Comparative proteomes, immunoreactivities and neutralization of procoagulant activities of Calloselasma rhodostoma (Malayan pit viper) venoms from four regions in Southeast Asia.

The intraspecific geographical venom variations of Calloselasma rhodostoma from Malaysia (CR-M), Indonesia (CR-I), Thailand (CR-T) and Vietnam (CR-V) were investigated through 1D SDS-PAGE and nano-ESI-LCMS/MS. The venom antigenicity, procoagulant activities and neutralization using Thai C. rhodostoma Monovalent Antivenom (CRMAV) were also investigated. SDS-PAGE patterns of the venoms were relatively similar with minor variations. Proteomic analysis revealed that snake venom metalloproteinases (SVMPs, particularly P-I class), serine proteases (SVSPs) and snaclecs dominated the venom protein composition (68.96-81.80%), followed by L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) (7.37-11.08% and 5.18-13.81%, respectively), corroborating C. rhodostoma envenoming effects (hemorrhage, consumptive coagulopathy, thrombocytopenia and local tissue necrosis). Other proteins of lower abundances (2.82-9.13%) identified include cysteine-rich secretory proteins (CRISP), phospholipase B, phosphodiesterase, nerve growth factor, 5'-nucleotidase, aminopeptidase and hyaluronidase. All four venoms exhibited strong procoagulant effects which were neutralized by CRMAV to different extents. CRMAV immunoreactivity was high toward venoms of CR-M, CR-I and CR-T but relatively low for CR-V venom. Among the venom samples from different locales, CR-V venom proteome has the smallest SVMP composition while SVSP, PLA2 and phosphodiesterase were more abundant in the venom. These variations in C. rhodostoma venom protein composition could partly explain the differences seen in immunoreactivity. (198 words).

A single-domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor.

BACKGROUND: Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF). OBJECTIVE: We aimed to generate a single-domain antibody (sdAb) that recognizes free FVIIa rather than TF-bound FVIIa. METHODS: A llama-derived phage library was used to screen for anti-FVIIa sdAbs. RESULTS: One sdAb, KB-FVIIa-004, bound to FVIIa, but not to its precursor FVII or to homologous proteins (prothrombin, factor X, or their activated derivatives). FVIIa amidolytic activity was inhibited by KB-FVIIa-004 (Ki  = 28-45 nM) in a competitive manner. KB-FVIIa-004 also inhibited FVIIa-mediated FX activation (Ki  = 26 nM). In contrast, KB-FVIIa-004 was inefficient in prolonging the clotting time of the prothrombin time-test, which was prolonged by a maximum of 10 s at high sdAb concentrations (10 µM). Furthermore, FVIIa/TF amidolytic activity or FVIIa/TF-mediated FX activation remained unaffected up to a 50-fold to 1000-fold molar excess of KB-FVIIa-004. These data suggest that KB-FVIIa-004 loses its inhibitory activity in the presence of TF. A KB-FVIIa-004/albumin fusion-protein (004-HSA) was generated for in vivo testing. By using 004-HSA, we observed that this sdAb blocked the therapeutic capacity of FVIIa to correct bleeding in FVIII-deficient mice. DISCUSSION: This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF-independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF-bound and TF-free FVIIa.

Effects of atorvastatin on ADP-, arachidonic acid-, collagen-, and epinephrine-induced platelet aggregation.

Objective Atorvastatin reduces the incidence of cardiovascular events. However, the effects of atorvastatin on platelet aggregation are unknown. Methods Blood samples were obtained from 126 healthy volunteers. Prepared isolated platelet suspensions were adjusted with saline to three different concentrations of 100 × 109, 300 × 109, and 600 × 109 platelets/L. Platelet samples were incubated with atorvastatin (10-7 mol/L, 10-6 mol/L or 10-5 mol/L), and stimulated with ADP (10 µmol/L), arachidonic acid (0.5 mmol/L), collagen (2 µg/mL), and epinephrine (1 mg/mL). The maximal amplitude of aggregation and the curve slope were measured by electric impedance aggregometry. Results Atorvastatin inhibited platelet aggregation at moderate (300 × 109/L) and high (600 × 109/L) concentrations. However, an inhibitory effect of atorvastatin at low concentrations (100 × 109/L) was not observed. Conclusions The study shows that atorvastatin inhibits platelet aggregation in vitro, and this inhibitory effect is related to platelet concentrations.

