Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex

Article

Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex X. Frank Zhang,1,* Wei Zhang,1 M. Edward Quach,2 Wei Deng,2 and Renhao Li2,* 1 Department of Bioengineering, Department of Mechanical Engineering & Mechanics, Lehigh University, Bethlehem, Pennsylvania and 2Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia

ABSTRACT In platelets, the glycoprotein (GP) Ib-IX receptor complex senses blood shear flow and transmits the mechanical signals into platelets. Recently, we have discovered a juxtamembrane mechanosensory domain (MSD) within the GPIba subunit of GPIb-IX. Mechanical unfolding of the MSD activates GPIb-IX signaling into platelets, leading to their activation and clearance. Using optical tweezer-based single-molecule force measurement, we herein report a systematic biomechanical characterization of the MSD in its native, full-length receptor complex and a recombinant, unglycosylated MSD in isolation. The native MSD unfolds at a resting rate of 9
103 s1. Upon exposure to pulling forces, MSD unfolding accelerates exponentially over a force scale of 2.0 pN. Importantly, the unfolded MSD can refold with or without applied forces. The unstressed refolding rate of MSD is 17 s1 and slows exponentially over a force scale of 3.7 pN. Our measurements confirm that the MSD is relatively unstable, with a folding free energy of 7.5 kBT. Because MSD refolding may turn off GPIb-IX’s mechanosensory signals, our results provide a mechanism for the requirement of a continuous pulling force of >15 pN to fully activate GPIb-IX.

INTRODUCTION The mechanical shear force generated by blood flow in the vasculature is an important factor in mediating physiological hemostasis and pathological thrombosis. The induction of platelet aggregation by elevated shear stress requires the association of plasma protein von Willebrand factor (VWF) with the glycoprotein (GP)Ib-IX receptor complex on platelets (1–3). VWF in circulation or immobilized at the damaged vessel wall responds to shear stress, exposing and activating its A1 domain, which is the ligand for GPIb-IX (4–7). Ligation of immobilized VWF to GPIb-IX recruits circulating platelets to the site of injury and transmits a signal into the adherent platelet that eventually leads to its activation (3,8–10). Ligation of soluble VWF to platelet GPIb-IX cross-links platelets in circulation, which can also transmit a signal, leading to their rapid clearance (11–13). Recognized as a major shear sensor and shear receptor on the platelet surface more than 20 years ago (14), GPIb-IX is a highly integrated transmembrane receptor complex (Fig. 1 a). Each of its four subunits—GPIba,

Submitted September 24, 2018, and accepted for publication March 29, 2019. *Correspondence: [email protected] or [email protected] Editor: Thomas Perkins. https://doi.org/10.1016/j.bpj.2019.03.037 Ó 2019 Biophysical Society.

1960 Biophysical Journal 116, 1960–1969, May 21, 2019

two GPIbb, and GPIX—interact with one another through its transmembrane helices and juxtamembrane extracellular domains (15–18). GPIba, the major subunit in GPIb-IX, contains an N-terminal ligand-binding domain (LBD) for the A1 domain of VWF and many other ligands present in circulation (Fig. 1 a) (6,19). Recently, we performed the first single-molecule force measurement of an intact GPIb-IX complex (20). Pulling on the immobilized GPIb-IX with recombinant A1 induced unfolding of a previously unidentified structural domain in the juxtamembrane stalk region of GPIba before dissociating A1 from GPIb-IX (20). This mechanosensory domain (MSD) can be unfolded mechanically by physiologically relevant forces (10–15 pN) (11,21). Force-induced unfolding of the MSD could conceivably convert a mechanical signal into a change in protein conformation. Indeed, physiological shear stress exerted on GPIb-IX through its LBD triggers platelet signaling, leading to increased intracellular calcium concentration and the significant exposure of phosphatidylserine (PS) lipids, P-selectin, and b-galactose on the platelet surface, the latter of which has been identified as a ‘‘clear-me’’ sign for platelet clearance (11,21–25). Although the functional significance of MSD unfolding is increasingly understood, its biomechanical properties, which allow the MSD to exert its mechanosensing and

Platelet GPIb-IX Biomechanics

a GPIbα

b

Optical trap

Ligandbinding domain

Biotin Streptavidin WM 23 Fab

GPIb-IX WM23 binding site

macroglycopeptide

MSD

GPIX

2-μm polystyrene bead

transmembrane biotin cytoplasmic

Retract

c

FIGURE 1 Single-molecule force measurement of pulling mAb on GPIb-IX to induce the unfolding of MSD. (a) A diagram of the GPIb-IX construct used in this study is shown. The biotag that is biotinylated by E. coli biotin ligase was appended to the GPIX cytoplasmic domain. The binding epitope of WM23 is indicated. (b) WM23 Fab was covalently attached to a DNA handle. The other end of the DNA handle was immobilized to a 2-mm polystyrene bead via biotin-streptavidin linkage, which was controlled by the optical trap. The biotinylated GPIb-IX was captured by another streptavidin bead. This bead was held by a fixed micropipette. (c) Successive cycles of stretching and relaxation with MSD unfolding and refolding events was arrowed, with the pulling speed at 150 nm/s. To see this figure in color, go online.

