Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module.

ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbß antibody derivative.

BACKGROUND: Glycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb-IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R-IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb-IX-mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild-type mice. Previously, an anti-GPIbß antibody RAM.1 was developed. RAM.1 inhibits GPIbα-dependent platelet signaling and activation. Further, RAM.1 also inhibits anti-GPIbα antibody-mediated filopodia formation. OBJECTIVE: To investigate whether RAM.1 can ameliorate trigger sequence exposure-mediated platelet clearance. METHODS: Spontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. RESULT: Transfected Chinese hamster ovary cells stably expressing the same chimeric IL4R-Ibα protein complex as in IL4R-IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R-IbαTg platelets. CONCLUSION: These results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb-IX-mediated thrombocytopenia.

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

Von 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.