Characterization of the Binding Between the Polymeric Platelet Adhesive Proteins, Multimerin 1 and Von Willebrand Factor

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1144-1144
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Nola Fuller ◽  
J. Evan Sadler ◽  
Philip G de Groot ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
A Domains ◽  
Adhesive Proteins ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 1144 Introduction: Multimerin 1 (MMRN1) is a massive variably-sized homopolymeric protein that is stored in platelet and endothelial cell secretion granules, for release with vascular injury. Recently, MMRN1 was identified to support platelet adhesion in vitro and in vivo. At high shear, MMRN1 supports platelet adhesion by a von Willebrand factor (VWF)-dependent, but integrin-independent mechanism, involving platelet glycoprotein (GP) Ibα. Direct binding of MMRN1 to GP Ibα has not been demonstrated. These data led us to postulate that VWF binds MMRN1 at site(s) distinct from the GP Ibα binding site, and test the roles of VWF A domains in MMRN1 binding. Methods: Modified enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were used to assess binding interactions between wildtype (WT) MMRN1 and WT or domain deleted VWF constructs, and VWF polypeptides. Protein constructs tested included: multimeric VWF deletion constructs ΔA1A2A3-VWF, ΔA1A3-VWF, and ΔA1-VWF, and monomeric VWF polypeptides A1A2A3, A1A2, A1 and A3. Bovine serum albumin (BSA) coated surfaces were used as the negative control. Results: Unlike WT-VWF, VWF lacking the A domains (ΔA1A2A3-VWF) or the combination of the A1 and A3 domains (ΔA1A3-VWF) did not detectably bind to MMRN1 (p < 0.001). VWF lacking the A1 domain (ΔA1-VWF) showed MMRN1 binding comparable to WT-VWF (p = 0.39), excluding the possibility that MMRN1 binding site is located in VWF A1 domain (the region that binds GP Ibα). VWF polypeptides A1A2A3, A1A2 and A3 bound to MMRN1 (p < 0.001), unlike the VWF polypeptide A1 (p = 0.137), although the A1A2 polypeptide showed reduced binding compared to A1A2A3 (p < 0.001). SPR analyses confirmed that MMRN1 binding was supported by VWF peptides containing the A3 and/or A2 domains. Conclusions: The regions of VWF that support MMRN1 includes the A3, and possibly A2 domains, which respectively contain binding sites for collagen and ADAMTS-13. Our data suggest that the mechanism by which GP Ibα and VWF support platelet adhesion to MMRN1 at high shear include: VWF binding to GP Ibα via the A1 domain, and to MMRN1 via the A3 and possibly A2 domains. These findings have implications for the molecular mechanisms that support platelet adhesion at sites of vessel injury. Disclosures: No relevant conflicts of interest to declare.