Activation of glycoprotein VI (GPVI) and C-type lectin-like receptor-2 (CLEC-2) underlies platelet activation by diesel exhaust particles and other charged/hydrophobic ligands.

Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)-FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.

New predictive approaches for ITI treatment.

Immune tolerance induction (ITI) therapy in patients with haemophilia A and inhibitors constitutes a huge burden for affected patients and families and poses a large economic burden for a chronic disease. Concerted research efforts are attempting to optimize the therapeutic approach to the prevention and eradication of inhibitors. The Italian ITI Registry has provided data on 110 patients who completed ITI therapy as at July 2013. Analysis of independent predictors of success showed that, together with previously recognized factors - namely inhibitor titre prior to ITI, historical peak titre and peak titre on ITI - the type of causative FVIII gene mutation also contributes to the identification of patients with good prognosis and may be useful to optimize candidate selection and treatment regimens. Numerous studies have demonstrated that inhibitor reactivity against different FVIII products varies and is lower against concentrates containing von Willebrand factor (VWF). An Italian study compared inhibitor titres against a panel of FVIII concentrates in vitro and correlated titres with the capacity to inhibit maximum thrombin generation as measured by the thrombin generation assay (TGA). Observations led to the design of the PredictTGA study which aims to correlate TGA results with epitope specificity, inhibitor reactivity against different FVIII concentrates and clinical data in inhibitor patients receiving FVIII in the context of ITI or as prophylactic/on demand treatment. At the immunological level, it is known that T cells drive inhibitor development and that B cells secrete FVIII-specific antibodies. As understanding increases about the immunological response in ITI, it is becoming apparent that modulation of T-cell- and B-cell-mediated responses offers a range of potential new and specific approaches to prevent and eliminate inhibitors as well as individualize ITI therapy.

Intraspecific differences in the immunochemical reactivity and neutralization of venom from Argentinean Bothrops (Rhinocerophis) alternatus by specific experimental antivenoms.

The venoms of Bothrops (Rhinocerophis) alternatus (B.a.) from different regions of Argentina have shown biochemical, toxicological and immunological variations. Considering these variations, we produced nine experimental antisera (rabbit, IgG) against venoms from snakes of nine different regions and a pool of venom, comprised of equal amounts of venoms from each region. The immunologic studies (ELISA, Westernblot) showed significant cross reactivity among all regional antivenoms with all regional venoms, with no significant differences regarding the specificity of the immunogens used for the production of antivenom. Neutralization of hemorrhage was variable (although all the antivenoms neutralized this activity in all venoms) and the neutralization of coagulant and phospholipase activities were evident in all cases. Some antivenoms neutralized toxic activities that were absent or very low in the venoms used as immunogen, on other non-homologous venoms (e.g. thrombin like activity). Despite the different toxic potencies of regional venoms, antivenoms developed using venoms of snakes from a particular region showed high immunochemical reactivity and cross-neutralizing capacity on snake venoms from different and distant regions, in occasions over those of the homologous antivenoms. These findings could be used to improve the generation of pools of venoms for the production of antivenoms.

Successful management of factor IX inhibitor-associated nephrotic syndrome in a hemophilia B patient.

BACKGROUND: Nephrotic syndrome (NS) is a recognized complication of immune tolerance induction (ITI) therapy, a treatment strategy used to treat inhibitors in patients with hemophilia B receiving factor IX concentrate. CASE DIAGNOSIS/TREATMENT: We present a 4-year-old boy with hemophilia B and an inhibitor who underwent ITI, and developed NS 19 months into this therapy. A percutaneous renal biopsy was safely performed with factor IX (FIX) concentrate administration both preceding and following the procedure. The patient's inhibitor level had increased to 1.4-1.6 Bethesda Units just prior to the onset of proteinuria. Histology confirmed segmental membranous nephropathy (MGN). The patient was continued on FIX concentrate as ITI and also received 4 weekly doses of rituximab and ongoing immunosuppression with mycophenolate mofetil. This resulted in the complete resolution of his inhibitor and his NS. He continues with a modified ITI regimen and remains inhibitor-free without proteinuria >12 months post-biopsy. CONCLUSIONS: Hemophilia B patients undergoing ITI should be regularly screened for NS. At first detection of proteinuria, with proper precautions, a percutaneous kidney biopsy can be performed safely in patients with low levels of inhibitor. Our patient had segmental MGN with complete remission of NS.

