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Journal of Thrombosis and Haemostasis 10/2010; 8(12):2839-41. · 5.73 Impact Factor
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ABSTRACT: Oral estrogen use is associated with changes in plasma levels of many coagulation proteins.
To gain more insight into the underlying mechanism of estrogen-induced changes in coagulation.
Ovariectomized 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.
Ten 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 α(2) -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 2.5-5 h after EE administration, suggesting a short chain of events leading to its final effects.
Oral 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.
Journal of Thrombosis and Haemostasis 08/2010; 8(8):1838-46. · 5.73 Impact Factor
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ABSTRACT: Pregnancy, 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.
To 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.
APC 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.
In 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.
Pregnancy 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.
Journal of Thrombosis and Haemostasis 12/2008; 7(2):312-8. · 5.73 Impact Factor
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Journal of Thrombosis and Haemostasis 12/2007; 5(11):2301-4. · 5.73 Impact Factor
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ABSTRACT: Mouse 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.
To 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.
Thrombin generation was monitored with calibrated automated thrombography (CAT) using a low-affinity fluorogenic substrate for thrombin.
To overcome the higher activity of coagulation inhibitors in mouse plasma as compared with human plasma, the reaction temperature was lowered to 33 degrees 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 CaCl(2) 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).
We 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.
Journal of Thrombosis and Haemostasis 11/2007; 5(10):2079-86. · 5.73 Impact Factor
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Journal of Thrombosis and Haemostasis 10/2007; 5(11):2301 - 2304. · 5.73 Impact Factor
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ABSTRACT: In vitro studies implicate that the low-density lipoprotein receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of lipoproteins.
We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3+ T cells were observed in lesions from macrophage specific LRP-deficient mice.
Our data demonstrate that independent of its role in lipoprotein uptake, absence of LRP in macrophages resulted in more advanced atherosclerosis and in lesions that contained more collagen and less CD3+ T cells. In contrast to previous in vitro studies, we conclude that macrophage LRP has an atheroprotective potential and may modulate the extracellular matrix in the atherosclerotic lesions.
Arteriosclerosis Thrombosis and Vascular Biology 01/2007; 26(12):2710-5. · 6.37 Impact Factor
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L S M Boesten,
S M Zadelaar,
S De Clercq,
S Francoz,
A van Nieuwkoop,
E A L Biessen,
F Hofmann,
S Feil,
R Feil,
A G Jochemsen,
C Zurcher,
L M Havekes, B J M van Vlijmen,
J-C Marine
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ABSTRACT: p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.
Cell Death and Differentiation 01/2007; 13(12):2089-98. · 8.85 Impact Factor
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ABSTRACT: Coagulation factor VIII (FVIII) is a heavily glycosylated heterodimeric plasma protein that consists of a heavy (domains A1-A2-B) and light chain (domains A3-C1-C2). It has been well established that the clearance of FVIII from the circulation involves mechanisms that are sensitive to the low-density lipoprotein receptor (LDLR) family antagonist receptor-associated protein (RAP), including LDLR-related protein. Because FVIII clearance in the presence of a bolus injection of RAP still occurs fairly efficient, also RAP-independent mechanisms are likely to be involved.
In the present study, we investigated the interaction of FVIII with the endocytic lectin asialoglycoprotein receptor (ASGPR) and the physiological relevance thereof.
Surface plasmon resonance studies demonstrated that FVIII dose-dependently bound to ASGPR with high affinity (Kd approximately 2 nM). FVIII subunits were different in that only the heavy chain displayed high-affinity binding to ASGPR. Studies employing a FVIII variant that lacks the B domain revealed that FVIII-ASGPR complex assembly is driven by structure elements within the B domain of the heavy chain. The FVIII heavy chain-ASGPR interaction required calcium ions and was inhibited by soluble D-galactose. Furthermore, deglycosylation of the FVIII heavy chain by endoglycosidase F completely abrogated the interaction with ASGPR. In clearance experiments in mice, the FVIII mean residence time was prolonged by the ASGPR-antagonist asialo-orosomucoid (ASOR).
We conclude that asparagine-linked oligosaccharide structures of the FVIII B domain recognize the carbohydrate recognition domains of ASGPR and that an ASOR-sensitive mechanism, most likely ASGPR, contributes to the catabolism of coagulation FVIII in vivo.
Journal of Thrombosis and Haemostasis 07/2005; 3(6):1257-65. · 5.73 Impact Factor
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ABSTRACT: THE PROPOSED HYPOTHESIS will address the pathophysi-ologic link between the metabolic syndrome (MS) and atherosclerosis. Vis-ceral obesity is a prominent feature of the syndrome and has been shown to associate with hepatic insulin resistance. Based on epidemiologic, clinical and experimental data we propose that the metabolic syndrome if associated with hepatic insulin resistance results in a dysfunction of the hepatic low-density lipoprotein receptor-related protein (LRP) clearance pathway. This pathway is responsible for the clearance and regulation of plasma levels of more than 35 pro-atherothrombotic and antifibrinolytic proteins. Many of these factors are known to be increased, or to play a role in MS and diabetes on the one hand, and on atherosclerosis on the other. Examples are apolipoprotein E, a key apoprotein in the clearance of very low density lipoprotein, metalloprotease-9 important in plaque vulnerability, and PAI-1 a key regulator of fibrinolysis. In addition, hepatic LRP deficiency resulted in increased measures of atherosclerosis in an experimental setting. Dysregulation and accumulation of LRP ligands in MS-associated hepatic insulin resistance likely is a pathophysiologic link to atherosclerosis.
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