scholarly journals Von Willebrand factor A1 domain affinity for GPIbα and stability are differentially regulated by its O-glycosylated N-linker and C-linker

2022 ◽  
Author(s):  
Roxana Iacob ◽  
Klaus Bonazza ◽  
Nathan Hudson ◽  
Jing Li ◽  
Chafen Lu ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Intermediate State ◽  
Energy Gap ◽  
Tensile Force ◽  
Hydrodynamic Drag ◽  
Arterial Circulation ◽  
Hydrogen Deuterium ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.

Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association

Blood ◽  
2021 ◽  
Vol 138 (23) ◽  
pp. 2425-2434
Author(s):  
Hongxia Fu ◽  
Yan Jiang ◽  
Wesley P. Wong ◽  
Timothy A. Springer
Keyword(s):  
Von Willebrand Factor ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Tensile Force ◽  
Sharp Decrease ◽  
Hydrodynamic Drag ◽  
High Tension ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) is an ultralong concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to 0 at the downstream free end. Using single-molecule fluorescence microscopy, we directly visualized the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules. We showed that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF, reaches half maximum at a characteristic tension of ∼10 pN, and plateaus above ∼25 pN. Association is reversible and hence noncovalent; a sharp decrease in shear flow results in rapid dissociation of bound VWF. Tethered primary VWF molecules can recruit more than their own mass of secondary VWF molecules from the flow stream. Kinetics show that instead of accelerating, the rate of accumulation decreases with time, revealing an inherently self-limiting self-association mechanism. We propose that this may occur because multiple tether points between secondary and primary VWF result in lower tension on the secondary VWF, which shields more highly tensioned primary VWF from further association. Glycoprotein Ibα (GPIbα) binding and VWF self-association occur in the same region of high tension in tethered VWF concatemers; however, the half-maximal tension required for activation of GPIbα is higher, suggesting differences in molecular mechanisms. These results have important implications for the mechanism of platelet plug formation in hemostasis and thrombosis.

Platelet-VWF complexes get the chop

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 475-475
Author(s):  
Michael C. Berndt ◽  
Robert K. Andrews
Keyword(s):  
Ligand Binding ◽  
Von Willebrand Factor ◽  
Tensile Force ◽  
Inhibitory Effect ◽  
Platelet Glycoprotein ◽  
A1 Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Binding Subunit

In this issue of Blood, Shim and colleagues define a dual role for platelet glycoprotein (GP)Ibα (the major ligand-binding subunit of the GPIb-IX-V complex) in regulating ADAMTS13-mediated cleavage of von Willebrand factor (VWF) under shear: it alleviates an inhibitory effect of the VWF A1 domain on cleavage of the A2 domain,1 and it allows tensile force to be exerted on the A2 domain through at least 2 platelets binding per VWF multimer via the A1 domain (see figure).

scholarly journals Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicholas A. Arce ◽  
Wenpeng Cao ◽  
Alexander K. Brown ◽  
Emily R. Legan ◽  
Moriah S. Wilson ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Single Molecule ◽  
Tensile Force ◽  
Conformational Dynamics ◽  
Subsequent Activation ◽  
Mechanically Stabilized ◽  
Responsive Element ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractVon Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.

