Population pharmacokinetics of a triple-secured fibrinogen concentrate administered to afibrinogenaemic patients: Observed age- and body weight-related differences and consequences for dose adjustment in children.

AIMS: The pharmacokinetics (PK) of a triple-secured fibrinogen concentrate (FC) was assessed in patients ≥40 kg by noncompartmental analysis over a period of 14 days with multiple blood samples. Limited PK time point assessments in children lead to consideration of using Bayesian estimation for paediatric data. The objectives were (i) to define the population PK of FC in patients with afibrinogenaemia; (ii) to detect age- and body weight-related differences and consequences for dose adjustment. METHODS: A population PK model was built using plasma fibrinogen activity data collected in 31 patients aged 1 to 48 years who had participated in a single-dose PK study with FC 0.06 g kg-1 . RESULTS: A 1-compartment model with allometric scaling accounting for body weight was found to best describe the kinetics of FC. Addition of age and sex as covariates did not improve the model. Incremental in vivo recovery assessed at the end of infusion with the predicted maximal concentrations was lower, weight-adjusted clearance was higher, and fibrinogen elimination half-life was shorter in patients <40 kg than patients ≥40 kg. Interpatient variability was similar in both groups. CONCLUSION: Dosing in patients ≥40 kg based on the previous empirical finding using noncompartmental analysis where FC 1 g kg-1 raises the plasma fibrinogen activity by 23 g L-1 was confirmed. In patients <40 kg, (covering the age range from birth up to about 12 years old) FC 1 g kg-1 raises the plasma fibrinogen by 19 g L-1 . Dosing should be adapted accordingly unless therapy is individualized.

Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae.

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.

The Rho3 and Rho4 small GTPases interact functionally with Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae.

Rgd1, a GTPase-activating protein, is the only known negative regulator of the Rho3 and Rho4 small GTPases in the yeast Saccharomyces cerevisiae. Rho3p and Rho4p are involved in regulating cell polarity by controlling polarized exocytosis. Co-inactivation of RGD1 and WSC1, which is a cell wall sensor-encoding gene, is lethal. Another plasma membrane sensor, Mid2p, is known to rescue the rgd1Deltawsc1Delta synthetic lethality. It has been proposed that Wsc1p and Mid2p act upstream of the protein kinase C (PKC) pathway to function as mechanosensors of cell wall stress. Analysis of the synthetic lethal phenomenon revealed that production of activated Rho3p and Rho4p leads to lethality in wsc1Delta cells. Inactivation of RHO3 or RHO4 was able to rescue the rgd1Deltawsc1Delta synthetic lethality, supporting the idea that the accumulation of GTP-bound Rho proteins, following loss of Rgd1p, is detrimental if the Wsc1 sensor is absent. In contrast, the genetic interaction between RGD1 and MID2 was not due to an accumulation of GTP-bound Rho proteins. It was proposed that simultaneous inactivation of RGD1 and WSC1 constitutively activates the PKC-mitogen-activated protein kinase (MAP kinase) pathway. Moreover, it was shown that the activity of this pathway was not involved in the synthetic lethal interaction, which suggests the existence of another mechanism. Consistent with this idea, it was found that perturbations in Rho3-mediated polarized exocytosis specifically impair the abundance and processing of Wsc1 and Mid2 proteins. Hence, it is proposed that Wsc1p participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the PKC-MAP kinase pathway.

Nonclassical PITPs activate PLD via the Stt4p PtdIns-4-kinase and modulate function of late stages of exocytosis in vegetative yeast.

Phospholipase D (PLD) is a PtdCho-hydrolyzing enzyme that plays central signaling functions in eukaryotic cells. We previously demonstrated that action of a set of four nonclassical and membrane-associated Sec14p-like phosphatidylinositol transfer proteins (PITPs) is required for optimal activation of yeast PLD in vegetative cells. Herein, we focus on mechanisms of Sfh2p and Sfh5p function in this regulatory circuit. We describe several independent lines of in vivo evidence to indicate these SFH PITPs regulate PLD by stimulating PtdIns-4,5-P2 synthesis and that this stimulated PtdIns-4,5-P2 synthesis couples to action of the Stt4p PtdIns 4-kinase. Furthermore, we provide genetic evidence to suggest that specific subunits of the yeast exocyst complex (i.e. a component of the plasma membrane vesicle docking machinery) and the Sec9p plasma membrane t-SNARE are regulated by PtdIns(4,5)P2 and that Sfh5p helps regulate this interface in vivo. The collective in vivo and biochemical data suggest SFH-mediated stimulation of Stt4p activity is indirect, most likely via a substrate delivery mechanism.

Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex.

Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.

Functional interactions between the VRP1-LAS17 and RHO3-RHO4 genes involved in actin cytoskeleton organization in Saccharomyces cerevisiae.

The RGD1 gene from Saccharomyces cerevisiae, which encodes a GTPase-activating protein for the Rho3 and Rho4 small G proteins, exhibits synthetic lethality with the VRP1 and LAS17 genes. Their products are proline-rich proteins that interact with both actin and myosins to ensure polarized growth. By testing functional links, we found that the VRP1 and LAS17 genes are potent suppressors of the rho3Delta mutation. In particular, they restore the polarization of actin patches in rho3Delta cells. Moreover, the vrp1Delta and las17Delta mutations were found to display a similar pattern of genetic interactions with specific actin-linked genes. These mutations also increase the sensitivity to activated forms of both Rho3p and Rho4p. These data support our working model, in which the VRP1 and LAS17 genes define a cellular complex that works in concert with the RHO3-RHO4 signaling pathway in yeast polarized growth. In addition, other observations lead us to propose that Rvs167p may act as a linking protein between the two cellular elements.