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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: J Thromb Haemost. 2021 May 20;19(8):2044–2055. doi: 10.1111/jth.15359

Differential regulation of the platelet GPIb-IX complex by anti-GPIbβ antibodies

M Edward Quach a, Wenchun Chen a, Yingchun Wang a, Hans Deckmyn b, Francois Lanza c, Bernhard Nieswandt d, Renhao Li a
PMCID: PMC8324530  NIHMSID: NIHMS1701211  PMID: 33915031

Abstract

Background:

Platelets’ initial recognition of endothelial damage proceeds through the interaction between collagen, plasma von Willebrand factor (VWF), and the platelet glycoprotein (GP)Ib-IX complex (CD42). GPIb-IX complex consists of one GPIbα, one GPIX, and two GPIbβ subunits. Once platelets are immobilized to the subendothelial matrix, shear generated by blood flow unfolds a membrane-proximal mechanosensory domain (MSD) in GPIbα, exposing a conserved trigger sequence and activating the receptor. Currently, GPIbα appears to solely facilitate ligand-induced activation, as it contains both the MSD and the binding sites for all known ligands to GPIb-IX. Despite being positioned directly adjacent to the MSD, the roles of GPIbβ and GPIX in signal transduction remain murky.

Objectives:

To characterize a novel rat monoclonal antibody 3G6 that binds GPIbβ.

Methods:

Effects of 3G6 on activation of GPIb-IX are characterized in platelets and Chinese hamster ovary cells expressing GPIb-IX (CHO-Ib-IX) and compared to those of an inhibitory anti-GPIbβ antibody, RAM.1.

Results:

Both RAM.1 and 3G6 bind to purified GPIbβ and GPIb-IX with high affinity. 3G6 potentiates GPIb-IX-associated filopodia formation in platelets or CHO-Ib-IX when they adhere VWF or antibodies against the ligand-binding domain (LBD) of GPIbα. Pre-treatment with 3G6 also increased anti-LBD antibody induced GPIb-IX activation. Conversely, RAM.1 inhibits nearly all GPIb-IX-related signaling in platelets and CHO-Ib-IX cells.

Conclusions:

These data represent the first report of a positive modulator of GPIb-IX activation. The divergent modulatory effects of 3G6 and RAM.1, both targeting GPIbβ, strongly suggest that changes in the conformation of GPIbβ underlie outside-in activation via GPIb-IX.

Keywords: Platelet activation, Glycoprotein Ib-IX complex, Filopodia, Biomechanics, Microscopy

Introduction

Platelets serve a vital role in primary hemostasis as first responders to the site of vascular injury. Platelets’ recognition of endothelial damage proceeds through the glycoprotein (GP)Ib-IX complex (CD42). When the vasculature is damaged, plasma von Willebrand factor (VWF) binds to collagen in the exposed extracellular matrix, undergoes a shear-induced conformational change, and binds GPIb-IX to arrest platelets [1]. GPIb-IX is a highly expressed surface receptor complex consisting of one GPIbα subunit covalently linked to two GPIbβ subunits via disulfide bonds, and tightly associated to GPIX via transmembrane interactions [2–4]. GPIbα is the largest subunit of the complex, responsible for binding to all of GPIb-IX’s known ligands, including thrombin, αMβ2, and plasma von Willebrand factor (VWF) [5–7].

In addition to its role in tethering platelets, GPIb-IX is a major mechanoreceptor complex that mediates platelet activation and aggregation. GPIb-IX activation initiates several platelet phenomena including inside-out activation of αIIbβ3 [8–10], formation of platelet microparticles [11, 12], degranulation [13, 14], desialylation via NEU1 [15, 16], and other procoagulant phenomena [17]. Several intracellular mediators of these signals have been implicated in GPIb-IX signaling, including calcium [8, 18, 19], Src family kinases [20, 21]; PLCγ2 [22], PI3K [21, 23]; mitogen-activated protein kinase (MAPK) pathway [20, 24]; and 14-3-3-ζ [25, 26]. However, despite the establishment of VWF ligation as a key step in GPIb-IX activation and a growing understanding of intracellular mediators of signaling, a unified mechanism by which the activation signal is physically transduced by GPIb-IX remains elusive.

It was recently reported that GPIbα contains a quasi-stable mechanosensory domain (MSD) between the sialomucin region and the transmembrane domain [27]. When VWF engages GPIbα and exerts a pulling force, the MSD becomes unfolded, exposing the trigger sequence therein, and leading to GPIb-IX activation [28–30]. Under the recently proposed “trigger model”, GPIb-IX activation proceeds through a continuous, sustained pulling force applied to the MSD [31]. A ligand that binds to GPIbα with high affinity but low unbinding force (the force threshold an interaction can withstand without rupturing) may become unbound under shear and thus be unable to facilitate MSD unfolding [28]. In contrast with a previous model involving lateral clustering of GPIb-IX in the membrane, the trigger model of GPIb-IX activation offers robust explanations for the diverse functional effects of some anti-GPIb-IX antibodies as well as the requirement of shear for activation [28, 32]. However, the steps following unfolding of the MSD remain unclear.

It is well established that expression of GPIbα, GPIbβ and GPIX is required for surface trafficking of the GPIb-IX complex [2, 33–35]. Although it is possible that GPIbβ and GPIX serve only to stabilize GPIb-IX, reports utilizing mutational analyses and/or monoclonal antibodies (MAbs) against GPIbβ suggest that it participates in GPIb-IX activation and platelet activity [29, 36, 37]. However, it is difficult to disentangle these effects from the overall importance of GPIbβ to the organization and stabilization of the complex. Each copy of GPIbβ in GPIb-IX makes different contacts with the extracellular domain of GPIX and potentially with the MSD of GPIbα [38], and the extracellular domain of GPIbβ can undergo conformational changes in response to changes in these inter-subunit contacts [39]. While no binding sites for endogenous ligands have been identified on the extracellular domains of GPIbβ or GPIX, there are reports that RAM.1, a MAb targeting GPIbβ, can inhibit GPIb-IX activation including associated morphological changes, intracellular Ca2+ spikes, collagen-induced thrombin generation, and thrombus formation [37, 40].

