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J. E. Sadler - One of the best experts on this subject based on the ideXlab platform.
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Type IIb mutation His-505-->Asp implicates a new segment in the control of von Willebrand factor binding to platelet Glycoprotein Ib.
The Journal of biological chemistry, 1993Co-Authors: I Rabinowitz, Elodee A. Tuley, A M Randi, Kenneth S. Shindler, P K Rustagi, J. E. SadlerAbstract:Abstract Type IIb von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet Glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet Glycoprotein Ib. A new mutation was identified in a patient with type IIb von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhIbited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIb vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIb vWD mutations and implicates a new segment of vWF in the regulation of platelet Glycoprotein Ib binding.
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von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet Glycoprotein Ib.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: I Rabinowitz, Elodee A. Tuley, David J. Mancuso, A M Randi, B G Firkin, M A Howard, J. E. SadlerAbstract:Abstract von Willebrand factor (vWF) is a multimeric Glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet Glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antIbiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed Glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to Glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhIbited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the Glycoprotein Ib binding function of vWF.
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Molecular basis of von Willebrand disease type IIb. Candidate mutations cluster in one disulfide loop between proposed platelet Glycoprotein Ib binding sequences.
The Journal of clinical investigation, 1991Co-Authors: A M Randi, I Rabinowitz, David J. Mancuso, Pier Mannuccio Mannucci, J. E. SadlerAbstract:Abstract Many variants of von Willebrand disease (vWD) with qualitatively abnormal von Willebrand factor (vWF) are recognized. In vWD type IIb, the abnormal protein displays enhanced affinity for a platelet vWF receptor, the Glycoprotein Ib-IX complex. 14 patients from 7 unrelated families with vWD type IIb were studied to determine the molecular basis for this phenotype. Specific oligonucleotide primers were used to amplify portions of vWF exon 28 encoding a domain that interacts with the platelet Glycoprotein Ib-IX complex. Candidate missense mutations were identified for all 14 patients by DNA sequencing, allele specific oligonucleotide hybridization, and restriction endonuclease digestion. These sequence changes occur in an 11 amino acid segment within a single disulfide loop bounded by Cys(509) and Cys(695). All of these sequence changes are C----T transitions within CG dinucleotides. Six patients from two unrelated families were heterozygous for the encoded sequence Arg(543)----Trp. Seven patients from four unrelated families were heterozygous for the encoded sequence Arg(545)----Cys; this sequence change appears to have occurred independently three times, once as a new spontaneous mutation. One patient with apparently sporadic vWD type IIb was heterozygous for the encoded sequence Val(553)----Met, and this appears to be a new mutation. None of these sequence changes was found in 100 normal alleles. These findings suggest that vWD type IIb may be caused by relatively few distinct mutations, that these mutations may cluster within a specific region of one disulfide loop in vWF domain A1, and that this region can modulate the affinity of vWF for the platelet Glycoprotein Ib-IX complex.
Robert I. Handin - One of the best experts on this subject based on the ideXlab platform.
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mapping the Glycoprotein Ib binding site in the von willebrand factor a1 domain
Journal of Biological Chemistry, 2000Co-Authors: Jonas Emsley, Miguel A. Cruz, Robert C Liddington, Thomas G Diacovo, Robert I. HandinAbstract:The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet Glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396–10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhIbit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations descrIbed here.
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Transient expression of recombinant Glycoprotein Ib alpha polypeptides in COS cells that inhIbit von Willebrand factor binding to the platelet Glycoprotein Ib/IX complex.
