Concizumab improves clot formation in hemophilia A under flow.

BACKGROUND: Inhibition of tissue factor pathway inhibitor (TFPI) is an emerging therapeutic strategy for treatment of hemophilia. Concizumab is a monoclonal antibody that binds TFPI and blocks its inhibition of factor (F)Xa thereby extending the initiation of coagulation and compensating for lack of FVIII or FIX. OBJECTIVES: The objective of this in vitro study was to evaluate how concizumab affects clot formation in hemophilia A under flow. METHODS: Blood was collected from normal controls or people with hemophilia A. An anti-FVIII antibody was added to normal controls to simulate hemophilia A with inhibitory antibodies to FVIII. Whole blood and recombinant activated FVII (rFVIIa, 25 nM) or concizumab (200, 1000, and 4000 ng/mL) were perfused at 100 s-1 over a surface micropatterned with tissue factor (TF) and collagen-related peptide. Platelet and fibrin(ogen) accumulation were measured by confocal microscopy. Static thrombin generation in plasma was measured in response to rFVIIa and concizumab. RESULTS: Concizumab (1000 and 4000 ng/mL) and rFVIIa both rescued (93%-101%) total platelet accumulation, but only partially rescued (53%-63%) fibrin(ogen) incorporation to normal control levels in simulated hemophilia A. Results using congenital hemophilia A blood confirmed effects of rFVIIa and concizumab. While these 2 agents had similar effect on clot formation under flow, concizumab enhanced thrombin generation in plasma under static conditions to a greater extent than rFVIIa. CONCLUSION: TFPI inhibition by concizumab enhanced activation and aggregation of platelets and fibrin clot formation in hemophilia A to levels comparable with that of rFVIIa.

Treatment with recombinant ADAMTS13, alleviates hypoxia/reoxygenation-induced pathologies in a mouse model of human sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is an inherited red blood cell disorder with a causative substitution in the beta-globin gene that encodes beta-globin in hemoglobin. Furthermore, the ensuing vasculopathy in the microvasculature involves heightened endothelial cell adhesion, inflammation, and coagulopathy, all of which contribute to vaso-occlusive crisis (VOC) and the sequelae of SCD. In particular, dysregulation of the von Willebrand factor (VWF) and a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in human SCD pathology. OBJECTIVES: To investigate the beneficial potential of treatment with recombinant ADAMTS13 (rADAMTS13) to alleviate VOC. METHODS: Pharmacologic treatment with rADAMTS13 in vitro or in vivo was performed in a humanized mouse model of SCD that was exposed to hypoxia/reoxygenation stress as a model of VOC. Then, pharmacokinetic, pharmacodynamic, and behavioral analyses were performed. RESULTS: Administration of rADAMTS13 to SCD mice dose-dependently increased plasma ADAMTS13 activity, reduced VWF activity/antigen ratios, and reduced baseline hemolysis (free hemoglobin and total bilirubin) within 24 hours. rADAMTS13 was administered in SCD mice, followed by hypoxia/reoxygenation stress, and reduced VWF activity/antigen ratios in parallel to significantly (p < .01) improved recovery during the reoxygenation phase. Consistent with the results in SCD mice, we demonstrate in a human in vitro system that treatment with rADAMTS13 counteracts the inhibitory activity of hemoglobin on the VWF/ADAMTS13-axis. CONCLUSION: Collectively, our data provide evidence that relative ADAMTS13 insufficiency in SCD mice is corrected by pharmacologic treatment with rADAMTS13 and provides an effective disease-modifying approach in a human SCD mouse model.

Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease.

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events.

Heterogeneity in Bleeding Tendency and Arthropathy Development in Individuals with Hemophilia.