In this study, we report a novel anti-GPIbβ antibody, 3G6, which amplifies the activation of GPIb-IX by VWF and antibodies targeting the LBD. This is the first report of a potentiating modulator of GPIb-IX, and the second MAb targeting GPIbβ that alters GPIb-IX’s activation state. We also demonstrate the ability of RAM.1 to inhibit morphological, cell adhesion, and platelet-clearance effects downstream of GPIb-IX. These data strongly suggest that a change in orientation or conformation of the GPIbβ extracellular domain is a critical step in mechano-transduction by GPIb-IX.

Methods

Materials:

FITC-conjugated Erythrina cristagalli lectin (ECL) (Vector Laboratories), APC-conjugated anti-P-selectin antibody (Biolegend), full-length VWF (Haematologic Technologies), IV.3 (Stem Cell Technologies), and AK2 (GeneTex) were purchased commercially. Antibodies RAM.1 [41] and 6B4 [42] were previously described. Recombinant human GPIbβ extracellular domain (GPIbβE), recombinant human GPIb-IX complex, and Chinese hamster ovary (CHO) cells expressing human GPIb-IX have been previously described [29, 38]. Where CHO-Ib-IX cells were used, high expression of GPIb-IX was confirmed by FACS every ~8 passages. Human GPIb-IX complex, glycocalicin, and snake venom botrocetin were purified as previously described [29, 43, 44].

The 3G6 antibody is a rat IgG1 that was raised against mouse platelets, following a procedure as described before [45]. The single clone was identified by binding to mouse and human platelets, immunoprecipitation of mouse and human GPIb-IX, and by binding to mouse and human GPIbβ in a Western blot analysis of platelet lysates. The antibody was produced by standard cultivation of hybridomas, purified by protein G sepharose affinity chromatography, and stored at −80°C.

Mice:

Transgenic mice expressing only human GPIbα (hTg) have been previously described [46]. Both male and female mice were equally represented. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of Emory University and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Human Platelets:

All procedures using donor-derived human platelets have been approved by the Institutional Review Board of Emory University/Children’s Healthcare of Atlanta (Study No. 00006228). Platelets were isolated from healthy, consenting volunteers including both men and women across a variety of ethnic identities. To obtain washed platelets, platelet rich plasma (PRP) was isolated from whole blood collected in 3.2% sodium citrate by centrifugation. PRP was diluted 1:10 with modified Tyrode’s buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose, 12 mM NaHCO3, 20 mM HEPES, pH 7.35) supplemented with 0.3 μM prostaglandin E1 (PGE1) and centrifuged again to pellet platelets prior to resuspension.

Platelet Aggregometry:

Platelet aggregation was monitored in a dual-channel Chrono-Log aggregometer largely as described previously [47]. Briefly, human PRP in Tyrode’s buffer at 2.5 × 108 platelets/ml was added to the cuvette at 37°C for each measurement. It was incubated with the noted antibody for 15–30 s followed by 0.75 mg/ml ristocetin or buffer control. The aggregation was recorded for 10 min.

Gel Electrophoresis and Western Blot:

Purified GPIbβE or full-length GPIb-IX were denatured via incubation at 95°C for 5 minutes in buffer with or without 2.5% β-mercaptoethanol (reducing vs nonreducing conditions). Following gel electrophoresis on a gradient (4 – 20%) polyacrylamide gel, protein was transferred to a nitrocellulose membrane and blotted with either RAM.1 or 3G6 overnight. Either antibody was then detected with an IRDye-conjugated polyclonal goat-anti-rat Fc secondary antibody (LI-COR). Blot images were captured on an Odyssey CLx imager (LI-COR).

ELISA:

For indirect ELISA, GPIbβE or full-length GPIb-IX were immobilized (2 μg/mL) to a plate overnight at 4°C. RAM.1 or 3G6 were added at a range of concentrations, and detected with an HRP-conjugated polyclonal anti-Rat secondary antibody (BioLegend).

Filopodia Assay:

Siliconized glass cover slips were coated with antibodies (4 μg/ml) or VWF (10 μg/ml) in PBS overnight at 4°C. Coverslips were blocked with 1% bovine serum albumin at 22°C for 1 hour. CHO-Ib-IX cells or washed platelets were resuspended in modified Tyrode’s buffer (described above) supplemented with EDTA (10mM). In experiments with 3G6 or RAM.1 in solution, both MAbs were added to cells at 10 μg/ml 5 minutes prior to incubation on coverslips. Where VWF was the coating ligand, resuspension buffer was also supplemented with Botrocetin (1.5 μg/ml) immediately prior to incubation on coverslips. Cells were incubated on coverslips for 30 minutes at 37°C. Nonadherent cells were washed off with PBS. Samples were fixed for 10 minutes in 4% PFA, permeabilized for 15 minutes with 0.1% Triton X-100, and stained with 1 μg/ml TRITC-Phalloidin (Thermo-Fisher) for 30 minutes. Slides were sealed and stored at 4°C overnight before imaging.

Microscopy and Image Analysis:

Images were collected on an Olympus FluoView FV1000 confocal microscope and all analysis was performed in ImageJ. For quantitative analysis of filopodia, we wrote a short macro. Briefly, the median function followed by thresholding demarcates cell bodies. This allows thresholding of filopodia on the edges of cells, quantitated via the skeletonize function (script available upon request). For classifications of platelet morphology, images were provided to volunteers blinded to the experimental conditions. Volunteers classified each platelet in an image as resting, projecting filopodia, or spreading.

Uniform Shear Assay:

Uniform shear assay and subsequent flow cytometry analyses were performed largely as previously described [28, 48]. Briefly, platelets in PRP are adjusted to 250,000 per μl with pooled human plasma (Precision BioLogic) and incubated with an agonist or antibody of interest for 5 min. Where indicated, either RAM.1 or 3G6 was added simultaneously, at 10 μg/ml. Following incubation, the sample is exposed to the indicated shear level on a CAP 2000+ cone-plate rotational viscometer (Brookfield) for 5 minutes, fixed in 4% PFA for 20 minutes, and interrogated via flow cytometry.

Software and Statistics:

Unless otherwise indicated, statistical analyses were performed in Graphpad Prism and Fiji (ImageJ). Significance determined by one-way ANOVA with post-hoc Tukey test, as indicated. Image analysis was performed in Fiji (ImageJ). Flow cytometry data was analyzed in FlowJo.

