Dominique Baruch - Academia.edu (original) (raw)

Papers by Dominique Baruch

Research paper thumbnail of Method for Producing Platelets

Research paper thumbnail of A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation

Scientific Reports, 2016

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Wi... more von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

Research paper thumbnail of Method for producing platelets from megakaryocytes

Research paper thumbnail of Activation of integrin alphaIIbbeta3 expressed in Chinese hamster ovary cells is required for interaction with solid-phase von Willebrand factor

Brit J Haematol, 2002

Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to p... more Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-alphaIIbbeta3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-alphaIIbbeta3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-alphaIIbbeta3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-alphaIIbbeta3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated alphaIIbbeta3 is absolutely required for spread cells to resist detachment and strengthens the importance of the alphaIIbbeta3 activation step for adhesion and spreading to VWF.

Research paper thumbnail of Role of av integrins in mesangial cell adhesion to vitronectin and von Willebrand factor

Kidney Int, 1997

Role of αv integrins in mesangial cells adhesion to vitronectin and von Willebrand factor. This s... more Role of αv integrins in mesangial cells adhesion to vitronectin and von Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to αv, β3, and αvβ3 and αvβ5 complexes) that αvβ3, the vitronectin receptor, and αvβ5 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to

Research paper thumbnail of The integrin α2β1 (GPIa/IIa) I-domain inhibits platelet–collagen interaction

Thrombosis and Haemostasis

ABSTRACT

Research paper thumbnail of Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

PLOS ONE, 2015

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowi... more Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

Research paper thumbnail of von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

Blood, 2004

Fibrin is actively involved in platelet reactions essential for thrombus growth, in which von Wil... more 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 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...

Research paper thumbnail of Activation of recombinant alphaIIbbeta3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to alphaIIbbeta3

British journal of haematology, 2002

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with reco... more We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin alphaIIbbeta3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce alphaIIbbeta3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of alphaIIbbeta3. By comparing the effects of two potent platelet alphaIIbbeta3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated alphaIIbbeta3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no eff...

Research paper thumbnail of Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III

Thrombosis research, 1985

We have investigated the antithrombin III independent effect of crude heparin, two heparin fracti... more We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute...

Research paper thumbnail of Inhibition of Thrombin-Catalyzed Reactions in Blood Coagulation and Platelet Activation by Heparin Fractions in the Absence of Antithrombin III

Pathophysiology of Haemostasis and Thrombosis, 1986

The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyze... more The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyzed reactions: activation of purified plasma factor V and partially purified plasma factor VIII:C, generation of factor Va from the platelets and, in the presence of collagen, of the platelet procoagulant activity. Five heparin fractions and a heparinoid were compared to crude heparin. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of heparin (fractions) appeared to be the result of the formation of a complex between heparin and thrombin that alters the specificity of thrombin towards high molecular weight substrates. The inhibition of these thrombin-dependent feedback reactions in blood coagulation might be of importance in the mechanisms for the dissociation between the antithrombotic and hemorrhagic properties of low molecular weight heparins.

Research paper thumbnail of Role of αv integrins in mesangial cell adhesion to vitronectin and von Willebrand factor

Kidney International, 1997

Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell ... more Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to a, f3, and aI3 and aj35 complexes) that avf3, the vitronectin receptor, and Ov13 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to 8th passages. Antibodies to a, 13 and 0V133 respectively precipitated an afl heterodimer with molecular weights of 140 and 97 kDa. We analyzed the role of the various integrins in HMC interactions with vitronectin, and with fIbronectin and von Willebrand factor (vWf), which are synthetized respectively by mesangial and endothelial cells. Cell adhesion increased in a dose dependent manner with the concentration of plastic-coated matrix protein and vWf. Inhibition of cell attachment with monoclonal antibodies to integrins indicated that HMC adhesion to vWf primarily involves a(33, and that aj35 may also contribute to cell binding to vWf. Adhesion to vitronectin involves both zj33 and c3 complexes. In contrast, adhesion to fibronectin was not affected by monoclonal antibodies to a(33 and a13 complexes. We propose that integrins av(33 and af35, present on HMC, could mediate an interaction between mesangial and endothelial cells by binding to vWf, released at the basal site of endothelial cells.

Research paper thumbnail of CHO cells expressing the high affinity alphaIIbbeta3T562N integrin demonstrate enhanced adhesion under shear

Journal of Thrombosis and Haemostasis, 2006

To cite this article: Legendre P, Salsmann A, Rayes J, Trassard O, Kieffer N, Baruch D. CHO cells... more To cite this article: Legendre P, Salsmann A, Rayes J, Trassard O, Kieffer N, Baruch D. CHO cells expressing the high affinity a IIb b z3 T562N integrin demonstrate enhanced adhesion under shear. J Thromb Haemost 2006; 4: 236-46.