A Short N-Terminal Fragment in Front of the A1 Domain of Von Willebrand Factor Prevents the A1 from Binding the Platelet By Masking a Region Clustered By Type 2B VWD Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 553-553
Author(s):  
Wei Deng ◽  
Yingchun Wang ◽  
Pete Lollar ◽  
Renhao Li
Keyword(s):  
Blood Vessel ◽  
Von Willebrand Factor ◽  
Deuterium Exchange ◽  
The Other ◽  
Hydrogen Deuterium Exchange ◽  
Gain Of Function ◽  
Hydrogen Deuterium ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Blood glycoprotein von Willebrand Factor (VWF) is constantly circulating in the blood vessel, with its platelet GPIb-IX recognizing unit-the A1 domain-being masked to avoid its adventitious binding and activating platelets. This "self-mask" mechanism, however, remains poorly understood. The impediment in identifying the self-mask ligand is to have the right pair of VWF fragments with opposite binding behavior for comparison. Here we characterize using hydrogen-deuterium exchange mass spec a pair of VWF fragments containing the A1 domain with opposite association to platelets. The results suggest that the short N-terminal region (1238-1260) in front of the A1 domain might involve in protecting the VWF A1 domain by masking a critical region near the binding site for GPIb. We expressed and purified using baby hamster kidney cells two C-terminally His-tagged proteins: both contain the entire A1 domain (1273-1453), with one has more extra N-terminal residues (1238-1472, named lA1) than the other (1261-1472, named sA1). Both proteins seem to be glycosylated and stable without obvious deformation for over one week when stored at 4°C. The analytical ultracentrifugation analysis determined that both proteins are primarily in the monomeric form: the sedimentation coefficient (S) and the derived apparent molecular weight for lA1 are 2.50 S and 36.6 kDa, and those for sA1 are 1.37 S and 27.4 kDa. Interaction of lA1 and sA1 to GPIb, as determined by flow cytometry, is surprisingly opposite: only sA1, but not lA1, efficiently binds washed platelets at the concentration similar to the physiological concentration of vWF in the blood vessel. Increasing lA1 concentration 10 times did not improve its binding. Replacing the platelets with CHO cells that express human GPIb-IX complex exhibited similar result. Implied in these results are that either lA1 adopt a different conformation that disadvantages its binding or the binding site on A1 is masked by the extra amino acids in lA1 compared to sA1. Protein hydrogen-deuterium exchange is a sensitive assay to determine the local conformational change and the existence of the masked region. We acquired hydrogen-deuterium exchange data for both proteins and got nearly 100% sequences coverage from over 150 overlaid peptides for each protein, with on average over 10 redundancy per residue, which allows us to compare the hydrogen-deuterium exchange rate/extent of the A1 domain in lA1 and sA1 in good resolution. The results show that the global hydrogen-deuterium exchange pattern is the same between the two, indicating that the tertiary structures of the two proteins are similar. However, a contiguous region in lA1 is clearly protected from hydrogen-deuterium exchange compared to that in sA1 (indicated by the colorful region in figure 1). The protected region is located at the bottom of the globular A1 domain, including three loop regions (1272-1276; 1304-1310; and 1332-1340) and a part of the a1 helix (1289-1300). Compared to sA1, in this protected region of lA1 the deuterium exchange extents are reduced by 20-60% even after 3-hour incubation. Furthermore, 11 of out of 15 identified "gain-of-function" mutation sites of type 2B VWD are located in this masked region (P1266, H1268, C1272, M1304, R1306, R1308, I1309, S1310, W1313, P1337, R1341); the other 4 are located adjacently (V1314, V1316, L1460, A1461). This good alignment implies that the "gain-of-function" might associate with the detachment of the identified masking ligand in this region. Based on our findings, we propose that this short region in front of the A1 domain may involve in masking the A1 domain from interacting with platelet GPIb-IX. If confirmed, this internal masking ligand might offer new clues on how the mutations on A1 domain affect its role -as "gain-of-function" in type 2B VWD and "loss-of-function" in type 2M VWD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease

2020 ◽  
Vol 432 (2) ◽  
pp. 305-323 ◽  
Author(s):  
Alexander Tischer ◽  
Maria A. Brehm ◽  
Venkata R. Machha ◽  
Laurie Moon-Tasson ◽  
Linda M. Benson ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study

2021 ◽  
Vol 6 (1) ◽  
pp. e000703
Author(s):  
Taleen A MacArthur ◽  
Julie Goswami ◽  
Laurie Moon Tasson ◽  
Alexander Tischer ◽  
Kent R Bailey ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Acute Phase ◽  
Von Willebrand Factor ◽  
Thrombin Generation ◽  
Trauma Patients ◽  
Phase Reaction ◽  
Time Points ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

BackgroundVon Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous VWF–platelet interaction. After trauma, ULVWF is released into circulation as part of the acute phase reaction. We hypothesized that trauma patients would have increased levels of VWF and decreased levels of ADAMTS-13 and that these patients would have accelerated thrombin generation.MethodsWe assessed plasma concentrations of VWF antigen and ADAMTS-13 antigen, the Rapid Enzyme Assays for Autoimmune Diseases (REAADS) activity of VWF, which measure exposure of the platelet-binding A1 domain, and thrombin generation kinetics in 50 samples from 30 trauma patients and an additional 21 samples from volunteers. Samples were analyzed at 0 to 2 hours and at 6 hours from the time of injury. Data are presented as median (IQR) and Kruskal-Wallis test was performed between trauma patients and volunteers at both time points.ResultsREAADS activity was greater in trauma patients than volunteers both at 0 to 2 hours (190.0 (132.0–264.0) vs. 92.0 (71.0–114.0), p<0.002) and at 6 hours (167.5 (108.0–312.5.0) vs. 92.0 (71.0–114.0), p<0.001). ADAMTS-13 antigen levels were also decreased in trauma patients both at 0 to 2 hours (0.84 (0.51–0.94) vs. 1.00 (0.89–1.09), p=0.010) and at 6 hours (0.653 (0.531–0.821) vs. 1.00 (0.89–1.09), p<0.001). Trauma patients had accelerated thrombin generation kinetics, with greater peak height and shorter time to peak than healthy volunteers at both time points.DiscussionTrauma patients have increased exposure of the VWF A1 domain and decreased levels of ADAMTS-13 compared with healthy volunteers. This suggests that the VWF burst after trauma may exceed the proteolytic capacity of ADAMTS-13, allowing circulating ULVWF multimers to bind platelets, potentially contributing to trauma-induced coagulopathy.Level of evidenceProspective case cohort study.