Thrombosis and haemostasis, 1992Co-Authors: Eefke J Petersen, Robert I. HandinAbstract:The cDNA encoding the Glycoprotein Ib alpha polypeptide has been expressed in COS cells. Transfection with full-length cDNA and a cDNA truncated at an internal XbaI site produced a recombinant polypeptide doublet with estimated molecular weights of 48 and 46 kDa (rGpIb alpha L318) which could be resolved into a single band of molecular weight 36 kDa following digestion with endoglycosidase F. A portion of the truncated polypeptide was retained in the endoplasmic reticulum of COS cells and slowly released. A second fraction was rapidly secreted into COS cell-conditioned medium and could be used for functional studies. Soluble rGpIb alpha L318 harvested from COS cell-conditioned medium inhIbited ristocetin-dependent binding of [125I]-vWF to fixed washed human platelets (IC50 20 nM). Binding was inhIbited by reduction and alkylation of "rGp1b alpha L318" suggesting the need for a critical disulfide bond to maintain biological activity of the recombinant polypeptide. We conclude that the recombinant polypeptides produced by transfection of GpIb alpha cDNA into heterologous cells are secreted, soluble, and can inhIbit vWF binding to platelet GpIb/IX.
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Functional analysis of a recombinant Glycoprotein Ib alpha polypeptide which inhIbits von Willebrand factor binding to the platelet Glycoprotein Ib-IX complex and to collagen.
The Journal of biological chemistry, 1992Co-Authors: Miguel A. Cruz, Eefke J Petersen, S M Turci, Robert I. HandinAbstract:Abstract By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the Glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhIbited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhIbited the binding of 125I-vWF to immobilized type I and III collagen. InhIbition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhIbition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhIbited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhIbits vWF interaction with two important substrates, platelet GpIb/IX and collagen.
I Rabinowitz - One of the best experts on this subject based on the ideXlab platform.
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Type IIb mutation His-505-->Asp implicates a new segment in the control of von Willebrand factor binding to platelet Glycoprotein Ib.
The Journal of biological chemistry, 1993Co-Authors: I Rabinowitz, Elodee A. Tuley, A M Randi, Kenneth S. Shindler, P K Rustagi, J. E. SadlerAbstract:Abstract Type IIb von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet Glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet Glycoprotein Ib. A new mutation was identified in a patient with type IIb von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhIbited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIb vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIb vWD mutations and implicates a new segment of vWF in the regulation of platelet Glycoprotein Ib binding.
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von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet Glycoprotein Ib.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: I Rabinowitz, Elodee A. Tuley, David J. Mancuso, A M Randi, B G Firkin, M A Howard, J. E. SadlerAbstract:Abstract von Willebrand factor (vWF) is a multimeric Glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet Glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antIbiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed Glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to Glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhIbited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the Glycoprotein Ib binding function of vWF.
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Molecular basis of von Willebrand disease type IIb. Candidate mutations cluster in one disulfide loop between proposed platelet Glycoprotein Ib binding sequences.
The Journal of clinical investigation, 1991Co-Authors: A M Randi, I Rabinowitz, David J. Mancuso, Pier Mannuccio Mannucci, J. E. SadlerAbstract:Abstract Many variants of von Willebrand disease (vWD) with qualitatively abnormal von Willebrand factor (vWF) are recognized. In vWD type IIb, the abnormal protein displays enhanced affinity for a platelet vWF receptor, the Glycoprotein Ib-IX complex. 14 patients from 7 unrelated families with vWD type IIb were studied to determine the molecular basis for this phenotype. Specific oligonucleotide primers were used to amplify portions of vWF exon 28 encoding a domain that interacts with the platelet Glycoprotein Ib-IX complex. Candidate missense mutations were identified for all 14 patients by DNA sequencing, allele specific oligonucleotide hybridization, and restriction endonuclease digestion. These sequence changes occur in an 11 amino acid segment within a single disulfide loop bounded by Cys(509) and Cys(695). All of these sequence changes are C----T transitions within CG dinucleotides. Six patients from two unrelated families were heterozygous for the encoded sequence Arg(543)----Trp. Seven patients from four unrelated families were heterozygous for the encoded sequence Arg(545)----Cys; this sequence change appears to have occurred independently three times, once as a new spontaneous mutation. One patient with apparently sporadic vWD type IIb was heterozygous for the encoded sequence Val(553)----Met, and this appears to be a new mutation. None of these sequence changes was found in 100 normal alleles. These findings suggest that vWD type IIb may be caused by relatively few distinct mutations, that these mutations may cluster within a specific region of one disulfide loop in vWF domain A1, and that this region can modulate the affinity of vWF for the platelet Glycoprotein Ib-IX complex.
