scholarly journals Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress

2020 ◽  
Author(s):  
Harrison E. R. O’Brien ◽  
X. Frank Zhang ◽  
Maximo Sanz‐Hernandez ◽  
Alain Chion ◽  
Susan Shapiro ◽  
...  
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

1996 ◽  
Vol 75 (04) ◽  
pp. 655-660 ◽  
Author(s):  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Paola Pradella ◽  
Adriana Masotti ◽  
Francesco I Pareti
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Transmembrane Flux ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

scholarly journals Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3792-3799 ◽  
Author(s):  
Hilde Depraetere ◽  
Nadine Ajzenberg ◽  
Jean-Pierre Girma ◽  
Catherine Lacombe ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
Rotational Viscometer ◽  
Induce Platelet Aggregation ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

von Willebrand Factor Ristocetin Cofactor Activity Correlates with Platelet Function in a High Shear Stress System

2000 ◽  
Vol 84 (10) ◽  
pp. 727-728 ◽  
Author(s):  
Agnès Veyradier ◽  
Edith Fressinaud ◽  
Catherine Boyer-Neumann ◽  
Marc Trossaert ◽  
Dominique Meyer
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Stress System ◽  
High Shear ◽  
High Shear Stress ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

Shear stress-induced binding of von willebrand factor to platelets

Biorheology ◽  
1997 ◽  
Vol 34 (1) ◽  
pp. 57-71 ◽  
Author(s):  
K KONSTANTOPOULOS ◽  
T CHOW ◽  
N TURNER ◽  
J HELLUMS ◽  
J MOAKE
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Binding of ADAMTS13 to Von Willebrand Factor Under Static Conditions and Under Fluid Shear Stress

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 258-258
Author(s):  
Hendrik B Feys ◽  
Patricia J Anderson ◽  
J. Evan Sadler
Keyword(s):  
Shear Stress ◽  
Von Willebrand Factor ◽  
Time Course ◽  
Fluid Shear Stress ◽  
Proteolytic Processing ◽  
Full Length ◽  
Fluid Shear ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 is a plasma metalloprotease that is essential for the normal proteolytic processing of von Willebrand factor (VWF). Dysfunctional ADAMTS13 may lead to thrombotic thrombocytopenic purpura, as uncleaved and unusually large VWF multimers accumulate in the blood and cause intravascular platelet aggregation. Many studies indicate that proteolysis of multimeric VWF involves conformational changes in the VWF A2 domain that expose the Y1605-M1606 scissile bond and also allow substrate binding to multiple exosites on ADAMTS13. For example, VWF is resistant to proteolysis by ADAMTS13 unless the VWF is subjected to fluid shear stress, mild denaturation with guanidine or urea, or adsorption onto a surface. However, the functional interactions between shear stress, various ADAMTS13 binding sites and VWF cleavage are not understood. Therefore, we investigated the effect of fluid shear stress and ADAMTS13 structure on ADAMTS13-VWF binding and VWF cleavage. Upon mixing recombinant VWF (rVWF) and ADAMTS13 in a physiological buffer (50 mM HEPES, 5 mM CaCl2, 1 μM ZnCl2, 150 mM NaCl, pH 7.4), we found that immunoprecipitation with anti-VWF also pulled down substantial amounts of ADAMTS13. Although less striking, a similar result was obtained with purified plasma VWF. Therefore, ADAMTS13 can bind VWF without gaining access to the cleavage site in VWF domain A2. When fluid shear stress was applied for 2 min with a bench-top vortexer, ADAMTS13 binding increased 3-fold and VWF was also cleaved. Lowering the ionic strength markedly increased the rate of VWF cleavage but did not affect ADAMTS13 binding, which suggests that cleavage and binding depend on distinct VWF-ADAMTS13 interactions. Shear-induced binding was reversible slowly upon removal of unbound ADAMTS13 or rapidly by addition of SDS. ADAMTS13-VWF binding was stable for at least 24 h after cessation of shear stress, indicating that the structural change in VWF that promotes binding was not readily reversible. Using a catalytically inactive ADAMTS13 variant to simplify the analysis of binding assays, 30 nM ADAMTS13(E231Q) bound to 30 μg/ml rVWF (120 nM subunits) with a stoichiometry of 0.012 ± 0.004 under static conditions and 0.098 ± 0.023 after shearing (mean ± SD, n = 3, P = 0.019). With 120 nM ADAMTS13(E231Q) the stoichiometry increased to 0.086 ± 0.036 under static conditions and 0.469 ± 0.033 after shearing for 2 min. Recombinant ADAMTS13 truncated after TSP-1 repeat 8 (lacking the C-terminal CUB domains, delCUB), or truncated after the Spacer domain (consisting of domains MDTCS), did not bind rVWF under static conditions, implicating the CUB domains in binding to VWF. In contrast, full-length ADAMTS13, delCUB and MDTCS bound similarly to rVWF after shearing. In a previous study, delCUB and MDTCS did not cleave VWF subjected to fluid shear stress (Zhang et al, Blood2007; 110: 1887–1894). However, under the conditions employed in these experiments, MDTCS and delCUB displayed significant proteolytic activity, cleaving VWF at a rate comparable to that of full length ADAMTS13 when shear stress was applied over a time course of 0–160 sec. We conclude that ADAMTS13 CUB domains contribute to binding a few sites on multimeric VWF under static conditions, whereas ADAMTS13 MDTCS domains are sufficient to bind many sites in an altered conformation of VWF that is induced by fluid shear stress. Binding of ADAMTS13 to unsheared VWF multimers may facilitate the cleavage of VWF within a growing thrombus.