Research paper thumbnail of Human neutrophil integrin  9 1: up-regulation by cell activation and synergy with  2 integrins during adhesion to endothelium under flow

Journal of Leukocyte Biology, 2010

Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this stu... more Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this study. alpha9beta1 and alpha5beta1 appeared here as the main beta1 integrins expressed on the membrane of resting platelet-depleted neutrophils-alpha6beta1 representing <15% and alpha2beta1 undetectable. Neutrophil activation slightly enhanced alpha5 expression, did not change alpha6, but resulted in a two- to threefold increase of alpha9beta1, which then became the major beta1 integrin of the neutrophil membrane. alpha9beta1 was the only beta1 integrin to be up-regulated after transendothelial migration across TNF-alpha-activated HUVECs. As alpha9beta1 binds VCAM-1, we analyzed its participation to neutrophil adhesion to TNF-alpha-activated endothelial cells. Blocking anti-alpha9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti-beta2 mAb. Under flow conditions, the anti-alpha9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti-beta2 antibodies significantly and further enhanced the velocity of beta2-blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up-regulation of alpha9beta1 expression among all beta1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between alpha9beta1 and beta2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

Research paper thumbnail of Activation of recombinant alphaIIbbeta3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to alphaIIbbeta3

British Journal of Haematology, 2002

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with reco... more We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin aIIbb3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce aIIbb3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of aIIbb3. By comparing the effects of two potent platelet aIIbb3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated aIIbb3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated aIIbb3-dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT-activated aIIbb3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.

Research paper thumbnail of Activation of integrin alphaIIbbeta3 expressed in Chinese hamster ovary cells is required for interaction with solid-phase von Willebrand factor

British Journal of Haematology, 2002

Dithiothreitol (DTT) is known to induce an active conformation of aIIbb3 integrin and to promote ... more Dithiothreitol (DTT) is known to induce an active conformation of aIIbb3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-aIIbb3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-aIIbb3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-aIIbb3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-aIIbb3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated aIIbb3 is absolutely required for spread cells to resist detachment and strengthens the importance of the aIIbb3 activation step for adhesion and spreading to VWF.

Research paper thumbnail of Exposure of human megakaryocytes to high shear rates accelerates platelet production

Blood, 2009

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct... more Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation. (Blood.

Research paper thumbnail of beta 3 receptor v RGD motif that mediates endothelial cell adhesion through the alpha Solidphase von Willebrand factor contains a conformationally active

HE INTEGRITY OF the endothelium is an absolute T requirement for maintaining a nonthrombogenic st... more HE INTEGRITY OF the endothelium is an absolute T requirement for maintaining a nonthrombogenic state of blood vessels. Its structural and functional integrity depends on the firm attachment of endothelial cells to the subendothelial extracellular matrix. To strengthen their an- chorage, endothelial cells establish cell-cell and cell-matrix interactions, via integrin and nonintegrin receptors. Most receptors involved in the interactions between endothelial cells and extracellular matrix proteins belong to the integrin family.' The a,P3 integrin present at the endothelial cell surface is a promiscuous receptor that binds a number of adhesive proteins such as vitronectin, fibronectin, fibrino- gen, thrombospondin, and von Willebrand factor (vWF).~,~ vWF is synthesized by endothelial cells as a pro-vWF, containing a 74 1 amino acid propeptide and a mature vWF subunit of 2,050 amino acids.4 It is released into the plasma and deposited on the s~bendothelium.~ Mature vWF, the largest soluble protein found in plasma, is assembled in multimers with a molecular mass extending from 500 kD (dimers) to more than 15,000 kD. A major role is to mediate platelet adhesion to the subendothelium, especially under conditions of high shear Two specific platelet recep- tors are known to bind vWF, glycoprotein (GP) Ib for initial platelet contact and GPIIb/IIIa (aIrbP3) for subsequent plate- let aggregation and spreading.'-13 However, no spontaneous interaction occurs between soluble plasma vWF and unacti- vated platelets in the absence of high shear rate. A confor- mational change of vWF is required, which takes place on

Research paper thumbnail of Link between ADAMTS-13 and troponin levels? Authors' reply

Critical Care Medicine, 2008

Research paper thumbnail of ACTIVATION OF pp125FAK BY TYPE 2B RECOMBINANT VON WILLEBRAND FACTOR BINDING TO PLATELET GPIb AT HIGH SHEAR RATE OCCURS

