Cécile V Denis

Publications of Cécile V Denis

• von Willebrand factor: at the crossroads of bleeding and thrombosis.

  Authors: Cécile V Denis, Peter J Lenting

  International journal of hematology. 04/2012;

  Hemostasis and thrombosis represent two sides of the same coin. Hemostasis maintains blood fluidity in the vascular system while allowing for rapid thrombus formation to prevent excessive hemorrhageHemostasis and thrombosis represent two sides of the same coin. Hemostasis maintains blood fluidity in the vascular system while allowing for rapid thrombus formation to prevent excessive hemorrhage after blood vessel injury. Thrombosis is a pathologic extension of the normal hemostatic mechanism, occurring when unwanted clot formation develops in certain pathological situations. The molecular mechanisms underlying both phenomena are fundamentally identical. One of the key players in both processes is the plasma glycoprotein von Willebrand factor, which perfectly illustrates this duality between hemostatic and thrombotic mechanisms. The purpose of this review is to discuss novel findings on the role of von Willebrand factor at this interface, and how some of these findings may help develop new therapeutic strategies.

• Macrophage LRP1 contributes to the clearance of von Willebrand factor.

  Authors: Ghasem Rastegarlari, Julie N Pegon, Caterina Casari, Soline Odouard, Ana-Maria Navarrete, Nathalie Saint-Lu, Bart J van Vlijmen, Paulette Legendre, Olivier D Christophe, Cécile V Denis, Peter J Lenting

  Blood. 03/2012; 119(9):2126-34.

  The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interactionThe relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1(-)) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1(-) mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1(-) mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1(-) mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1(+) mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm(2), implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving β2-integrins that binds both VWF and LRP1 also is implicated because inhibition of β2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1(-) mice (0.08 ± 0.15 U/mL).

• Identification of Galectin-1 and Galectin-3 as Novel Partners for Von Willebrand Factor.

  Authors: Nathalie Saint-Lu, Beatrijs D Oortwijn, Julie N Pegon, Soline Odouard, Olivier D Christophe, Philip G de Groot, Cécile V Denis, Peter J Lenting

  Arteriosclerosis, thrombosis, and vascular biology. 01/2012;

  OBJECTIVE: Although von Willebrand factor (VWF) is a heavily glycosylated protein, its potential to associate with glycan-binding proteins is poorly investigated. Here, we explored its interactionOBJECTIVE: Although von Willebrand factor (VWF) is a heavily glycosylated protein, its potential to associate with glycan-binding proteins is poorly investigated. Here, we explored its interaction with the glycan-binding proteins galectin-1 and galectin-3. METHODS AND RESULTS: Immunofluorescence analysis using Duolink proximity ligation assays revealed that VWF colocalizes with galectin-1 and galectin-3 in endothelial cells, both before and after stimulation of endothelial cells. Moreover, galectin-1 was found along the typical VWF bundles that are released by endothelial cells. Galectin-1 and galectin-3 could be coprecipitated with VWF from plasma in immunoprecipitation assays, whereas plasma levels of galectin-1 and galectin-3 were significantly reduced in VWF-deficient mice. Binding studies using purified proteins confirmed that VWF could directly interact with both galectins, predominantly via its N-linked glycans. In search of the physiological relevance of the VWF-galectin interaction, we found that inhibition of galectins in in vitro perfusion assays was associated with increased VWF-platelet string formation, a phenomenon that was reproduced in galectin-1/galectin-3 double-deficient mice. These mice were also characterized by a more rapid formation of initial thrombi following ferric chloride-induced injury. CONCLUSIONS: We have identified galectin-1 and galectin-3 as novel partners for VWF, and these proteins may modulate VWF-mediated thrombus formation.

• Novel function of tenascin-C, a matrix protein relevant to atherosclerosis, in platelet recruitment and activation under flow.

