scholarly journals Identification of functional domains on von Willebrand factor by binding of tryptic fragments to collagen and to platelets in the presence of ristocetin

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1498-1503
Author(s):  
WP Houdijk ◽  
ME Schiphorst ◽  
JJ Sixma
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Functional Domains ◽  
Collagen Binding ◽  
Platelet Binding ◽  
Single Fragment ◽  
Direct Binding ◽  
Reducing Substances ◽  
Von Willebrand ◽  
Willebrand Factor

With the use of monoclonal antibodies that inhibit the ristocetin- induced binding of von Willebrand factor (VWF) to platelets and the binding to collagen, we have previously identified two distinct tryptic fragments. To prove that these fragments contain the platelet binding or the collagen binding domain, we investigated the direct binding of tryptic fragments of 125I-VWF to platelets in the presence of ristocetin and to collagen fibrils. During the course of the tryptic digestion, there was a rapid and parallel decrease in binding to platelets and collagen. In the first ten minutes, binding decreased greater than 50%; a further decrease to 19% and 29%, respectively, was noted at 90 minutes, but no further decrease was observed thereafter. The bound fragments were eluted from platelets and collagen and analyzed on polyacrylamide gradient gels. The fragments bound to the platelets appeared to be reduced, probably by endogenous reducing substances from the platelets. This was prevented by addition of N- ethylmaleimide during the incubation. After 24 hours of digestion, platelets predominantly bound fragments of 116 kd and collagen bound a single fragment of 48 kd. These fragments are similar to those previously identified with the monoclonal antibodies.

scholarly journals Identification of functional domains on von Willebrand factor by binding of tryptic fragments to collagen and to platelets in the presence of ristocetin

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1498-1503 ◽  
Author(s):  
WP Houdijk ◽  
ME Schiphorst ◽  
JJ Sixma
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Functional Domains ◽  
Collagen Binding ◽  
Platelet Binding ◽  
Single Fragment ◽  
Direct Binding ◽  
Reducing Substances ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract With the use of monoclonal antibodies that inhibit the ristocetin- induced binding of von Willebrand factor (VWF) to platelets and the binding to collagen, we have previously identified two distinct tryptic fragments. To prove that these fragments contain the platelet binding or the collagen binding domain, we investigated the direct binding of tryptic fragments of 125I-VWF to platelets in the presence of ristocetin and to collagen fibrils. During the course of the tryptic digestion, there was a rapid and parallel decrease in binding to platelets and collagen. In the first ten minutes, binding decreased greater than 50%; a further decrease to 19% and 29%, respectively, was noted at 90 minutes, but no further decrease was observed thereafter. The bound fragments were eluted from platelets and collagen and analyzed on polyacrylamide gradient gels. The fragments bound to the platelets appeared to be reduced, probably by endogenous reducing substances from the platelets. This was prevented by addition of N- ethylmaleimide during the incubation. After 24 hours of digestion, platelets predominantly bound fragments of 116 kd and collagen bound a single fragment of 48 kd. These fragments are similar to those previously identified with the monoclonal antibodies.

scholarly journals Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 797-800 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
BS Coller
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Adenosine Diphosphate ◽  
Glycoprotein Ib ◽  
Human Fibrinogen ◽  
Factor Binding ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

scholarly journals Type 2M von Willebrand Disease: F606I and I662F Mutations in the Glycoprotein Ib Binding Domain Selectively Impair Ristocetin- but not Botrocetin-Mediated Binding of von Willebrand Factor to Platelets

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1572-1581 ◽  
Author(s):  
Cheryl A. Hillery ◽  
David J. Mancuso ◽  
J. Evan Sadler ◽  
Jay W. Ponder ◽  
Mary A. Jozwiak ◽  
...  
Keyword(s):  
Amino Acid ◽  
Missense Mutation ◽  
Von Willebrand Factor ◽  
Amino Acid Substitution ◽  
Von Willebrand Disease ◽  
Glycoprotein Ib ◽  
Collagen Binding ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstractvon Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T → A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A → T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

scholarly journals Type 2M von Willebrand Disease: F606I and I662F Mutations in the Glycoprotein Ib Binding Domain Selectively Impair Ristocetin- but not Botrocetin-Mediated Binding of von Willebrand Factor to Platelets

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1572-1581 ◽  
Author(s):  
Cheryl A. Hillery ◽  
David J. Mancuso ◽  
J. Evan Sadler ◽  
Jay W. Ponder ◽  
Mary A. Jozwiak ◽  
...  
Keyword(s):  
Amino Acid ◽  
Missense Mutation ◽  
Von Willebrand Factor ◽  
Amino Acid Substitution ◽  
Von Willebrand Disease ◽  
Glycoprotein Ib ◽  
Collagen Binding ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T → A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A → T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

scholarly journals Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1356-1366 ◽  
Author(s):  
JP Girma ◽  
M Kalafatis ◽  
G Pietu ◽  
JM Lavergne ◽  
MW Chopek ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Structural Defects ◽  
Membrane Glycoproteins ◽  
Collagen Binding ◽  
Functional Sites ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin- induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino- terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.

scholarly journals Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 797-800 ◽  
Author(s):  
HR Gralnick ◽  
SB Williams ◽  
BS Coller
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Adenosine Diphosphate ◽  
Glycoprotein Ib ◽  
Human Fibrinogen ◽  
Factor Binding ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

Enhancement of Factor VIII-von Willebrand Factor Ristocetin Cofactor Activity by Monoclonal Antibodies

1985 ◽  
Vol 54 (02) ◽  
pp. 510-524 ◽  
Author(s):  
V Hornsey ◽  
L R Micklem ◽  
M C McCann ◽  
K James ◽  
J Dawes ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Cross Linking ◽  
Platelet Binding ◽  
Specific Affinity ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

SummaryFive monoclonal antibodies to human von Willebrand factor were selected for characterization from 18 produced in murine hybridomas. All showed a high and specific affinity for human von Willebrand factor (vWf) but exhibited little if any crossreaction with sera from other species. The antibodies defined four epitopes on vWf, none of which were involved in platelet binding. Binding of two distinct antibodies at one of these epitopes was associated with enhancement of the rate of vWf-dependent platelet agglutination in the presence of ristocetin. This effect was more noticeable when cryosupernatant plasma was used in place of normal plasma as the source of vWf, and was not explicable simply in terms of antibody-induced cross-linking of vWf.

scholarly journals Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1356-1366
Author(s):  
JP Girma ◽  
M Kalafatis ◽  
G Pietu ◽  
JM Lavergne ◽  
MW Chopek ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Structural Defects ◽  
Membrane Glycoproteins ◽  
Collagen Binding ◽  
Functional Sites ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin- induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino- terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.

The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

1992 ◽  
Vol 68 (04) ◽  
pp. 464-469 ◽  
Author(s):  
Y Fujimura ◽  
S Miyata ◽  
S Nishida ◽  
S Miura ◽  
M Kaneda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Normal Individuals ◽  
Rag 1 ◽  
The One ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

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