Von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1741-1746 ◽  
Author(s):  
Jeffrey F. W. Keuren ◽  
Dominique Baruch ◽  
Paulette Legendre ◽  
Cécile V. Denis ◽  
Peter J. Lenting ◽  
...  
Keyword(s):  
Shear Rate ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
High Shear Rate ◽  
High Shear ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractFibrin is actively involved in platelet reactions essential for thrombus growth, in which von Willebrand factor (VWF) might be an important mediator. The aim of this study was to localize VWF domains that bind to fibrin and to determine their relevance in platelet adhesion. VWF binds specifically to fibrin with an apparent Kd of 2.2 μg/mL. Competition in the presence of 2 complementary fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), indicated that the high affinity binding site for fibrin is located in the C-terminal part, thus distinct from the A domains. Comparison of 2 deleted rVWF (ΔD4B-rVWF, ΔC1C2-rVWF) suggested that the C1C2 domains contained a fibrin binding site. This site is distinct from RGD, as shown by binding of D1746G-rVWF to fibrin. Perfusion studies at high shear rate demonstrated that C1C2 domains were required for optimal platelet adhesion to fibrin. With the use of a VWF-deficient mouse model, it was found that plasma VWF is critical for platelet tethering and adhesion to fibrin. These results suggest a dual role of fibrin-bound VWF in thrombus formation: first, fibrin-bound VWF is critical in the recruitment of platelets by way of glycoprotein (GP) Ib, and, second, it contributes to stationary platelet adhesion by way of binding to activated αIIbβ3.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Vimentin Exposed On Platelets Serves As an Adhesive Receptor for Von Willebrand Factor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 259-259
Author(s):  
Miguel A. Cruz ◽  
K. Vinod Vijayan
Keyword(s):  
Binding Site ◽  
Platelet Adhesion ◽  
Flow Conditions ◽  
Platelet Receptor ◽  
Coated Surface ◽  
Initial Interaction ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 259 Platelet adhesion, activation, and aggregation in the vasculature are necessary events in both life-saving hemostasis and pathological thrombosis. Thrombosis may occur in patients presenting with several clinical conditions including atherosclerosis, cardiovascular disease, and inflammation. Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in mediating platelet adhesion, activation, and aggregation on the exposed subendothelium in order to maintain hemostasis under arterial flow conditions. On the other hand, VWF permits the stabilization of platelets adherent to components of ruptured atherosclerotic plaques, leading to artery-occluding thrombus formation. The initial interaction of activated or hyperadhesive VWF with platelets occurs via the interaction between the A1 domain of VWF and the platelet receptor glycoprotein (GP)Ibα. This engagement is responsible for reducing the velocity of rapidly flowing platelets, allowing the rolling platelets to interact with the second binding site on VWF for the platelet receptor GPIIb/IIIa; a binding site that is located within the C domains of VWF. Therefore, the hyperadhesive property of VWF apparently relies on the synchronized interaction of the two platelet surface receptors, GPIbα and GPIIb/IIIa. Despite this concept, we and others have speculated that other binding site in VWF synergistically works with the A1 domain to quickly capture the extremely fast flowing platelets. We have obtained interesting results from studies using a monomeric A1A2A3 domain protein that lacks the binding site for GPIIb/IIIa. For example, the rolling velocity of platelets over an A1A2A3-coated surface was markedly lower than that seen with use of the single A1 domain. This observation suggests the possibility of an additional binding site in the A domains for platelets. Given the similar hyperadhesive features of the A1A2A3 protein and plasma VWF, we proposed to look for a potential receptor on platelets with a recognition site within the A domains of VWF. We suggested examining vimentin because, it was identified as a binding protein for the isolated A2 domain of VWF in our laboratory, and vimentin has been found on the surface of platelets. First, both full length VWF and recombinant A1A2A3 proteins efficiently bound to human vimentin only in the presence of the modulator ristocetin, indicating that vimentin preferably interact with the active conformation of VWF. In fact, a constitutively active A1A2A3 protein (containing a gain-of-function mutation in A1 domain) had a binding activity for vimentin higher than that of wild type (WT) A1A2A3 in the absence of ristocetin. Second, anti-vimentin monoclonal antibody blocked the interaction of that mutant A1A2A3 to activated washed platelets using flow cytometry. Third, we then examined the effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3-coated surface at high shear stress. In comparison to whole blood incubated with irrelevant IgG molecule as a negative control, the anti-vimentin antibody blocked 75% platelet adhesion to the triple-A domain protein. Finally, whole blood from vimentin-deficient or WT mice was perfused over a surface coated with murine VWF at high shear rate. In comparison to platelets from WT mice, vimentin-deficient platelets had a significant reduced platelet adhesion to VWF (25% of WT). Similarly, vimentin-deficient platelets had a reduced platelet adhesion to collagen (20% of WT) under high flow conditions. This platelet-collagen interaction is initially mediated by VWF. These interesting results indicate that vimentin on platelets serves as a receptor for VWF, and this binding may participate in the initial interaction of circulating platelets with VWF under flow conditions. Disclosures: No relevant conflicts of interest to declare.

