Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and a factor V-stabilizing fab.

INTRODUCTION: The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. AIMS: To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. METHODS: Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0 nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300 nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). RESULTS: With 1 and 2.5 nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300 nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. CONCLUSIONS: The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.

Concizumab, an anti-tissue factor pathway inhibitor antibody, induces increased thrombin generation in plasma from haemophilia patients and healthy subjects measured by the thrombin generation assay.

AIMS: Concizumab, a humanized monoclonal antibody against tissue factor pathway inhibitor (TFPI), is being developed as a subcutaneously (s.c.) administered treatment for haemophilia. It demonstrated a concentration-dependent procoagulant effect in functional TFPI assays; however, global haemostatic assays, such as the thrombin generation assay (TGA), offer a more complete picture of coagulation. We investigated how concizumab affects thrombin generation following ex vivo spiking in plasma from haemophilia patients using the TGA, and if the assay can detect the effect of multiple s.c. concizumab doses in healthy subjects. METHODS: For the ex vivo spiking study, platelet-poor plasma (PPP) from 18 patients with severe haemophilia was spiked with 0.001-500 nm concizumab. For the multiple-dosing study, four healthy males received concizumab 250 µg kg-1 s.c. every other day for eight doses; blood was collected before and after dosing and processed into PPP. In both studies, thrombin generation was measured using a Calibrated Automated Thrombogram® system with 1 pm tissue factor. RESULTS: In spiked samples from haemophilia patients, peak thrombin and endogenous thrombin potential (ETP) increased concentration dependently, reaching near-normal levels at concizumab concentrations >10 nm. Repeated s.c. doses of concizumab in healthy subjects increased both peak thrombin and ETP; these effects were sustained throughout the dosing interval. CONCLUSIONS: Thrombin generation assay demonstrated increased thrombin generation with concizumab after ex vivo spiking of haemophilia plasma and multiple s.c. doses in healthy subjects, supporting both the utility of the TGA in evaluating concizumab treatment and the potential of s.c. concizumab as a novel haemophilia therapy.

Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive.

BACKGROUND: Conventional coagulation factor assays are associated with certain limitations, as they do not always reflect the clinical heterogeneity of bleeding in hemophilic patients or correctly reflect the individual patient response to treatment with bypassing agents or novel factor concentrates. The thrombin generation assay (TGA) is currently being assessed as a possible method for characterizing bleeding phenotypes in individuals with hemophilia. OBJECTIVES: This study assessed the robustness and sensitivity of the TGA for measuring the activity of recombinant factor VIII (rFVIII), recombinant factor IX (rFIX) and their glycoPEGylated derivatives, N8-GP and N9-GP, in vitro. METHODS: Factor-deficient plasma was spiked with 0.13-130 IU dL(-1) rFVIII or N8-GP (hemophilia A [HA] plasma), or rFIX or N9-GP (hemophilia B [HB] plasma). A calibrated automated thrombogram triggered with tissue factor (TF) or activated FXI (FXIa) was used to measure thrombin generation over time. Endogenous thrombin potential, peak thrombin, velocity index, lag time and time to peak thrombin were analyzed. RESULTS: FXIa-triggered assays were not affected by glycoPEGylation and were sufficiently sensitive to differentiate between spiked samples mimicking severe and moderate HB and HA; TF-triggered assays were not sufficiently sensitive for this distinction in HA. Both FXIa-triggered and TF-triggered assays had an acceptable level of variability (≤ 20%), although TF-triggered assays were associated with greater variability. CONCLUSIONS: FXIa-triggered TGA reactions produced more robust and sensitive results than TF-triggered TGA reactions, and have the potential for use in monitoring patients treated with glycoPEGylated or non-PEGylated coagulation factor concentrates. These promising results merit confirmation with clinical samples to correlate in vitro and in vivo data.

Aptamer BAX 499 mediates inhibition of tissue factor pathway inhibitor via interaction with multiple domains of the protein.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multidomain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)-activated factor VII-activated FX (FXa) complex during TF-mediated coagulation initiation. The aptamer BAX 499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both in vitro and in vivo models of hemophilia. OBJECTIVES: This study sought to identify the regions of TFPI that are critical for BAX 499 binding, and to determine how binding mediates aptamer inhibition of TFPI. METHODS AND RESULTS: In vitro biochemical methods were used to evaluate the BAX 499 interaction with and inhibition of TFPI. Binding experiments indicated that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicated the Kunitz 1, Kunitz 3 and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicated that aptamer and FXa can bind simultaneously to TFPI. In enzymatic assays, BAX 499 inhibited TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of the TFPI cofactor protein S. CONCLUSIONS: These studies demonstrate that BAX 499 binds to TFPI via multiple domains of the protein in a manner that is distinct from other TFPI inhibitors, mediating a mechanism of inhibition that does not involve direct competition with FXa. With this unique inhibitory mechanism, BAX 499 provides a useful tool for studying TFPI biology in health and disease.

