TWO-DIMENSIONAL MULTIMERIC ANALYSIS OF PLASMA AND PLATELET VON WILLEBRAND FACTOR (VWF) WITH IDENTIFICATION OF PLATELET VWF FRAGMENTS EXPRESSING A NEO-ANTIGEN

SDS-agarose electrophoresis of von Willebrand factor (vWF) was followed by reduction, second dimension SDS-polyacrylamide gel electrophoresis and immunoblotting with monoclonal anti-vWF antibodies. The multiple bands in each multimer of plasma vWF from normal and IIA von Willebrand disease (vWD) patients were shown to contain varying proportions of the intact 225 kDa vWF subunit and fragments of 189, 176, and 140 kDa. Only one relatively minor band in each multimer was composed entirely of the intact 225 kDa subunit. Repeating bands in successively larger multimers up to the thirteenth, exhibited similar compositions, whereas the largest multimers contained only the intact 225 kDa subunit. Thus the complex multimeric pattern of plasma vWF is the result, at least in part, of proteolytic degradation, and smaller multimers may derive from proteolytic degradation of larger species. In contrast, none of the fragments present in plasma vWF were seen in the vWF derived from platelets. Rather, fragments of 172 and 182 kDa were present in the smallest one or two multimers, whereas the larger multimers contained only the intact subunit. The fragments of platelet vWF reacted only with one monoclonal antibody (K14) of the 80 tested. This antibody did not react with unreduced plasma vWF nor with the unreduced fragments generated by Staphylococcus aureus V8 protease digestion of plasma vWF and reacted very poorly with reduced intact vWF subunit. Thus, the monoclonal antibody K14 recognized a neo-antigenic epitope expressed on at least two fragments of normal platelet, but not plasma, vWF.

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

Blood ◽

10.1182/blood.v70.6.1804.1804 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1804-1809 ◽

Cited By ~ 11

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.

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

Blood ◽

10.1182/blood.v70.6.1804.bloodjournal7061804 ◽

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

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.

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PRODUCTION OF ANTIBODIES TO HUMAN VON WILLEBRAND FACTOR IN LAYING HENS. ISOLATION OF IMMUNOGLOBULINS AND APPLICATIONS TO THE DETECTION OF MOLECULAR DEFECTS OF VON WILLEBRAND FACTOR

10.1055/s-0038-1644084 ◽

1987 ◽

Author(s):

F Toti ◽

A Stierlé ◽

M L Wiesel ◽

A Schwartz ◽

J M Freyssinet ◽

...

Keyword(s):

Von Willebrand Factor ◽

Laying Hens ◽

Protein A ◽

Von Willebrand Disease ◽

Primary Hemostasis ◽

Normal Platelet ◽

Electrophoretic Patterns ◽

Egg Yolks ◽

Von Willebrand ◽

Willebrand Factor

Von Willebrand disease (vWD) is an inherited disorder of primary hemostasis caused by deficiency or structural abnormalities of von Willebrand factor (vWF). VWF circulates in plasma and is also present in platelets. Plasma vWF, the carrier protein for factor VIII, is a large multimeric glycoprotein composed of identical subunits linked by disulfide bridges. Plasma and platelet vWF display distinct multimeric electrophoretic patterns. The different vWD subtypes can be classified either by the determination of vWFantigen (vWFíAg) and/or by multimer distribution. Antibodies to human vWF were raised in laying hens by intramuscular injections of purified human vWF. Immunoglobulins were isolated from egg yolks by selective polyethylene glycol and ammonium sulfate precipitations. These antibodies appeared to be monospecific, as they did not react with the plasma proteins of a patient with severe vWD. The pullets received weekly 50 μg vWF for 4 weeks and then had monthly injections. The antibodies occurred as early as the third injection, the yield being 300 to 500 mg of immunoglobulin per week (6-7 eggs). The titre could be constant over periods greater than 1 year. These immunoglobulins to vWF were tested in vWFíAg electroimmunoassays and for the multimer analysis of plasma and platelet vWF by electrophoresis and immunoblotting techniques. In no case could a difference be detected between assays performed with rabbit monospecific antiserum or with yolk immunoglobulins to human vWF. Ten to 12 multimers could be revealed for normal plasma vWF and up to 12 to 14 bands for normal platelet vWF (1.7% agarose). In the case of vWD, the electrophoresis patterns were identical with both antibodies. Thus, antibodies to vWF raised in laying hens are a suitable tool to detect and to characterize vWD. Although they do not interact with protein A, yolk antibodies are certainly advantageous to produce, as they do not contain IgM or IgA. Immunoglobulin fractions can contain up to 10 % of specific antibodies. Since they are available in larger quantities and are easy to isolate, larger homogeneous batches of antibodies can be obtained. This method may easily be applied to develop antibodies to a variety of antigens.