Results

RAM.1 and 3G6 exhibit specific binding to the extracellular domain of GPIbβ

RAM.1 is a MAb targeting GPIbβ previously reported to inhibit ristocetin-induced platelet aggregation, reduce botrocetin-induced binding of VWF to platelets, and alter adhesion of GPIb-IX-expressing CHO cells to a VWF surface under flow [41]. Although later work did not support an effect on GPIb-IX adhesion to VWF [37, 43], RAM.1 does appear to inhibit GPIb-IX signaling [29, 37, 41]. 3G6 is a new MAb also raised against mouse GPIbβ and appears to accelerate ristocetin-induced platelet aggregation (Fig. S1). In order to confirm binding of both RAM.1 and 3G6 to GPIbβ, immobilized recombinant human GPIbβ extracellular domain (GPIbβE) or purified human GPIb-IX was detected via immunoblot by either MAb. GPIbβE is a 22-kDa protein and appears as a clear single band on the blot. While both RAM.1 and 3G6 were able to blot for GPIbβE and GPIb under non-reducing conditions, blotting was abolished under reducing conditions, suggesting that both MAbs recognize epitopes stabilized by or dependent on disulfide bonds (Fig. 1A). In order to assess the affinities of RAM.1 and 3G6 for the GPIbβ subunit, apparent Kds were determined via indirect ELISA using either GPIbβE or the full GPIb-IX complex as bait. Both 3G6 and RAM.1 bound to immobilized GPIbβE with low nanomolar affinity (3G6 Kd= 20.3 nM; RAM.1 Kd= 11.5 nM) (Fig. 1B). Interestingly, the affinity of both MAbs for the GPIb-IX complex was higher than affinity for GPIbβE alone, by an order of magnitude (3G6 Kd= 2.4 nM; RAM.1 Kd= 0.25 nM). Given that RAM.1 or 3G6 had little to no binding to the extracellular domain of GPIbα, known as glycocalicin (Fig. S2), interaction of either MAb with GPIbα likely does not account for this difference in affinities. Together, these data suggest conformational epitopes for both RAM.1 and 3G6 within the GPIbβ extracellular domain.

Figure 1. RAM.1 and 3G6 bind to GPIbβ with high affinity.

Figure 1.

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(A) Western blot analysis detecting purified GPIbβ extracellular domain (IbβE) and GPIb-IX complex with 3G6 (top) or RAM.1 (bottom) under reducing (R) or nonreducing (NR) conditions. In each gel, molecular weight ladders are marked on the left, and the protein band are marked on the right. (B) Quantitative ELISA measurements of 3G6 (top) or RAM.1 (bottom) binding to the immobilized extracellular domain of GPIbβ (IbβE, red) or the full GPIb-IX complex (blue). Apparent Kd for each interaction is listed on the respective graph.

RAM.1 and 3G6 differentially modulate GPIb-IX activation in CHO cells

Platelets and cells expressing human GPIb-IX undergo morphological changes, including protruding filopodia, when the receptor is activated [29, 37, 49, 50]. In an earlier study, where CHO cells expressing GPIb-IX (CHO-Ib-IX) were allowed to adhere to VWF, RAM.1 inhibited filopodia extension, which is a readout of receptor activation [37]. Like VWF, MAbs against the ligand-binding domain (LBD) of GPIbα are also capable of activating GPIb-IX, provided that the unbinding force of the MAb from the LBD is greater than the force required to unfold the mechanosensory domain (MSD) (~15 pN) [28, 31]. Using surfaces coated with anti-LBD MAbs instead of VWF rules out potential off-target interactions between VWF and CHO-Ib-IX cells/platelets and ensures that observed effects are specifically due to GPIb-IX activation. Furthermore, by using anti-LBD MAbs with different unbinding forces, we can determine the force-sensitivity of GPIb-IX’s interactions with anchored ligands. It follows that antibodies with unbinding forces below the unfolding force of the MSD would have attenuated effects compared to those with high unbinding forces.

6B4 is an anti-LBD MAb with a high unbinding force from the LBD (~40–50 pN) [28, 42]. When seeded on a surface coated with 6B4 or 6B4’s Fab fragment, CHO-Ib-IX cells undergo morphological changes including robust filopodia extensions (Fig. 2A). Alternatively, when seeded on a surface coated with AK2, an anti-LBD antibody with an unbinding force insufficient to unfold the MSD and activate GPIb-IX [28], little to no filopodial protrusions were observed (Fig. 2A). The mean length and number of filopodia per cell was quantified via a standardized macro in Fiji/ImageJ software. Compared to 6B4, AK2 induced the formation of, on average, 84% fewer filopodia, and the filopodia that did form were 59% shorter on average (Fig. 2B). In addition to extending filopodia, CHO-Ib-IX cells also extended membrane projections closer in morphology to lamellipodia. This phenomenon mimics platelet activation, wherein platelets initially protrude filopodia before adopting a “spreading” morphology [51]. The higher frequency of lamellipodia-like projections induced by 6B4 and 6B4 Fab compared to AK2 is reflected in the mean cell area of adherent CHO-Ib-IX cells (Fig. 2B). Both 6B4 and 6B4 Fab coated surfaces induced similar filopodia projection and spreading in CHO-Ib-IX cells (Fig 2B), implying that the observed activation was not due to Fc-mediated effects.

Figure 2. 6B4 and 6B4 Fab, but not AK2, activate CHO-Ib-IX cells.

Figure 2.

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(A) Representative confocal microscopy images of CHO-Ib-IX cells adhering to immobilized AK2, 6B4, or 6B4 Fab. Cells are stained with TRITC-Phalloidin. Each image is a max intensity projection of several z-stacks spanning the height of the cells. Scale bar = 10 μm. (B) Quantitation of mean length of filopodia, # of filopodia per cell, and cell area. Filopodia were defined as protrusions ≥ 1.5 μm in length. Each point represents the mean value of ≥30 cells from an independent experiment. All values obtained via analysis by a uniform ImageJ macro. Error bars represent mean ± SD. Significance determined by one-way ANOVA with post-hoc Tukey. p≤0.0001; ****.

In order to test the effects of RAM.1 and 3G6 on GPIb-IX signaling, CHO-Ib-IX cells were adhered to MAb-coated surfaces in the presence of soluble 3G6 or RAM.1. Compared to CHO-Ib-IX cells alone, cells adhering to AK2 in the presence of 3G6 extended more filopodia, longer filopodia, and had a higher mean cell area (Fig. 3). 3G6 also positively modulated GPIb-IX activation via 6B4 or 6B4 Fab, although the effect size was modest compared to the effect on AK2. Addition of 3G6 increased the number and mean length of filopodia extended by CHO-Ib-IX cells on 6B4 Fab surfaces by 24% when compared to those surfaces alone (Fig. 3). In contrast, the addition of RAM.1 robustly inhibited filopodia formation on surfaces coated with AK2, 6B4, or 6B4 Fab (Fig. 3).

