Proc. NatI. Acad. Sci. USA Vol. 89, pp. 9846-9849, October 1992 Medical Sciences
von Willebrand disease type B: A missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein lb IAN RABINOWITZ*t, ELODEE A. TULEY*, DAVID J. MANCUSO*t, ANNA M. RANDIt, BARRY G. FIRKIN§, MARGARET A. HOWARD§, AND J. EVAN SADLER*t¶II *Howard Hughes Medical Institute, and Departments of tMedicine and lBiochemistry and Molecular Biophysics, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO 63110; and §Department of Medicine, Monash Medical School, Box Hill Hospital, Box Hill, Victoria 3128, Australia
Communicated by Earl W. Davie, July 16, 1992
relationships of the interaction between vWF and platelet gplb. The molecular basis of vWD type B was studied by the sequencing of genomic DNA. The affected patient was heterozygous for a missense mutation in exon 28 that resulted in the Gly-561 -- Ser change within the proposed gplb binding domain. The corresponding mutant recombinant protein was expressed and shown to reproduce the functional characteristics of the patient's plasma vWF. These results 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 platelet gplb by different mechanisms.
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 lb binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetininduced, binding to glycoprotein lb. 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 ristocetininduced binding of rvWF(G561S) suggest that the primay defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein lb binding function of vWF.
MATERIALS AND METHODS The patient was described in detail in previous reports (6, 11). Genomic DNA was extracted from peripheral blood, DNA from exon 28 of the gene encoding vWF was amplified by the polymerase chain reaction (PCR) (12), and the amplified DNA fragments were subcloned and sequenced as described (13, 14). Allele-Specific Oligonucleotide Hybridization. Allelespecific oligonucleotide hybridization was performed as described (15). The vWF 17-nucleotide segments used for detection of the normal and mutant alleles began at position 3962 and were, respectively, ACC ACG ACG GCT CCC AC [wild type (wt)] and ACC ACG ACA GCT CCC AC with the
von Willebrand factor (vWF) is a multimeric plasma glycoprotein involved in platelet adhesion to the subendothelium (reviewed in ref. 1). This function depends upon the interaction of vWF with the platelet membrane glycoprotein lb (gplb) and with components of the vessel wall. The binding site for platelet gpIb has been localized within vWF to a tryptic fragment extending from residue Val-449 to Lys-728 (2). In vitro, the binding of vWF to gplb is induced by the antibiotic ristocetin (3) or by the snake venom protein botrocetin (4, 5). There is evidence that ristocetin and botrocetin mediate the binding of vWF to platelet gplb through different mechanisms (6-9). von Willebrand disease (vWD) is the most common inherited bleeding disorder (10). Most patients have a quantitative deficiency of vWF which is referred to as vWD type I, but -20%o produce a qualitatively abnormal protein and are said to have vWD type II. A patient with a variant of vWD type II, termed "type B," was previously reported whose plasma vWF exhibited normal botrocetin-induced binding but no ristocetin-induced binding to gpIb. The patient's plasma vWF contained a full range of multimers (6, 11). Therefore, vWF type B displayed an aberrant interaction with the gplb platelet receptor that seemed to be independent of multimeric structure. Identification ofthe molecular defect causing vWD type B could provide insight into the structure-function
G -- A mutation at 3970. Nucleotides of vWF cDNA are numbered from the ATG of the initiator codon. The final washings were performed at 50°C. Plasmid Constructs. Plasmid pSVHVWF1 (16) contains a full-length cDNA insert for normal human vWF cloned into the expression vector pSV7D (17). Plasmid pSVHG561S contains a G -+ A transition at nucleotide 3970, resulting in the substitution of serine for the normal glycine at amino acid position 561 of the mature vWF subunit and was derived from pSVHVWF1 as follows. A plasmid containing the sequence from the patient's mutant allele was digested with TthlllI and Kpn I, and the resulting 0.9-kilobase (kb) fragment (nucleotides 3829-4740 of vWF cDNA) was purified on GeneClean glass beads (Bio 101, La Jolla, CA). Similarly, plasmid pSVHVWF1 was digested with TthlllI and Kpn I, resulting in 0.9-kb and 10.5-kb fragments. The 10.5-kb fragAbbreviations: vWF, von Willebrand factor; rvWF, recombinant vWF; rvWF(G561S), rvWF with missense mutation Gly-561 -. Ser; gplb, glycoprotein Ib; vWD, von Willebrand disease; wt, wild type; mAb, monoclonal antibody; FFP, formalin-fixed platelets. tPresent address: The Blood Center of Southeastern Wisconsin, 1701 West Wisconsin Avenue, Milwaukee, WI 53233. To whom reprint requests should be addressed at: Howard Hughes Medical Institute, Washington University, 660 South Euclid, Box 8045, St. Louis, MO 63110.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9846