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A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

Blood ◽

10.1182/blood.v92.12.4663 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4663-4670 ◽

Cited By ~ 28

Author(s):

S. Jorieux ◽

C. Gaucher ◽

J. Goudemand ◽

C. Mazurier

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Stop Codon ◽

Novel Mutation ◽

Von Willebrand Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Sequencing Analysis ◽

Normal Platelet ◽

Von Willebrand ◽

Willebrand Factor

Abstract In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient’s vWF gene showed, at heterozygous state, a G→A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C→T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient’s vWF. The monoclonal antibody 31H3 against D’ domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D’ domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

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Reduced von Willebrand factor survival in type Vicenza von Willebrand disease

Blood ◽

10.1182/blood.v99.1.180 ◽

2002 ◽

Vol 99 (1) ◽

pp. 180-184 ◽

Cited By ~ 95

Author(s):

Alessandra Casonato ◽

Elena Pontara ◽

Francesca Sartorello ◽

Maria Grazia Cattini ◽

Maria Teresa Sartori ◽

...

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Bleeding Tendency ◽

Normal Platelet ◽

Cofactor Activity ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor ◽

Ristocetin Cofactor Activity ◽

Original Type

Type Vicenza variant of von Willebrand disease (VWD) is characterized by a low plasma von Willebrand factor (VWF) level and supranormal VWF multimers. Two candidate mutations, G2470A and G3864A at exons 17 and 27, respectively, of the VWF gene were recently reported to be present in this disorder. Four additional families, originating from northeast Italy, with both mutations of type Vicenza VWD are now described. Like the original type Vicenza subjects, they showed a mild bleeding tendency and a significant decrease in plasma VWF antigen level and ristocetin cofactor activity but normal platelet VWF content. Unlike the original patients, ristocetin-induced platelet aggregation was found to be normal. Larger than normal VWF multimers were also demonstrated in the plasma. Desmopressin (DDAVP) administration increased factor VIII (FVIII) and VWF plasma levels, with the appearance of even larger multimers. However, these forms, and all VWF oligomers, disappeared rapidly from the circulation. The half-life of VWF antigen release and of elimination was significantly shorter than that in healthy counterparts, so that at 4 hours after DDAVP administration, VWF antigen levels were close to baseline. Similar behavior was demonstrated by VWF ristocetin cofactor activity and FVIII. According to these findings, it is presumed that the low plasma VWF levels of type Vicenza VWD are mainly attributed to reduced survival of the VWF molecule, which, on the other hand, is normally synthesized. In addition, because normal VWF-platelet GPIb interaction was observed before or after DDAVP administration, it is proposed that type Vicenza VWD not be considered a 2M subtype.

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A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

Blood ◽

10.1182/blood.v92.12.4663.424k06_4663_4670 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4663-4670 ◽

Cited By ~ 1

Author(s):

S. Jorieux ◽

C. Gaucher ◽

J. Goudemand ◽

C. Mazurier

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Stop Codon ◽

Novel Mutation ◽

Von Willebrand Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Sequencing Analysis ◽

Normal Platelet ◽

Von Willebrand ◽

Willebrand Factor

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient’s vWF gene showed, at heterozygous state, a G→A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C→T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient’s vWF. The monoclonal antibody 31H3 against D’ domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D’ domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

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Heterogeneity of type I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor

Blood ◽

10.1182/blood.v66.4.796.bloodjournal664796 ◽

1985 ◽

Vol 66 (4) ◽

pp. 796-802 ◽

Cited By ~ 1

Author(s):

PM Mannucci ◽

R Lombardi ◽

R Bader ◽

L Vianello ◽

AB Federici ◽

...