Figure 3. 3G6 and RAM.1 differentially modulate CHO-Ib-IX activation by anti-LBD MAbs and VWF.

Figure 3.

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(A) Mean length of filopodia extended by CHO-Ib-IX cells on AK2, 6B4, 6B4 Fab, or VWF + botrocetin (Bc), ±3G6/RAM.1 in solution. (B) Mean # of filopodia (≥ 1.5 μm) per cell for each condition in (A). (C) Quantitation of cell area for each condition in (A). For all graphs, each point represents the mean value of ≥30 cells from an experimental replicate. Error bars are mean ± SEM. Significance determined by one-way ANOVA with post-hoc Tukey. p≤0.05; *, p≤0.01; **, p≤0.001; ***, p≤0.0001; ****.

3G6 amplifies GPIb-IX activation in platelets, whereas RAM.1 inhibits

Thus far, our study of the effects of the anti-GPIbβ MAbs RAM.1 and 3G6 has relied on the CHO-Ib-IX system to assay GPIb-IX activation. This system provides a ready source of GPIb-IX expressing cells with functional readouts linked to receptor activation and has been employed in several studies of GPIb-IX activity and function [37, 40, 52, 53]. Next, we investigated the effects of RAM.1 and 3G6 on GPIb-IX activation in washed human platelets from healthy donors. Platelets were adhered to AK2 or 6B4 coated surfaces in the presence of 3G6 or RAM.1. Upon adhesion to 6B4 (without addition of 3G6 or RAM.1), platelets adopt a mature activated phenotype, with upwards of 80% of platelets presenting a spread morphology. Adhesion to AK2 also induced some platelet spreading, with 26% of observed platelets in the spread morphology (Fig 4A, C, E). Some of this is likely attributable to background platelet activation during isolation [13]. The effects of 6B4 or AK2 on platelet morphology are not due to FcγRIIA activation, as they (i) persist in the presence of IV.3, an inhibitory antibody against FcγRIIA, and (ii) are recapitulated in mouse platelets, which do not express FcγRIIA (Fig. S3). Some studies report that RAM.1 diminishes affinity for or adhesion to VWF in platelets and cells expressing GPIb-IX [40, 41]. A more recent study showed no effect for RAM.1 on the affinity of the purified complex for VWF [43]. Although some of our RAM.1-treated samples appeared to trend toward a lower cell density, no significant changes were induced by the addition of 3G6 or RAM.1 (Fig S4).

Figure 4. 3G6 and RAM.1 differentially modulate platelet activation by anti-LBD MAbs or VWF.

Figure 4.

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(A-B) Representative images of human platelets adhering to 6B4/AK2 (A) or VWF (B) with or without RAM.1 or 3G6 in solution. For all images, cells were stained with TRITC-Phalloidin and each image represents a max intensity projection of several z-stacks spanning the height of the cells. Scale bars = 10 μm. (C-D) Stacked bar graphs showing the ratio of platelets adopting a spread, filopodia-extending, or resting morphology under the conditions in (A) and (B), respectively. Morphological classification was performed by individuals blinded to experimental conditions. Error bars represent mean ± SD, n=4. (E-F) Individual graphs of each morphology in (C) and (D), respectively. Filopodia were defined as protrusions ≥0.5 μm in length. Significance determined by one-way ANOVA with post-hoc Tukey. p≤0.05; *, p≤0.01; **, p≤0.0001; ****.

When RAM.1 is added with platelets in solution, the number of platelets adhering to 6B4 which were at rest greatly increased (from 11% to over 55%) compared to platelets on a 6B4 surface alone (Fig 4A, C, E). RAM.1 also significantly decreased the percentage of platelets which spread on an AK2 surface (Fig 4A, C, E). In contrast, in the presence of 3G6, a much smaller percentage of platelets on a 6B4 surface remained at rest (1% vs 11%), accompanying an increase in platelets extending filopodia. 3G6 also increased the percentage of platelets on an AK2 surface which adopted a spread morphology and reduced the percentage of resting platelets (Fig 4A, C, E).

In order to determine whether the effects of RAM.1 and 3G6 also extended to activation of GPIb-IX by VWF, platelets were adhered to a VWF surface in the presence of botrocetin as described previously [37]. On VWF surfaces, the majority (64%) of platelets extended filopodia, with only about 8% exhibiting a spread phenotype, and the remainder at rest (Fig 4B, E, F). With the addition of RAM.1, the percentage of platelets with filopodia drops from 64% to 11% and the percentage of platelets with a spread phenotype is vanishingly small. When 3G6 was added to platelets on a VWF surface, the percentage of platelets at rest was unchanged, but the percentage of platelets exhibiting a spread phenotype increased significantly, from 8% to 40% (Fig 4B, E, F).

3G6 and RAM.1 modulate platelet activation downstream of GPIb-IX

Activation of GPIb-IX induces signaling that leads to overall platelet activation. Thus far, we have used changes in cell morphology (filopodia extension and membrane spreading) to report on the effects of GPIb-IX signaling. When platelets are activated, they undergo a host of processes not limited to the morphological changes necessary to form the platelet plug, including the surface exposure of adhesion molecules, specific phospholipids, and glycan modifications. We utilized a uniform shear assay to apply shear stress to platelets preincubated with either AK2 or 6B4, as previously described [28, 31, 48], and detected two surface markers of platelet activation: P-selectin and β-galactose as readouts to assess the effects of RAM.1 and 3G6 on GPIb-IX activation. In previous studies, many anti-LBD MAbs (including 6B4) were able to activate GPIb-IX in solution by crosslinking platelets under shear. Unlike most anti-LBD MAbs, AK2’s low unbinding force from the LBD precludes it from robustly activating GPIb-IX, and this was again observed in the present study (Fig. 5). The addition of RAM.1 to the solution decreased expression of P-selectin and β-galactose even further. On the other hand, when 3G6 was added along with AK2, the percentage of events positive for P-selectin or double-positive for P-selectin and β-galactose increased 2–3-fold across several levels of shear. Across all shear levels, 6B4 induced expression of both P-selectin and β-galactose in a higher percentage of platelets than AK2. The addition of RAM.1 reduced 6B4-induced P-selectin and β-galactose expression five-fold at 30 dyn/cm2. 3G6 positively modulated 6B4’s ability to activate GPIb-IX in platelets and to induce expression of both surface markers, especially at higher shear levels (Fig. 5).