  Authors: Mathieu Schaff, Nicolas Receveur, Catherine Bourdon, Virginie Wurtz, Cécile V Denis, Gertraud Orend, Christian Gachet, François Lanza, Pierre H Mangin

  Arteriosclerosis, thrombosis, and vascular biology. 01/2011; 31(1):117-24.

  The identification of platelet-reactive proteins exclusively present in atherosclerotic plaques could provide interesting targets for effective and safe antithrombotic strategies. In this context, weThe identification of platelet-reactive proteins exclusively present in atherosclerotic plaques could provide interesting targets for effective and safe antithrombotic strategies. In this context, we explored platelet adhesion and activation to tenascin-C (TN-C), a matrix protein preferentially found within atheroma. We show that platelets efficiently adhere to TN-C under both static and flow conditions. Videomicroscopy revealed a unique behavior under flow, with platelets exhibiting stationary adhesion to TN-C; in contrast, platelets rolled over von Willebrand factor and detached from fibrinogen. Platelet interaction with TN-C was predominantly supported by integrin α(2)β(1) under static conditions, whereas under high shear, it was dependent on both the α(2)β(1) integrin and the glycoprotein Ib-IX complex. Integrin α(IIb)β(3) appeared to play a secondary role but only at low shear rates. The glycoprotein Ib-IX-dependent interaction was indirect, relying on von Willebrand factor, and increased as a function of wall shear rate. Von Willebrand factor bound directly to TN-C, as shown by ELISA and coimmunoprecipitation, suggesting that it acts as a bridge between TN-C and platelets. The adhesion of platelets to TN-C triggered their activation, as demonstrated by a shape change and increases in intracellular calcium level. This study provides evidence that TN-C serves as a novel adhesive matrix for platelets in a context that is relevant to atherothrombosis.

• Binding of von Willebrand factor to collagen and glycoprotein Ibalpha, but not to glycoprotein IIb/IIIa, contributes to ischemic stroke in mice--brief report.

  Authors: Simon F De Meyer, Tobias Schwarz, Hans Deckmyn, Cécile V Denis, Bernhard Nieswandt, Guido Stoll, Karen Vanhoorelbeke, Christoph Kleinschnitz

  Arteriosclerosis, thrombosis, and vascular biology. 10/2010; 30(10):1949-51.

  To unravel crucial von Willebrand factor (VWF) interactions that are detrimental in stroke development. VWF(-/-) mice received gene transfer to express mutants of VWF defective either in binding toTo unravel crucial von Willebrand factor (VWF) interactions that are detrimental in stroke development. VWF(-/-) mice received gene transfer to express mutants of VWF defective either in binding to fibrillar collagen, glycoprotein (GP)Ibα or GPIIb/IIIa, and underwent 60 minutes of transient middle cerebral artery occlusion. In VWF(-/-) mice reconstituted with VWF mutants defective in binding to collagen or GPIbα, protection against stroke was sustained, whereas VWF lacking the GPIIb/IIIa binding site restored full susceptibility similar to normal VWF. VWF-collagen and VWF-GPIbα (but not VWF-GPIIb/IIIa) interactions are instrumental in thrombus formation after transient middle cerebral artery occlusion, and their inhibition could be a promising target for stroke treatment.

• Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B.

  Authors: Julie Rayes, Martine J Hollestelle, Paulette Legendre, Isabelle Marx, Philip G de Groot, Olivier D Christophe, Peter J Lenting, Cécile V Denis

  Blood. 03/2010; 115(23):4870-7.

  Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increasedVon Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

• Efficient inhibition of collagen-induced platelet activation and adhesion by LAIR-2, a soluble Ig-like receptor family member.

  Authors: Peter J Lenting, Geertje H A Westerlaken, Cécile V Denis, Jan Willem Akkerman, Linde Meyaard

  PloS one. 01/2010; 5(8):e12174.

  LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen andLAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s(-1); IC(50) = 18 microg/ml) and high shear rate (1500 s(-1); IC(50) = 30 microg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and alpha2beta1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis.

