Publications (11)54.95 Total impact
-
Article: Monocytes downregulate the early stage of collagen-induced platelet activation by a PECAM-1-dependent mechanism.
[show abstract] [hide abstract]
ABSTRACT: Blood vessel damage results in exposure of the subendothelial matrix, to which platelets adhere. Monocytes are recruited and activated at the site of injury. Here we studied the effect of monocytes on platelet activation induced by exposure to fibrillar collagen. Washed platelets and isolated monocytes (100/1) were coincubated with type I collagen in static adhesion conditions or in suspension. Platelet activation was assessed by measuring RANTES production and alpha-granule secretion. Platelet adherence on immobilized collagen was analyzed by fluorescence confocal microscopy. Cell-cell contacts were prevented by incubating platelets and monocytes in transwell coculture dishes. Experiments were also performed in the presence of soluble recombinant platelet endothelial cell adhesion molecule-1 (PECAM-1) or of antibodies to PECAM-1. Unexpectedly, unstimulated monocytes limited the initial phase of platelet activation by fibrillar collagen. In adhesion conditions, monocytes reduced the secretion by platelets of the inflammatory chemokine RANTES and of beta-thromboglobulin and the formation of platelet aggregates. The inhibitory effect of monocytes on platelet activation required direct cell-cell contacts between platelets and monocytes. Monocytes also inhibited collagen-induced platelet activation in suspension conditions as assessed by the reduction of P-selectin exposure and RANTES secretion. A recovery of platelet responses was observed in the presence of soluble PECAM-1 and of PECAM-1.3 Fab, indicating that PECAM-1 is involved in monocyte-triggered downregulation of platelet reactivity. Our data provide the first evidence that unstimulated monocytes limit the initial phase of platelet activation by collagen via a mechanism that is, at least in part, PECAM-1-dependent.Journal of Thrombosis and Haemostasis 11/2008; 7(1):143-51. · 5.73 Impact Factor -
Article: A monoclonal antibody directed against human von Willebrand factor induces type 2B-like alterations.
[show abstract] [hide abstract]
ABSTRACT: We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin-induced platelet aggregation (RIPA) and induces a preferential binding of the high-molecular-weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s(-1) could now demonstrate that shear-induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti-GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s(-1), using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N-terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.Journal of Thrombosis and Haemostasis 10/2004; 2(9):1622-8. · 5.73 Impact Factor -
Article: Defect of heparin binding in plasma and recombinant von Willebrand factor with type 2 von Willebrand disease mutations.
[show abstract] [hide abstract]
ABSTRACT: The aim of our study was to characterise heparin-binding properties of mutated von Willebrand factor (VWF) in 24 patients plasmas with type 2 von Willebrand disease (VWD). and in 15 recombinant VWF (rVWF) with the corresponding mutations. Binding of mutated rVWF or plasma VWF was compared to that of WT-rVWF or normal pool plasma VWF. Four mutations, at positions C509, V551, R552 and R611 lead to significantly decreased binding to heparin in both plasma and rVWF. Interestingly, whereas these four residues are distant in the primary structure of VWF-A1domain, they are close to each other in its three-dimensional structure. Structural analysis suggested how folding problems and destabilisation due to these mutations could induce reorganisation of surface regions involved in heparin binding. In contrast, no heparin-binding defect was found associated with different type 2 VWF mutants, at positions G561, E596, I662, R543, R545, V553, R578 or L697.Thrombosis and Haemostasis 01/2002; 86(6):1459-65. · 5.04 Impact Factor -
Article: Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation.
[show abstract] [hide abstract]
ABSTRACT: The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds(-1)) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds(-1). Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds(-1) confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD. (Blood. 2000;95:3796-3803)Blood 07/2000; 95(12):3796-803. · 9.90 Impact Factor -
Article: von Willebrand factor binding to heparin in various types of von Willebrand disease.