Keyword(s):

Von Willebrand Factor ◽

Plasma Concentrations ◽

Sodium Dodecyl ◽

Von Willebrand Disease ◽

Type I ◽

Normal Platelet ◽

Complete Set ◽

Von Willebrand ◽

Willebrand Factor ◽

Factor Antigen

Type I von Willebrand disease (vWD) is characterized by equally low plasma concentrations of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RiCof) and by the presence of all vWF multimers in sodium dodecyl sulfate (SDS)-agarose gel electrophoresis. For 17 patients (13 kindreds) diagnosed with these criteria, we have studied the platelet contents of vWF:Ag and RiCof and the changes of these in plasma after DDAVP infusion. Platelet vWF:Ag and RiCof were normal in four kindreds (called “platelet normal” subgroup); following 1-deamino- 8-D-arginine vasopressin; plasma vWF:Ag, RiCof and the bleeding time (BT) became normal. In six kindreds, platelet vWF:Ag and RiCof were equally low (platelet low); after DDAVP, plasma vWF:Ag and RiCof remained low, and the BT was prolonged. In three additional kindreds, platelets contained normal concentrations of vWF:Ag, but RiCof was very low (platelet discordant); even though a complete set of multimers was found in plasma and platelets, there was a relatively small amount of large multimers. After DDAVP, plasma vWF:Ag became normal, but RiCof remained low and the BT was very prolonged. These findings demonstrated that there can be an abnormal vWF (RiCof less than vWF:Ag) even in type I vWD, coexisting with a complete set of vWF multimers (platelet discordant); that the abnormal vWF can be shown more clearly in platelets than in plasma or else in plasma after DDAVP infusion; and that DDAVP normalizes the BT only in those patients with normal platelet levels of both vWF:Ag and RiCof (platelet normal).

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Heterogeneity of type I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor

Blood ◽

10.1182/blood.v66.4.796.796 ◽

1985 ◽

Vol 66 (4) ◽

pp. 796-802 ◽

Cited By ~ 121

Author(s):

PM Mannucci ◽

R Lombardi ◽

R Bader ◽

L Vianello ◽

AB Federici ◽

...

Keyword(s):

Von Willebrand Factor ◽

Plasma Concentrations ◽

Sodium Dodecyl ◽

Von Willebrand Disease ◽

Type I ◽

Normal Platelet ◽

Complete Set ◽

Von Willebrand ◽

Willebrand Factor ◽

Factor Antigen

Abstract Type I von Willebrand disease (vWD) is characterized by equally low plasma concentrations of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RiCof) and by the presence of all vWF multimers in sodium dodecyl sulfate (SDS)-agarose gel electrophoresis. For 17 patients (13 kindreds) diagnosed with these criteria, we have studied the platelet contents of vWF:Ag and RiCof and the changes of these in plasma after DDAVP infusion. Platelet vWF:Ag and RiCof were normal in four kindreds (called “platelet normal” subgroup); following 1-deamino- 8-D-arginine vasopressin; plasma vWF:Ag, RiCof and the bleeding time (BT) became normal. In six kindreds, platelet vWF:Ag and RiCof were equally low (platelet low); after DDAVP, plasma vWF:Ag and RiCof remained low, and the BT was prolonged. In three additional kindreds, platelets contained normal concentrations of vWF:Ag, but RiCof was very low (platelet discordant); even though a complete set of multimers was found in plasma and platelets, there was a relatively small amount of large multimers. After DDAVP, plasma vWF:Ag became normal, but RiCof remained low and the BT was very prolonged. These findings demonstrated that there can be an abnormal vWF (RiCof less than vWF:Ag) even in type I vWD, coexisting with a complete set of vWF multimers (platelet discordant); that the abnormal vWF can be shown more clearly in platelets than in plasma or else in plasma after DDAVP infusion; and that DDAVP normalizes the BT only in those patients with normal platelet levels of both vWF:Ag and RiCof (platelet normal).

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