Conformational Changes in the A3 Domain of von Willebrand Factor Modulate the Interaction of the A1 Domain With Platelet Glycoprotein Ib

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 1959-1968 ◽  
Author(s):  
Bernadette Obert ◽  
Anne Houllier ◽  
Dominique Meyer ◽  
Jean-Pierre Girma
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Platelet Glycoprotein ◽  
Terminal Part ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Dose Dependent

Bitiscetin has recently been shown to induce von Willebrand factor (vWF)-dependent aggregation of fixed platelets (Hamako J, et al,Biochem Biophys Res Commun 226:273, 1996). We have purified bitiscetin from Bitis arietans venom and investigated the mechanism whereby it promotes a form of vWF that is reactive with platelets. In the presence of bitiscetin, vWF binds to platelets in a dose-dependent and saturable manner. The binding of vWF to platelets involves glycoprotein (GP) Ib because it was totally blocked by monoclonal antibody (MoAb) 6D1 directed towards the vWF-binding site of GPIb. The binding also involves the GPIb-binding site of vWF located on the A1 domain because it was inhibited by MoAb to vWF whose epitopes are within this domain and that block binding of vWF to platelets induced by ristocetin or botrocetin. However, in contrast to ristocetin or botrocetin, the binding site of bitiscetin does not reside within the A1 domain but within the A3 domain of vWF. Thus, among a series of vWF fragments, 125I-bitiscetin only binds to those that overlap the A3 domain, ie, SpIII (amino acid [aa] 1-1365), SpI (aa 911-1365), and rvWF-A3 domain (aa 920-1111). It does not bind to SpII corresponding to the C-terminal part of vWF subunit (aa 1366-2050) nor to the 39/34/kD dispase species (aa 480-718) or T116 (aa 449-728) overlapping the A1 domain. In addition, bitiscetin that does not bind to DeltaA3-rvWF (deleted between aa 910-1113) has no binding site ouside the A3 domain. The localization of the binding site of bitiscetin within the A3 domain was further supported by showing that MoAb to vWF, which are specific for this domain and block the interaction between vWF and collagen, are potent inhibitors of the binding of bitiscetin to vWF and consequently of the bitiscetin-induced binding of vWF to platelets. Thus, our data support the hypothesis that an interaction between the A1 and A3 domains exists that may play a role in the function of vWF by regulating the ability of the A1 domain to bind to platelet GPIb.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Background It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. Results Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. Conclusions These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

An Anti-von Willebrand Factor Aptamer Reduces Platelet Adhesion Among Patients Receiving Aspirin and Clopidogrel in an Ex Vivo Shear-Induced Arterial Thrombosis

2010 ◽  
Vol 17 (6) ◽  
pp. E70-E78 ◽  
Author(s):  
Dabit Arzamendi ◽  
Firas Dandachli ◽  
Jean-François Théorêt ◽  
Gregory Ducrocq ◽  
Mark Chan ◽  
...  
Keyword(s):  
Antiplatelet Therapy ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Glycoprotein ◽  
Artery Disease ◽  
A Site ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 106/cm2, P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet−leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.

Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1375-1383 ◽  
Author(s):  
Arnaud Bonnefoy ◽  
Hiroshi Yamamoto ◽  
Chantal Thys ◽  
Morikazu Kito ◽  
Jos Vermylen ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Amino Acid Sequences ◽  
Platelet Glycoprotein ◽  
Surface Distribution ◽  
Perfusion Studies ◽  
Mechanistic Basis ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIbα. Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the VWF A1 domain in several species, expressing VWF recognized by the blocking monoclonal antibody AJvW-2, suggested 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIbα-binding site. Glutathione-S-transferase (GST)–human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin- or ristocetin-mediated VWF binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIbα binding, their mutation-impairing platelet recruitment. In agreement with the surface distribution of VWF type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIbα-binding site, located around the front pocket of the A1 domain and defined by strands β3, β4, and helix α3, and it provides a mechanistic basis for VWF neutralization by this antibody.

