Effect of von Willebrand Factor on the Pharmacokinetics of Recombinant Human Platelet Glycoprotein Ibα-Immunoglobulin G1 Chimeric Proteins (original) (raw)
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GUINEA PIG BLOOD: A MODEL FOR THE PHARMACOLOGIC MODULATION OF THE GPIb/IX-VWF AXIS
Thrombosis Research, 1996
Antithrombotic activity of two recombinant GPIb-binding fragments of vWF, RG12986 (residues 445-733), and VCL (residues 504-728), were assessed m an ex vivo capillary perfusion chamber exposing human type 111collagen to native nonanticoagulated guinea pig blood. Platelet adhesion and thrombus formation were evaluated by computer assisted morphometry for two shear rates (650 and 1800 s-') and for two perfusion times (1.5 and 4 rein). At 1800 s-' and 4 min of perfusion, platelet adhesion decreased from 63~7°/0for control, to 46 i 4°/0 for 20 mglkg RG12986, and to 29 t 5Y0for 4 mglkg VCL, and the mean thrombus height dropped from 40~8 Km to 24~3 um and 7.5~1 km, respectively. The two doses did not change bleeding time values. Our results suggest that guinea pig blood and the circular perfusion chamber represent a good model for the evaluation of limited amount of GPIb/IX-vWF axis inhibitors. Von Willebrand factor (vWF) Ma multimeric glycoprotein synthesized by endothelial cells and megakaryocytes (1-4). It plays a key role in the thrombotic process (i.e. platelet/ thrombogenic surface interaction and platelet/platelet interaction) which develops at the site of vascular wall damage at high shear rates (5-7). Platelet adhesion at such rates is made possible by a spatial confirmational change of vWF once it is bound to a thrombogenic surface which then binds directly and specifically to the platelet membrane glycoprotein (GP)IbflX complex (5).
Blood, 2001
Phenotype changes resulting in high-affinity binding of von Willebrand factor http://bloodjournal.hematologylibrary.org/cgi/content/full/98/6/1812 Updated information and services can be found at: (2435 articles) Hemostasis, Thrombosis, and Vascular Biology (790 articles) Cell Adhesion and Motility collections: Blood Articles on similar topics may be found in the following http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub\_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/subscriptions/index.dtl Information about subscriptions and ASH membership may be found online at: . Hematology; all rights reserved To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIb␣ polypeptide chain have been identified in these patients-a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentra-tions of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gainof-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIb␣ chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIb␣, thereby influencing the stability of the GPIb␣-vWf interaction. (Blood. 2001; 98:1812-1818)
Biochemical and Biophysical Research Communications, 1997
reside within a limited area of the amino-terminal half von Willebrand factor (vWf) is known to interact of the molecule called the A1 loop domain , with the two b 3 integrins, a IIb b 3 and a v b 3 , in an RGDwhile that for the a IIb b 3 receptor has been localized to dependent manner. We characterized a novel murine an area containing an Arg-Gly-Asp (RGD) sequence in monoclonal antibody to human vWf, GUR76-23, the carboxy portion of the ligand . vWf is which recognized a site within the carboxy-terminal also known to serve as a substrate for the other b 3 half of the molecule containing the RGD sequence. integrin (a v b 3 ) on vascular endothelial cells This antibody inhibited high shear-induced platelet which, previously recognized as a vitronectin receptor, aggregation and blocked adhesion of ADP plus epihas been shown to have an important role in angiogenenephrine-stimulated platelets to vWf, indicating that sis or vascular wall remodeling (13). The binding site it interferes with the interaction with a IIb b 3 . Unlike for a v b 3 has also been localized to the region containing antibodies against the RGD site, however, the antithe RGD sequence on the carboxy half of vWf. However, body was without effect on adhesion of cultured huit remains unknown whether exactly the same RGD man umbilical vein endothelial cells to vWf, a phesite is responsible for receptor binding of the two b 3 nomenon known to involve the interaction with a v b 3 .
