with hemophilia A, hemophilia B and VWD requires further investigation.

Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex.

The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIII(WT) and FVIII(Arg562Ala) showed catalytic rate constant (k(cat)) values of approximately 60 minute(-1) in the presence of phospholipid, whereas the hemophilia A-associated mutants showed k(cat) values ranging from 3.3 minute(-1) to 7.5 minute(-1). In the absence of phospholipid, all k(cat) values were reduced but FVIII(WT) and FVIII(Arg562Ala) retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.

Total thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and pro-TAFI in patients with haemophilia A.

Pro-thrombin-activatable fibrinolysis inhibitor (pro-TAFI), also known as TAFI, procarboxypeptidase U, or procarboxypeptidase B, is a relatively recently described plasma glycoprotein synthesized in the liver. It can be catalysed into its active form, TAFI (TAFIa, carboxypeptidase U or B) by a complex of thrombin/thrombomodulin. TAFI can potentially inhibit fibrinolysis by removing carboxyterminal lysine residues from partially degraded fibrin, decreasing plasminogen binding on the surface of fibrin, which thereby results in a decrease of the fibrinolytic activity. As TAFI represents a connection between coagulation and fibrinolysis, it can be expected that TAFI levels are altered in different thrombotic and haemorrhagic diseases, such as haemophilia A. Total TAFI antigen (including pro-TAFI, TAFI and the inactive form of TAFI [TAFIi]) and pro-TAFI were determined in 17 patients with haemophilia A. Thirteen healthy age-matched volunteers served as controls. No significant difference in levels of total TAFI antigen was observed between controls and patients with haemophilia, although it was slightly decreased in patients with haemophilia. Pro-TAFI was significantly reduced in haemophilia patients compared to controls (P=0.0113). TAFI antigen levels similar to controls have already been described in different clinical conditions, including haemophilia A. Decrease of pro-TAFI in haemophilia A can be an additional factor, together with decrease in thrombin generation, which induces impaired activation of pro-TAFI to TAFI, and could cause accelerated fibrinolysis. This supports the validity of usage of antifibrinolytics in the treatment of haemophilia A. In this paper we use new nomenclature for TAFI, and we believe that this recommended terminology for different forms of TAFI can simplify further standardization in TAFI investigation.

CPR-Total (TAFI and activated TAFI) levels in plasma/serum of hemophiliacs.

Arginine carboxypeptidase (CPR) is a single-chain plasma protein generated during coagulation from a precursor (proCPR). proCPR is the same molecule as thrombin activable fibrinolysis inhibitor (TAFI), which retards fibrin clot lysis in vitro and most likely modulates fibrinolysis in vivo. In this study, the amount of CPR-total, which includes proCPR (TAFI) and CPR (activated TAFI), in hemophiliac patients was evaluated using a newly developed enzyme linked immunosorbent assay (ELISA). The amount of CPR-total in plasma or serum of most of the hemophiliac patients was in the range of healthy individuals. There was no significant difference in hemophiliac patients with or without HIV-1 infection. However, two out of the 74 hemophiliac patients showed a significantly high level. The upregulation of CPR-total might contribute to compensate for inefficient coagulation in some hemophiliac individuals.

Catalytic activity of antibodies against factor VIII in patients with hemophilia A.

Hemophilia A is an X chromosome-linked recessive disorder resulting in defective or deficient factor VIII (FVIII) molecules, which, in its severe form, is a life-threatening and crippling hemorrhagic disease. Infusion of homologous FVIII to patients with severe hemophilia A results, in 25% of patients, in the emergence of alloantibodies against FVIII (inhibitors)( ref. 1) that inhibit FVIII procoagulant activity by steric hindrance of the interaction of FVIII either with stabilizing molecules, with molecules essential for its activity or with activating molecules. Here, we report on the proteolysis of FVIII by alloantibodies of two patients with severe hemophilia A, demonstrating a previously unknown mechanism by which FVIII inhibitors may prevent the pro-coagulant function of FVIII. The kinetic parameters of FVIII hydrolysis indicate a functional role for the catalytic immune response in the inactivation of FVIII in vivo. The characterization of alloantibodies against FVIII as site-specific proteases may provide new approaches to the treatment of FVIII inhibitors.

Expression of dipeptidylaminopeptidase IV/CD26 in peripheral blood lymphocytes of hemophilic subjects.

CD26 antigen, a 110 kDa membrane glycoprotein with exopeptidase activity (DAP IV), is an activation marker of T lymphocytes preferentially expressed on CD4+ memory cells and involved in T cell proliferation and IL-2 production after antigenic stimulation. We employed cytochemical and immunocytochemical techniques to study DAP IV/CD26 expression in circulating lymphocytes from 40 hemophilic patients, chronically treated with coagulation factors, in order to verify the possible involvement of this molecule in the immunological alterations of hemophilia. In all the hemophiliacs DAP IV activity was significantly lower than in the controls, independently of the quantity of blood transfused and previous exposure to viruses. This reduction may be responsible for the impaired proliferative response of lymphocytes to antigens and mitogens, notoriously observed in hemophilia. Whereas in the group of HIV- patients CD26 expression was similar to that of normal controls, in the 8 HIV+ hemophilic patients both percentages of positive lymphocytes and intensity of staining were significantly lower. In only 4 of the 8 cases was this deficit associated with CD4+ cell depletion. The significant selective loss of CD26 expression observed in HIV+ patients is probably an early event after HIV infection and seems to occur even before CD4 cell depletion. In conclusion, evaluation of DAP IV/CD26 might be a useful option for monitoring the immunological alterations of all hemophilic patients, HIV positive or not, chronically treated with coagulation factors.