• Models for prediction of factor VIII half-life in severe haemophiliacs: distinct approaches for blood group O and non-O patients.

  Authors: Kathelijn Fischer, Ronan Pendu, Carina J van Schooten, Karin van Dijk, Cécile V Denis, H Marijke van den Berg, Peter J Lenting

  PloS one. 02/2009; 4(8):e6745.

  BACKGROUND: Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWFBACKGROUND: Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life. METHODOLOGY: Standardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15-44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated. PRINCIPAL FINDINGS: FVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5+/-2.6 h versus 14.3+/-3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%). CONCLUSION: Our approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.

• Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related protein.

  Authors: Patricia P Spijkers, Cécile V Denis, Anna M Blom, Peter J Lenting

  European journal of immunology. 04/2008; 38(3):809-17.

  C4b-binding protein (C4BP) is a protein acting as a complement inhibitor and a carrier protein for anticoagulant protein S. Previously, we reported that the in vivo clearance of C4BP involves CD91,C4b-binding protein (C4BP) is a protein acting as a complement inhibitor and a carrier protein for anticoagulant protein S. Previously, we reported that the in vivo clearance of C4BP involves CD91, and that a CD91-interactive site overlaps the heparin-binding site within C4BP alpha-chains 26. Here, we investigated the C4BP-CD91 interaction in more detail. Binding of C4BP to CD91 was unaffected by protein S, which associates with C4BP beta-chain. Second, mutagenesis of cationic residues within C4BP alpha-chains impaired CD91 binding, reducing the affinity of triple mutant C4BPalpha/R39Q-R64Q-R66Q by 20-fold (Kd= 10 nM versus 214 nM for wild-type and mutant C4BP, respectively). Accordingly, intracellular degradation of this mutant by CD91-expressing cells was reduced to levels of CD91-deficient cells. Moreover, C4BPalpha/R39Q-R64Q-R66Q displayed a 3-fold prolonged survival compared to normal C4BP in in vivo clearance experiments. Since these residues also contribute to heparin binding, we explored the role of heparin-sulfate proteoglycans (HSPG) in the endocytosis of C4BP. The absence of HSPG was associated with a near complete absence of cell binding and intracellular degradation of C4BP. Apparently, the cellular uptake of C4BP depends on both HSPG and CD91, involving interactions with positively charged residues within C4BP alpha-chain.

• Correction of bleeding symptoms in von Willebrand factor-deficient mice by liver-expressed von Willebrand factor mutants.

  Authors: Isabelle Marx, Peter J Lenting, Thure Adler, Ronan Pendu, Olivier D Christophe, Cécile V Denis

  Arteriosclerosis, thrombosis, and vascular biology. 04/2008; 28(3):419-24.

  OBJECTIVE: von Willebrand Factor (vWF) structure-function relationship has been studied only in vitro. To investigate the physiological importance of particular vWF domains, we have introducedOBJECTIVE: von Willebrand Factor (vWF) structure-function relationship has been studied only in vitro. To investigate the physiological importance of particular vWF domains, we have introduced mutations into murine vWF (mvWF) cDNA inhibiting vWF binding to glycoprotein (Gp) Ib, GpIIbIIIa, and to fibrillar collagen. METHODS AND RESULTS: We delivered wild-type (WT) or mutant mvWF cDNA into vWF-deficient (Vwf-/-) mice using hydrodynamic injection and assessed whether hemorrhagic symptoms could be corrected. Hydrodynamic gene transfer resulted in high expression of plasma mvWF 24 hours after injection (438+/-63% for 50 microg of cDNA). Factor VIII activity was normalized in Vwf-/- mice injected with mvWF cDNA and multimerization was achieved. Bleeding time was corrected after injection of WT mvWF cDNA in Vwf-/- mice whereas noninjected mice did not stop bleeding. Injection of the GpIIbIIIa and the collagen binding mutants in Vwf-/- mice also resulted in a correction of bleeding time whereas mice injected with the GpIb binding mutant were bleeding for as long they were observed, although blood loss was decreased compared with noninjected mice (61+/-21 microL versus 232+/-63 microL). CONCLUSIONS: Our model allows the rapid in vivo evaluation of specific mutations on plasma vWF function.