Journal of Clinical Investigation, 1993
We developed a monoclonal antibody, 1C1E7, against vWf that increases ristocetin-induced platelet aggregation in a dose-dependent manner and lowers the threshold concentration of ristocetin needed to obtain a full aggregatory response. The platelet aggregatory effect of asialo vWf (ASvWf) also is enhanced by 1CIE7, in the presence or absence of glycoprotein (GP) Ilb/IlIa receptor antagonism. In the presence of ristocetin, both intact 1ClE7 and its Fab fragments enhance specific binding of '25I-vWf to platelets. With 1ClE7, the intermediate and higher molecular weight multimers of vWf are preferentially bound to both GP lb and GP IIb/IIIa. Thrombin-induced 125I-vWf binding to GP IIb/Illa also is increased by 1ClE7. Maximal binding of 1C1E7 to vWf corresponds to 0.97 mol/mol vWf monomer with a Kd of 4.7 x 10-10 M. 1ClE7 reacts with a 34 /36-kD tryptic fragment (III-T4) and a 34-kD plasmic fragment (P34), which localizes the epitope between amino acid residues 1 and 272; this was confirmed by NH2-terminal amino acid sequencing. Finally, platelet aggregation by ASvWf was associated with a sharp rise in intracellular Ca2+ only in the presence of 1C1E7. An antibody-mediated conformational change of vWf may result in an improved presentation of the GP Iband GP IIb/ IIIa-binding domains of mainly the larger multimers; the increased density of vWf on the platelet surface leads to platelet activation. The antibody may thus recognize a domain of relevance for vWf physiology. (J. Clin. Invest. 1993. 91:273-282.)
Journal of Thrombosis and Haemostasis, 2007
Summary. Background: von Willebrand factor (VWF) does not interact with circulating platelets unless it is induced to expose the binding site for platelet glycoprotein (GP)Ibα in the A1 domain by high shear stress, immobilization, and/or a modulator. Previous studies have implied indirectly that the A2 domain may be involved in regulating A1–GPIbα binding. Objective and methods: Because the relationship between the A1 and A2 domains has not been defined, we have investigated the effect of the A2 domain on the binding activity of the A1 domain using recombinant A domain polypeptides, multimeric VWF, and monoclonal antibodies (mAb). Results: The A2 domain polypeptide bound specifically to the immobilized A1 domain polypeptide or full-length VWF, with half-maximal binding being obtained at 60 or 168 nm, respectively. This A1–A2 interaction was inhibited by mAb against the A2 or A1 domain and by the A1 domain polypeptide. The A2 domain polypeptide effectively blocked GPIbα-mediated platelet adhesion under high flow conditions. The A2 domain polypeptide specifically recognizes the GPIbα-binding conformation in the A1 domain, as it only interacted with VWF activated by the modulator ristocetin or immobilized VWF. Furthermore, in contrast to plasma VWF, the ultra-large (UL)VWF multimers or a recombinant VWF–A1A2A3 polypeptide containing a gain-of-function mutation (R1308 L) of type 2B von Willebrand disease bound to the A2 domain polypeptide without the need for ristocetin. Conclusions: The recombinant A2 domain polypeptide specifically binds to the active conformation of the A1 domain in VWF and effectively blocks the interaction with platelet GPIbα under high-flow conditions.
Journal of Clinical …, 1993
We developed a monoclonal antibody, 1C1E7, against vWf that increases ristocetin-induced platelet aggregation in a dose-dependent manner and lowers the threshold concentration of ristocetin needed to obtain a full aggregatory response. The platelet aggregatory effect of asialo vWf (ASvWf) also is enhanced by 1CIE7, in the presence or absence of glycoprotein (GP) Ilb/IlIa receptor antagonism. In the presence of ristocetin, both intact 1ClE7 and its Fab fragments enhance specific binding of '25I-vWf to platelets. With 1ClE7, the intermediate and higher molecular weight multimers of vWf are preferentially bound to both GP lb and GP IIb/IIIa. Thrombin-induced 125I-vWf binding to GP IIb/Illa also is increased by 1ClE7. Maximal binding of 1C1E7 to vWf corresponds to 0.97 mol/mol vWf monomer with a Kd of 4.7 x 10-10 M. 1ClE7 reacts with a 34 /36-kD tryptic fragment (III-T4) and a 34-kD plasmic fragment (P34), which localizes the epitope between amino acid residues 1 and 272; this was confirmed by NH2-terminal amino acid sequencing. Finally, platelet aggregation by ASvWf was associated with a sharp rise in intracellular Ca2+ only in the presence of 1C1E7. An antibody-mediated conformational change of vWf may result in an improved presentation of the GP Iband GP IIb/ IIIa-binding domains of mainly the larger multimers; the increased density of vWf on the platelet surface leads to platelet activation. The antibody may thus recognize a domain of relevance for vWf physiology. (J. Clin. Invest. 1993. 91:273-282.)
Blood, 1999
Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo-and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.