Medical Sciences: Rabinowitz Natl. Acad. Sci. USA 89 (1992) al.al.Proc. Rabinowitz etet Medical Sciences: ment was purified on GeneClean glass beads. The 0.9-kb fragment containing the mutation was then ligated with the 10.5-kb fragment of pSVHVWF1 and used to transform Eseherichia coli strain XL1-Blue (Stratagene). Clones containing the desired insert were selected by hybridization with allele-specific oligonucleotides as above. The DNA sequence of the 0.9-kb fragment in pSVHG561S was confirmed by sequencing. Expression of Recombinant vWF (rvWF). COS-7 cells were transiently transfected with plasmids pSVHVWF1 or pSVHG561S by using a DEAE-dextran method (18). Conditioned media containing normal rvWF(wt) or mutant rvWF(G561S) were prepared and concentrated as described (19). Protein Characterization. SDS/polyacrylamide gel electrophoresis (20) and SDS/agarose multimeric analysis (21) of recombinant vWF were performed as described (19). Anti-vWF Monoclonal Antibody (mAb). Anti-vWF mAb 33E12 (a gift from C. Mazurier, Lille, France) recognizes the SpII fragment of vWF and interacts with all sizes of vWF multimers (22). The mAb interacts with native and rvWF (both wt and G561S) in a similar way and does not inhibit ristocetin- or botrocetin-induced binding of vWF to platelets. Ristocetin-Induced Binding of vWF to Platelets. Binding of vWF to platelets was performed as described by Nishio et al. (23) with minor modifications. Anti-vWF 33E12 (100 pgg/ml) was radioiodinated by the Iodo-Gen method to 0.8-1.0 .t~i/ttg (1 .tkCi = 37 kBq) (24). Preparations of vWF from
concentrated conditioned media were diluted to 1.5 ttg/ml in 50 mM Tris chloride, pH 7.5/150 mM NaCl (TBS) containing 3% (wt/vol) bovine serum albumin. Formalin-fixed platelets (FFP) (Biodata, Hatboro, PA) were resuspended in TBS containing 0.35% bovine serum albumin to a concentration of -200,000 per p1. Ristocetin (Sigma) was dissolved in water (15 mg/ml) and diluted to various concentrations. Samples of vWF (60 /.d) were incubated with 0.6 pAl of 1251-labeled mAb for 1 hr at room temperature (RT); 400 ttl of FFP and 50 1.d of ristocetin (final concentration, 0-2 mg/ml) were added and incubated for 40 min at RT. Four hundred microliters of the mixture was layered on 500 1.d of TBS containing 20% (wt/vol) sucrose and 3% bovine serum albumin. The samples were centrifuged 5 min at 12,500 x g, and the platelet pellet was assayed for bound radioactivity. Conditioned medium from mock-transfected cells was used as a control, and radioactivity in the platelet pellet was
von Willebrand disease type B: A missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein lb IAN RABINOWITZ*t, ELODEE A. TULEY*, DAVID J. MANCUSO*t, ANNA M. RANDIt, BARRY G. FIRKIN§, MARGARET A. HOWARD§, AND J. EVAN SADLER*t¶II *Howard Hughes Medical Institute, and Departments of tMedicine and lBiochemistry and Molecular Biophysics, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO 63110; and §Department of Medicine, Monash Medical School, Box Hill Hospital, Box Hill, Victoria 3128, Australia
Communicated by Earl W. Davie, July 16, 1992
relationships of the interaction between vWF and platelet gplb. The molecular basis of vWD type B was studied by the sequencing of genomic DNA. The affected patient was heterozygous for a missense mutation in exon 28 that resulted in the Gly-561 -- Ser change within the proposed gplb binding domain. The corresponding mutant recombinant protein was expressed and shown to reproduce the functional characteristics of the patient's plasma vWF. These results 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 platelet gplb by different mechanisms.
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 lb binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetininduced, binding to glycoprotein lb. 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 ristocetininduced binding of rvWF(G561S) suggest that the primay defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein lb binding function of vWF.
