B.J.M. van Vlijmen

Development of a calibrated automated thrombography based thrombin generation test in mouse plasma

Summary.u2002 Background:u2002Mouse models have become increasingly important in thrombosis research. However, only a limited number of assays are available for assessment of the coagulation system in mouse plasma. Objectives:u2002To quantify tissue factor‐initiated thrombin generation in murine platelet‐rich and platelet‐free plasma and to develop a test for measurement of resistance to activated protein C (APC) in mouse plasma. Methods:u2002Thrombin generation was monitored with calibrated automated thrombography (CAT) using a low‐affinity fluorogenic substrate for thrombin. Results:u2002To overcome the higher activity of coagulation inhibitors in mouse plasma as compared with human plasma, the reaction temperature was lowered to 33u2003°C and the assay was carried out at a 2‐fold higher final plasma dilution (1:3) than commonly used for CAT in human plasma. This increased the endogenous thrombin potential (ETP) 4‐ to 5‐fold and enabled reliable measurement of thrombin generation in both platelet‐free and platelet‐rich mouse plasma. For the APC resistance measurement, the reaction conditions were further optimized with respect to tissue factor, phospholipid, APC and CaCl2 concentrations. The test was validated using plasma of mice with different genetic background with respect to the factor V Leiden mutation (FV Leiden). Mice homozygous for FV Leiden had higher APC sensitivity ratios (mean 5.46; 95% CI 4.88–6.03) than heterozygous FV Leiden mice (mean 4.21; 95% CI 3.53–4.89) and than wild‐type mice (mean 2.71; 95%CI 2.15–3.27). Conclusions:u2002We have established reaction conditions for measurement of thrombin generation and APC resistance in mouse plasma. This assay enables evaluation of the coagulation system and the function of the protein C system in mouse models.

17α-Ethinylestradiol rapidly alters transcript levels of murine coagulation genes via estrogen receptor α

Summary.u2002 Background:u2002Oral estrogen use is associated with changes in plasma levels of many coagulation proteins. Objective:u2002To gain more insight into the underlying mechanism of estrogen‐induced changes in coagulation. Methods:u2002Ovariectomized female mice were used to study the impact of oral 17α‐ethinylestradiol (EE) on plasma coagulation, hepatic coagulation gene transcript levels, and dependence on estrogen receptor (ER) α and ERβ. Results:u2002Ten days of oral EE treatment resulted in significantly reduced plasma activity levels of factor (F)VIII, FXII, combined FII/FVII/FX and antithrombin, whereas FIX activity significantly increased. Regarding hepatic transcript levels, oral EE caused significant decreases in fibrinogen‐γ, FII, FV, FVII, FX, FXII, antithrombin, protein C, protein Z, protein Z inhibitor and heparin cofactor II mRNA levels, whereas FXI levels significantly increased and transcript levels of FVIII, FIX, protein S and α2‐antiplasmin remained unaffected. All EE‐induced coagulation‐related changes were neutralized by coadministration of the non‐specific ER antagonist ICI182780. In addition, ERα‐deficient mice lacked the EE‐induced changes in plasma coagulation and hepatic transcript profile, whereas ERβ‐deficient mice responded similarly to non‐deficient littermate controls. A crucial role for the ER was further demonstrated by its rapid effects on transcription, within 2.5–5u2003h after EE administration, suggesting a short chain of events leading to its final effects. Conclusions:u2002Oral EE administration has a broad impact on the mouse coagulation profile at the level of both plasma and hepatic mRNA levels. The effects on transcription are rapidly induced, mostly downregulatory, and principally mediated by ERα.

P-glycoprotein, CYP3A and plasma carboxylesterase determine brain and blood disposition of the mTOR inhibitor everolimus (Afinitor) in mice

Purpose: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. Experimental Design: We used wild-type, Abcb1a/1b−/−, Abcg2−/−, Abcb1a/1b;Abcg2−/−, and Cyp3a−/− mice to study everolimus oral bioavailability and brain accumulation. Results: Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b−/−and Abcb1a/1b;Abcg2−/−, but not Abcg2−/−mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a−/−, but not Abcb1a/1b−/−, Abcg2−/−, or Abcb1a/1b;Abcg2−/−mice, displayed highly (>5-fold) increased oral availability of everolimus. Conclusions: Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug–drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies. Clin Cancer Res; 20(12); 3133–45. ©2014 AACR.