The Platelet Glycoprotein Ib-von Willebrand Factor Interaction Activates the Collagen Receptor α2β1 and the Two Adhesive Receptors then Collaborate to Fully Activate Platelets to Form Thrombi.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1556-1556
Author(s):  
Junmei Chen ◽  
Jody L. Whitelock ◽  
Lisa D. Morales ◽  
Jose A. Lopez ◽  
Miguel A. Cruz
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Type I ◽  
High Shear ◽  
High Shear Stress ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Integrin α2β1 (GP Ia/IIa) is a major platelet receptor for collagen, containing its collagen binding site within the α2 I domain. α2β1 changes conformation upon platelet activation, increasing its affinity for collagen. The conformational changes are reflected in the markedly different crystal structures obtained for the α2 I domain depending on whether it is free or bound to a collagen peptide. However, it is not known whether in flowing blood α2β1 on platelets is activated before binding to collagen. To address this issue, we identified an antibody that has higher affinity for the activated α2β1. We found that two antibodies that bind within the α2 I domain, 12F1 and 6F1, bound preferentially to ADP-activated platelets, with 12F1 displaying the most marked increase in binding with activation. We corroborated this result for 12F1 by showing that it binds with higher affinity to a gain-of-function I domain mutant than to either the wild-type I domain or to a loss-of-function mutant. In addition, when whole blood was perfused over a surface coated with 12F1, the antibody did not support the adhesion of unstimulated platelets. Because thrombus formation on collagen at a high shear stress is initiated by the binding of the platelet glycoprotein Ib-IX-V complex (GP Ib) to von Willebrand factor (VWF), we tested whether this interaction can activate α2β1, using 12F1 as a probe for integrin activation. We perfused blood over a surface coated with a mixture of VWF A1 domain (a GP Ib ligand) and 12F1, or VWF A1 and mouse IgG. Platelets rolled and did not attach stably on the A1/IgG surface, but they firmly bound and covered the A1/12F1 surface. The fact that 12F1 alone failed to capture resting platelets under flow but supported firm platelet adhesion if GP Ib interacted with VWF A1 strongly suggests that GP Ib ligation by VWF induces signals that activate α2β1 and increase its affinity for collagen. The two receptors (GP Ib and α2β1) then cooperate in platelet adhesion to collagen, which was demonstrated by perfusing, at a high shear stress, reconstituted blood lacking VWF and fibrinogen over surfaces coated with collagen or A1/collagen. The A1/collagen surface contained more firmly adherent platelets than the collagen surface; firm adhesion was blocked by 6F1. We then tested whether the signals from GP Ib and α2β1 cooperate to fully activate platelets and allow thrombus growth. For this, we perfused whole blood over a mixed matrix of A1 and the α2β1-specific type I collagen-derived triple-helical peptide, CP10. We observed that platelets not only firmly adhered to this surface, they also formed thrombi, similar to those seen on collagen surfaces. Thrombus formation was inhibited by either the αIIbβ3 antibodies or blocking the A1/CP10 surface with the recombinant α2 I domain. Together, our data indicate that platelets adhere to collagen in a stepwise fashion, beginning with the interaction of GP Ib with VWF, which rapidly activates α2β1 to engage collagen. The combination of adhesive ligand-receptor interactions induces the activation of integrin αIIbβ3, which enables thrombus formation.

scholarly journals Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

2020 ◽  
Author(s):  
Matthias F. Schneider ◽  
Mohammad A. Fallah ◽  
Christian Mess ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Platelet Glycoprotein ◽  
Aggregate Formation ◽  
Cooperative Phenomena ◽  
Interference Contrast Microscopy ◽  
A Domains ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Background It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. Results Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. Conclusions These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

Sign in / Sign up

Export Citation Format

Share Document