ALBERT

Description: 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 αIIbβ3. 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 αIIbβ3. 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 αIIbβ3. 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 αIIbβ3. 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 Fcγ 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.

Description: 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 GPIb, GPIbβ, and GPIX subunits (CHO-GPIbβ/IX cells). We found that CHO-GPIbβ/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbβ/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-GPIbβ/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 GPIb 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 GPIb 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-GPIbβ/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbβ/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.

Description: 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 GPIb, GPIbβ, and GPIX subunits (CHO-GPIbβ/IX cells). We found that CHO-GPIbβ/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbβ/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-GPIbβ/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 GPIb 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 GPIb 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-GPIbβ/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbβ/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.

Description: 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 αIIbβ3. 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 αIIbβ3. 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 αIIbβ3. 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 αIIbβ3. 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 Fcγ 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.

Description: The negative regulation of transcription of the human von Willebrand factor (vWF) gene was investigated in human umbilical vein endothelial cells (HUVECs) and HeLa cells. A fragment spanning −89 to +244 nucleotides (nt), containing the first exon, is active in HUVECs only but not in HeLa cells. The activity of this promoter is sharply reduced by mutagenesis of the GATA binding site at +221. Extension of the upstream sequences from nt −89 to −142 and to −496 results in progressive reduction of the activity of the −89 to +244 promoter identifying a negative regulatory element between nt −142 and −89. A factor present in nuclear extracts from endothelial and nonendothelial cells binds to an AT-rich sequence located between nt −133 and −125. Mutagenesis of the AT-rich sequence interferes with nuclear protein binding and restores the activity of the −142 to +244 fragment to the level of the −89 to +244 promoter. Binding of the nuclear protein to the vWF AT-rich sequence in mobility shift assays is inhibited by competition with a consensus Oct-1 binding site and with a silencer octamer-like sequence from the vascular cell adhesion molecule-1 (VCAM-1) promoter. Subsequent supershift experiments identified Oct-1 as the transcription factor that binds to vWF and VCAM-1 silencer elements. These results indicate that Oct-1 acts as a transcriptional repressor of promoters of genes expressed in endothelial cells.© 1998 by The American Society of Hematology.

Description: Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

Description: Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

Description: The negative regulation of transcription of the human von Willebrand factor (vWF) gene was investigated in human umbilical vein endothelial cells (HUVECs) and HeLa cells. A fragment spanning −89 to +244 nucleotides (nt), containing the first exon, is active in HUVECs only but not in HeLa cells. The activity of this promoter is sharply reduced by mutagenesis of the GATA binding site at +221. Extension of the upstream sequences from nt −89 to −142 and to −496 results in progressive reduction of the activity of the −89 to +244 promoter identifying a negative regulatory element between nt −142 and −89. A factor present in nuclear extracts from endothelial and nonendothelial cells binds to an AT-rich sequence located between nt −133 and −125. Mutagenesis of the AT-rich sequence interferes with nuclear protein binding and restores the activity of the −142 to +244 fragment to the level of the −89 to +244 promoter. Binding of the nuclear protein to the vWF AT-rich sequence in mobility shift assays is inhibited by competition with a consensus Oct-1 binding site and with a silencer octamer-like sequence from the vascular cell adhesion molecule-1 (VCAM-1) promoter. Subsequent supershift experiments identified Oct-1 as the transcription factor that binds to vWF and VCAM-1 silencer elements. These results indicate that Oct-1 acts as a transcriptional repressor of promoters of genes expressed in endothelial cells. © 1998 by The American Society of Hematology.

Description: Bitiscetin has recently been shown to induce von Willebrand factor (vWF)-dependent aggregation of fixed platelets (Hamako J, et al,Biochem Biophys Res Commun 226:273, 1996). We have purified bitiscetin from Bitis arietans venom and investigated the mechanism whereby it promotes a form of vWF that is reactive with platelets. In the presence of bitiscetin, vWF binds to platelets in a dose-dependent and saturable manner. The binding of vWF to platelets involves glycoprotein (GP) Ib because it was totally blocked by monoclonal antibody (MoAb) 6D1 directed towards the vWF-binding site of GPIb. The binding also involves the GPIb-binding site of vWF located on the A1 domain because it was inhibited by MoAb to vWF whose epitopes are within this domain and that block binding of vWF to platelets induced by ristocetin or botrocetin. However, in contrast to ristocetin or botrocetin, the binding site of bitiscetin does not reside within the A1 domain but within the A3 domain of vWF. Thus, among a series of vWF fragments, 125I-bitiscetin only binds to those that overlap the A3 domain, ie, SpIII (amino acid [aa] 1-1365), SpI (aa 911-1365), and rvWF-A3 domain (aa 920-1111). It does not bind to SpII corresponding to the C-terminal part of vWF subunit (aa 1366-2050) nor to the 39/34/kD dispase species (aa 480-718) or T116 (aa 449-728) overlapping the A1 domain. In addition, bitiscetin that does not bind to DeltaA3-rvWF (deleted between aa 910-1113) has no binding site ouside the A3 domain. The localization of the binding site of bitiscetin within the A3 domain was further supported by showing that MoAb to vWF, which are specific for this domain and block the interaction between vWF and collagen, are potent inhibitors of the binding of bitiscetin to vWF and consequently of the bitiscetin-induced binding of vWF to platelets. Thus, our data support the hypothesis that an interaction between the A1 and A3 domains exists that may play a role in the function of vWF by regulating the ability of the A1 domain to bind to platelet GPIb.

Description: We have evaluated the performance of a new analyzer using high shear stress, the PFA-100 (Platelet Function Analyzer, Dade International, Massy, France), for screening of patients with von Willebrand disease (vWD). Whole citrated blood is aspirated through a capillary to the central aperture of a membrane coated with collagen and with a platelet agonist (either epinephrine or adenosine diphosphate [ADP]). The time required to obtain occlusion of the aperture by a platelet plug is defined as the closure time (CT). We studied 60 patients with different types of vWD and 96 normal subjects. Fourteen subjects with hemophilia and 15 patients with a platelet disorder were also analyzed. When omitting results from two patients with type 2N, the 58 other patients with type 1, type 2A, type 2B, type 3, or acquired vWD all exhibited an abnormal occlusion with collagen-ADP (sensitivity, 100%) and 56 of 58 had an abnormal CT with collagen-epinephrine (sensitivity, 96.5%). Only two patients with mild type 1 were not detected with collagen-epinephrine. In comparison, the bleeding time (BT) was normal in 20 patients: 17 with type 1, two with type 2A, and one with acquired vWD (sensitivity, 65.5%). The specificity of the PFA-100 was over 95% with both types of cartridges. Thus, the analyzer is well adapted to routine testing, as it has the advantages of simplicity and ease of execution, and demonstrates a high sensitivity, clearly superior to that of BT, for the screening of patients with vWD.