Figure 5. 3G6 and RAM.1 modulate platelet activation downstream of GPIb-IX.

Figure 5.

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Graphs of percentage of platelets positive for P-selectin (top) or both P-selectin and β-galactose (bottom) in platelets treated with AK2 (left) or 6B4 (right) over the indicated shear range. Platelets treated with AK2/6B4 alone (black), AK2/6B4 and 3G6 (red), AK2/6B4 and RAM.1 (blue), RAM.1 alone (green), or 3G6 alone (purple). Error bars represent mean ± SD, n=3. Significance determined by one-way ANOVA with post-hoc Tukey. p≤0.01; **, p≤0.001; ***, p≤0.0001; ****. ECL (Erythrina cristagalli lectin), PSel (anti-P-Selectin MAb).

Discussion

Current models of GPIb-IX activation do not take into account the role of either GPIbβ or GPIX in signal transduction. It has been previously suggested that GPIb-IX is activated by lateral clustering on the platelet surface induced by binding of VWF multimers and enriched in lipid rafts [54, 55]. This clustering model explains the observation that anti-LBD antibodies or their divalent F(ab’)2 fragments, but not their monovalent Fab fragments, could activate GPIb-IX and subsequently induce platelet clearance [42, 56]. However, it cannot account for other observations related to GPIb-IX, including the well-documented requirement of shear in VWF-mediated activation of GPIb-IX [57], as well as the inability of certain anti-GPIbα, many anti-GPIbβ and anti-GPIX antibodies to activate GPIb-IX and induce platelet clearance [28, 29, 36, 37, 58, 59]. Although the trigger model offers explanations for both of these observations, there is still a significant knowledge gap regarding the contribution of GPIbβ to GPIb-IX activation. Here, we report that 3G6, a novel MAb targeting the relatively small GPIbβ subunit, potentiates GPIb-IX activation. The ability of 3G6 to amplify the effects of GPIb-IX agonists stands in stark contrast to the effects of another anti-GPIbβ MAb, RAM.1.

In this study, we utilize several MAbs against the LBD, previously demonstrated to activate GPIb-IX in solution [28], as adherent ligands. One of these anti-LBD MAbs, AK2, forms a weak bond to the LBD and does not activate GPIb-IX under shear, in contrast to MAbs with higher unbinding force to the LBD, such as 6B4 [28]. Here, we show that 6B4, but not AK2, induces robust filopodia formation in CHO-Ib-IX cells. Given that the key difference between 6B4 and AK2 is the ability to facilitate force-induced MSD unfolding, the observed morphological changes in CHO-Ib-IX cells should proceed specifically from MSD unfolding.

Both CHO cells and platelets readily probe their microenvironments, applying significant contractile force to conjugated or unconjugated substrata [60–63]. Of note, in previous studies, while 6B4 induced GPIb-IX activation, the monovalent 6B4 Fab had little effect. This was possibly due to 6B4 Fab’s inability to cluster GPIb-IX or, alternatively, its inability to crosslink platelets via GPIbα. In this study we demonstrate that, when anchored to a surface, 6B4 Fab does activate GPIb-IX. This is likely because surface-conjugated 6B4 Fab provides an anchored ligand for CHO cells and platelets to “pull” via GPIbα. However, it is difficult to imagine that 6B4 Fab clusters GPIb-IX on the surface, while the bivalent AK2 (which induces very little activation) does not. Soluble RAM.1 inhibits GPIb-IX signaling in the case of 6B4, 6B4 Fab, and even AK2. However, while 3G6 significantly potentiates the CHO-Ib-IX response to AK2, 3G6’s effect on 6B4/6B4 Fab mediated activation is only mild potentiation. Given that the CHO-Ib-IX response elicited by 6B4/6B4 Fab is already quite robust, with almost all cells extending filopodia, potentiation in the form of further increases in the response may be difficult to detect.

It is commonly accepted that the VWF-GPIb-IX interaction, and thus GPIb-IX activation, leads to weak platelet activation, including activation of integrins, which in turn facilitate integrin-dependent platelet adhesion/aggregation [8, 64–66]. Surprisingly, in this study, >75% of platelets underwent robust spreading upon adhesion to 6B4 surfaces, even in the presence of EDTA. This response does not require the Fc region of antibodies and was independent of platelet FcγRIIA. RAM.1 significantly reduced platelet spreading on surfaces coated with 6B4, AK2, or VWF. Alternatively, in the presence of soluble 3G6, platelets adopted more mature morphologies on all surfaces. In previous studies, AK2 induced little-to-no activation of GPIb-IX in solution [28, 31]. Remarkably, in the presence of 3G6, shear-induced GPIb-IX activation by both AK2 and 6B4 was significantly amplified. These data suggest that the modulatory effects of both anti-GPIbβ antibodies affect a broad spectrum of GPIb-IX associated phenomena including morphological changes, degranulation, and surface desialylation.

Both RAM.1 and 3G6 have a higher affinity for GPIb-IX than GPIbβ alone. This is likely a function of stoichiometry/avidity; each GPIb-IX complex contains two GPIbβ subunits, and binding of one Fab within the same IgG molecule likely makes binding of the second Fab more favorable. We observed that neither RAM.1 nor 3G6 could blot reduced GPIbβ (Fig. 1A), suggesting that their epitopes are conformational-sensitive. RAM.1 was previously reported to blot reduced GPIbβ [41]. Comparison of blotting procedures reveals a difference in the reducing condition during SDS sample preparation (2.5% β-mercaptoethanol in this study vs. 5 mM dithiothreitol or 0.5% β-mercaptoethanol in the earlier study). Thus, it appears that a harsher reducing condition reduces blotting by RAM.1.

