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

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.

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

Blood ◽

10.1182/blood.2021012595 ◽

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.

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Correlating Conformational Dynamics with the Von Willebrand Factor Reductase Activity of Factor H Using Single Molecule Force Measurements

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.8b06153 ◽

2018 ◽

Vol 122 (47) ◽

pp. 10653-10658 ◽

Cited By ~ 1

Author(s):

Sithara S. Wijeratne ◽

Leticia Nolasco ◽

Jingqiang Li ◽

Kevin Jiang ◽

Joel L. Moake ◽

...

Keyword(s):

Von Willebrand Factor ◽

Single Molecule ◽

Reductase Activity ◽

Conformational Dynamics ◽

Factor H ◽

Force Measurements ◽

Von Willebrand ◽

Willebrand Factor

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Platelet-VWF complexes get the chop

Blood ◽

10.1182/blood-2007-10-116012 ◽

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).

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Activation of A1 Domain Adhesiveness in von Willebrand Factor by Elongational Force

Blood ◽

10.1182/blood.v120.21.sci-16.sci-16 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-16-SCI-16

Author(s):

Timothy A. Springer ◽

Jongseong Kim

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Von Willebrand Factor ◽

Single Molecule ◽

Conflicts Of Interest ◽

Stable State ◽

Receptor Ligand ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract SCI-16 Multiple mechanisms may contribute to activation of von Willebrand factor (vWF) adhesiveness by elongational flow at sites of hemostasis, including enhancement of A1 domain exposure within vWF concatamers and conformational change within the A1 domain or its complex with GPIbα. A receptor and ligand in a single molecule (ReaLiSM) containing the A1 domain, a flexible linker, and GPIbα fused in a single polypeptide and suspended between beads using DNA handles was interrogated with a laser trap. Two pathways for unbinding representing flexed and extended states were previously reported, with the flexed, more stable state predominantly at forces above 10 pN. vWD type 2B mutations in the A1 domain selectively stabilize the extended, high affinity state, whereas platelet-type vWD mutations in GPIbα stabilize both states. With ReaLiSM, we can also measure the kinetics and force-dependence of receptor-ligand binding. Remarkably, we also see two on-rates for receptor-ligand binding, with the faster on-rate predominating above 10 pN and the slower on-rate predominating below 10 pN. vWD type 2B mutations in the A1 domain selectively increased the fast on-rate, whereas platelet-type mutations in GPIbα increased both on-rates. Our results support force-dependent conformational change as one of the mechanisms that activates vWF in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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

10.1101/2022.01.13.476208 ◽

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.

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

Journal of Molecular Biology ◽

10.1016/j.jmb.2019.09.022 ◽

2020 ◽

Vol 432 (2) ◽

pp. 305-323 ◽

Cited By ~ 1

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

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

Trauma Surgery & Acute Care Open ◽

10.1136/tsaco-2021-000703 ◽

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.

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Humanized GPIbα–von Willebrand factor interaction in the mouse

Blood Advances ◽

10.1182/bloodadvances.2018023507 ◽

2018 ◽

Vol 2 (19) ◽

pp. 2522-2532 ◽

Cited By ~ 6

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.

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

Blood ◽

10.1182/blood.v94.12.4186 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4186-4194 ◽

Cited By ~ 9

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.

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