scholarly journals Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2322-2328 ◽  
Author(s):  
KA Cooney ◽  
D Ginsburg
Keyword(s):  
Von Willebrand Factor ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Surface Glycoprotein ◽  
Platelet Surface ◽  
Disease Mutations ◽  
A Domains ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete “on” or “off” conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

scholarly journals The defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor

Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 1013-1021 ◽  
Author(s):  
PA Kroner ◽  
PA Foster ◽  
SA Fahs ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Solid Phase ◽  
Disease Patient ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Mutant Proteins ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
Willebrand Factor

In this report we describe the further investigation of the von Willebrand factor (vWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by one-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing recombinant FVIII. Sequence analysis of the FVIII-binding domain encoded by the vWF mRNA of the affected individual identified mutations in both vWF alleles. In allele A, the mutations C2344T and T2451A result in the substitution of Trp for Arg19 (R19W) and of G1n for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported polymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding domain (delta G2515) that results in the premature termination of translation. Analysis of the binding of FVIII by full- length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q substitutions are the cause of the defective vWF/FVIII interaction in this patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins are recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12, and 31H3, which block the binding of FVIII by vWF, indicating that amino acids Arg19, Thr26, and His54 are not critical residues in the epitopes of these antibodies.

Alteration in GPIIb/IIIa Binding of VWD-Associated von Willebrand Factor Variants with C-Terminal Missense Mutations

2019 ◽  
Vol 119 (07) ◽  
pp. 1102-1111
Author(s):  
Gesa König ◽  
Tobias Obser ◽  
Olivier Marggraf ◽  
Sonja Schneppenheim ◽  
Ulrich Budde ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Active Form ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Missense Mutations ◽  
Primary Hemostasis ◽  
Platelet Receptor ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThe platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and β3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF–GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.

scholarly journals Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1738-1743 ◽  
Author(s):  
AS Ribba ◽  
JM Lavergne ◽  
BR Bahnak ◽  
A Derlon ◽  
G Pietu ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Functional Domains ◽  
Gene Coding ◽  
Gradient Gel Electrophoresis ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5′ end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.

scholarly journals Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1787-1791 ◽  
Author(s):  
SD Russell ◽  
GJ Roth
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Single Amino Acid ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Platelet Surface ◽  
Normal Individuals ◽  
Alpha Gene ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.

On the Mechanism of Plasmin-induced Aggregation of Human Platelets: Implication of Secreted von Willebrand Factor

1998 ◽  
Vol 79 (06) ◽  
pp. 1191-1198 ◽  
Author(s):  
S. Rabhi-Sabile ◽  
C. de Romeuf ◽  
D. Pidard
Keyword(s):  
Von Willebrand Factor ◽  
Shape Change ◽  
Human Platelets ◽  
Lag Phase ◽  
Distribution Analysis ◽  
Platelet Surface ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

SummaryPlasmin triggers a strong metabolic activation in human platelets, leading to shape change and granule exocytosis. However, its capacity to induce cell aggregation remains discussed and, when observed, this aggregation is preceded by a remarkable lag phase. We have thus investigated the effect of plasmin on the adhesive proteins which can be secreted by isolated platelets and mediate cell-to-cell interactions, but are also substrates for the enzyme. Immunoblot analysis of fibrinogen (Fg), thrombospondin-1 (TSP-1), fibronectin (Fn) and von Willebrand factor (vWf) was performed on extracts of platelets exposed under stirring to increasing concentrations of plasmin for up to 10 min at 37° C. Under conditions leading to formation of large aggregates, Fg, Fn and TSP-1 are extensively degraded concomitantly with their secretion, and readily lost from the surface of aggregated cells. Part of the monomers in the platelet vWf are cleaved during secretion into two main fragments with M r ≈180,000 and ≈145,000. However, multimer distribution analysis shows only a slight decrease in the very high molecular weight multimers, and most of the fragmented as well as intact vWf remains associated with the platelet surface when aggregation is maximal. That indeed vWf largely supports plasmin-induced aggregation is suggested by the observation that platelets from a patient with type 3 von Willebrand’s disease, who totally lacks vWf, show little aggregation in response to the enzyme. Finally, plasmin-induced aggregation can be totally inhibited by antagonists of the αIIbβ3 integrin. The present study thus indicates a major role for secreted vWf in platelet aggregation induced by plasmin, through its likely interaction with the multifunctional receptor αIIbβ3.Presented in part at the European Platelet Group Meeting, Erfurt, Germany, May 1996

scholarly journals Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2788-2794 ◽  
Author(s):  
T Englender ◽  
A Lattuada ◽  
PM Mannucci ◽  
JE Sadler ◽  
A Inbal
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Patient Characteristics ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Disease Mutations ◽  
Normal Transport ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Intracellular Proteolysis

Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90- 2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation.

Defect of Heparin Binding in Plasma and Recombinant von Willebrand Factor with Type 2 von Willebrand Disease Mutations

2001 ◽  
Vol 86 (12) ◽  
pp. 1459-1465 ◽  
Author(s):  
Ghassem Rastegar-Lari ◽  
Nadine Ajzenberg ◽  
Anne-Sophie Ribba ◽  
Valérie Vereycken-Holler ◽  
Paulette Legendre ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Three Dimensional ◽  
Von Willebrand Disease ◽  
Dimensional Structure ◽  
Heparin Binding ◽  
Disease Mutations ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe 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-A1 domain, 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.

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