More than a decade of international experience with a pdFVIII/VWF concentrate in immune tolerance.

Approximately 20-30% of patients with severe haemophilia A develop alloantibodies ('inhibitors') to infused FVIII rendering use of such replacement therapy ineffective. Once an inhibitor emerges, immune tolerance induction (ITI) is the standard treatment. ITI involves giving regular doses of FVIII concentrate to eradicate the inhibitor and achieve immunogenic acceptance of administered FVIII. In the early 2000s, a retrospective analysis of inhibitor patients treated at a single centre in Germany indicated that success rates were higher when patients were treated with von Willebrand factor (VWF)-containing plasma-derived FVIII (pdFVIII/VWF) concentrate compared with recombinant or non-VWF-containing pdFVIII products. Importantly, pdFVIII/VWF as rescue therapy was able to convert 8 of 10 patients who had failed primary ITI with recombinant or non-VWF-containing pdFVIII product. A subsequent study from Italy in patients with poor prognostic factors for ITI success also reported good success rates with pdFVIII/VWF as rescue therapy (53% success; 41% partial success). The Grifols-Immune Tolerance Induction (G-ITI) Study represents the largest group of haemophilia A inhibitor patients treated with a single pdFVIII/VWF concentrate (Alphanate(®)/Fanhdi(®)) to be reported to date. Data have been collected for 95 patients who underwent primary or rescue ITI at 46 centres in Europe and the US. Currently, published data are available for 33 patients in the US cohort (11 centres), and data from the European cohort are being analysed. Both groups contained patients with poor prognostic factors and most patients received a high-dose regimen (≥ 100 IU pdFVIII/VWF kg(-1) daily). As expected, the success rate was better for primary vs. rescue ITI and for patients with good vs. poor prognostic factors. However, more than half the patients in the US cohort receiving rescue ITI achieved success (33% complete success; 20% partial success). These results should encourage clinicians to consider the use of pdFVIII/VWF concentrates for rescue ITI. Published outcomes data from the total global G-ITI cohort (95 patients) are awaited with anticipation.

The immune tolerance induction (ITI) dose debate: does the International ITI Study provide a clearer picture?

Among the proposed predictors for immune tolerance induction (ITI) outcome, the therapeutic regimen - specifically the dose and frequency of administered factor VIII (FVIII) as well as FVIII product type - is intensely debated. Are there any advantages for low-dose regimens (50 IU FVIII kg(-1) three times a week) over high-dose regimens (200 IU FVIII kg day(-1)) or vice versa? Are von Willebrand factor (VWF)-containing plasma-derived concentrates superior to recombinant FVIII concentrates for tolerance induction? A review of the available literature indicates that patients with good prognostic factors can achieve success with either low-dose or high-dose ITI regimens. Retrospective data suggest that patient characteristics such as maximum historical inhibitor titres and pre-ITI inhibitor titres are better predictors of treatment success than dose. Results of the prospective International ITI Study have recently become available. In inhibitor patients with good prognosis, success rates were similar between low-dose (50 IU FVIII kg(-1) three times a week) and high-dose (200 IU FVIII kg(-1) daily) regimens. However, patients receiving low-dose ITI took longer to achieve various ITI milestones and had a significantly higher bleed rate per month compared with the high-dose group (0.62 vs. 0.28; P = 0.00024), findings with important clinical implications. Inhibitor patients with poor prognostic features should be treated with a high-dose protocol. This conclusion is supported by a meta-analysis of the International Immune Tolerance Registry and North American Immune Tolerance Registry and by data from Germany showing good success rates with the high-dose, high-frequency Bonn protocol in poor prognosis patients. Type of concentrate also appears to have an influence on ITI success rates in this patient subgroup, with evidence suggesting an advantage for VWF-containing plasma-derived FVIII concentrates over recombinant or VWF-free concentrates. The ongoing prospective studies REScue Immunotolerance STudy and Observational Immune Tolerance Induction are evaluating ITI outcome with respect to product type and are expected to answer this important clinical question as well as provide greater insight into patient- and therapy-related variables in inhibitor patients with poor prognostic features.

Comprehensive care of the patient with haemophilia and inhibitors undergoing surgery: practical aspects.