People with hemophilia (PWH) have an increased tendency to bleed, often into their joints, causing debilitating joint disease if left untreated. To reduce the incidence of bleeding events, PWH receive prophylactic replacement therapy with recombinant factor VIII (FVIII) or FIX. Bleeding events in PWH are typically proportional to their plasma FVIII or IX levels; however, in many PWH, bleeding tendency and the likelihood of developing arthropathy often varies independently of endogenous factor levels. Consequently, many PWH suffer repeated bleeding events before correct dosing of replacement factor can be established. Diagnostic approaches to define an individual's bleeding tendency remain limited. Multiple modulators of bleeding phenotype in PWH have been proposed, including the type of disease-causing variant, age of onset of bleeding episodes, plasma modifiers of blood coagulation or clot fibrinolysis pathway activity, interindividual differences in platelet reactivity, and endothelial anticoagulant activity. In this review, we summarize current knowledge of established factors modulating bleeding tendency and discuss emerging concepts of additional biological elements that may contribute to variable bleeding tendency in PWH. Finally, we consider how variance in responses to new gene therapies may also necessitate consideration of patient-specific tailoring of treatment. Cumulatively, these studies highlight the need to reconsider the current "one size fits all" approach to treatment regimens for PWH and consider therapies guided by the bleeding phenotype of each individual PWH at the onset of therapy. Further characterization of the biological bases of bleeding heterogeneity in PWH, combined with the development of novel diagnostic assays to identify those factors that modulate bleeding risk in PWH, will be required to meet these aspirations.

A bispecific antibody demonstrates limited measurability in routine coagulation assays.

: Accurate monitoring of coagulation, needed for optimal management of patients with haemophilia A with inhibitors, presents a challenge for treating physicians. Although global haemostatic assays may be used in this population, their utility with nonfactor therapies has yet to be established in the clinical setting. The aim of this study was to assess options for potential haemostatic activity monitoring and feasibility for factor VIII (FVIII)-equivalency measurement with a sequence identical analogue (SIA) to emicizumab using different coagulation assays. SIA was analysed using five commercial chromogenic assays and activated partial thromboplastin time (aPTT) assays including clot waveform analysis using five different triggers. Recombinant FVIII served as a comparator in all assays. Thrombin generation in haemophilia A plasma was measured using extrinsic and intrinsic trigger conditions (tissue factor or Factor XIa). Of the five chromogenic assays, a concentration-dependent increase in Factor Xa was observed with one assay, with human Factor IXa and X reagents. The SIA dose-response signal plateaued at therapeutically relevant concentrations and was nonparallel with FVIII reference, thereby not permitting FVIII-equivalence assessment. aPTT varied between reagents, with aPTT normalization occurring at low and below-therapeutic SIA concentrations. SIA [600 nmol/l (90 µg/ml)] only partially restored thrombin generation in individual haemophilia A patient plasma. FVIII-equivalence of SIA could not be determined using standard FVIII protocols and was found to be highly influenced by assay type, analytical conditions and parameters used for calculation. New and/or modified methodology and standard reagents specific for use with nonfactor therapies are required for their utilization in the clinical setting.

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.

Role of exosite binding modulators in the inhibition of Fxa by TFPI.

Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 µM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2-1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1-150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25-0.5 nM TFPI) inhibit FXa with a t1/2 between 3-15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

UNLABELLED: BACKGROUND & METHODS: Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & DISCUSSION: The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. CONCLUSIONS: From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Correcting thrombin generation ex vivo using different haemostatic agents following cardiac surgery requiring the use of cardiopulmonary bypass.

Recently, lower thrombin generation has been associated with excess bleeding post-cardiopulmonary bypass (CPB). Therefore, treatment to correct thrombin generation is a potentially important aspect of management of bleeding in this group of patients. The objective of the present study was to investigate the effects of fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), prothrombin complex concentrate (PCC) and tissue factor pathway inhibitor (TFPI) inhibition on thrombin generation when added ex vivo to the plasma of patients who had undergone cardiac surgery requiring CPB. Patients undergoing elective cardiac surgery were recruited. Blood samples were collected before administration of heparin and 30 min after its reversal. Thrombin generation was measured in the presence and absence of different concentrations of FFP, rFVIIa, PCC and an anti-TFPI antibody. A total of 102 patients were recruited. Thrombin generation following CPB was lower compared with pre-CPB (median endogenous thrombin potential pre-CPB 339 nmol/l per min, post-CPB 155 nmol/l per min, P < 0.0001; median peak thrombin pre-CPB 35 nmol/l, post-CPB 11 nmol/l, P < 0.0001). Coagulation factors and anticoagulants decreased, apart from total TFPI, which increased (55-111 ng/ml, P < 0.0001), and VWF (144-170 IU/dl, P < 0.0001). Thrombin generation was corrected to pre-CPB levels by the equivalent of 15 ml/kg FFP, 45 µg/kg rFVIIa and 25 U/kg of PCC. Inhibition of TFPI resulted in an enhancement of thrombin generation significantly beyond pre-CPB levels. This study shows that FFP, rFVIIa, PCC and inhibition of TFPI correct thrombin generation in the plasma of patients who have undergone surgery requiring CPB. Inhibition of TFPI may be a further potential therapeutic strategy for managing bleeding in this group of patients.