• Clearance of von Willebrand factor.

  Authors: Cécile V Denis, Olivier D Christophe, Beatrijs D Oortwijn, Peter J Lenting

  Thrombosis and haemostasis. 03/2008; 99(2):271-8.

  The life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of the VWF gene in endothelial cells and megakaryocytes to the removal ofThe life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of the VWF gene in endothelial cells and megakaryocytes to the removal of VWF from the circulation. The various aspects of VWF clearance have been the objects of intense research in the last few years, stimulated by observations that VWF clearance is a relatively common component of the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, improving the survival of VWF is now considered as a viable therapeutic strategy to prolong the half-life of factor VIII in order to optimise treatment of haemophilia A. The present review aims to provide an overview of recent findings with regard to the molecular basis of VWF clearance. A number of parameters have been identified that influence VWF clearance, including its glycosylation profile and a number of VWF missense mutations. In addition, in-vivo studies have been used to identify cells that contribute to the catabolism of VWF, providing a starting point for the identification of receptors that mediate the cellular uptake of VWF. Finally, we discuss recent data describing chemically modification of VWF as an approach to prolong the half-life of the VWF/FVIII complex.

• Platelet adhesion receptors and their ligands in mouse models of thrombosis.

  Authors: Cécile V Denis, Denisa D Wagner

  Arteriosclerosis, thrombosis, and vascular biology. 05/2007; 27(4):728-39.

  Platelet adhesion and aggregation at sites of vascular injury are two key events in hemostasis and thrombosis. Because of exciting advances in genetic engineering, the mouse has become an importantPlatelet adhesion and aggregation at sites of vascular injury are two key events in hemostasis and thrombosis. Because of exciting advances in genetic engineering, the mouse has become an important and frequently used model to unravel the molecular mechanisms underlying the multistep process leading to the formation of a stable platelet plug. In gene-targeted mice, the crucial importance of platelet adhesion receptors such as glycoprotein Ib alpha or the alphaIIb beta3 integrin has been confirmed and further clarified. Their absence leads to highly impaired thrombus formation, independent of the model used to induce vascular injury. In contrast, the relative contribution of other receptors, such as glycoprotein VI, or of various platelet ligands may be regulated by the severity of injury, the type of vessel injured, and the signaling pathways that are generated. Murine models have also helped improve understanding of the second wave of events that leads to stabilization of the platelet aggregate. Despite the current limitations due to lack of standardization and the virtual absence of thrombosis models in diseased vessels, there is no doubt that the mouse will play a key role in the discovery and characterization of the next generation of antithrombotic agents. This review focuses on key findings about the molecular mechanisms supporting hemostasis and thrombosis that have been obtained with genetically engineered mouse models deficient in various platelet adhesion receptors and ligands. Combination of these models with sophisticated methods allowing direct visualization of platelet-vessel wall interactions after injury greatly contributed to recent advances in the field.

• Correction of the bleeding time in von Willebrand factor (VWF)-deficient mice using murine VWF.

  Authors: Peter J Lenting, Philip G de Groot, Simon F De Meyer, Karen Vanhoorelbeke, Cynthia Pruss, David Lillicrap, Isabelle Marx, Cécile V Denis

  Blood. 04/2007; 109(5):2267-8.

• Variations in glycosylation of von Willebrand factor with O-linked sialylated T antigen are associated with its plasma levels.

  Authors: Carina J M van Schooten, Cécile V Denis, Ton Lisman, Jeroen C J Eikenboom, Frank W Leebeek, Jenny Goudemand, Edith Fressinaud, H Marijke van den Berg, Philip G de Groot, Peter J Lenting

  Blood. 03/2007; 109(6):2430-7.