A M Randi - One of the best experts on this subject based on the ideXlab platform.
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Type IIb mutation His-505-->Asp implicates a new segment in the control of von Willebrand factor binding to platelet Glycoprotein Ib.
The Journal of biological chemistry, 1993Co-Authors: I Rabinowitz, Elodee A. Tuley, A M Randi, Kenneth S. Shindler, P K Rustagi, J. E. SadlerAbstract:Abstract Type IIb von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet Glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet Glycoprotein Ib. A new mutation was identified in a patient with type IIb von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhIbited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIb vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIb vWD mutations and implicates a new segment of vWF in the regulation of platelet Glycoprotein Ib binding.
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von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet Glycoprotein Ib.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: I Rabinowitz, Elodee A. Tuley, David J. Mancuso, A M Randi, B G Firkin, M A Howard, J. E. SadlerAbstract:Abstract von Willebrand factor (vWF) is a multimeric Glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet Glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antIbiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed Glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to Glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhIbited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the Glycoprotein Ib binding function of vWF.
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Molecular basis of von Willebrand disease type IIb. Candidate mutations cluster in one disulfide loop between proposed platelet Glycoprotein Ib binding sequences.
The Journal of clinical investigation, 1991Co-Authors: A M Randi, I Rabinowitz, David J. Mancuso, Pier Mannuccio Mannucci, J. E. SadlerAbstract:Abstract Many variants of von Willebrand disease (vWD) with qualitatively abnormal von Willebrand factor (vWF) are recognized. In vWD type IIb, the abnormal protein displays enhanced affinity for a platelet vWF receptor, the Glycoprotein Ib-IX complex. 14 patients from 7 unrelated families with vWD type IIb were studied to determine the molecular basis for this phenotype. Specific oligonucleotide primers were used to amplify portions of vWF exon 28 encoding a domain that interacts with the platelet Glycoprotein Ib-IX complex. Candidate missense mutations were identified for all 14 patients by DNA sequencing, allele specific oligonucleotide hybridization, and restriction endonuclease digestion. These sequence changes occur in an 11 amino acid segment within a single disulfide loop bounded by Cys(509) and Cys(695). All of these sequence changes are C----T transitions within CG dinucleotides. Six patients from two unrelated families were heterozygous for the encoded sequence Arg(543)----Trp. Seven patients from four unrelated families were heterozygous for the encoded sequence Arg(545)----Cys; this sequence change appears to have occurred independently three times, once as a new spontaneous mutation. One patient with apparently sporadic vWD type IIb was heterozygous for the encoded sequence Val(553)----Met, and this appears to be a new mutation. None of these sequence changes was found in 100 normal alleles. These findings suggest that vWD type IIb may be caused by relatively few distinct mutations, that these mutations may cluster within a specific region of one disulfide loop in vWF domain A1, and that this region can modulate the affinity of vWF for the platelet Glycoprotein Ib-IX complex.
Elodee A. Tuley - One of the best experts on this subject based on the ideXlab platform.
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Type IIb mutation His-505-->Asp implicates a new segment in the control of von Willebrand factor binding to platelet Glycoprotein Ib.
The Journal of biological chemistry, 1993Co-Authors: I Rabinowitz, Elodee A. Tuley, A M Randi, Kenneth S. Shindler, P K Rustagi, J. E. SadlerAbstract:Abstract Type IIb von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet Glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet Glycoprotein Ib. A new mutation was identified in a patient with type IIb von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhIbited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIb vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIb vWD mutations and implicates a new segment of vWF in the regulation of platelet Glycoprotein Ib binding.
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von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet Glycoprotein Ib.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: I Rabinowitz, Elodee A. Tuley, David J. Mancuso, A M Randi, B G Firkin, M A Howard, J. E. SadlerAbstract:Abstract von Willebrand factor (vWF) is a multimeric Glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet Glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antIbiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed Glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to Glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhIbited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the Glycoprotein Ib binding function of vWF.