Research paper thumbnail of Method for Producing Platelets

Research paper thumbnail of A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation

Scientific Reports, 2016

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Wi... more von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

Research paper thumbnail of Method for producing platelets from megakaryocytes

Research paper thumbnail of Activation of integrin alphaIIbbeta3 expressed in Chinese hamster ovary cells is required for interaction with solid-phase von Willebrand factor

Brit J Haematol, 2002

Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to p... more Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-alphaIIbbeta3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-alphaIIbbeta3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-alphaIIbbeta3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-alphaIIbbeta3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated alphaIIbbeta3 is absolutely required for spread cells to resist detachment and strengthens the importance of the alphaIIbbeta3 activation step for adhesion and spreading to VWF.

Research paper thumbnail of Role of av integrins in mesangial cell adhesion to vitronectin and von Willebrand factor

Kidney Int, 1997

Role of αv integrins in mesangial cells adhesion to vitronectin and von Willebrand factor. This s... more Role of αv integrins in mesangial cells adhesion to vitronectin and von Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to αv, β3, and αvβ3 and αvβ5 complexes) that αvβ3, the vitronectin receptor, and αvβ5 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to

Research paper thumbnail of The integrin α2β1 (GPIa/IIa) I-domain inhibits platelet–collagen interaction

Thrombosis and Haemostasis

ABSTRACT

Research paper thumbnail of Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

PLOS ONE, 2015

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowi... more Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

Research paper thumbnail of von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

Blood, 2004

Fibrin is actively involved in platelet reactions essential for thrombus growth, in which von Wil... more 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 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...

Research paper thumbnail of Activation of recombinant alphaIIbbeta3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to alphaIIbbeta3

British journal of haematology, 2002

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with reco... more We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin alphaIIbbeta3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce alphaIIbbeta3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of alphaIIbbeta3. By comparing the effects of two potent platelet alphaIIbbeta3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated alphaIIbbeta3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no eff...

Research paper thumbnail of Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III

Thrombosis research, 1985

We have investigated the antithrombin III independent effect of crude heparin, two heparin fracti... more We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute...

Research paper thumbnail of Inhibition of Thrombin-Catalyzed Reactions in Blood Coagulation and Platelet Activation by Heparin Fractions in the Absence of Antithrombin III

Pathophysiology of Haemostasis and Thrombosis, 1986

The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyze... more The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyzed reactions: activation of purified plasma factor V and partially purified plasma factor VIII:C, generation of factor Va from the platelets and, in the presence of collagen, of the platelet procoagulant activity. Five heparin fractions and a heparinoid were compared to crude heparin. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of heparin (fractions) appeared to be the result of the formation of a complex between heparin and thrombin that alters the specificity of thrombin towards high molecular weight substrates. The inhibition of these thrombin-dependent feedback reactions in blood coagulation might be of importance in the mechanisms for the dissociation between the antithrombotic and hemorrhagic properties of low molecular weight heparins.

Research paper thumbnail of Role of αv integrins in mesangial cell adhesion to vitronectin and von Willebrand factor

Kidney International, 1997

Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell ... more Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to a, f3, and aI3 and aj35 complexes) that avf3, the vitronectin receptor, and Ov13 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to 8th passages. Antibodies to a, 13 and 0V133 respectively precipitated an afl heterodimer with molecular weights of 140 and 97 kDa. We analyzed the role of the various integrins in HMC interactions with vitronectin, and with fIbronectin and von Willebrand factor (vWf), which are synthetized respectively by mesangial and endothelial cells. Cell adhesion increased in a dose dependent manner with the concentration of plastic-coated matrix protein and vWf. Inhibition of cell attachment with monoclonal antibodies to integrins indicated that HMC adhesion to vWf primarily involves a(33, and that aj35 may also contribute to cell binding to vWf. Adhesion to vitronectin involves both zj33 and c3 complexes. In contrast, adhesion to fibronectin was not affected by monoclonal antibodies to a(33 and a13 complexes. We propose that integrins av(33 and af35, present on HMC, could mediate an interaction between mesangial and endothelial cells by binding to vWf, released at the basal site of endothelial cells.

Research paper thumbnail of CHO cells expressing the high affinity alphaIIbbeta3T562N integrin demonstrate enhanced adhesion under shear

Journal of Thrombosis and Haemostasis, 2006

To cite this article: Legendre P, Salsmann A, Rayes J, Trassard O, Kieffer N, Baruch D. CHO cells... more To cite this article: Legendre P, Salsmann A, Rayes J, Trassard O, Kieffer N, Baruch D. CHO cells expressing the high affinity a IIb b z3 T562N integrin demonstrate enhanced adhesion under shear. J Thromb Haemost 2006; 4: 236-46.