Compromised Proteolytic Cleavage of Von Willebrand Factor Type 2N Variants by ADAMTS13 in the Presence of Factor VIII (and Platelets) Under Fluid Shear Stress.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 28-28
Author(s):  
Christopher G. Skipwith ◽  
Sandra L. Haberichter ◽  
Wenjing Cao ◽  
Ashley L. Gehrand ◽  
X. Long Zheng
Keyword(s):  
Shear Stress ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Proteolytic Cleavage ◽  
Binding Activity ◽  
Clotting Factor ◽  
Fluid Shear ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 28 von Willebrand Factor (VWF) is a large, multimeric adhesive glycoprotein that is involved in the formation of a platelet plug after vascular injury. In addition, VWF functions as a carrier protein for clotting factor VIII (FVIII) which prevents rapid clearance of plasma FVIII. Decreased levels of VWF or defects in VWF function are found in patients with von Willebrand disease (VWD). Quantitative defects of VWF protein in type 1 and 3 VWD affect plasma levels of both VWF and FVIII, whereas qualitative defects in type 2 VWD result in abnormal binding of VWF to platelets such as in type 2A, 2B, and 2M, or to FVIII such as in type 2N. Previous studies have demonstrated that FVIII binds VWF, which dramatically accelerates the proteolytic cleavage of multimeric VWF by ADAMTS13 under mechanically-induced shear stresses. This rate-enhancing effect of FVIII on VWF proteolysis appears to depend on the ability of FVIII to bind VWF, as a FVIII variant lacking the A3 acidic region fails to exhibit the cofactor activity that accelerates VWF proteolysis. To determine whether reduced FVIII binding in VWF type 2N variants affects VWF proteolysis, we examined the proteolytic cleavage of recombinant VWF type 2N variants in the presence of FVIII (and lyophilized platelets) for variants with a moderate VWD phenotype (Arg854Gln and His817Gln) or severe VWD phenotype (Arg763Gly, Arg782Trp, Thr791Met, and Arg782Trp + His817Gln). Recombinant VWF type 2N variants (37.5 μg/ml or 150 nM) were incubated with ADAMTS13 (25 nM) in the absence and the presence of various concentrations of FVIII (0-40 nM) with or without lyophilized platelets (0-600×103/μl) under fluid shear stress. The proteolytic cleavage products (350 kDa) were determined by 5% SDS-PAGE and Western blot under denaturing but non-reducing conditions. We show that the proteolytic cleavage of VWF type 2N variants by ADAMTS13 under these conditions was variably reduced as compared that of wild type VWF. The reduction in the cleavage rate was proportional to the degree of reduction in VWF FVIII binding activity, which was assessed with a microtiter assay, with the least cleavage by ADAMTS13 of the variants with the lowest FVIII binding activity. This reduced cleavage of the type 2N variants was not correlated with the binding affinity between the type 2N variants and ADAMTS13 protease. These results provide further evidence that binding of FVIII to VWF, which may alter VWF conformation, is necessary to accelerate VWF proteolysis by ADAMTS13 under fluid shear stress. This variability in ADAMTS13 cleavage may contribute to the heterogeneity of bleeding phenotype of type 2N VWD variants. The bleeding phenotype may be modulated not only by plasma FVIII levels, but also the extent of VWF proteolysis by ADAMTS13 under physiological fluid shear stress. Disclosures: No relevant conflicts of interest to declare.