Screening of complex fucoidans from four brown algae species as procoagulant agents.

Fucoidans are complex sulfated polysaccharides extracted from brown algae. Depending on the concentration, they have been shown to stimulate and inhibit blood coagulation in vitro. Promotion of coagulation is mediated by blocking tissue factor pathway inhibitor (TFPI). We screened fucoidan extracts from four brown algae species in vitro with respect to their potential to improve coagulation in bleeding disorders. The fucoidans' pro- and anticoagulant activities were assessed by global hemostatic and standard clotting assays. Results showed that fucoidans improved coagulation parameters. Some fucoidans also activated the contact pathway of coagulation, an undesired property reported for sulfated glycosaminoglycans. Chemical evaluation of fucoidans' complex and variable structure included molecular weight (Mw), polydispersity (polyD), structural heterogeneity, and organic and inorganic impurities. Herewith, we describe a screening strategy that facilitates the identification of crude fucoidan extracts with desired biological and structural properties for improvement of compromised coagulation like in hemophilia.

Small peptides blocking inhibition of factor Xa and tissue factor-factor VIIa by tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded ß-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Plasmatic tissue factor pathway inhibitor is a major determinant of clotting in factor VIII inhibited plasma or blood.

Tissue factor pathway inhibitor (TFPI) is a major inhibitor of coagulation. We therefore hypothesised that high plasmatic TFPI levels are associated with impaired ex vivo clotting in a model of acquired haemophilia. Blood samples were collected in a prospective clinical study from 30 healthy volunteers. Coagulation in normal or factor VIII (FVIII)-inhibited human blood or plasma was measured by the calibrated automated thrombogram (CAT) and rotational thromboelastometry (ROTEM). Both methods are global haemostatic assays that provide insight into the whole coagulation process. Monoclonal mouse antibodies raised against either the C-terminus or the Kunitz domain 2 of TFPI were used to determine full-length (fl-) and total TFPI by an enzyme-immunoassay. Clotting times and parameters of thrombin generation correlated with TFPI levels. Subjects with low fl-TFPI levels had significantly shorter clotting times and a higher endogenous thrombin potential (ETP) compared to those with high fl-TFPI levels (p≤0.005 for all). An even stronger effect was seen in FVIII-inhibited blood/plasma: ROTEM clotting time was 26% shorter (p=0.01) and the ETP assessed by CAT was >2-fold higher in subjects with low fl-TFPI levels (p≤0.0001). Plasmatic TFPI is a major determinant of coagulation in global haemostatic tests particularly when FVIII is missing. Thus, inhibition of TFPI might be a promising novel treatment approach, especially in haemophilia patients with FVIII inhibitors.

Neutralization of macrophage migration inhibitory factor (MIF) by fully human antibodies correlates with their specificity for the ß-sheet structure of MIF.

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a ß-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this ß-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

von Willebrand factor-cleaving protease ADAMTS13 reduces ischemic brain injury in experimental stroke.

Stroke is a leading cause of death and disability. The only therapy available is recombinant tissue plasminogen activator, but side effects limit its use. Platelets play a crucial role during stroke, and the inflammatory reaction promotes neurodegeneration. von Willebrand factor (VWF), an adhesion molecule for platelets, is elevated in patients with acute stroke. The activity of VWF is modulated by ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) that cleaves VWF to smaller less-active forms. We recently documented that ADAMTS13 negatively regulates both thrombosis and inflammation. We report that deficiency or reduction of VWF reduces infarct volume up to 2-fold after focal cerebral ischemia in mice, thus showing the importance of VWF in stroke injury. In contrast, ADAMTS13 deficiency results in larger infarctions, but only in mice that have VWF. Importantly, infusion of a high dose of recombinant human ADAMTS13 into a wild-type mouse immediately before reperfusion reduces infarct volume and improves functional outcome without producing cerebral hemorrhage. Furthermore, recombinant ADAMTS13 did not enhance bleeding in a hemorrhagic stroke model. Our findings show the importance of VWF in regulating infarction and suggest that recombinant ADAMTS13 could be considered as a new therapeutic agent for prevention and/or treatment of stroke.