MATERIALS AND METHODS The patient was described in detail in previous reports (6, 11). Genomic DNA was extracted from peripheral blood, DNA from exon 28 of the gene encoding vWF was amplified by the polymerase chain reaction (PCR) (12), and the amplified DNA fragments were subcloned and sequenced as described (13, 14). Allele-Specific Oligonucleotide Hybridization. Allelespecific oligonucleotide hybridization was performed as described (15). The vWF 17-nucleotide segments used for detection of the normal and mutant alleles began at position 3962 and were, respectively, ACC ACG ACG GCT CCC AC [wild type (wt)] and ACC ACG ACA GCT CCC AC with the
von Willebrand factor (vWF) is a multimeric plasma glycoprotein involved in platelet adhesion to the subendothelium (reviewed in ref. 1). This function depends upon the interaction of vWF with the platelet membrane glycoprotein lb (gplb) and with components of the vessel wall. The binding site for platelet gpIb has been localized within vWF to a tryptic fragment extending from residue Val-449 to Lys-728 (2). In vitro, the binding of vWF to gplb is induced by the antibiotic ristocetin (3) or by the snake venom protein botrocetin (4, 5). There is evidence that ristocetin and botrocetin mediate the binding of vWF to platelet gplb through different mechanisms (6-9). von Willebrand disease (vWD) is the most common inherited bleeding disorder (10). Most patients have a quantitative deficiency of vWF which is referred to as vWD type I, but -20%o produce a qualitatively abnormal protein and are said to have vWD type II. A patient with a variant of vWD type II, termed "type B," was previously reported whose plasma vWF exhibited normal botrocetin-induced binding but no ristocetin-induced binding to gpIb. The patient's plasma vWF contained a full range of multimers (6, 11). Therefore, vWF type B displayed an aberrant interaction with the gplb platelet receptor that seemed to be independent of multimeric structure. Identification ofthe molecular defect causing vWD type B could provide insight into the structure-function
G -- A mutation at 3970. Nucleotides of vWF cDNA are numbered from the ATG of the initiator codon. The final washings were performed at 50°C. Plasmid Constructs. Plasmid pSVHVWF1 (16) contains a full-length cDNA insert for normal human vWF cloned into the expression vector pSV7D (17). Plasmid pSVHG561S contains a G -+ A transition at nucleotide 3970, resulting in the substitution of serine for the normal glycine at amino acid position 561 of the mature vWF subunit and was derived from pSVHVWF1 as follows. A plasmid containing the sequence from the patient's mutant allele was digested with TthlllI and Kpn I, and the resulting 0.9-kilobase (kb) fragment (nucleotides 3829-4740 of vWF cDNA) was purified on GeneClean glass beads (Bio 101, La Jolla, CA). Similarly, plasmid pSVHVWF1 was digested with TthlllI and Kpn I, resulting in 0.9-kb and 10.5-kb fragments. The 10.5-kb fragAbbreviations: vWF, von Willebrand factor; rvWF, recombinant vWF; rvWF(G561S), rvWF with missense mutation Gly-561 -. Ser; gplb, glycoprotein Ib; vWD, von Willebrand disease; wt, wild type; mAb, monoclonal antibody; FFP, formalin-fixed platelets. tPresent address: The Blood Center of Southeastern Wisconsin, 1701 West Wisconsin Avenue, Milwaukee, WI 53233. To whom reprint requests should be addressed at: Howard Hughes Medical Institute, Washington University, 660 South Euclid, Box 8045, St. Louis, MO 63110.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
9846
Medical Sciences: Rabinowitz Natl. Acad. Sci. USA 89 (1992) al.al.Proc. Rabinowitz etet Medical Sciences: ment was purified on GeneClean glass beads. The 0.9-kb fragment containing the mutation was then ligated with the 10.5-kb fragment of pSVHVWF1 and used to transform Eseherichia coli strain XL1-Blue (Stratagene). Clones containing the desired insert were selected by hybridization with allele-specific oligonucleotides as above. The DNA sequence of the 0.9-kb fragment in pSVHG561S was confirmed by sequencing. Expression of Recombinant vWF (rvWF). COS-7 cells were transiently transfected with plasmids pSVHVWF1 or pSVHG561S by using a DEAE-dextran method (18). Conditioned media containing normal rvWF(wt) or mutant rvWF(G561S) were prepared and concentrated as described (19). Protein Characterization. SDS/polyacrylamide gel electrophoresis (20) and SDS/agarose multimeric analysis (21) of recombinant vWF were performed as described (19). Anti-vWF Monoclonal Antibody (mAb). Anti-vWF mAb 33E12 (a gift from C. Mazurier, Lille, France) recognizes the SpII fragment of vWF and interacts with all sizes of vWF multimers (22). The mAb interacts with native and rvWF (both wt and G561S) in a similar way and does not inhibit ristocetin- or botrocetin-induced binding of vWF to platelets. Ristocetin-Induced Binding of vWF to Platelets. Binding of vWF to platelets was performed as described by Nishio et al. (23) with minor modifications. Anti-vWF 33E12 (100 pgg/ml) was radioiodinated by the Iodo-Gen method to 0.8-1.0 .t~i/ttg (1 .tkCi = 37 kBq) (24). Preparations of vWF from
concentrated conditioned media were diluted to 1.5 ttg/ml in 50 mM Tris chloride, pH 7.5/150 mM NaCl (TBS) containing 3% (wt/vol) bovine serum albumin. Formalin-fixed platelets (FFP) (Biodata, Hatboro, PA) were resuspended in TBS containing 0.35% bovine serum albumin to a concentration of -200,000 per p1. Ristocetin (Sigma) was dissolved in water (15 mg/ml) and diluted to various concentrations. Samples of vWF (60 /.d) were incubated with 0.6 pAl of 1251-labeled mAb for 1 hr at room temperature (RT); 400 ttl of FFP and 50 1.d of ristocetin (final concentration, 0-2 mg/ml) were added and incubated for 40 min at RT. Four hundred microliters of the mixture was layered on 500 1.d of TBS containing 20% (wt/vol) sucrose and 3% bovine serum albumin. The samples were centrifuged 5 min at 12,500 x g, and the platelet pellet was assayed for bound radioactivity. Conditioned medium from mock-transfected cells was used as a control, and radioactivity in the platelet pellet was
