Type 2B “Malmo/New York” Is Not So Rare among Italian Patients with von Willebrand Disease: The Role of P1266L/Q Mutations of the von Willebrand Factor Gene in Seven Cases.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 712-712
Author(s):  
Luciano Baronciani ◽  
Silvia La Marca ◽  
Giancarlo Castaman ◽  
Margherita Punzo ◽  
Rosanna Garavaglia ◽  
...  
Keyword(s):  
New York ◽  
Gene Conversion ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Large Population ◽  
Von Willebrand Disease ◽  
Nucleotide Substitutions ◽  
Nucleotide Mutation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: Type 2B von Willebrand disease (VWD) is due to a unique gain-of-function variant of von Willebrand factor (VWF) that spontaneously interacts with circulating platelets resulting in loss of VWF high molecular weight multimers (HMWM) in plasma and reduced platelet counts, in most cases. Diagnosis of 2B is based on increased ristocetin-induced platelet aggregation (RIPA) in patient’s platelet rich plasma (PRP). Type 2B “Malmo or New York” is caused by the mutation P1266L that is associated with increased RIPA, but with normal multimeric distribution in plasma and no thrombocytopenia. Aims: To determine the incidence and the molecular defects of this rare VWF variant among a large population of VWD patients regularly followed at two Northern Italian Hemophilia Centers. Methods: Criteria for VWD type 2B were those recommended by the ISTH-SSC-SC on VWF. RIPA and multimeric analysis were performed according to standardized methods. Bleeding severity score (BSS) was calculated in all patients enrolled in this study. Sequence analysis of the portion of exon 28 encoding for the A1 domain was performed to identify nucleotide substitutions in all cases where RIPA was <0.8 mg/ml. Results: 74 patients from 16 different families satisfied the criteria for VWD type 2B. Seven patients from 3 unrelated families clearly showed normal multimeric distribution in plasma and no thrombocytopenia also after stimuli. Clinical and laboratory parameters of the seven affected members are reported as mean values (ranges): BSS=2.5 (2–4); RIPA=0.56 (0.5–0.7) mg/ml; platelet count=255 x 10^9/L (172–353); VWF:RCo=46 U/dL (21–66); VWF:Ag=53 U/dL (30–67); FVIII=61 U/dL (54–76). Patients from family A showed five distinct substitutions (3686T>G, 3692A>C, 3735G>A, 3789G>A, 3797C>T), patient B1 two (3789G>A, 3797C>T) and patient from family C three (3692A>C, 3789G>A, 3797C>A). Two of these were silent, whereas the other caused the following amino acid changes: three in family A (V1229G, N1231T, P1266L), one in B1 (P1266L) and two in family C (N1231T, P1266Q). Mutations V1229G and N1231T have already been reported, whereas mutation P1266Q is novel. The substitutions found in families A and B clearly correspond to the pseudogene sequence, suggesting gene conversion into VWF gene. In family C, the nucleotide mutation causing P1266Q substitution is not consistent with this mechanism: moreover the patients do not show the substitution 3735G>A found in the pseudogene. Conclusion: We confirm that the substitution of proline 1266 with leucine appears to be the main cause of type 2B “Malmo/New York” phenotype while its replacements with a glutamine apparently results in lower VWF activities in family C. Based on these data, gene conversion appears to be more frequent than expected and capable to generate different set of mutations.

scholarly journals Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1804-1809 ◽  
Author(s):  
JL Miller ◽  
ZM Ruggeri ◽  
VA Lyle
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Normal Platelet ◽  
High Affinity Binding Site ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Formalin Fixed

Abstract The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

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 Identification of two mutations (Arg611Cys and Arg611His) in the A1 loop of von Willebrand factor (vWF) responsible for type 2 von Willebrand disease with decreased platelet-dependent function of vWF [see comments]

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1010-1018 ◽  
Author(s):  
L Hilbert ◽  
C Gaucher ◽  
C Mazurier
Keyword(s):  
Von Willebrand Factor ◽  
Functional Characterization ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
In Vitro Mutagenesis ◽  
Nucleotide Substitutions ◽  
Dependent Function ◽  
Von Willebrand ◽  
Willebrand Factor

We report the identification of von Willebrand factor (vWF) gene mutations within exon 28 occurring in three unrelated families with an infrequent form of type 2 von Willebrand disease (vWD). A C-->T transition and a G-->A transition, both at the codon for arginine 611 of the mature vWF subunit, were found. They result in either a cysteine or an histidine substitution, respectively. Patients were found to be heterozygous for these substitutions and the vWD was transmitted dominantly. These substitutions have been reproduced by in vitro mutagenesis of full-length cDNA of vWF and transiently expressed in Cos- 7 cells. The corresponding recombinant vWFs (rvWF) exhibited decreased expression and a significant decrease in the high molecular weight multimeric forms. In addition, ristocetin- and botrocetin-induced binding of mutated rvWFs to platelets were markedly decreased as compared with that for the wild-type rvWFs. Thus, the structural and functional characterization of both mutated rvWFs confirmed that the two nucleotide substitutions identified at position 611 of the mature subunit of vWF are real mutations. Although they are located in the A1 loop containing most of the type 2B mutations inducing increased affinity of vWF for platelet glycoprotein Ib, they are responsible for abnormal vWF with decreased platelet-dependent function.