Similar to the previously reported divergent effects of anti-GPIbα antibodies [28, 58], it is unclear how the contrasting effects of RAM.1 and 3G6 in this study fit into the clustering model. Moreover, 3G6 alone does not have a noticeable effect on receptor signaling. Rather, 3G6’s effect was only observed when combined with a separate ligand targeting the LBD and scales with shear, reflecting its modulation of shear-dependent GPIb-IX activation. The trigger model accommodates the contrasting effects of RAM.1 and 3G6. GPIbβ is positioned proximal to the MSD. Since GPIbβ and GPIX are structurally malleable [39], GPIbβ may change conformation in response to unfolding of the adjoining MSD and the exposure of the trigger sequence. Thus, it is not entirely unexpected that a MAb targeting GPIbβ could stabilize a specific conformation of GPIbβ and thereby modulate GPIb-IX activation. It is nevertheless striking that two MAbs targeting the small extracellular domain of GPIbβ have such opposite modulatory effects. One potential mechanism for the effects of 3G6 could be that 3G6 binds favorably to the conformation GPIbβ adopts upon MSD unfolding and thereby holds the receptor in an “on” state for longer. This would explain its ability to potentiate responses even to a weak ligand like AK2, which rarely sustains enough force to unfold the MSD. It is unlikely, based on previous reports, that RAM.1 prevents MSD unfolding [29], but it is possible that RAM.1 binding prevents the conformational changes in GPIbβ which normally result from MSD unfolding. The precise mechanism of GPIb-IX modulation via anti-GPIbβ MAbs warrants further investigation, as an understanding of this step is integral to a complete mechanism of GPIb-IX activation.

Overall, this study provides evidence that GPIbβ plays a significant role in modulation and/or outside-in activation of GPIb-IX. We report the first potentiator of GPIb-IX, the novel MAb 3G6. Along with RAM.1, 3G6 is the second MAb against GPIbβ to alter GPIb-IX’s activation state. Our results expand the current understanding of the mechanism of GPIb-IX activation.

Supplementary Material

si1

Essentials:

  1. Anti-GPIbβ antibody RAM.1 has been reported to inhibit GPIb-IX-mediated signal transduction, via an unclear mechanism.

  2. Like RAM.1, a new rat monoclonal antibody 3G6 binds human or mouse GPIbβ with high affinity.

  3. Unlike RAM.1, 3G6 increases GPIb-IX-mediated activation or morphological change in platelets or transfected cells.

  4. The divergent effects of 3G6 and RAM.1 suggest a conformational change in GPIbβ during outside-in activation of GPIb-IX.

Acknowledgements

We thank Dr. Neil Anthony and William Giang at the Emory Integrated Cellular Imaging Core for their technical assistance with image analysis and quantification. We thank Wolfgang Bergmeier for helpful discussion. This work is partly supported by NIH grants HL082808, HL146299 (RL) and HL134241 (MEQ).

Footnotes

Disclosures

The authors declare no conflicts of interest.

