Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

Effect of Type IIB von Willebrand Disease Mutation Arg(545)Cys on Platelet Glycoprotein Ib Binding – Studies with Recombinant von Willebrand Factor

1993 ◽  
Vol 70 (06) ◽  
pp. 1058-1062 ◽  
Author(s):  
Aida Inbal ◽  
Nurit Kornbrot ◽  
Paul Harrison ◽  
Anna M Randi ◽  
J Evan Sadler
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Wild Type ◽  
Binding Studies ◽  
Function Type ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein lb (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristic gain-of-function type IIB vWD phenotype.

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3796-3803 ◽  
Author(s):  
Nadine Ajzenberg ◽  
Anne-Sophie Ribba ◽  
Ghassem Rastegar-Lari ◽  
Dominique Meyer ◽  
Dominique Baruch
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
High Shear ◽  
Shear Rates ◽  
High Shear Rates ◽  
Consistent Increase ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

scholarly journals Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2048-2055 ◽  
Author(s):  
PA Kroner ◽  
ML Kluessendorf ◽  
JP Scott ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

Modulation of von Willebrand Factor A1 Domain-Mediated Platelet Aggregation/Agglutination by α-Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 628-628
Author(s):  
Grazia Loredana Mendolicchio ◽  
Reha Celikel ◽  
Kottayil I. Varughese ◽  
Brian Savage ◽  
Zaverio M. Ruggeri
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Amino Terminal ◽  
Low Responder ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Evaluation of the crystal structures of the amino terminal domain of platelet glycoprotein (GP) Ibα bound to the von Willebrand factor A1 domain (VWFA1) or to α-thrombin indicate the absence of significant steric hindrance in a putative triple complex of the two ligands interacting with the same receptor molecule. Superposition of the models reveals that intermolecular contacts may be established between VWFA1 and α-thrombin concurrently bound to GP Ibα, and suggests that these additional interactions could stabilize the intrinsically low affinity binding of the VWF A1 domain. To verify the predictions of the model, we used gel electrophoresis under native conditions and purified components in solution to demonstrate directly the formation of a triple complex. We then sought to evaluate whether α-thrombin could influence the functional effects of the VWF-GP Ibα interaction. For this purpose, we established a model of platelet agglutination/aggregation dependent on the interaction between recombinant dimeric VWFA1 domain, purified from the culture medium of stably transfected D. melanogaster cell lines, and GP Ibα. In this assay, platelet rich plasma prepared from individual donor blood collected with the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK) as an anticoagulant (80 μM) was mixed with varying concentrations of dimeric VWFA1 (0.5-10 μg/ml) and exposed to variable shear rate levels in a cone-and-plate viscometer. Platelet aggregation was observed at shear rates between 6 and 108 dyn/cm2. The response in different normal controls was reproducible but variable in extent, and individuals could be assigned to one of two categories, low responder and high responder. An agglutination response was observed after platelets were treated with 10 μM prostaglandin E1 to block activation, and the distinction between low and high responders remained true under these conditions. For simplicity, agglutinated platelets were still defined as “aggregates”. With activation blocked platelets, aggregates were stable up to a shear rate of 30 dyn/cm2, but began to dissipate at higher levels. The addition of α-thrombin with the active site irreversibly blocked by PPACK at concentrations between 5 and 10 μg/ml substantially increased the extent of the platelet response. This was demonstrated by a faster rate of platelet agglutination/aggregation, a greater stability of aggregates at higher shear rates, and an overall increase in the size of aggregates formed. To demonstrate the latter, samples were exposed to shear stress under selected conditions and immediately fixed with 1% glutaraldehyde for quantitative image analysis. Maximum aggregate size was increased several fold in the presence of α-thrombin, and the difference was particularly evident in low responder individuals in whom dimeric VWFA1 alone caused the formation of small and unstable aggregates. PPACK-blocked thrombin by itself had no effect on platelet aggregate formation at any shear rate tested. Our findings delineate a mechanism through which α-thrombin may stabilize platelet-platelet contacts by mediating a tighter association between VWF A1 domain and GP Ibα receptor. Such a function, independent of proteolytic activity, may enhance platelet deposition at sites of vascular injury.