Research paper thumbnail of Human neutrophil integrin  9 1: up-regulation by cell activation and synergy with  2 integrins during adhesion to endothelium under flow

Journal of Leukocyte Biology, 2010

Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this stu... more Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this study. alpha9beta1 and alpha5beta1 appeared here as the main beta1 integrins expressed on the membrane of resting platelet-depleted neutrophils-alpha6beta1 representing <15% and alpha2beta1 undetectable. Neutrophil activation slightly enhanced alpha5 expression, did not change alpha6, but resulted in a two- to threefold increase of alpha9beta1, which then became the major beta1 integrin of the neutrophil membrane. alpha9beta1 was the only beta1 integrin to be up-regulated after transendothelial migration across TNF-alpha-activated HUVECs. As alpha9beta1 binds VCAM-1, we analyzed its participation to neutrophil adhesion to TNF-alpha-activated endothelial cells. Blocking anti-alpha9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti-beta2 mAb. Under flow conditions, the anti-alpha9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti-beta2 antibodies significantly and further enhanced the velocity of beta2-blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up-regulation of alpha9beta1 expression among all beta1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between alpha9beta1 and beta2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

Research paper thumbnail of Activation of recombinant alphaIIbbeta3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to alphaIIbbeta3

British Journal of Haematology, 2002

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with reco... more We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin aIIbb3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce aIIbb3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of aIIbb3. By comparing the effects of two potent platelet aIIbb3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated aIIbb3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated aIIbb3-dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT-activated aIIbb3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.

Research paper thumbnail of Activation of integrin alphaIIbbeta3 expressed in Chinese hamster ovary cells is required for interaction with solid-phase von Willebrand factor

British Journal of Haematology, 2002

Dithiothreitol (DTT) is known to induce an active conformation of aIIbb3 integrin and to promote ... more Dithiothreitol (DTT) is known to induce an active conformation of aIIbb3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-aIIbb3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-aIIbb3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-aIIbb3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-aIIbb3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated aIIbb3 is absolutely required for spread cells to resist detachment and strengthens the importance of the aIIbb3 activation step for adhesion and spreading to VWF.

Research paper thumbnail of Exposure of human megakaryocytes to high shear rates accelerates platelet production

Blood, 2009

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct... more Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation. (Blood.

Research paper thumbnail of beta 3 receptor v RGD motif that mediates endothelial cell adhesion through the alpha Solidphase von Willebrand factor contains a conformationally active

HE INTEGRITY OF the endothelium is an absolute T requirement for maintaining a nonthrombogenic st... more HE INTEGRITY OF the endothelium is an absolute T requirement for maintaining a nonthrombogenic state of blood vessels. Its structural and functional integrity depends on the firm attachment of endothelial cells to the subendothelial extracellular matrix. To strengthen their an- chorage, endothelial cells establish cell-cell and cell-matrix interactions, via integrin and nonintegrin receptors. Most receptors involved in the interactions between endothelial cells and extracellular matrix proteins belong to the integrin family.' The a,P3 integrin present at the endothelial cell surface is a promiscuous receptor that binds a number of adhesive proteins such as vitronectin, fibronectin, fibrino- gen, thrombospondin, and von Willebrand factor (vWF).~,~ vWF is synthesized by endothelial cells as a pro-vWF, containing a 74 1 amino acid propeptide and a mature vWF subunit of 2,050 amino acids.4 It is released into the plasma and deposited on the s~bendothelium.~ Mature vWF, the largest soluble protein found in plasma, is assembled in multimers with a molecular mass extending from 500 kD (dimers) to more than 15,000 kD. A major role is to mediate platelet adhesion to the subendothelium, especially under conditions of high shear Two specific platelet recep- tors are known to bind vWF, glycoprotein (GP) Ib for initial platelet contact and GPIIb/IIIa (aIrbP3) for subsequent plate- let aggregation and spreading.'-13 However, no spontaneous interaction occurs between soluble plasma vWF and unacti- vated platelets in the absence of high shear rate. A confor- mational change of vWF is required, which takes place on

Research paper thumbnail of Link between ADAMTS-13 and troponin levels? Authors' reply

Critical Care Medicine, 2008

Research paper thumbnail of ACTIVATION OF pp125FAK BY TYPE 2B RECOMBINANT VON WILLEBRAND FACTOR BINDING TO PLATELET GPIb AT HIGH SHEAR RATE OCCURS