Structure and dynamics of the platelet integrin-binding C4 domain of von Willebrand factor.
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ABSTRACT:
SUBMITTER: Xu ER
PROVIDER: S-EPMC6450055 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCollagen interactions with von Willebrand factor (VWF) perform an important role in initiation of hemostasis.ObjectivesWe hypothesized that in addition to collagen, other extracellular matrix (ECM) proteins such as fibronectin can bind VWF.MethodsFibronectin-VWF interactions were measured by ELISA using both plasma-derived and recombinant VWF-containing variants in specific domains. Inhibition was measured by antibody competition using antibodies directed against both VWF and fibronectin. Binding affinities were measured by the Octet Biosensor for fibronectin and collagen IV.ResultsFibronectin was able to bind both plasma-derived and recombinant wild-type VWF. This interaction was inhibited by both anti-VWF antibodies and collagen types III and IV. Several VWF A1 domain variants in the region of the collagen IV binding site also demonstrated absent fibronectin binding, as did variants with defects in high-molecular-weight multimers. Binding affinity testing showed fibronectin has a strong affinity for VWF, in a range similar to that of collagen IV. Fibronectin binds VWF via a restricted region of the A1 domain. This interaction requires high-molecular-weight multimers and is similar to that seen with vascular collagens.ConclusionsTherefore, VWF would appear to be the common factor linking platelet adhesion to various ECM proteins and facilitating hemostasis under conditions of ECM exposure.
| S-EPMC8178691 | biostudies-literature
Project description:Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY:Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.
| S-EPMC5949882 | biostudies-literature
Project description:Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIb?.
| S-EPMC3367877 | biostudies-other
Project description:von Willebrand factor (vWF) is a multimeric plasma glycoprotein with three tandem A domains. Domains A1 and A3 bind to platelet glycoprotein Ibalpha (GPIbalpha) and collagen, respectively. Domain A2 contains the Tyr-1605-Met-1606 bond that is cleaved by the metalloprotease ADAMTS13, and this reaction inhibits platelet thrombus growth. Fluid shear stress increases the rate of cleavage, suggesting that productive interaction with ADAMTS13 requires conformational changes within or near domain A2. The influence of the adjacent A1 and A3 domains was assessed by mutagenesis of a recombinant substrate consisting of domains A1A2A3. Deletion of domain A3 did not affect cleavage by ADAMTS13, whereas deletion of domain A1 increased the rate of cleavage approximately 10-fold. Similar effects were observed with plasma ADAMTS13 and recombinant ADAMTS13 truncated after the spacer domain. Digestion of A1A2A3 by plasma ADAMTS13 was enhanced to a similar extent by a recombinant mutant fragment of platelet GPIbalpha that binds with high affinity to domain A1 or by heparin. Heparin also increased the digestion of purified plasma vWF. Neither GPIbalpha nor heparin increased the cleavage of substrate A2A3 that lacks domain A1. The results suggest that vWF domain A1 inhibits the cleavage of domain A2, and that inhibition can be relieved by interaction of domain A1 with platelet GPIbalpha or certain glycosaminoglycans. Thus, binding of vWF to its major physiological ligands may promote the feedback inhibition of platelet adhesion by stimulating the cleavage of domain A2 by ADAMTS13 independent of fluid shear stress.
| S-EPMC489977 | biostudies-literature
Project description:The von Willebrand factor (VWF) A1 and A3 domains are structurally isomorphic yet exhibit distinct mechanisms of unfolding. The A1 domain, responsible for platelet adhesion to VWF in hemostasis, unfolds through a molten globule intermediate in an apparent three-state mechanism, while A3 unfolds by a classical two-state mechanism. Inspection of the sequences or structures alone does not elucidate the source of this thermodynamic conundrum; however, the three-state character of the A1 domain suggests that it has more than one cooperative substructure yielding two separate unfolding transitions not present in A3. We investigate the extent to which structural elements contributing to intermediate conformations can be identified using a residue-specific implementation of the structure-energy-equivalence-of-domains algorithm (SEED), which parses proteins of known structure into their constituent thermodynamically cooperative components using protein-group-specific, transfer free energies. The structural elements computed to contribute to the non-two-state character coincide with regions where Von Willebrand disease mutations induce misfolded molten globule conformations of the A1 domain. This suggests a mechanism for the regulation of rheological platelet adhesion to A1 based on cooperative flexibility of the α2 and α3 helices flanking the platelet GPIbα receptor binding interface.