Congenital haemophilia is a rare and complex condition for which dedicated specialized and comprehensive care has produced measurable improvements in clinical outcomes and advances in patient management. Among these advances is the ability to safely perform surgery in patients with inhibitor antibodies to factors VIII and IX, in whom all but the most necessary of surgeries were once avoided due to the risk for uncontrollable bleeding due to ineffectiveness of replacement therapy. Nevertheless, surgery continues to pose a major challenge in this relatively rare group of patients because of significantly higher costs than in patients without inhibitors, as well as a high risk for bleeding and other complications. Because of the concentration of expertise and experience, it is recommended that any surgery in patients with haemophilia and inhibitors be planned in conjunction with a haemophilia treatment centre (HTC) and performed in a hospital that incorporates a HTC. Coordinated, standard pre-, intra- and postoperative assessments and planning are intended to optimize surgical outcome and utilization of resources, including costly factor concentrates and other haemostatic agents, while minimizing the risk for bleeding and other adverse consequences both during and after surgery. This article will review the special considerations for patients with inhibitors as they prepare for and move through surgery and recovery, with an emphasis on the roles and responsibilities of individual members of the multidisciplinary team in facilitating this process.

Antidote strategies to reverse anticoagulation with TB-402, a long-acting partial inhibitor of factor VIII.

BACKGROUND: TB-402 is a partially inhibiting antibody of factor VIII that is under development as a long-acting anticoagulant. PATIENTS AND METHODS: The reversibility of FVIII inhibition by TB-402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma-derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg(-1) rhFVIII 48 h after a single dose of 620 µg kg(-1) TB-402. TB-402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. RESULTS: In spiked samples, TB-402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 µg mL(-1) TB-402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB-402. The inhibitory effect of TB-402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half-life (t(1/2) ) of TB-402 was 14.2 days. TB-402 lowered the endogenous thrombin potential by 23% for ~ 35 days. Infusion of 35 IU kg(-1) rhFVIII had a marginal effect, whereas 70 IU kg(-1) rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ~ 3 h. CONCLUSIONS: TB-402 resulted in a stable long-term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB-402 temporarily, and may be effective antidotes for future clinical practice.

Aronia melanocarpa extract suppresses the biotoxicity of homocysteine and its metabolite on the hemostatic activity of fibrinogen and plasma.

OBJECTIVE: Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances, and it has been shown to have various biological activities. Berries of A. melanocarpa (chokeberry) have been supposed to be beneficial for the prevention of cardiovascular events. In this study the influence of aronia extract on the clot formation (using human plasma and purified fibrinogen) and the fibrin lysis during the model of hyperhomocysteinemia was investigated. METHODS: Hyperhomocysteinemia was induced using a reduced form of Hcys (at final dose of 0.1mM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (HTL, 1 µM). The aim of our study in vitro was also to investigate the modifications of human plasma total proteins and the oxidative stress (by measuring the total antioxidant level - TAS) in plasma after incubation with Hcys, HTL and/or aronia extract. The biological properties of aronia extract were compared with the action of a well characterized antioxidative commercial polyphenol - resveratrol (3,4',5- trihydroxystilbene). RESULTS: The HTL, like its precursor, Hcys stimulated polymerization of fibrinogen. The results also demonstrated that Hcys (0.1mM) and HLT at lower doses than Hcys (1 µM) reduced the fibrin lysis in human plasma. Moreover, Hcys and HTL change the level of thiol and amino groups in plasma total proteins and induce the oxidative stress in plasma. Our results indicate that aronia extract reduced the biotoxicity action of Hcys and HTL on hemostatic properties of fibrinogen or plasma, suggesting its possible protective properties in hyperhomocysteinemia - induced cardiovascular diseases. Moreover, our results showed that the extract from berries of A. melanocarpa due to antioxidant action, significantly reduced the oxidative stress (measured by TAS) in plasma during the model of hyperhomocysteinemia. CONCLUSION: In the comparative studies, the extract from berries of A. melanocarpa and reseveratrol had similar protective properties. It gives hopes for development of diet supplements, which may be preventing thrombosis in pathological states where plasma procoagulant activity and oxidative stress are observed e.g. in hyperhomocysteinemia.

Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin.

NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.

Inhibition of the inflammatory and coagulant action of Bothrops atrox venom by the plant species Marsypianthes chamaedrys.