Regulation of Von Willebrand Factor A1 Domain by Mechanical Force and Neighboring Domains

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2117-2117
Author(s):  
Wendy E Thomas ◽  
Rebecca A Penkala ◽  
Elaine Hillenmeyer ◽  
Matthew Whitfield ◽  
An-yue Tu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Shear Stress ◽  
Von Willebrand Factor ◽  
Mechanical Force ◽  
Platelet Glycoprotein ◽  
Bond Rupture ◽  
C Terminus ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2117 Regulation of the bond between platelet glycoprotein (GP) Ibα of the GPIb-IX-V complex, and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and inhibited by neighboring domains in VWF, but the role of neighboring domains in the shear-dependence remained unknown. Here it is shown that platelet aggregation required shear stress in the presence of VWF proteins that contain the neighboring D′D3 domain (Plus D′D3 or plasma VWF) but that platelets aggregate spontaneously with a protein that lacks this region (Delta D′D3). Moreover, platelets and microspheres coated with the N-terminal 300 amino acids of GPIbα (GC300) bind to immobilized VWF in a shear-enhanced manner for Plus D′D3 but not for Delta D′D3. In single-molecule force spectroscopy experiments, the D′D3 domain decreased the number of GPIbα-A1 bonds that formed, but did not alter bond rupture force, consistent with the hypothesis that D′D3 shields the A1 domain. By expressing recombinant VWF fragments that contain the A1 domain and various lengths of the N-terminal region, we determined that most of the inhibition by the D′D3 domain was conferred by 23 amino acids in the linker between the A1 domain and the D′D3 domain. By anchoring the fragments to the surface in an oriented manner, we demonstrated that binding was much stronger when force was applied between GPIbα and the A1 C-terminus, than when force was applied between GPIbα and the A1 N-terminus, similar to what has been observed for integrins. Based on these results, we propose the following model for regulation of VWF by mechanical force. When multimeric VWF is stretched in flow, the D′D3 domains are pulled away from the A1 domains, exposing the latter to bind platelets. When force is applied between GPIbα and the C-terminus of A1, it induces an activating conformational change that could be analogous to that seen in integrins. Disclosures: No relevant conflicts of interest to declare.

Shear Stress-Induced Unfolding of Von Willebrand Factor Accelerates Oxidation of Key Methionine Residues In the A1A2A3 Region

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2112-2112
Author(s):  
Xiaoyun Fu ◽  
Ryan P. Gallagher ◽  
Dominic Chung ◽  
Junmei Chen ◽  
José A. López
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Von Willebrand Factor ◽  
Cleavage Site ◽  
Elongational Flow ◽  
Shear Stresses ◽  
Inflammatory Conditions ◽  
Methionine Residues ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2112 The interaction between von Willebrand factor (VWF) and the platelet glycoprotein Ib-IX-V complex mediates the first step of platelet adhesion to the vessel wall at sites of injury in the hemostatic response to blood loss. This interaction is also involved in pathologic thrombosis, the most extreme case being thrombotic thrombocytopenic purpura, but the interaction has been proposed to have important pathogenic roles in disparate syndromes such as sepsis, HELLP syndrome, antiphospholipid syndrome, acute lung injury, sickle cell anemia, and cerebral malaria. These syndromes have in common an association with severe inflammation, one of the consequences of which is production of oxidants, in particular by neutrophils. We recently showed that one of the most potent neutrophil oxidants, hypochlorous acid (HOCl), which is produced by the myeloperoxidase-catalyzed reaction of H2O2 with chloride ion, markedly reduces ADAMTS13 proteolysis of VWF by oxidizing M1606 at the ADAMTS13 cleavage site within the A2 domain of VWF (Blood, 115(3) 706-12, 2010). In that study, M1606 present in a substrate A2 peptide was readily oxidized by HOCl, but only minimally oxidized in multimeric plasma VWF, except in the presence of the denaturing agent urea. As this requirement resembled the requirement of urea for ADAMTS13 proteolysis of plasma VWF, we wondered whether the application of shear stress would similarly enhance M1606 oxidation by HOCl. Using a system containing 25 nM MPO (a plasma concentration often seem in inflammatory conditions) and varying concentrations of H2O2, we found that application of 0.6 dynes/cm2 shear stress through a closed circuit of plastic tubing rendered M1606 much more sensitive to oxidation: 80% oxidized within 1 hr. This suggestion of shear-induced unfolding and enhanced oxidation was verified when we examined 7 other methionine residues in the A1A2A3 region of VWF, the region containing the binding sites for platelets and collagen and the ADAMTS13 cleavage site. The Met residues were variably sensitive to oxidation, but all became increasingly oxidized over time in the presence of shear stress. Although the shear stresses we used in this experiment are far below the shear stress considered necessary to unfold even very large VWF multimers, the VWF solution also experienced constant elongational flow generated by a peristaltic pump, necessitating flow acceleration through the region narrowed by the rollers. Elongational flow can impart up to 100-fold more tensile stress to suspended VWF than the constant shear stress (Biophys. J., 98 L35, 2010). Two other findings favor the interpretation that oxidation of the A1A2A3 region is facilitated by domain unfolding. First, we further separated the oxidized VWF by gel-filtration into large, intermediate, and small multimeric fractions and found that methionine oxidation was much more prevalent in the fraction with the largest multimers and rare in the fraction with the smallest multimers. Second, we found that ristocetin, a VWF modulator that simulates the effect of shear stress on VWF, also accelerated oxidation of M1606. In functional tests, we found that HOCl-oxidized plasma VWF agglutinated fixed platelets at concentrations of ristocetin that induced minimal agglutination using unoxidized VWF. These findings have several important clinical implications. First, inflammatory conditions will not only activate endothelial cells and induce release of VWF, especially the largest and most adhesive forms (ultralarge VWF), the oxidants produced from endothelial cells themselves and from the neutrophil respiratory burst will render the VWF resistant to proteolysis. Second, these same oxidants will also convert the largest preexisting plasma VWF multimers that were previously rendered quiescent by ADAMTS13, into hyperfunctional and uncleavable forms. All of these mechanisms converge to generate a highly prothrombotic state, perhaps initially evolved as a mechanism to trap and isolate microorganisms, but which also has the potential to cause tremendous harm to those affected by these inflammatory conditions. Disclosures: No relevant conflicts of interest to declare.