scholarly journals A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2028-2033
Author(s):  
A Casonato ◽  
L De Marco ◽  
M Mazzucato ◽  
V De Angelis ◽  
D De Roia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Bleeding Tendency ◽  
Binding Studies ◽  
Spontaneous Platelet Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.

scholarly journals Identification of two mutations (Arg611Cys and Arg611His) in the A1 loop of von Willebrand factor (vWF) responsible for type 2 von Willebrand disease with decreased platelet-dependent function of vWF [see comments]

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1010-1018 ◽  
Author(s):  
L Hilbert ◽  
C Gaucher ◽  
C Mazurier
Keyword(s):  
Von Willebrand Factor ◽  
Functional Characterization ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
In Vitro Mutagenesis ◽  
Nucleotide Substitutions ◽  
Dependent Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We report the identification of von Willebrand factor (vWF) gene mutations within exon 28 occurring in three unrelated families with an infrequent form of type 2 von Willebrand disease (vWD). A C-->T transition and a G-->A transition, both at the codon for arginine 611 of the mature vWF subunit, were found. They result in either a cysteine or an histidine substitution, respectively. Patients were found to be heterozygous for these substitutions and the vWD was transmitted dominantly. These substitutions have been reproduced by in vitro mutagenesis of full-length cDNA of vWF and transiently expressed in Cos- 7 cells. The corresponding recombinant vWFs (rvWF) exhibited decreased expression and a significant decrease in the high molecular weight multimeric forms. In addition, ristocetin- and botrocetin-induced binding of mutated rvWFs to platelets were markedly decreased as compared with that for the wild-type rvWFs. Thus, the structural and functional characterization of both mutated rvWFs confirmed that the two nucleotide substitutions identified at position 611 of the mature subunit of vWF are real mutations. Although they are located in the A1 loop containing most of the type 2B mutations inducing increased affinity of vWF for platelet glycoprotein Ib, they are responsible for abnormal vWF with decreased platelet-dependent function.

scholarly journals Fibrin-Dependent Platelet Procoagulant Activity Requires GPIb Receptors and von Willebrand Factor

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 564-570 ◽  
Author(s):  
S. Béguin ◽  
R. Kumar ◽  
I. Keularts ◽  
U. Seligsohn ◽  
B.S. Coller ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Procoagulant Activity ◽  
Increased Risk ◽  
Coagulation Proteins ◽  
A Minor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by ≈ 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + vβ3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from vβ3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.

Homozygous gene conversion in von Willebrand factor gene as a cause of type 3 von Willebrand disease and predisposition to inhibitor development

Blood ◽  
2001 ◽  
Vol 98 (1) ◽  
pp. 248-250 ◽  
Author(s):  
Gurcharan K. Surdhar ◽  
Mohammad S. Enayat ◽  
Sarah Lawson ◽  
Michael D. Williams ◽  
Frank G. H. Hill
Keyword(s):  
Gene Conversion ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Factor Gene ◽  
Inhibitor Development ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

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

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 Cosegregation of von Willebrand factor gene polymorphisms and possible germinal mosaicism in type IIB von Willebrand disease

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1476-1483 ◽  
Author(s):  
EW Murray ◽  
AR Giles ◽  
PJ Bridge ◽  
IR Peake ◽  
DP Lillicrap
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Nucleotide Substitutions ◽  
Gene Encoding ◽  
Single Nucleotide ◽  
Unaffected Parent ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Germinal Mosaicism ◽  
Significant Linkage

Abstract Recent reports of the mutations resulting in von Willebrand disease (vWD) have indicated that some cases of type IIA vWD are caused by single nucleotide substitutions in the gene encoding von Willebrand factor (vWF). However, the molecular pathogenesis of type IIB vWD remains unresolved and, with the complex posttranslational processing required for fully functional vWF, the mutations responsible for this phenotype may occur at loci other than the vWF gene. This study has used six intragenic vWF polymorphisms to assess the linkage of type IIB vWD to this gene in three families (48 individuals). The results of these studies indicate that there is significant linkage between the vWF gene and the type IIB phenotype (logarithm of the odds ratio of 7.2 at theta = 0), suggesting that the mutations responsible for this disorder frequently occur at this locus. Results from one of these families indicates that the disorder has been transmitted from an unaffected parent to two children who have inherited the same vWF gene as seven unaffected siblings. This finding is suggestive of the presence of germinal mosaicism for the mutation in the father.

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