[show abstract] [hide abstract]
ABSTRACT: The purpose was to study von Willebrand factor (vWF) binding to heparin in different types of von Willebrand disease (vWD). Plasma samples from 92 patients were representative of most vWD subtypes as they included 13 type 1, ten type 2N, 27 type 2A, 23 type 2B, and 19 type 2M patients. We selected assay conditions suitable for the screening of plasma vWF concentrations as low as 15 U/dl vWF:Ag. We determined the range of vWF concentrations in plasma where the percentage of (125)I-MAb/vWF complexes bound to heparin-agarose beads was constant. This range of dilution allowed circumvention of potential competition by other plasma heparin-binding proteins. The multimeric composition of vWF had hardly any influence on the ability of vWF to bind to heparin. Results were expressed as the ratio of heparin-binding capacity of patients' plasma to that of normal pool plasma. We found a ratio of 0.99+/-0.004 (mean+/-s.e.m.) for 23 normal individual donors. Furthermore, when comparing the mean values of plasma vWF-heparin binding ratios by ANOVA F-test in the six groups (one normal and five vWD), we found significant differences between them (P<0.0001). Pairwise comparison of multiples by the Scheffe's test indicated that the mean values of ratios in type 2A on the one hand and type 2M on the other, were significantly lower than in normal plasma, type 2N, type 2B and type 1. Our data suggest a relationship between the ability of vWF to bind to heparin and to the platelet GPIb receptor, since type 2B and 2N patients have an increased or normal ability to bind to GPIb whereas type 2A and 2M patients have an impaired interaction with that receptor.The Hematology Journal 02/2000; 1(3):190-8. · 1.86 Impact Factor -
Article: Modulation by heparin of the interaction of the A1 domain of Von Willebrand factor with glycoprotein Ib.
[show abstract] [hide abstract]
ABSTRACT: The conformation of the A1 domain of von Willebrand factor (vWF) is a critical determinant of its interaction with the glycoprotein (GP) Ib/V/IX complex. To better define the regulatory mechanisms of vWF A1 domain binding to the GPIb/V/IX complex, we studied vWF-dependent aggregation properties of a cell line overexpressing the GPIbalpha, GPIbbeta, and GPIX subunits (CHO-GPIbalphabeta/IX cells). We found that CHO-GPIbalphabeta/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbalphabeta/IX cell aggregation was completely inhibited by the recombinant VCL fragment of vWF that contains the A1 domain. Surprisingly, the substitution of heparin for ristocetin resulted in the formation of CHO-GPIbalphabeta/IX cell aggregates. Using monoclonal antibodies blocking vWF interaction with GPIb/V/IX or mocarhagin, a venom metalloproteinase that removes the amino-terminal fragment of GPIbalpha extending from aa 1 to 282, we demonstrated that both ristocetin- and heparin-induced aggregations involved an interaction between the A1 domain of vWF and the GPIbalpha subunit of the GPIb/V/IX complex. The involvement of heparin in cell aggregation was also demonstrated after treatment of heparin with heparinase that abolished CHO-GPIbalphabeta/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbalphabeta/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.Blood 01/2000; 94(12):4186-94. · 9.90 Impact Factor -
Article: Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor.
[show abstract] [hide abstract]
ABSTRACT: Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.Blood 05/1998; 91(10):3792-9. · 9.90 Impact Factor -
Article: Distinct sequences of the glycoprotein Ib-binding domain of von Willebrand factor involved in shear induced platelet aggregation.
[show abstract] [hide abstract]
ABSTRACT: Shear-induced platelet aggregation (SIPA) requires von Willebrand factor (vWF) binding to the platelet receptors GPIb and alphaIIbbeta3. In order to determine the vW F sequences involved in SIPA at 4000/s, we studied the llb 3 effect of three monoclonal antibodies (mabs) 724, 713 and 328 to the A1 domain of vWF. We found that mab 724 induced an enhanced SIPA via a Fc gamma-receptor independent mechanism. In contrast, mab 713 and mab 328 could inhibit SIPA by 52 and 91% , respectively. Based on distinct effects on SIPA, we can propose the following working model for the interaction between vWF and GPIb: mabs 713 and 328, which block SIPA, may recognize an epitope that is involved in binding to GPIb, whereas mab 724, which increases SIPA in the presence of vWF, may mimic the effect of botrocetin when binding to vWF, by inducing an active conformation of vWF, which may be more sensitive to high shear rate.Platelets 02/1998; 9(3-4):151-3. · 1.85 Impact Factor -
Article: Binding of heparin fractions to von Willebrand factor: effect of molecular weight and affinity for antithrombin III.