| S-EPMC4621621 | biostudies-literature
Project description:The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.
| S-EPMC8876638 | biostudies-literature
Project description:BackgroundA molecular basis for von Willebrand factor (VWF) self-inhibition has been proposed by which the N-terminal and C-terminal flanking sequences of the globular A1 domain disulfide loop bind to and suppress the conformational dynamics of A1. These flanking sequences are rich in O-linked glycosylation (OLG), which is known to suppress platelet adhesion to VWF, presumably by steric hindrance. The inhibitory mechanism remains unresolved as to whether inhibition is due to steric exclusion by OLGs or a direct self-association interaction that stabilizes the domain.ObjectivesThe platelet adhesive function, thermodynamic stability, and conformational dynamics of the wild-type and type 2M G1324S A1 domain lacking glycosylation (Escherichia coli) are compared with the wild-type glycosylated A1 domain (HEK293 cell culture) to decipher the self-inhibitory mechanism.MethodsSurface plasmon resonance and analytical rheology are utilized to assess Glycoprotein Ibα (GPIbα) binding at equilibrium and platelet adhesion under shear flow. The conformational stability is assessed through a combination of protein unfolding thermodynamics and hydrogen-deuterium exchange mass spectrometry (HXMS).ResultsA1 glycosylation inhibits both GPIbα binding and platelet adhesion. Glycosylation increases the hydrodynamic size of A1 and stabilizes the thermal unfolding of A1 without changing its equilibrium stability. Glycosylation does not alter the intrinsic conformational dynamics of the A1 domain.ConclusionsThese studies invalidate the proposed inhibition through conformational suppression since glycosylation within these flanking sequences does not alter the native state stability or the conformational dynamics of A1. Rather, they confirm a mechanism by which glycosylation sterically hinders platelet adhesion to the A1 domain at equilibrium and under rheological shear stress.
| S-EPMC6940534 | biostudies-literature
Project description:Binding of the A1 domain of von Willebrand factor (vWF) to glycoprotein Ibα (GPIbα) results in platelet adhesion, activation, and aggregation that initiates primary hemostasis. Both the elevated shear stress and the mutations associated with type 2B von Willebrand disease enhance the interaction between A1 and GPIbα. Through molecular dynamics simulations for wild-type vWF-A1 and its eight gain of function mutants (R543Q, I546V, ΔSS, etc.), we found that the gain of function mutations destabilize the N-terminal arm, increase a clock pendulum-like movement of the α2-helix, and turn a closed A1 conformation into a partially open one favoring binding to GPIbα. The residue Arg(578) at the α2-helix behaves as a pivot in the destabilization of the N-terminal arm and a consequent dynamic change of the α2-helix. These results suggest a localized dynamics-driven affinity regulation mechanism for vWF-GPIbα interaction. Allosteric drugs controlling this intrinsic protein dynamics may be effective in blocking the GPIb-vWF interaction.
| S-EPMC3772212 | biostudies-literature
Project description:BACKGROUND:von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. OBJECTIVES:We report here on several mutations that exclusively alter binding to type VI collagen. PATIENTS/METHODS:Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. RESULTS:Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. CONCLUSIONS:VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.
| S-EPMC3809952 | biostudies-literature
Project description:It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s(-1)) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf (-/-) mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf (-/-) and Vwf (+/+) blood, exposed to VWF at 1800 s(-1) in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf(-/-) mice than from Vwf(+/+) mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf(-/-) mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s(-1) on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.
| S-EPMC3667798 | biostudies-literature