  The glycosylation profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarilyThe glycosylation profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of sialylated T antigen (NeuAc(alpha2-3)Gal-(beta1-3)-[NeuAc(alpha2-6)]GalNAc). It is not yet known whether O-linked carbohydrates affect VWF levels. We developed an immunosorbent assay based on neuraminidase incubation allowing subsequent binding of peanut agglutinin (PNA) to desialylated O-linked T antigen on VWF. An inverse relation was found between PNA binding and VWF antigen levels in healthy individuals (n = 111; Pearson rank = -0.43; P < .001). A similar inverse association was observed in randomly selected plasma samples from our diagnostic laboratory: 252% +/- 125% for VWF levels less than 0.5 U/mL (n = 15); 131% +/- 36% for VWF levels between 0.5 and 1.5 U/mL (n = 32); and 92% +/- 40% for VWF levels more than 1.5 U/mL (n = 19). Reduced or increased PNA binding was also observed in patients with increased (liver cirrhosis) or reduced (von Willebrand disease [VWD] type 1) VWF antigen levels, respectively. VWD type 1 patients further displayed increased ratios of propeptide over mature VWF antigen levels (0.38 +/- 0.18 versus 0.17 +/- 0.03 for patients and controls, respectively; P < .001), which is indicative of reduced VWF survival in these patients. Of interest, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank = 0.47), suggesting a potential association between O-linked glycosylation and VWF survival. Finally, we detected a marked decrease in PNA binding in post-DDAVP (1-deamino-8-D-arginine vasopressin) samples from various patients, indicating that the O-linked glycosylation profile of VWF stored in endothelial storage organelles may differ from circulating VWF.

• Role of von Willebrand factor in tumor metastasis.

  Authors: Virginie Terraube, Isabelle Marx, Cécile V Denis

  Thrombosis research. 01/2007; 120 Suppl 2:S64-70.

  Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in primary hemostasis, allowing the adhesion of platelets to the exposed subendothelium. The key role playedVon Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in primary hemostasis, allowing the adhesion of platelets to the exposed subendothelium. The key role played by VWF in platelet adhesion suggests a potential implication in various pathologies where this process is involved. In cancer metastasis development, tumor cells interact with platelets and the vessel wall to extravasate from the circulation. A number of potential receptors for VWF have been identified on tumor cells such as glycoprotein Ib or the alpha(IIb)beta(3) and alpha(v)beta(3) integrins and direct interactions between VWF and tumor cells have been reported. To address the role of VWF in an experimental metastasis model, we compared the formation of pulmonary metastatic foci in C57BL/6J wild-type and VWF-null mice following I.V. injection of murine melanoma B16-BL6 cells or Lewis lung carcinoma cells. Surprisingly we found a significant increase in the number of pulmonary metastatic foci in VWF-null mice. Restoration of VWF plasma levels by co-injection of VWF with the tumor cells led to the correction of this pro-metastatic phenotype. In vitro analysis revealed that VWF did not influence tumor cell proliferation or invasion but induced cellular death. This result was confirmed in vivo where analysis of the early survival of tumor cells in the lungs revealed that the presence of VWF led to a decreased survival of these cells during the first 24 hours after injection. Our results suggest that VWF plays a role in tumor metastasis, independently of its role in hemostasis.

• In vivo clearance of human protein S in a mouse model: influence of C4b-binding protein and the Heerlen polymorphism.

  Authors: Cécile V Denis, Sarah J Roberts, Tilman M Hackeng, Peter J Lenting

  Arteriosclerosis, thrombosis, and vascular biology. 10/2005; 25(10):2209-15.