A multicenter pharmacosurveillance study for the evaluation of the efficacy and safety of recombinant factor VIII in the treatment of patients with hemophilia A. German Kogenate Study Group.

In this open multicenter study the safety and efficacy of recombinant factor VIII (rFVIII) was assessed in 39 previously treated patients with hemophilia A (factor VIII basal activity < or = 15%). Recombinant FVIII was administered for prophylaxis and treatment of bleeding episodes and for surgical procedures. A total of 3679 infusions of rFVIII were given. Efficacy of rFVIII as assessed by subjective evaluation of response to infusion and mean annual consumption of rFVIII was comparable to that of plasma derived FVIII concentrates. The incremental recovery of FVIII (2.4 +/- 0.83%/IU/kg, 2.12 +/- 0.61%/IU/kg, resp.) was within the expected range. No clinical significant FVIII inhibitor was detected in this trial. Five of 16 susceptible patients showed a seroconversion for parvovirus B19. However, the results are ambiguous in two cases and might be explained otherwise in one further case. Thus, in two patients a reliable seroconversion for parvovirus B19 was observed.

Activity of granzyme A, a serine protease in the killing granules of cytotoxic T lymphocytes, is reduced in cells from HIV-infected hemophiliacs.

Cytotoxic CD8+ lymphocytes (CTLs) kill virally infected target cells by releasing cytotoxic granules. The primary objective of this study was to determine whether the activity of granzyme A, a serine protease in the killing granules of CTLs is altered in HIV-infected hemophiliacs. A sensitive colorimetric assay that measures cleavage of a synthetic substrate, N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), was used to quantitate granzyme A activity. Granzyme A activities from hemophiliacs were normalized to to granzyme A activities of healthy donors run concurrently. Granzyme A activity in CD8+ T cells from HIV-seropositive hemophiliacs was significantly lower than granzyme A activity in cells from HIV-seronegative hemophiliacs (0.48 units +/- 0.086/CD8+ T cell and 1.573 +/- 0.434 units/CD8+ T cell, respectively; p < 0.005). These results indicate that cytotoxic cells in HIV-infected hemophiliacs have reduced granzyme A activity, which may result in a defect in CTL-mediated cell killing in these patients.

Clinical significance of serum 2,5-oligoadenylate synthetase and soluble interleukin-2 receptor in hemophiliacs positive and negative for human immunodeficiency virus type 1.

We measured serum 2,5-oligoadenylate synthetase (2,5-AS) levels and soluble interleukin-2 receptor (sIL-2R) levels in human immune deficiency virus type 1 (HIV-1)-positive and HIV-1-negative hemophiliacs in order to clarify the clinical significance of these parameters in hemophiliacs. Serum 2,5-AS levels were measured by a radioimmunosorbent assay, and sIL-2R levels were measured by an enzyme-linked immunosorbent assay. The mean serum 2,5-AS levels were higher in AIDS-related-complex and AIDS patients, asymptomatic carriers, and HIV-1-negative hemophiliacs than in hepatitis C virus-positive patients and healthy controls. Serial determinations showed that the 2,5-AS levels tended to increase in HIV-1-positive patients, especially those with AIDS-related complex or AIDS, although it showed a substantial decrease in the terminal stage. The serum sIL-2R levels were higher in HIV-1-positive patients, HIV-1-negative patients, and hepatitis C virus-positive patients than in controls. Serial studies showed little change in the HIV-1-positive and HIV-1-negative groups, although sIL-2R levels showed a tendency to decrease with zidovudine treatment. On the basis of the present results, we may well conclude that 2,5-AS and sIL-2R are not specific markers for hemophiliacs with HIV-1 infection. However, serial measurement of these markers can still be useful for assessing the progression of AIDS and the prognosis for patients with AIDS, as well as for monitoring the response to zidovudine.

Interferon alfa for chronic hepatitis C in haemophiliacs.

Chronic hepatitis C virus (HCV) associated liver disease is an important cause of morbidity and mortality in haemophilia. Recombinant interferon alfa-2b was used in a randomised controlled liver biopsy trial to treat haemophiliacs with chronic HCV. All 18 patients entered had antibodies to HCV. During the first year of the study, 10 patients were randomised on the basis of histology to receive interferon alfa-2b, 3 million units subcutaneously, thrice weekly and eight to receive no treatment (control group). After 12 months, all patients had a second liver biopsy and the control group patients were offered interferon at the same dosage but for only six months. The alanine aminotransferase (ALT) activity had returned to normal in four of 10 patients treated for one year and five of six patients treated for six months, compared with none of the eight patients in the control group (p < 0.01). Although the histological scores of the two groups were similar at entry into the study, after one year the biopsy specimens in the treated group showed significant improvement compared with controls (p < 0.01). It is concluded that interferon alfa-2b is effective in returning ALT values to normal and improving liver histology in at least 50% of patients treated.

Proteolysis of factor VIII heavy chain polypeptides in plasma and concentrates.

Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases. FVIII samples were incubated with labelled affinity-purified anti-FVIII Fab' fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification. Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250-300 kD, and the smallest at 80-90 kD: the bands visualized correspond to the 90-210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma. Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250-300 kD and other intermediate bands, intensified then removed the 80-90 kD polypeptide and produced a band at 40-50 kD. Thrombin-associated rise and fall in FVIII clotting activity by one-stage assay correlated with intensity of the 80-90 kD polypeptide. A polypeptide of Mr 40-50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure. FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately 'activated' product.