Immobilized transition metal ions stimulate contact activation and drive factor XII-mediated coagulation.

BACKGROUND: Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn(2+) is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces. OBJECTIVES: To investigate whether transition metal ions immobilized on liposome surfaces can initiate coagulation via the contact pathway. METHODS AND RESULTS: Liposomes containing a metal ion-chelating lipid, 1,2-dioleoyl-sn-glycero-3-{(N[5-amino-1-carboxypentyl]iminodiacetic acid)succinyl} ammonium salt (DOGS-NTA), were prepared by membrane extrusion (20% DOGS-NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni(2+) immobilized on such liposomes accelerated clotting in normal plasma, but not factor XI (FXI)-deficient or FXII-deficient plasma. The results were similar to those obtained with a commercial activated partial thromboplastin time reagent. Charging such liposomes with other transition metal ions revealed differences in their procoagulant capacity, with Ni(2+) > Cu(2+) > Co(2+) and Zn(2+). Plasma could be depleted of FXI, FXII and HK by adsorption with Ni(2+) -containing beads, resulting in longer clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni(2+) or Cu(2+) with high affinity as determined by surface plasmon resonance. In the presence of Ni(2+) -bearing liposomes, K(m) and k(cat) values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values obtained in the presence of dextran sulfate. CONCLUSIONS: Immobilized Ni(2+) and Cu(2+) bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII-mediated coagulation. Activation of the contact system by immobilized transition metal ions may have implications during pathogenic infection or in individuals exposed to high levels of pollution.

Aggregation and competitive exclusion: explaining the coexistence of human Papillomavirus types and the effectiveness of limited vaccine conferred cross-immunity.

Human Papillomavirus (HPV) types are sexually transmitted infections that cause a number of human cancers. According to the competitive exclusion principle in ecology, HPV types that have lower transmission probabilities and shorter durations of infection should be outcompeted by more virulent types. This, however, is not the case, as numerous HPV types co-exist, some which are less transmissible and more easily cleared than others. This paper examines whether this exception to the competitive exclusion principle can be explained by the aggregation of infection with HPV types, which results in patchy spatial distributions of infection, and what implications this has for the effect of vaccination on multiple HPV types. A deterministic transmission model is presented that models the patchy distribution of infected individuals using Lloyd's mean crowding. It is first shown that higher aggregation can result in a reduced capacity for onward transmission and reduce the required efficacy of vaccination. It is shown that greater patchiness in the distribution of lower prevalence HPV types permits co-existence. This affirms the hypothesis that the aggregation of HPV types provides an explanation for the violation of the competitive exclusion principle. Greater aggregation of lower prevalence types has important implications where type-specific HPV vaccines also offer cross-protection against non-target types. It is demonstrated that the degree of cross-protection can be less than the degree of vaccine protection conferred against directly targeted types and still result in the elimination of non-target types when these non-target types are patchily distributed.

Validation of fixed sample size plans for monitoring lepidopteran pests of Brassica oleracea crops in North Korea.

The combined action of two lepidoteran pests, Plutella xylostella L. (Plutellidae) and Pieris rapae L. (Pieridae),causes significant yield losses in cabbage (Brassica oleracea variety capitata) crops in the Democratic People's Republic of Korea. Integrated pest management (IPM) strategies for these cropping systems are in their infancy, and sampling plans have not yet been developed. We used statistical resampling to assess the performance of fixed sample size plans (ranging from 10 to 50 plants). First, the precision (D = SE/mean) of the plans in estimating the population mean was assessed. There was substantial variation in achieved D for all sample sizes, and sample sizes of at least 20 and 45 plants were required to achieve the acceptable precision level of D < or = 0.3 at least 50 and 75% of the time, respectively. Second, the performance of the plans in classifying the population density relative to an economic threshold (ET) was assessed. To account for the different damage potentials of the two species the ETs were defined in terms of standard insects (SIs), where 1 SI = 1 P. rapae = 5 P. xylostella larvae. The plans were implemented using different economic thresholds (ETs) for the three growth stages of the crop: precupping (1 SI/plant), cupping (0.5 SI/plant), and heading (4 SI/plant). Improvement in the classification certainty with increasing sample sizes could be seen through the increasing steepness of operating characteristic curves. Rather than prescribe a particular plan, we suggest that the results of these analyses be used to inform practitioners of the relative merits of the different sample sizes.

Differential stimulation of monocytic cells results in distinct populations of microparticles.

BACKGROUND: Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. METHODS: We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. RESULTS: Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. CONCLUSION: We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.