[show abstract] [hide abstract]
ABSTRACT: To investigate the influence of the structure of heparin on its binding to vWF, we compared heparin fractions of different molecular weight (MW) or affinity for antithrombin III (ATIII). We studied the interaction of purified 125I-vWF or plasma vWF, labeled with a pool of 125I-monoclonal antibodies to vWF, with unfractionated heparin immobilized on agarose beads. Fractions were compared as competitors of these interactions and their effect was quantitated by their half-maximal inhibition (IC50). When the MW of the fractions decreased, especially below 7500, their IC50 increased, indicating that the affinity of the fractions for vWF decreased with their MW. Using heparin-derived oligosaccharides, we also demonstrated that a minimal chain length of 18 monosaccharides was required for heparin binding to vWF. In addition, different fractions with low affinity for ATIII were compared as competitors of 125I-vWF binding to heparin-agarose. Despite a very low content of ATIII binding sites, some fractions retained a low IC50. Thus, heparin interaction with vWF is independent of the presence of the ATIII binding site and is mostly dependent on the length of the heparin chain. These data suggest that unfractionated heparin is a more potent inhibitor of vWF-dependent functions than low MW heparin fractions.Thrombosis and Haemostasis 02/1994; 71(1):141-6. · 5.04 Impact Factor -
Article: Localization of von Willebrand factor binding domains to endothelial extracellular matrix and to type VI collagen.
[show abstract] [hide abstract]
ABSTRACT: We have recently shown that von Willebrand factor (vWF) binds to endothelial and fibroblastic extracellular matrixes (ECM) in a dose-dependent, specific, and saturable way. To localize the domain on the vWF subunit responsible for this interaction, purified proteolytic fragments of vWF were compared for their ability to inhibit 125I-vWF binding to ECM. A tryptic dimeric fragment of 116 kD (T116), extending from amino acid (aa) residues 449 to 728, produced a significant inhibition of 125I-vWF binding to the ECM. In contrast, P34 (aa 1-272), SpI (aa 911-1,365), and SpII (aa 1,366-2,050) had no significant effect on 125I-vWF binding to the ECM. Using an immunofluorescence technique, we identified type VI collagen and heparan sulfate in the endothelial ECM. 125I-vWF was found to bind specifically to purified type VI collagen. Unlabeled vWF and SpIII were able to completely inhibit 125I-vWF binding to type VI collagen. T116 and SpI appeared as competitors of this interaction, whereas P34 and SpII were not. Our data suggest that vWF binds to the endothelial ECM through the T116 fragment and that T116 and SpI each contain a binding site for type VI collagen. Heparin is known to be a vWF ligand, but did not appear as a competitor of vWF binding to the ECM, nor did heparan sulfate.Arteriosclerosis and thrombosis: a journal of vascular biology / American Heart Association 04/1993; 13(3):398-406. -
Article: Characterization of murine anti-glycoprotein Ib monoclonal antibodies that differentiate between shear-induced and ristocetin/botrocetin-induced glycoprotein Ib-von Willebrand factor interaction.
[show abstract] [hide abstract]
ABSTRACT: Platelet adhesion to vascular subendothelium under conditions of high shear stress is mediated by the platelet glycoprotein (GP) Ib-von Willebrand Factor (vWF) interaction. The aim of this study was to characterize the murine monoclonal antibodies (MoAbs) 27A10 and 28E6, both raised against purified GPIb. The MoAb 27A10 is a potent inhibitor of shear-induced platelet adhesion to collagen type I in a flow chamber at shear rates of 1,300 and 2,700 s(-1). 20 microg/ml of MoAb 27A10, furthermore, could completely block shear-induced aggregation in a modified Couette viscometer at shear rates of 1,000 and 4,000 s(-1). On the other hand, MoAb 27A10 had a negligible effect on botrocetin-induced GPIb-vWF binding and is only a poor inhibitor of the ristocetin-dependent interaction. In contrast, MoAb 28E6 did abolish both the ristocetin- and botrocetin-induced GPIb-vWF binding, whereas it did not block the shear-induced interaction. Thus, we identify here two anti-GPIb MoAbs 27A10 and 28E6 that either preferentially inhibit the shear-induced or the ristocetin/botrocetin-induced platelet-vWF interaction. With these tools it should be possible to more clearly define the mechanisms by which platelets bind to vWF in vivo.Haemostasis 30(3):139-48.