AIM OF THE STUDY: This study investigated the efficacy of Marsypianthes chamaedrys Vahl (Lamiaceae) inflorescence and leaf extracts in inhibiting the inflammatory and coagulant actions of Bothrops atrox venom. MATERIALS AND METHODS: Marsypianthes chamaedrys, which is used in Brazil as a folk medicine to treat snakebites and local inflammatory reactions, was tested in vitro to determine its ability to block indirect phospholipase A(2) and direct coagulant activities and in vivo to determine its ability to inhibit leukocyte migration and cytokine release. RESULTS: In vitro, Marsypianthes chamaedrys showed antiphospholipase A(2) and anticoagulant activities; the latter activity was also confirmed by prothrombin time (PT) and activated partial thromboplastin time (aPTT) in the absence of venom. Of the extracts used, those obtained from the crushed plant had the greater inhibitory activity in in vitro tests, showing that biological activity is affected by the way extracts are obtained. In vivo, Marsypianthes chamaedrys inhibited leukocyte migration and the release of the proinflammatory cytokines IL-6 and TNF-α without altering the concentration of the anti-inflammatory cytokine IL-10. CONCLUSIONS: As specific antivenoms are not effective in neutralizing the local action of Bothrops venoms, characterization of the anti-inflammatory mechanisms induced by Marsypianthes chamaedrys is of vital importance if the extracts of this plant species are to be used in future as adjuvants in the treatment of snakebites.

Review of antihemophilic factor injection for the routine prophylaxis of bleeding episodes and risk of joint damage in severe hemophilia A.

Individuals with severe factor VIII deficiency experience recurrent hemorrhages and develop progressive joint damage. Large retrospective, nonrandomized studies of patient cohorts followed over decades show that factor prophylaxis initiated at an early age before the onset of recurrent bleeding reduces the incidence of hemophilic arthropathy. Two recent prospective, multicenter, randomized trials conducted in Europe (the ESPRIT study) and the USA (the Joint Outcome Study) confirm the efficacy of prophylaxis in the prevention of hemarthroses and arthropathy. Regular prophylaxis initiated in early childhood enhances the quality of life for patients with severe hemophilia and reduces the risk of inhibitor development. The substantial costs of such preventative therapy may be offset by the reduced expenditures that the care of degenerative joint disease in adult hemophilia patients would otherwise require.

Generation and biochemical characterization of glycoPEGylated factor VIIa derivatives.

Prophylaxis with 2-4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient, recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface, TF, FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylationtrade mark technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.

Occurrence of inhibitors in previously untreated or minimally treated patients with haemophilia A after exposure to a plasma-derived solvent-detergent factor VIII concentrate.

A solvent-detergent virus-inactivated plasma-derived FVIII concentrate (SD-pdFVIII) has been employed for treatment of Italian patients with haemophilia A for 15 years. This product is a non-monoclonally purified, high purity FVIII concentrate, containing large amounts of von Willebrand factor (VWF). A retrospective survey was carried out in Italy in order to evaluate the immunogenicity of SD-pdFVIII in previously untreated patients (PUPs) or in minimally treated patients (MTPs), i.e. previously exposed for up to 5 days only to other plasma-derived concentrates. The survey included 99 patients with ages ranging from 6 to 64 years (median=21.3) of whom 31 PUPs and 68 MTPs, the latter with a median of four exposure days (EDs; range 1-5) to other plasma products. Surveyed patients had been exposed to SD-pdFVIII for a median of 83 EDs (range 21-1580). Seven patients (three PUPs and four MTPs), all with severe haemophilia, had developed inhibitors [7.1%, 95%; confidence interval: 3-14%] after a median of 11 EDs (range 4-22). Of them, two were low responders (

Gene expression in the salivary complexes from Haementeria depressa leech through the generation of expressed sequence tags

A survey of the transcriptional profile of Haementeria depressa Ringuelet, 1972 (Annelida, Hirudinea) salivary complexes was produced through expressed sequence tag (EST). Sequences from 898 independent clones were assembled in 555 clusters, representing the transcript profile of this tissue. The repertoire of possible proteins involved in feeding and host interaction processes of the leech corresponded to 10.6% of all identified transcripts (67 clusters), being the carbonic anhydrases (30%), several coagulation inhibitors (25%) and hemerythrin-like molecules (19%), the major components. Among the 387 clusters matching cellular proteins, the majority represents molecules involved in gene and protein expression, reflecting a high specialization of this tissue for protein synthesis. Our H. depressa dbEST was also compared to those from other blood-feeding organisms, providing evidences that among the secreted proteins, the coagulation inhibitors present a profile very characteristic of this animal class