No evidence for a direct effect of von Willebrand factor's ABH blood group antigens on von Willebrand factor clearance

One of the major determinants of von Willebrand factor (VWF) plasma levels is ABO blood group status, and individuals with blood group O have ~ 25% lower plasma levels. The exact mechanism behind this relationship remains unknown, although effects on clearance have been postulated.

Pregnancy‐associated changes in the hemostatic system in wild‐type and factor V Leiden mice

Summary.u2002 Background:u2002Pregnancy, oral contraceptive (OC) use and hormone replacement therapy (HRT) are established risk factors for venous thrombosis. Acquired resistance to activated protein C (APC) has been proposed to contribute to the increased thrombosis risk. Mouse models are often used for preclinical testing of newly developed hormone preparations. However, it is not known whether hormone‐induced APC resistance is also observed in laboratory animals. Objectives:u2002To investigate whether hormonal changes modulate APC resistance in mice, we used pregnant mice as a model of hormone‐induced APC resistance. The effect of pregnancy on APC resistance was studied in wild‐type and factor (F)V Leiden mice. Methods:u2002APC resistance was determined in mouse plasma using a thrombin generation‐based APC resistance test. APC resistance determinants, i.e. prothrombin, FV, FX, antithrombin and protein S levels, and of tissue factor pathway inhibitor (TFPI) activity were evaluated in plasma from non‐pregnant and pregnant mice. Results:u2002In contrast to humans, pregnancy induced a decrease in APC resistance in wild‐type and in FV Leiden mice. Pregnant mice had higher levels of prothrombin, FV, FX, protein S and TFPI activity as compared with non‐pregnant mice. Conclusions:u2002Pregnancy causes a decrease in APC resistance in mice, which can be explained by the elevation of protein S levels and increased TFPI activity in plasma. Our findings show species specificity in the effects of pregnancy on the major determinants of the protein C system and suggest that protein S and TFPI play an important role in the development of pregnancy‐induced APC resistance in humans.

Long-term estrogen treatment of mice with a prothrombotic phenotype induces sustained increases in thrombin generation without affecting tissue fibrin deposition

A. C . A . CLEUR EN ,* R . VAN OERLE , P . H . RE I TSMA, * H . M. SPRONK and B . J . M . VAN VL I JMEN* *Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden; and Laboratory for Clinical Thrombosis and Hemostasis, Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands

The role of hepatocyte nuclear factor 4α in regulating mouse hepatic anticoagulation and fibrinolysis gene transcript levels

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Correction of a dominant-negative von Willebrand factor multimerization defect by small interfering RNA-mediated allele-specific inhibition of mutant von Willebrand factor

Essentials Substitution therapy for von Willebrand (VW) disease leaves mutant VW factor (VWF) unhindered. Presence of mutant VWF may negatively affect phenotypes despite treatment. Inhibition of VWF by allele‐specific siRNAs targeting single‐nucleotide polymorphisms is effective. Allele‐specific inhibition of VWF p.Cys2773Ser improves multimerization.

Plasma plasminogen activator inhibitor-1 level is not regulated by the hepatic low-density lipoprotein receptor-related protein

L . HU,* N . BOVENSCHEN, L . M. HAVEKES ,* B . J . M . VAN VL I JMEN§– and J . T . TA MSMA * *Department of Endocrinology and Metabolic Diseases, General Internal Medicine, Leiden University Medical Center, Leiden; Department of Pathology, University Medical Center Utrecht, Utrecht; TNO-Quality of Life, Gaubius Laboratory, Leiden; §Einthoven Laboratory for Experimental Vascular Medicine; and –Department of Haemostasis and Thrombosis, Leiden University Medical Center, Leiden, the Netherlands