Ristocetin Exposes the ADAMTS13 Cleavage Site of Von Willebrand Factor and Enhances VWF Cleavage

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4317-4317
Author(s):  
Junmei Chen ◽  
Min hua Ling ◽  
José A. López ◽  
Dominic W. Chung
Keyword(s):  
Shear Stress ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Cleavage Site ◽  
Tensile Force ◽  
The Other ◽  
Peptide Antibiotic ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 4317 Ristocetin, a peptide antibiotic from the soil bacterium Nocardia lurida, has been used for decades as a tool to diagnose deficiency or dysfunction of von Willebrand factor (VWF) in von Willebrand disease. Ristocetin is able to assess the functional state of VWF because it induces the interaction of VWF with the platelet glycoprotein (GP) Ib-IX-V complex in the absence of shear stress or VWF immobilization, conditions normally required in vivo for their interaction. Presumably, ristocetin is able to do this by inducing an allosteric change in VWF that exposes the binding site for GPIbα. Ristocetin is one of two widely used modulators of the VWF–GPIb α interaction (the other being botrocetin), and the one that induces an interaction that most closely mimics shear-induced platelet adhesion and aggregation. Recently, Shim et al, (Blood, 2008;111(2):651-7) demonstrated that VWF bound to platelets was a better substrate for the plasma metalloprotease ADAMTS13, raising the possibility that exposure of the GPIbα binding site on VWF could be coupled to exposure of the ADAMTS13 cleavage site. Another possibility would be that the tensile force experienced by a VWF strand with multiple bound platelets in a shear field would be sufficient to stretch VWF and expose the ADAMTS13 cleavage site. We therefore evaluated whether ristocetin alone could enhance ADAMTS13 cleavage of VWF in the absence of shear force. We used four VWF sources for these experiments: plasma; purified, multimeric VWF from plasma; a recombinant fragment encompassing the three A domains (A1A2A3); and two recombinant A2 domains, one containing a previously identified ristocetin-binding site between D1459 and P1465, and the other lacking it. Ristocetin at 1.0 mg/ml induced the cleavage of VWF by ADAMTS13 in plasma or of the purified multimeric form as efficiently as did 1.5 M urea, the standard reagent and concentration used for this assay. Similarly, ristocetin accelerated cleavage of the monomeric A1A2A3 fragment. Finally, and somewhat surprisingly, ristocetin accelerated cleavage of the isolated A2 domain, but only when the D1459–P1465 sequence was included in the construct. Vancomycin, a related antibiotic, did not have this effect. Our data suggest that exposure of the ADAMTS13 cleavage site is not only induced by tensile force in vivo, but also by other more subtle biochemical forces. These findings also indicate that exposure of the binding site for GPIbα is coupled to exposure of the ADAMTS13 cleavage site in VWF, perhaps providing part of the explanation for why platelet-bound VWF is a better ADAMTS13 substrate and for why newly released ultralarge VWF is both capable of spontaneously binding platelets and of being cleaved rapidly by ADAMTS13 in the presence of minimal shear stress. Finally, our findings also suggest that ristocetin might be a more specific reagent to evaluate the activity of ADAMTS13 for cleaving multimeric VWF in vitro. Disclosures: No relevant conflicts of interest to declare.

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