Publications

    De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8
    Roy, A., et al. De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8. Nat Commun 14, 1, 5660 (2023).Abstract
    The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
    Structural insights into MIC2 recognition by MIC2-associated protein in Toxoplasma gondii
    Zhang, S., et al. Structural insights into MIC2 recognition by MIC2-associated protein in Toxoplasma gondii. Commun Biol 6, 1, 895 (2023).Abstract
    Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.
    A recombinant technique for mapping functional sites of hetero-trimeric collagen helices: collagen IV CB3 fragment as a prototype for integrin binding
    Boudko, S.P., et al. A recombinant technique for mapping functional sites of hetero-trimeric collagen helices: collagen IV CB3 fragment as a prototype for integrin binding. J Biol Chem 104901 (2023).Abstract
    Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 hetero-trimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The hetero-trimeric fragments encompassed the CB3 trimeric peptide of collagen IV which harbors the binding motifs for α1β1 and α2β1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high yield production of hetero-trimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.
    A specialized integrin-binding motif enables proTGF-β2 activation by integrin αVβ6 but not αVβ8
    Le, V.Q., et al. A specialized integrin-binding motif enables proTGF-β2 activation by integrin αVβ6 but not αVβ8. Proc Natl Acad Sci U S A 120, 24, e2304874120 (2023).Abstract
    Activation of latent transforming growth factor (TGF)-β2 is incompletely understood. Unlike TGF-β1 and β3, the TGF-β2 prodomain lacks a seven-residue RGDLXX (L/I) integrin-recognition motif and is thought not to be activated by integrins. Here, we report the surprising finding that TGF-β2 contains a related but divergent 13-residue integrin-recognition motif (YTSGDQKTIKSTR) that specializes it for activation by integrin αVβ6 but not αVβ8. Both classes of motifs compete for the same binding site in αVβ6. Multiple changes in the longer motif underlie its specificity. ProTGF-β2 structures define interesting differences from proTGF-β1 and the structural context for activation by αVβ6. Some integrin-independent activation is also seen for proTGF-β2 and even more so for proTGF-β3. Our findings have important implications for therapeutics to αVβ6 in clinical trials for fibrosis, in which inhibition of TGF-β2 activation has not been anticipated.
    Conformational change of Plasmodium TRAP is essential for sporozoite migration and transmission
    Braumann, F., et al. Conformational change of Plasmodium TRAP is essential for sporozoite migration and transmission. EMBO Rep e57064 (2023).Abstract
    Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal.
    Single-molecule characterization of subtype-specific β1 integrin mechanics
    Jo, M.H., et al. Single-molecule characterization of subtype-specific β1 integrin mechanics. Nat Commun 13, 1, 7471 (2022).Abstract
    Although integrins are known to be mechanosensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin α4β1 and RGD-binding integrins (αVβ1 and α5β1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of α4β1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of αVβ1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.
    A general chemical principle for creating closure-stabilizing integrin inhibitors
    Lin, F.-Y., et al. A general chemical principle for creating closure-stabilizing integrin inhibitors. Cell 185, 19, 3533-3550.e27 (2022).Abstract
    Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbβ3 and α4β1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbβ3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4β1.
    Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange
    Anderson, J.R., Li, J., Springer, T.A. & Brown, A. Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange. Blood (2022).Abstract
    von Willebrand Factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excess bleeding of type 2A von Willebrand (VW) disease. Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report cryo-EM structures of complete VWF tubules before and after intermolecular disulfide-bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states prior to and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VW disease-causing mutations.
    Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state
    Feng, J., Dong, X., Su, Y., Lu, C. & Springer, T.A. Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state. Nat Commun 13, 1, 4064 (2022).Abstract
    Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.
    Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change
    Su, Y., Iacob, R.E., Li, J., Engen, J.R. & Springer, T.A. Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change. J Biol Chem 102323 (2022).Abstract
    Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.
    Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging
    Bergal, H.T., Jiang, Y., Yang, D., Springer, T.A. & Wong, W.P. Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging. Blood (2022).Abstract
    von Willebrand factor (VWF) is a multimeric blood protein that acts as a mechanical probe, responding to changes in flow to initiate platelet plug formation. Previously, our labs had shown using single-molecule imaging that shear stress can extend surface-tethered VWF, but paradoxically we found that the required shear stress was higher than reported for free-in-flow VWF-an observation inconsistent with basic physical principles. To resolve this inconsistency critical to VWF's molecular mechanism, we measured free VWF extension in shear flow using PULSIS-Pulsed Laser Stroboscopic Imaging of Single molecules. Here, laser pulses of different durations are used to capture multiple images of the same molecule within each frame, enabling accurate length measurements in the presence of motion blur. At high shear stresses, we observed a mean shift in VWF extension of less than 200 nm, much shorter than the multiple-micron extensions previously reported with no evidence for the predicted sharp globule-stretch conformational transition. Modeling VWF with a Brownian dynamics simulation, our results are consistent with VWF behaving as an uncollapsed polymer rather than the theorized compact ball. The muted response of free VWF to high shear rates implies that 1) the tension experienced by free VWF in physiological shear flow is lower than indicated by previous reports and 2) that tethering to platelets or the vessel wall is required to mechanically activate VWF adhesive function for primary hemostasis.
    Loss of LRRC33-dependent TGFβ1 activation enhances antitumor immunity and checkpoint blockade therapy
    Jiang, A., Qin, Y. & Springer, T.A. Loss of LRRC33-dependent TGFβ1 activation enhances antitumor immunity and checkpoint blockade therapy. Cancer Immunol Res 10, 4, 453-467 (2022).Abstract
    TGFβ has multiple roles and gene products (TGFβ1, -β2, and -β3), which make global targeting of TGFβ undesirable. Expression of TGFβ requires association with milieu molecules, which localize TGFβ to the surface of specific cells or extracellular matrices. Here, we found that LRRC33 was specifically associated with TGFβ1, not TGFβ2 and TGFβ3, and was required for surface display and activation of TGFβ1 on tumor-infiltrating myeloid cells. Loss of LRRC33-dependent TGFβ1 activation slowed tumor growth and metastasis by enhancing innate and adaptive antitumor immunity in multiple mouse syngeneic tumor models. LRRC33 loss resulted in a more immunogenic microenvironment, with decreased myeloid-derived suppressor cells, more active CD8+ T and NK cells, and more skewing toward tumor-suppressive M1 macrophages. LRRC33 loss and PD-1 blockade synergized in controlling B16.F10 tumor growth. Our results demonstrate the importance of LRRC33 in tumor biology and highlight the therapeutic potential of dual blockade of the LRRC33/TGFβ1 axis and PD-1/PD-L1 in cancer immunotherapy.
    Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
    Bonazza, K., et al. Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker. Elife 11, (2022).Abstract
    Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) 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.
    Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities
    Anderson, J.M., Li, J. & Springer, T.A. Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities. Mol Biol Cell mbcE21110536 (2022).Abstract
    Activation of integrins by Mn2+ is a benchmark in the integrin field, but how it works and whether it reproduces physiologic activation is unknown. We show that Mn2+ and high Mg2+ concentrations compete with Ca2+ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg2+ by Mn2+ at the MIDAS increases the intrinsic affinities of both the high affinity open and low affinity closed states of integrins, in agreement with stronger binding of Mn2+ than Mg2+ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on- and off-states, respectively.
    Protection of the Prodomain α1-Helix Correlates with Latency in the Transforming Growth Factor-β Family
    Le, V.Q., et al. Protection of the Prodomain α1-Helix Correlates with Latency in the Transforming Growth Factor-β Family. J Mol Biol 434, 5, 167439 (2022).Abstract
    The 33 members of the transforming growth factor beta (TGF-β) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-β1 and TGF-β2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-β family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.
    Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2
    Feng, J., et al. Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2. Elife 10, (2021).Abstract
    HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.
    Low affinity integrin states have faster ligand binding kinetics than the high affinity state
    Li, J., Yan, J. & Springer, T. Low affinity integrin states have faster ligand binding kinetics than the high affinity state. eLife 10, e73359, (2021). Publisher's VersionAbstract
    Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by "inside-out signaling".
    Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6
    Renella, R., et al. Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6. Am J Hematol (2021).Abstract
    Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.
    Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association
    Fu, H., Jiang, Y., Wong, W. & Springer, T.A. Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association. Blood 138, 23, 2425-2434 (2021). Publisher's VersionAbstract
    Von Willebrand factor (VWF) is an ultra-long 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 zero at the downstream, free end. Using single-molecule fluorescence microscopy, we directly visualize the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules and show 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 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 be 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. 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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