scholarly journals Humanized Von Willebrand Factor-Glycoprotein Ibα Interaction in Mouse Models of Hemostasis and Thrombosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 558-558 ◽  
Author(s):  
Sachiko Kanaji ◽  
Jennifer N Orje ◽  
Yuichi Kamikubo ◽  
Taisuke Kanaji ◽  
Jeremy Mattson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Mouse Strains ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Introduction: The interaction between von Willebrand Factor (VWF) and platelet glycoprotein (GP) Ibα is key for initiating the response to vascular injury that leads to hemostasis or, in pathological conditions, may be a cause of thrombosis. VWF binding to GPIbα occurs through the A1 domain (VWFA1) and its role in platelet adhesion and aggregation becomes progressively more important with increasing shear rates, i.e., in arterioles or pathologically stenosed arteries. Owing to the key role in platelet adhesion/aggregation under arterial flow conditions, VWFA1 has been considered an obvious target for antithrombotic intervention. However, efforts to develop this concept have been complicated by the lack of suitable animal models due to species-specificity in VWFA1-GPIb binding. To obviate the problem, we have generated new mouse strains with humanized VWF-GPIb interaction and characterized the resulting phenotypes in experimental ex vivo and in vivo models of hemostasis and thrombosis. Methods: In the human VWF gene, and in the mouse Vwf ortholog, exon 28 encodes domains A1 and A2, including the VWFA1 GPIb-binding site. We generated a knock-in mouse by targeted insertion of human VWF exon 28 (VWFh28) into the mouse Vwf exon28 locus such that mouse platelet GPIbα (M1) interacted with mouse VWF containing human A1 domain (HA); the strain was designated M1HA (Table). These mice were cross-bred with the previously described transgenic strain (mGPIbαnull;hIbαTg) in which human GPIbα is expressed on the platelet surface in the context of the mouse GPIb-IX-V complex (strain designation: H1MA). The resulting strain was thus designated H1HA, with humanized GPIb-VWF interaction. The unmodified wild type mouse strain used for reference was designated M1MA (Table). All mouse strains were in C57BL/6 genetic background. VWF plasma concentration was measured by ELISA and function ex vivo was evaluated by ristocetin-induced platelet aggregation. In vivo, we measured the tail bleeding time to gauge hemostatic efficiency as well as the propensity to support pathological thrombosis in the carotid artery injured by exposure to ferric chloride. Results: Plasma of VWFh28 mice expressing mouse or human platelet GPIbα had VWF levels (M1HA: 876.4 ± 209.5 mU/ml, n = 16; H1HA: 848.9 ± 121.0 mU/ml, n = 15) not significantly different from wild type mice (M1MA: 1022 ± 267.4 mU/ml, n = 23). Addition of 1.5 mg/ml ristocetin into platelet-rich plasma (PRP) from M1MA, M1HA and H1MA mice elicited no response; only in PRP of H1HA mice did ristocetin cause platelet aggregation that, as in human PRP, was inhibited by the anti-human VWFA1 monoclonal antibody, NMC-4. The tail bleeding time was abnormally prolonged in the M1HA strain expressing human VWFA1/mouse GPIbα, but normal in the H1HA strain expressing human VWFA1/human GPIbα (6.0 ± 3.8 min vs. 1.5 ± 0.9 min; n = 12); the latter was similar to the bleeding time in the M1MA strain (1.0 ± 0.1 min, n = 10). Thrombus formation (time to occlusion) following a carotid artery injury with 9% FeCl3∙6H2O was greatly delayed in the M1HA strain (1760.0 ± 538.5 s, n = 6) but similar to WT M1MA in the H1HA strain (485.2 ± 63.7 s vs. 598.3 ± 84.0 s, n = 6, respectively). Conclusions: Knock-in of human VWF exon 28 into the mouse Vwf locus led to successful biosynthesis of chimeric mouse-human VWF protein in vivo under endogenous promoter control, maintaining normal physiologic expression in endothelial cells and megakaryocytes/platelets. Tail bleeding time and in vivo thrombosis assays confirmed the normal functional interaction of mouse VWF containing human A1 domain with hGPIbα. Moreover, the normal response to ristocetin with platelet aggregation, and inhibition of the latter by the specific NMC4 antibody, indicates that the "humanized" H1HA mouse strain could be a powerful tool to select and develop new interventions for the diagnosis and treatment of hemostatic and thrombotic disorders. Table Table. Disclosures No relevant conflicts of interest to declare.

