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| 2. |
- Hillarp, Andreas, et al.
(författare)
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Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays
- 2011
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Ingår i: JOURNAL OF THROMBOSIS AND HAEMOSTASIS. - : Blackwell Publishing. - 1538-7933 .- 1538-7836. ; 9:1, s. 133-139
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Rivaroxaban is an oral direct factor Xa inhibitor developed for prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary but the dose-dependent effects on common reagents and assay procedures are largely unknown. Objectives: To investigate the effect of rivaroxaban on commonly used coagulation assays. Materials and Methods: Rivaroxaban was added to plasma from healthy subjects in the concentration range 0-1000 mu g L-1 and analyzed using different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, fibrinogen and activated protein C (APC) resistance assays. Results: At an expected peak concentration of rivaroxaban in clinical use, the APTTs were almost invariably prolonged but at lower concentrations the effect was weak. The concentration needed to double the APTT varied between 389 +/- 106 and 617 +/- 149 mu g L-1 for different reagents. The PT assays showed a marked degree of difference. In general, the Quick PT type assays were more sensitive compared with the Owren type PT assays. The results from antithrombin assays were dependent on the type of reagent, with the Xa-based assay being sensitive for rivaroxaban with an estimated increase of 0.09 IU mL-1 per 100 mu g L-1 rivaroxaban. There were only minor effects on fibrinogen assays based on thrombin reagents. The APTT-based assay for APC resistance is affected in a dose-dependent manner whereas an assay based on the activation of coagulation at the prothrombinase level was unaffected. Conclusions: Different assays, and even different reagents within an assay group, display variable effects by therapeutic concentrations of rivaroxaban.
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| 3. |
- Agersø, H, et al.
(författare)
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Recombinant human factor VIIa (rFVIIa) cleared principally by antithrombin following IV administration in haemophilia patients.
- 2011
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 9:2, s. 333-338
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Tidskriftsartikel (refereegranskat)abstract
- Objective: The objective of the present study was to evaluate the pharmacokinetics and the clearance pathways of rFVIIa after intravenous administration to haemophilia patients. Methods: Ten severe haemophilia patients were included in the study; all patients were intravenously administered a clinical relevant dose of 90 μg/kg (1.8 nmol/kg) rFVIIa. Blood samples were collected consecutively to describe the pharmacokinetics of rFVIIa. All samples were analysed using three different assays: a clot assay to measure the activity (FVIIa:C), an enzyme immunoassay (EIA) to measure the antigen levels (FVII:Ag), and a EIA (FVIIa-AT) to measure the FVIIa antithrombin III (AT) complex. Pharmacokinetic parameters were evaluated both by use of standard non-compartmental methods and by use of mixed effects methods. A population pharmacokinetic model was used to simultaneously model all three datasets. The total body clearance of rFVIIa:C was estimated to be 38 mL/h/kg. The rFVII:AT complex formation was responsible for 65% of the total rFVIIa:C clearance. The initial and the terminal half-life of rFVIIa:C was estimated to be 0.6 and 2.6 hours, respectively. The formation of rFVII-AT complex were able to explain the difference observed between the rFVIIa:C and the rFVII:Ag concentration. The non-compartmental analysis resulted in almost identical parameters.
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| 4. |
- Clark, C A, et al.
(författare)
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Endothelial cell protein C receptor-mediated redistribution and tissue-level accumulation of factor VIIa.
- 2012
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 10:11, s. 2383-2391
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recent studies show that FVIIa binds to endothelial cell protein C receptor (EPCR) on vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. Objective, The aim of the present study is to investigate the role of FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. Methods: Wild-type, EPCR-deficient or ECPR-over expressing mice were injected with human rFVIIa (120 μg/kg b.w.) via tail vein. At varying time intervals following rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. Results: The data reveal that, following intravenous injection, rFVIIa rapidly disappears from blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. FVIIa association with the endothelium was undetectable at time points exceeding 24 h post FVIIa administration. The levels of rFVIIa accumulated in tissue correlates with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. Conclusions: The observation that EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally-active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides. © 2012 International Society on Thrombosis and Haemostasis.
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| 5. |
- Ekman, Carl, et al.
(författare)
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Gas6 is complexed to soluble tyrosine kinase receptor Axl in human blood.