  OBJECTIVE: To explore the effect of the Heerlen polymorphism and C4b-binding protein (C4BP) on protein S catabolism in vitro and in vivo. METHODS AND RESULTS: Radiolabeled protein S was efficientlyOBJECTIVE: To explore the effect of the Heerlen polymorphism and C4b-binding protein (C4BP) on protein S catabolism in vitro and in vivo. METHODS AND RESULTS: Radiolabeled protein S was efficiently bound and intracellularly degraded by THP-1 macrophages, and both processes were strongly reduced in the presence of the protein S-carrier protein C4BP. To test whether C4BP displays a similar protective effect in vivo, survival experiments were performed in mice. In the absence of C4BP, radiolabeled human protein S disappeared in a biphasic manner (mean residence time [MRT] 2 hours). However, the presence of C4BP resulted in a 4-fold prolonged survival of protein S (MRT 8 hours; P<0.0001). We also applied this experimental model to recombinant protein S-Heerlen, a naturally occurring variant that contains a Ser460Pro substitution. These clearance experiments revealed a strongly decreased survival of recombinant protein S-S460P (MRT 0.6 hours; P=0.021), which could be compensated partially by C4BP (MRT 1.4 hours; P=0.012 compared with protein S-S460P). CONCLUSIONS: Protein S-S460P has a reduced survival in vivo, which may explain the low levels of free protein S in individuals carrying this polymorphism. Furthermore, C4BP prevents premature clearance of protein S and uses this ability to compensate the increased clearance of protein S-S460P.

• von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate.

  Authors: Jeffrey F W Keuren, Dominique Baruch, Paulette Legendre, Cécile V Denis, Peter J Lenting, Jean-Pierre Girma, Theo Lindhout

  Blood. 04/2004; 103(5):1741-6.

  Fibrin is actively involved in platelet reactions essential for thrombus growth, in which von Willebrand factor (VWF) might be an important mediator. The aim of this study was to localize VWF domainsFibrin is actively involved in platelet reactions essential for thrombus growth, in which von Willebrand factor (VWF) might be an important mediator. The aim of this study was to localize VWF domains that bind to fibrin and to determine their relevance in platelet adhesion. VWF binds specifically to fibrin with an apparent Kd of 2.2 microg/mL. Competition in the presence of 2 complementary fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), indicated that the high affinity binding site for fibrin is located in the C-terminal part, thus distinct from the A domains. Comparison of 2 deleted rVWF (DeltaD4B-rVWF, DeltaC1C2-rVWF) suggested that the C1C2 domains contained a fibrin binding site. This site is distinct from RGD, as shown by binding of D1746G-rVWF to fibrin. Perfusion studies at high shear rate demonstrated that C1C2 domains were required for optimal platelet adhesion to fibrin. With the use of a VWF-deficient mouse model, it was found that plasma VWF is critical for platelet tethering and adhesion to fibrin. These results suggest a dual role of fibrin-bound VWF in thrombus formation: first, fibrin-bound VWF is critical in the recruitment of platelets by way of glycoprotein (GP) Ib, and, second, it contributes to stationary platelet adhesion by way of binding to activated alphaIIbbeta3.

• An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation.

  Authors: Peter J Lenting, Erik Westein, Virginie Terraube, Anne-Sophie Ribba, Eric G Huizinga, Dominique Meyer, Philip G de Groot, Cécile V Denis

  The Journal of biological chemistry. 03/2004; 279(13):12102-9.

  To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection ofTo explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t((1/2))alpha = 13 min) and a slow secondary phase (t((1/2))beta = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D'-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D'-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D'-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D'-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.

• Elevated plasma von Willebrand factor in a murine model of severe hyperhomocysteinemia.

  Authors: Cécile V Denis, Virgine Terraube, Karine Robert, Nathalie Janel

  Thrombosis and haemostasis. 09/2003; 90(2):362-4.

• The clearance mechanism of chilled blood platelets.

  Authors: Karin M Hoffmeister, Thomas W Felbinger, Hervé Falet, Cécile V Denis, Wolfgang Bergmeier, Tanya N Mayadas, Ulrich H von Andrian, Denisa D Wagner, Thomas P Stossel, John H Hartwig

  Cell. 02/2003; 112(1):87-97.

  Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior toPlatelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.