References

  • 1.Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell. 1996; 84: 289–97. [DOI] [PubMed] [Google Scholar]
  • 2.Luo S-Z, Mo X, López JA, Li R. Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib-IX complex. J Thromb Haemost. 2007; 5: 2494–502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Luo SZ, Mo X, Afshar-Kharghan V, Srinivasan S, Lopez JA, Li R. Glycoprotein Ibalpha forms disulfide bonds with 2 glycoprotein Ibbeta subunits in the resting platelet. Blood. 2007; 109: 603–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Luo SZ, Li R. Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex. J Mol Biol. 2008; 382: 448–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Huizinga EG, Tsuji S, Romijn RA, Schiphorst ME, de Groot PG, Sixma JJ, Gros P. Structures of glycoprotein Iba and its complex with von Willebrand factor A1 domain. Science. 2002; 297: 1176–9. [DOI] [PubMed] [Google Scholar]
  • 6.Zarpellon A, Celikel R, Roberts JR, McClintock RA, Mendolicchio GL, Moore KL, Jing H, Varughese KI, Ruggeri ZM. Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I. Proc Natl Acad Sci U S A. 2011; 108: 8628–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wang Y, Gao H, Shi C, Erhardt PW, Pavlovsky A, D AS, Bledzka K, Ustinov V, Zhu L, Qin J, Munday AD, Lopez J, Plow E, Simon DI. Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbalpha. Nat Commun. 2017; 8: 15559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Mazzucato M, Pradella P, Cozzi MR, De Marco L, Ruggeri ZM. Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibalpha mechanoreceptor. Blood. 2002; 100: 2793–800. [DOI] [PubMed] [Google Scholar]
  • 9.Gu M, Xi X, Englund GD, Berndt MC, Du X. Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin αIIbβ3 using a reconstituted mammalian cell expression model. J Cell Biol. 1999; 147: 1085–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ju L, Chen Y, Li K, Yuan Z, Liu B, Jackson SP, Zhu C. Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species. Sci Rep. 2017; 7: 14185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bergmeier W, Piffath CL, Goerge T, Cifuni SM, Ruggeri ZM, Ware J, Wagner DD. The role of platelet adhesion receptor GPIbα far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis. Proc Natl Acad Sci U S A. 2006; 103: 16900–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Reininger AJ, Heijnen HF, Schumann H, Specht HM, Schramm W, Ruggeri ZM. Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress. Blood. 2006; 107: 3537–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Du X Signaling and regulation of the platelet glycoprotein Ib-IX-V complex. Curr Opin Hematol. 2007; 14: 262–9. [DOI] [PubMed] [Google Scholar]
  • 14.Liu J, Pestina TI, Berndt MC, Jackson CW, Gartner TK. Botrocetin/VWF-induced signaling through GPIb-IX-V produces TxA2 in an alphaIIbbeta3- and aggregation-independent manner. Blood. 2005; 106: 2750–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Li J, van der Wal DE, Zhu G, Xu M, Yougbare I, Ma L, Vadasz B, Carrim N, Grozovsky R, Ruan M, Zhu L, Zeng Q, Tao L, Zhai ZM, Peng J, Hou M, Leytin V, Freedman J, Hoffmeister KM, Ni H. Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nat Commun. 2015; 6: 7737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.van der Wal DE, Davis AM, Mach M, Marks DC. The role of neuraminidase 1 and 2 in glycoprotein Ibalpha-mediated integrin alphaIIbbeta3 activation. Haematologica. 2020; 105: 1081–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Li S, Wang Z, Liao Y, Zhang W, Shi Q, Yan R, Ruan C, Dai K. The glycoprotein Ibα-von Willebrand factor interaction induces platelet apoptosis. J Thromb Haemost. 2010; 8: 341–50. [DOI] [PubMed] [Google Scholar]
  • 18.Greco NJ, Tandon NN, Jones GD, Kornhauser R, Jackson B, Yamamoto N, Tanoue K, Jamieson GA. Contributions of glycoprotein Ib and the seven transmembrane domain receptor to increases in platelet cytoplasmic [Ca2+] induced by alpha-thrombin. Biochemistry. 1996; 35: 906–14. [DOI] [PubMed] [Google Scholar]
  • 19.Nesbitt WS, Kulkarni S, Giuliano S, Goncalves I, Dopheide SM, Yap CL, Harper IS, Salem HH, Jackson SP. Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow. J Biol Chem. 2002; 277: 2965–72. [DOI] [PubMed] [Google Scholar]
  • 20.Garcia A, Quinton TM, Dorsam RT, Kunapuli SP. Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets. Blood. 2005; 106: 3410–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wu Y, Asazuma N, Satoh K, Yatomi Y, Takafuta T, Berndt MC, Ozaki Y. Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase. Blood. 2003; 101: 3469–76. [DOI] [PubMed] [Google Scholar]
  • 22.Mangin P, Yuan Y, Goncalves I, Eckly A, Freund M, Cazenave JP, Gachet C, Jackson SP, Lanza F. Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA. J Biol Chem. 2003; 278: 32880–91. [DOI] [PubMed] [Google Scholar]
  • 23.Yap CL, Anderson KE, Hughan SC, Dopheide SM, Salem HH, Jackson SP. Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3). Blood. 2002; 99: 151–8. [DOI] [PubMed] [Google Scholar]
  • 24.Li Z, Zhang G, Feil R, Han J, Du X. Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin alphaIIb beta3. Blood. 2006; 107: 965–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chen Y, Ruggeri ZM, Du X. 14-3-3 proteins in platelet biology and glycoprotein Ib-IX signaling. Blood. 2018; 131: 2436–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Du X, Harris SJ, Tetaz TJ, Ginsberg MH, Berndt MC. Association of a phospholipase A2 (14-3-3 protein) with the platelet glycoprotein Ib-IX complex. J Biol Chem. 1994; 269: 18287–90. [PubMed] [Google Scholar]
  • 27.Zhang W, Deng W, Zhou L, Xu Y, Yang W, Liang X, Wang Y, Kulman JD, Zhang XF, Li R. Identification of a juxtamembrane mechanosensitive domain in the platelet mechanosensor glycoprotein Ib-IX complex. Blood. 2015; 125: 562–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Quach ME, Dragovich MA, Chen W, Syed AK, Cao W, Liang X, Deng W, De Meyer SF, Zhu G, Peng J, Ni H, Bennett CM, Hou M, Ware J, Deckmyn H, Zhang XF, Li R. Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets. Blood. 2018; 131: 787–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Deng W, Xu Y, Chen W, Paul DS, Syed AK, Dragovich MA, Liang X, Zakas P, Berndt MC, Di Paola J, Ware J, Lanza F, Doering CB, Bergmeier W, Zhang XF, Li R. Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor. Nat Commun. 2016; 7: 12863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Chen W, Druzak SA, Wang Y, Josephson CD, Hoffmeister KM, Ware J, Li R. Refrigeration-induced binding of von Willebrand factor facilitates fast clearance of refrigerated platelets. Arterioscler Thromb Vasc Biol. 2017; 37: 2271–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Zhang XF, Zhang W, Quach ME, Deng W, Li R. Force-regulated refolding of the mechanosensory domain in the platelet glycoprotein Ib-IX complex. Biophys J. 2019; 116: 1960–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Quach ME, Li R. Structure-function of platelet glycoprotein Ib-IX. J Thromb Haemost. 2020; 18: 3131–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Lopez JA, Leung B, Reynolds CC, Li CQ, Fox JEB. Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits. J Biol Chem. 1992; 267: 12851–9. [PubMed] [Google Scholar]
  • 34.Mo X, Lu N, Padilla A, López JA, Li R. The transmembrane domain of glycoprotein Ibβ is critical to efficient expression of glycoprotein Ib-IX complex in the plasma membrane. J Biol Chem. 2006; 281: 23050–9. [DOI] [PubMed] [Google Scholar]