Glycoprotein Ibα Forms Catch Bonds with von Willebrand Factor A1 Domain but Not with Mutant A1 Domains Exhibiting Properties of Type 2B von Willebrand Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 293-293
Author(s):  
Tao Wu ◽  
Tadayuki Yago ◽  
Jun Yang ◽  
Jonathan Miner ◽  
Leslie Coburn ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Arterial Blood Flow ◽  
Fluid Viscosity ◽  
Wild Type ◽  
Arterial Blood ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Catch Bonds

Abstract Interactions between glycoprotein Ibα (GPIbα) and von Willebrand factor (VWF) initiate platelet adhesion to injured vascular surfaces, which is enhanced by arterial blood flow. The flow requirement for adhesion is reduced (i.e., gain-of-function) by single-residue substitutions of the VWF-A1 domain, e.g., R543Q that occurs in some patients with type 2B von Willebrand disease (VWD) and R687E that was designed to exhibit type 2B VWD properties. Yet the mechanisms for flow-enhanced adhesion through GPIbα-VWF interactions are not understood. By probing single bonds with atomic force microscopy, we showed that lifetimes of GPIbα/VWF-A1 bonds first increased (catch) and then decreased (slip) with increasing force applied to the bond. Remarkably, the catch bond aspect of the GPIbα/VWF-A1 bonds was eliminated by the R543Q and R687E mutations, which exhibited slip bonds only with prolonged lifetimes at low forces. Flow chamber experiments showed that catch-slip transitional bonds governed flow-enhanced rolling of platelets and GPIbα-coated microspheres on wild-type A1, such that rolling velocities first decreased and then increased with increasing flow. By contrast, slip bonds governed rolling velocities on R543Q and R687E A1 mutants, which increased monotonically with increasing flow. We changed fluid viscosity by adding Ficoll to the medium, tether force by using microspheres of different radii, and platelet deformability by using a chemical fixative. The rolling velocity vs. flow curves aligned with tether force but not with transport parameters and were minimally affected by fixation, which respectively rule out transport-enhanced GPIbα/VWF-A1 bond formation and force-induced enlargement of platelet-surface contact area as the causes for flow-enhanced rolling. Flowing platelets agglutinated with microspheres bearing R543Q and R687E A1 mutants but not with those bearing wild-type A1, suggesting that GPIbα/VWF-A1 catch bonds prevent agglutination of circulating platelets via binding to VWF multimers and that platelet agglutination in patients with type 2B VWD may be explained by the prolonged lifetimes at low forces resulting from elimination of catch bonds in the interactions of GPIbα with VWF-A1 mutants.

A novel von Willebrand factor mutation (I1372S) associated with type 2B-like von Willebrand disease: An elusive phenotype and a difficult diagnosis

2007 ◽  
Vol 98 (12) ◽  
pp. 1182-1187 ◽  
Author(s):  
Francesca Sartorello ◽  
Elena Pontara ◽  
Lisa Gallinaro ◽  
Antonella Bertomoro ◽  
Maria Grazia Cattini ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Index Case ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Difficult Diagnosis ◽  
History Of ◽  
Spontaneous Platelet Aggregation ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMutations in the A1 domain of von Willebrand factor (VWF) may be associated with gain of function in theVWF-platelet GPIb interaction and consumption of largeVWF multimers, as seen in type 2B von Willebrand disease (VWD). We report a new VWF abnormality associated with greater VWF-GPIb interaction in the presence of all VWF multimers. The index case is a woman with a lifelong history of bleeding, found hyperresponsive to ristocetin with spontaneous platelet aggregation (SPA). She had normal factor VIII,VWF:Ag,VWF:RCo and VWF:CB levels, normal VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios, and a full panel of plasma and platelet VWF multimers. A missense mutation (4115T>G) was found in exon 28 of theVWF gene, which replaced a isoleucine with a serine at position 1372 of pre-pro-VWF (I1372S) at heterozygous level. Recombinant VWF carrying the I1372S mutation and showing a normal VWF multimer organisation was capable of inducing SPA on normal plateletrich plasma (unlike wild-type VWF), as well as a hyper-response to ristocetin in the same platelets (0.6 mg/ml ristocetin vs. 1.2 of wild-type VWF). The new I1372S VWF mutation, characterised by SPA and hyper-responsiveness to ristocetin thus has some of the features of type 2B VWD, but not the lack of large VWF multimers, so we defined this variant as type 2B-likeVWD. Why I1372SVWF is associated with bleeding symptoms, despite normalVWF levels and multimer organisation,remains to be seen.