Systemic antithrombotic effects of ADAMTS13.

The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13-/- mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in alphaIIbbeta3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.

Relation between ADAMTS13 activity and ADAMTS13 antigen levels in healthy donors and patients with thrombotic microangiopathies (TMA).

We have established a new, enzyme-linked immunosorbent assay (ELISA) for the detection of ADAMTS13 antigen using purified polyclonal rabbit anti-human ADAMTS13 IgG. Normal plasma ADAMTS13 antigen levels span a concentration of 740-1420 ng/ml (median 1080 ng/ml) resulting in an ADAMTS13 activity to antigen ratio of 0.48 to 1.68 U/mug. In a cohort of HUS patients, ADAMTS13 antigen was in the normal range, whereas in hereditary TTP patients antigen levels were low to undetectable, in concordance with severe deficient ADAMTS13 activity. Plasma of acquired TTP patients was found to contain free as well as autoantibody-bound ADAMTS13. We also present evidence for circulating anti-ADAMTS13 antibody/ADAMTS13 antigen immune complexes not only in acutely ill or actively treated patients but also in patients who have already achieved clinical remission. This new developed ADAMTS13 antigen ELISA assay allows rapid determination of ADAMTS13 antigen levels in human plasma but is of limited predictive value for the diagnosis or treatment of acquired TTP due to the detection of ADAMTS13 in antibody complexes.

Fluconazole upregulates sconC expression and inhibits sulphur metabolism in Microsporum canis.

Azole derivatives such as fluconazole are the mainstay of therapeutic agents for the treatment of fungal infections. Their mode of action involving alteration in the conversion of lanosterol to ergosterol is well established. Here we report the effect of fluconazole on the sulphur metabolism negative regulator gene (sconC) in Microsporum canis. Characterization of the M. canis sconC gene revealed that its ORF is comprised of 495bp interrupted by four introns of 47-70bp. Exposure of M. canis in suspension to fluconazole upregulates sconC mRNA level and protein expression as determined by Northern and Western blot analysis, respectively. Upregulation of sconC was accompanied by inhibition of sulphur metabolism of the fungus resulting in a greatly reduced incorporation of radioactive labelled sulphuric acid into fungal proteins. These data establish that in addition to its action on ergosterol synthesis, fluconazole acts on other biological pathways in fungal cells.

Stratum corneum-derived caspase-14 is catalytically active.

Caspase-14, a cysteine protease with restricted tissue distribution, is highly expressed in differentiated epidermal keratinocytes. Here, we extracted soluble proteins from stratum corneum (SC) of human epidermis and demonstrate that the extract cleaves tetrapeptide caspase substrates. The activity decreased to below 10% when caspase-14 was removed by immunodepletion showing that caspase-14 is the predominant caspase in SC. In contrast to normal SC, where caspase-14 was present exclusively in its processed form, incompletely matured SC of parakeratotic skin from psoriasis and seborrheic dermatitis contained both procaspase-14 and caspase-14 subunits. Fractionation of extract from parakeratotic SC revealed that the peak caspase activity coeluted with processed caspase-14 but not with procaspase-14. Our results suggest that during regular terminal keratinocyte differentiation, endogenous procaspase-14 is converted to caspase-14 subunits that are catalytically active in the outermost layers of normal human skin.

Ultrastructural localization of caspase-14 in human epidermis.

Caspase-14 has been implicated in the formation of stratum corneum because of its specific expression and activation in terminally differentiating keratinocytes. However, its precise physiological role and its protein substrate are elusive. We studied the ultrastructural localization of caspase-14 in human epidermis to compare its distribution pattern with that of well-characterized differentiation markers. Immunogold cytochemistry confirmed that caspase-14 is nearly absent in basal and spinous layers. In the granular, layer nuclei and keratohyalin granules were labeled with increasing intensity towards the transitional layer. Particularly strong caspase-14 labeling was associated with areas known to be occupied by involucrin and loricrin, whereas F-granules, occupied by profilaggrin/filaggrin, were much less labeled. A high density of gold particles was also present at the forming cornified cell envelope, including desmosomes. In corneocytes, intense labeling was both cytoplasmic and associated with nuclear remnants and corneodesmosomes. These observations will allow focusing efforts of biochemical substrate screening on a subset of proteins localizing to distinct compartments of terminally differentiated keratinocytes.