  • 35.Mo X, Luo S-Z, López JA, Li R. Juxtamembrane basic residues in glycoprotein Ibβ cytoplasmic domain are required for assembly and surface expression of glycoprotein Ib-IX complex. FEBS Lett. 2008; 582: 3270–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ravanat C, Strassel C, Hechler B, Schuhler S, Chicanne G, Payrastre B, Gachet C, Lanza F. A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbα N-terminal extracellular domain. Blood. 2010; 116: 1157–64. [DOI] [PubMed] [Google Scholar]
  • 37.Maurer E, Tang C, Schaff M, Bourdon C, Receveur N, Ravanat C, Eckly A, Hechler B, Gachet C, Lanza F, Mangin PH. Targeting platelet GPIbβ reduces platelet adhesion, GPIb signaling and thrombin generation and prevents arterial thrombosis. Arterioscler Thromb Vasc Biol. 2013; 33: 1221–9. [DOI] [PubMed] [Google Scholar]
  • 38.McEwan PA, Yang W, Carr KH, Mo X, Zheng X, Li R, Emsley J. Quaternary organization of GPIb-IX complex and insights into Bernard-Soulier syndrome revealed by the structures of GPIbβ and a GPIbβ/GPIX chimera. Blood. 2011; 118: 5292–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Zhou L, Yang W, Li R. Analysis of inter-subunit contacts reveals the structural malleability of extracellular domains in platelet glycoprotein Ib-IX complex. J Thromb Haemost. 2014; 12: 82–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Perrault C, Mangin P, Santer M, Baas MJ, Moog S, Cranmer SL, Pikovski I, Williamson D, Jackson SP, Cazenave JP, Lanza F. Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex. Blood. 2003; 101: 3477–84. [DOI] [PubMed] [Google Scholar]
  • 41.Perrault C, Moog S, Rubinstein E, Santer M, Baas MJ, de la Salle C, Ravanat C, Dambach J, Freund M, Santoso S, Cazenave JP, Lanza F. A novel monoclonal antibody against the extracellular domain of GPIbβ modulates vWF mediated platelet adhesion. Thromb Haemost. 2001; 86: 1238–48. [PubMed] [Google Scholar]
  • 42.Cauwenberghs N, Meiring M, Vauterin S, van Wyk V, Lamprecht S, Roodt JP, Novak L, Harsfalvi J, Deckmyn H, Kotze HF. Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates. Arterioscler Thromb Vasc Biol. 2000; 20: 1347–53. [DOI] [PubMed] [Google Scholar]
  • 43.Yan R, Mo X, Paredes AM, Dai K, Lanza F, Cruz MA, Li R. Reconstitution of platelet glycoprotein Ib-IX complex in phospholipid bilayer nanodiscs. Biochemistry. 2011; 50: 10598–606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Liang X, Russell SR, Estelle S, Jones LH, Cho S, Kahn ML, Berndt MC, Bunting ST, Ware J, Li R. Specific inhibition of ectodomain shedding of glycoprotein Ibalpha by targeting its juxtamembrane shedding cleavage site. J Thromb Haemost. 2013; 11: 2155–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Bergmeier W, Rackebrandt K, Schroder W, Zirngibl H, Nieswandt B. Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies. Blood. 2000; 95: 886–93. [PubMed] [Google Scholar]
  • 46.Ware J, Russell S, Ruggeri ZM. Generation and rescue of a murine model of platelet dysfunction: the Bernard-Soulier syndrome. Proc Natl Acad Sci U S A. 2000; 97: 2803–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Liang X, Russell SR, Estelle S, Jones LH, Cho S, Kahn ML, Berndt MC, Bunting ST, Ware J, Li R. Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site. J Thromb Haemost. 2013; 11: 2155–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Quach ME, Syed AK, Li R. A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry. J Vis Exp. 2019; 148: e59704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Mangin PH, Receveur N, Wurtz V, David T, Gachet C, Lanza F. Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets. J Thromb Haemost. 2009; 7: 1550–5. [DOI] [PubMed] [Google Scholar]
  • 50.Yuan Y, Kulkarni S, Ulsemer P, Cranmer SL, Yap CL, Nesbitt WS, Harper I, Mistry N, Dopheide SM, Hughan SC, Williamson D, de la Salle C, Salem HH, Lanza F, Jackson SP. The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells. J Biol Chem. 1999; 274: 36241–51. [DOI] [PubMed] [Google Scholar]
  • 51.Schurr Y, Sperr A, Volz J, Beck S, Reil L, Kusch C, Eiring P, Bryson S, Sauer M, Nieswandt B, Machesky L, Bender M. Platelet lamellipodium formation is not required for thrombus formation and stability. Blood. 2019; 134: 2318–29. [DOI] [PubMed] [Google Scholar]
  • 52.Mangin P, David T, Lavaud V, Cranmer SL, Pikovski I, Jackson SP, Berndt MC, Cazenave JP, Gachet C, Lanza F. Identification of a novel 14-3-3z binding site within the cytoplasmic tail of platelet glycoprotein Iba. Blood. 2004; 104: 420–7. [DOI] [PubMed] [Google Scholar]
  • 53.Cranmer SL, Ulsemer P, Cooke BM, Salem HH, de la Salle C, Lanza F, Jackson SP. Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear. J Biol Chem. 1999; 274: 6097–106. [DOI] [PubMed] [Google Scholar]
  • 54.Shrimpton CN, Borthakur G, Larrucea S, Cruz MA, Dong JF, Lopez JA. Localization of the adhesion receptor glycoprotein Ib-IX-V complex to lipid rafts is required for platelet adhesion and activation. J Exp Med. 2002; 196: 1057–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Jin W, Inoue O, Tamura N, Suzuki-Inoue K, Satoh K, Berndt MC, Handa M, Goto S, Ozaki Y. A role for glycosphingolipid-enriched microdomains in platelet glycoprotein Ib-mediated platelet activation. J Thromb Haemost. 2007; 5: 1034–40. [DOI] [PubMed] [Google Scholar]
  • 56.Nieswandt B, Bergmeier W, Rackebrandt K, Gessner JE, Zirngibl H. Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice. Blood. 2000; 96: 2520–7. [PubMed] [Google Scholar]
  • 57.Kroll MH, Hellums JD, McIntire LV, Schafer AI, Moake JL. Platelets and shear stress. Blood. 1996; 88: 1525–41. [PubMed] [Google Scholar]
  • 58.Liang X, Syed AK, Russell SR, Ware J, Li R. Dimerization of glycoprotein Ibα is not sufficient to induce platelet clearance. J Thromb Haemost. 2016; 14: 381–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Stolla M, Stefanini L, Andre P, Ouellette TD, Reilly MP, McKenzie SE, Bergmeier W. CalDAG-GEFI deficiency protects mice in a novel model of Fcgamma RIIA-mediated thrombosis and thrombocytopenia. Blood. 2011; 118: 1113–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Leader WM, Stopak D, Harris AK. Increased contractile strength and tightened adhesions to the substratum result from reverse transformation of CHO cells by dibutyryl cyclic adenosine monophosphate. J Cell Sci. 1983; 64: 1–11. [DOI] [PubMed] [Google Scholar]
  • 61.Qiu Y, Brown AC, Myers DR, Sakurai Y, Mannino RG, Tran R, Ahn B, Hardy ET, Kee MF, Kumar S, Bao G, Barker TH, Lam WA. Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation. Proc Natl Acad Sci USA. 2014; 111: 14430–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Kee MF, Myers DR, Sakurai Y, Lam WA, Qiu Y. Platelet mechanosensing of collagen matrices. PLoS One. 2015; 10: e0126624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Qiu Y, Ciciliano J, Myers DR, Tran R, Lam WA. Platelets and physics: How platelets “feel” and respond to their mechanical microenvironment. Blood reviews. 2015; 29: 377–86. [DOI] [PubMed] [Google Scholar]
  • 64.Li Z, Delaney MK, O’Brien KA, Du X. Signaling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol. 2010; 30: 2341–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Pabon D, Jayo A, Xie J, Lastres P, Gonzalez-Manchon C. Thrombin induces GPIb-IX-mediated fibrin binding to alphaIIbbeta3 in a reconstituted Chinese hamster ovary cell model. J Thromb Haemost. 2006; 4: 2238–47. [DOI] [PubMed] [Google Scholar]
  • 66.Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell. 1998; 94: 657–66. [DOI] [PubMed] [Google Scholar]

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