scholarly journals Humanized GPIbα–von Willebrand factor interaction in the mouse

2018 ◽  
Vol 2 (19) ◽  
pp. 2522-2532 ◽  
Author(s):  
Sachiko Kanaji ◽  
Jennifer N. Orje ◽  
Taisuke Kanaji ◽  
Yuichi Kamikubo ◽  
Yosuke Morodomi ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Von Willebrand Factor ◽  
Mouse Strain ◽  
Platelet Rich Plasma ◽  
Platelet Glycoprotein ◽  
Transgenic Mouse Strain ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Knockin Mouse

Abstract The interaction of platelet glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) initiates hemostasis after vascular injury and also contributes to pathological thrombosis. GPIbα binding to the VWF A1 domain (VWFA1) is a target for antithrombotic intervention, but attempts to develop pharmacologic inhibitors have been hindered by the lack of animal models because of the species specificity of the interaction. To address this problem, we generated a knockin mouse with Vwf exon 28–encoding domains A1 and A2 replaced by the human homolog (VWFh28). VWFh28 mice (M1HA) were crossbred with a transgenic mouse strain expressing human GPIbα on platelets (mGPIbαnull;hGPIbαTg; H1MA) to generate a new strain (H1HA) with humanized GPIbα-VWFA1 binding. Plasma VWF levels in the latter 3 strains were similar to those of wild-type mice (M1MA). Compared with the strains that had homospecific GPIbα-VWF pairing (M1MA and H1HA), M1HA mice of those with heterospecific pairing had a markedly greater prolongation of tail bleeding time and attenuation of thrombogenesis after injury to the carotid artery than H1MA mice. Measurements of GPIbα-VWFA1 binding affinity by surface plasmon resonance agreed with the extent of observed functional defects. Ristocetin-induced platelet aggregation was similar in H1HA mouse and human platelet-rich plasma, and it was comparably inhibited by monoclonal antibody NMC-4, which is known to block human GPIbα-VWFA1 binding, which also inhibited FeCl3-induced mouse carotid artery thrombosis. Thus, the H1HA mouse strain is a fully humanized model of platelet GPIbα-VWFA1 binding that provides mechanistic and pharmacologic information relevant to human hemostatic and thrombotic disorders.

Modulation by Heparin of the Interaction of the A1 Domain of von Willebrand Factor With Glycoprotein Ib

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4186-4194 ◽  
Author(s):  
Christelle Perrault ◽  
Nadine Ajzenberg ◽  
Paulette Legendre ◽  
Ghassem Rastegar-Lari ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Cell Aggregation ◽  
Cell Aggregates ◽  
Critical Determinant ◽  
Amino Terminal ◽  
A Cell ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Amino Terminal Fragment

Abstract The conformation of the A1 domain of von Willebrand factor (vWF) is a critical determinant of its interaction with the glycoprotein (GP) Ib/V/IX complex. To better define the regulatory mechanisms of vWF A1 domain binding to the GPIb/V/IX complex, we studied vWF-dependent aggregation properties of a cell line overexpressing the GPIb, GPIbβ, and GPIX subunits (CHO-GPIbβ/IX cells). We found that CHO-GPIbβ/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbβ/IX cell aggregation was completely inhibited by the recombinant VCL fragment of vWF that contains the A1 domain. Surprisingly, the substitution of heparin for ristocetin resulted in the formation of CHO-GPIbβ/IX cell aggregates. Using monoclonal antibodies blocking vWF interaction with GPIb/V/IX or mocarhagin, a venom metalloproteinase that removes the amino-terminal fragment of GPIb extending from aa 1 to 282, we demonstrated that both ristocetin- and heparin-induced aggregations involved an interaction between the A1 domain of vWF and the GPIb subunit of the GPIb/V/IX complex. The involvement of heparin in cell aggregation was also demonstrated after treatment of heparin with heparinase that abolished CHO-GPIbβ/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbβ/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.

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.

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