- 2010
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 8:4, s. 838-844
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Tidskriftsartikel (refereegranskat)abstract
- Summary Background: The vitamin K-dependent Gas6 protein (product of growth arrest specific gene 6) binds to, and activates the TAM receptor tyrosine kinases Tyro3, Axl, and Mer. Gas6 and the TAM receptors have been suggested to be important for primary platelet functions, but Gas6 cannot be found in human platelets. However, Gas6 is present in human plasma at a concentration of around 0.2 nM, which is 1,000-fold lower than that of the homologous protein S. The Axl and Mer receptors can be cleaved close to the cell membrane, yielding soluble molecules consisting of the extracellular parts of the receptors. Objective: To investigate if soluble Axl (sAxl) is present in human serum and plasma and if Gas6 circulates in complex with sAxl. Methods: We expressed recombinant sAxl, raised antibodies, developed and validated an ELISA for Axl. Serum and plasma were analyzed using ELISAs for Gas6, Axl, and sAxl-Gas6 complexes. Serum was gel filtered and fractions analyzed by the different ELISAs to determine if Gas6 in serum is free or complexed. Immunoprecipitation was used to investigate binding between Gas6 and sAxl in serum. Results: sAxl is present in serum and plasma at around 0.6 nM and all Gas6 is bound to sAxl. No complexes between Gas6 and the soluble forms of Mer and Tyro3 could be detected, indicating that sAxl is the physiological binder of Gas6 in human serum. Conclusions: Gas6 in circulation is bound to sAxl suggesting circulating Gas6 to be inhibited and incapable of stimulating the TAM receptors.
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| 6. |
- Gopalakrishnan, R., et al.
(författare)
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Bio-distribution of pharmacologically administered recombinant factor VIIa (rFVIIa)
- 2010
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 8:2, s. 301-310
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recent clinical studies suggest that the prophylactic use of recombinant factor VIIa (rFVIIa) markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Given the short biological half-life of rFVIIa, it is unclear how rFVIIa could be effective in prophylactic treatment. Objectives: To examine the extravascular distribution of pharmacologically administered rFVIIa to obtain clues on how rFVIIa could work in prophylaxis. Methods: Recombinant mouse FVIIa tagged with AF488 fluorophore (AF488-FVIIa) was administered into mice via the tail vein. At different time intervals following the administration, mice were exsanguinated and various tissues were collected. The tissue sections were processed for immunohistochemistry to evaluate distribution of rFVIIa. Results: rFVIIa, immediately following the administration, associated with the endothelium lining of large blood vessels. Within 1 h, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. In the liver, rFVIIa was localized to sinusoidal capillaries and accumulated in hepatocytes. In bone, rFVIIa was accumulated in the zone of calcified cartilage and some of it was retained there for a week. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to regions that contain TF expressing cells. Conclusions: The present study demonstrates that pharmacologically administered rFVIIa readily associates with the vascular endothelium and subsequently enters into extravascular spaces where it is likely to bind to TF and is retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa.
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| 7. |
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| 8. |
- Hillarp, Andreas, et al.
(författare)
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Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.
- 2010
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 8, s. 2216-2223
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Tidskriftsartikel (refereegranskat)abstract
- Summary Background, objectives and methods: An accurate, sensitive and precise assay for reliable determination of the ristocetin cofactor activity of von Willebrand factor (VWF:RCo) in plasma and von Willebrand Factor (VWF)-containing concentrates has been evaluated. The assay is based on a commercially available automated protocol with modifications including a combination of adding additional ristocetin and the use of two calibration curves for the high and low measuring ranges. Results: Addition of extra ristocetin resulted in improved measurement of VWF recoveries from various VWF-containing concentrates that were underestimated using the standard automated protocol. The modifications resulted in improved assay performance over an extended measuring range (2.00 - 0.03 IU/mL). Accuracy was tested using VWF deficiency plasma spiked with 1(st) international standard for VWF concentrate. Seven dilutions, ranging between 1.80 and 0.05 IU/mL, were analyzed and resulted in measured concentrations between 80% and 100% of the assigned potency of the standard. Linearity was determined from the regression plot of the same concentrate dilutions and resulted in a correlation coefficient of 0.998. The repeatability, expressed as coefficient of variation, was 2% in the normal range (0.90 IU/mL) and 8% at the level of 0.05 IU/mL. The corresponding reproducibility results were 2% and 15% at the normal and low measuring ranges, respectively. Conclusions: Analysis of patients with von Willebrand disease indicates that the modified automated BCS((R)) protocol has a superior discrimination power compared with the standard protocol. This is especially true in samples with low VWF, as in patients with type 3 von Willebrand disease.
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| 9. |
- Högberg, Carl, et al.
(författare)
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Succinate independently stimulates full platelet activation via cAMP and PI3β kinase signaling.
- 2011
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 9:2, s. 361-372
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Tidskriftsartikel (refereegranskat)abstract
- Background: The citric cycle intermediate succinate has recently been identified as ligand for the G-protein coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signalling pathways of this receptor in platelets. Methods and Results: Using RT-PCR, we could demonstrate that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y(1) receptor. Light transmission aggregation experiments showed a dose-dependent aggregation induced by succinate reaching a maximum response at 0.5mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry showing increased surface expression of activated GPIIb/IIIa, and P-selectin. Intracellular SUCNR1 signalling was found to result in decreased cAMP levels, Akt phosphorylation mediated by PI3Kβ activation and receptor desensitisation. Further, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A(2) and ATP release. The platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y(12) receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate-induced platelet aggregation depends on thromboxane A(2) generation, ATP release and P2Y(12) activation.
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