Characterization of a myeloma patient with a life-threatening hemorrhagic diathesis: presence of a lambda dimer protein inhibiting shear-induced platelet aggregation by binding to the A1 domain of vonWillebrand factor

2005 ◽  
Vol 93 (05) ◽  
pp. 889-896 ◽  
Author(s):  
Atsushi Shinagawa ◽  
Michael Berndt ◽  
Shin Kaneko ◽  
Kazumi Suzukawa ◽  
Yuichi Hasegawa ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Severe Bleeding ◽  
Hemorrhagic Diathesis ◽  
Bleeding Tendency ◽  
High Shear ◽  
Acquired Von Willebrand Disease ◽  
A1 Domain ◽  
Von Willebrand

SummaryWe have identified a patient with IgD λ-type multiple myeloma who was characterized by a severe bleeding tendency, especially after puncture of arterial vessels. Both the bleeding time (>25 min) and activated partial thromboplastin time (APTT) were prolonged. To clarify the underlying pathogenesis, we purified the APTT-prolonging activity from the patient’s serum. The purified protein was a highly negatively-charged homodimer of the λ light chain. The λ dimer protein (M-protein) inhibited ristocetin-and high shear-induced platelet aggregation, dependent on platelet glycoprotein Ibα (GPIbα), but not epinephrine-, collagen-, ADP-, thrombin-, or botrocetin-induced platelet aggregation. The λ dimer protein inhibited the binding of platelets to immobilized or ristocetin-treated von Willebrand factor (VWF). Furthermore, a 39/34 kD fragment of VWF encompassing the A1 domain specifically bound to the immobilized λ dimer protein in the presence of ristocetin, suggesting that the λ dimer protein directly binds to the A1 domain of VWF. To help elucidate the binding site within the A1 domain, binding of ristocetin-treated VWF to the immobilized λ dimer protein was assayed in the presence of various anti-A1 domain monoclonal antibodies. Based on these data, we conclude that the λ dimer protein binds to the region of the A1 domain composed of helices α3 and α4 and thus interferes with VWF-GPIbα interaction. The existence of a protein that inhibits high shear-induced platelet aggregation in acquired von Willebrand disease (VWD) has only rarely been reported. The results suggest that the hemostatic function in arteries with high shear force is profoundly disrupted if the binding of GPIbα to VWF is abrogated, supporting the relevance of shear-induced VWF interaction with GPIbα in the initiation of the hemostatic process.

Fc-receptor Dependent Platelet Aggregation Induced by Monoclonal Antibodies against Platelet Glycoprotein Ib or von Willebrand Factor

2001 ◽  
Vol 85 (04) ◽  
pp. 679-685 ◽  
Author(s):  
Nancy Cauwenberghs ◽  
Agotha Schlammadinger ◽  
Stephan Vauterin ◽  
Susan Cooper ◽  
Gretel Descheemaeker ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Fc Receptor ◽  
Platelet Glycoprotein ◽  
Protein Tyrosine Phosphorylation ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIn this paper we describe two pathways leading to platelet activation by crosslinking glycoprotein (GP) Ib to the platelet Fc-receptor (FcγRII). First the monoclonal antibody (MoAb) 9C8, raised against human platelet GPIbα, dose-dependently induced platelet aggregation of citrate-anticoagulated platelet-rich plasma, an effect that can be inhibited by several activation inhibitors. The FcγRII-inhibitory MoAb IV.3 was able to prevent the aggregatory effects of MoAb 9C8, indicating that crosslinking of the antigen GPIbαto the FcγII-receptor is necessary for the activating effect. Secondly we observed a synergistic activating effect of two anti-von Willebrand factor (vWF) MoAbs 1C1E7 and B724, both known to enhance vWF binding to GPIbαin the presence of shear or ristocetin. When these antibodies are added together to PRP, platelet aggregation is induced without further need for an additional modulator. This effect can be blocked by either MoAb IV.3 or an inhibitory anti-GPIbαMoAb, indicating that again the platelet activation results from signaling through FcγRII crosslinked to vWF bound to GPIbα. In addition, both the anti-GPIbαMoAb 9C8, or the two anti-vWF MoAbs 1C1E7 and B724 induce genuine platelet activation, as evidenced by the secretion of ATP and protein tyrosine phosphorylation. These findings with both anti-GPIbαand anti-vWF MoAbs add further proof to recent reports demonstrating an interaction between the platelet receptors GPIbαand FcγRII, suggesting a role for the FcγII-receptor in GPIb-related signaling.

scholarly journals Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2048-2055 ◽  
Author(s):  
PA Kroner ◽  
ML Kluessendorf ◽  
JP Scott ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

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