DNA handle

GPIbβ

Approach

Micropipette (fixed)

stretch relax

Force (pN)

30 20 10 0

50 nm 1

2

3

4

5

6

Trap position (nm)

mechanotransduction functions, have not been fully characterized. Our previous approach of using the A1 domain to capture GPIb-IX was not able to produce a pulling curve with sufficient force for these characterizations. In this study, using a stronger mechanical linkage to stretch GPIb-IX, we now report the force-dependent unfolding and refolding kinetics of the MSD. Our data illustrate how the MSD may be particularly suited to transduce mechanical force into biochemical signals in platelets. METHODS Materials Human fresh whole blood was obtained from healthy donors via venipuncture. Written informed consent was received from participants before their inclusion in studies, and all procedures using donor-derived human platelets were approved by the Institutional Review Board at Emory University. Recombinant human GPIb-IX complex in which the cytoplasmic domain of GPIX was biotinylated, a recombinant MSD expressed from Escherichia coli, recombinant A1 domain (residues 1261–1472) of VWF, botrocetin, GFP-fused lactadherin C2 domain (Lact-C2), monoclonal antibody 5G6, and its Fab fragment have been described (11,20,26,27). 5G6 Fab was conjugated with N-hydroxysuccinimide-Fluorescein (FITC) (Thermo Fischer Scientific, Waltham, MA) according to the manufacturer’s instruction (11). The WM23 antibody is kindly shared by Dr. Michael Berndt. Its Fab fragment was prepared using the Pierce Fab Preparation Kit (Thermo Fischer Scientific). The purified WM23 Fab was treated with Pierce Traut’s Reagent (Thermo Fischer Scientific) and coupled to the 2,20 -dithiodipyridine-activated, 800-bp DNA handle by overnight incubation at room tem-

perature. The coupled WM23 Fab-DNA handle construct was stored at 80 C.

Laser optical tweezer measurement Single-molecule force measurement was performed as described previously (20).

MSD unfolding analysis Unfolding was analyzed according to the Bell-Evans model, a theory to describe the influence of an external force on the rate of protein unfolding (28,29). According to this model, a pulling force, f, distorts the intermolecular potential of a protein complex, leading to a lower activation energy and an increase in the unfolding rate ku(f) as follows:




ku ðf Þ ¼ 1=tu ðf Þ ¼

ku0

 f gu exp ; kB T

(1)

where ku0 is the unfolding rate constant in the absence of a pulling force, gu is the distance to the transition state, T is the absolute temperature, and kB is the Boltzmann constant. For a constant loading rate Rf, the probability for the unfolding of the complex as a function of the pulling force f is given by




pð f Þ ¼

ku0

  0 
 f gu ku k B T f gu exp 1  exp exp ; (2) gu Rf kB T kB T

with the most probable unbinding force f*

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Zhang et al.


 kB T gu kB T f ¼ ln 0 lnðRf Þ þ gu gu ku kB T 

(3)

Hence, the Bell model predicts that the most probable unfolding force f* is a linear function of the logarithm of the loading rate. Experimentally, f* was determined from the mode of the unbinding force histograms (Fig. S1). The Bell-Evans model parameters ku0 and gu were determined by fitting Eq. 3 to the plot of f* versus ln(Rf). The fitted results are summarized in Table 1.

Refolding analysis The Bell-Evans model has been modified by Alemany et al. (30) to describe the refolding of protein barnase using an OT set up similar to the one used in the current study. In this modification, the distance to the transition state g has been defined as gf. Because in the refolding case, the loading rate is essentially an ‘‘unloading’’ rate carrying a minus sign (Rf), Eq. 3 can then be revised as:

!

 gf Rf kf0 kB T ! gf kB T kB T   ln 0 ln Rf ¼  gf gf kf kBT

kB T f ¼ ln gf 



(4)

In the current study, we used Eq. 4 to fit the plot of the most probable refolding force versus the natural logarithm of Rf. The Bell-Evans model parameters for refolding, kf0 and gf, were obtained from the slope and interset of the linear fit. In addition, refolding forces at different loading rates can be converted into refolding kinetics. According to a statistical model used by Kim et al. (31) to describe the kinetics of ligand-receptor rebinding, modified from an original work of Pierse and Dudko (32), association rate as a function of the force is given as:

krefold ðFÞ ¼

Rf
pðrefolding at FÞ ; Pðrefolding below FÞ

(5)

where p is the probability density of refolding at a given force, and P is the probability of refolding below this force. In practice, the refolding forces at different loading rates are binned and plotted as histograms. The refolding rate can be obtained by

krefold ðFk Þ ¼

hk Rf ðF0 þ ðk  1=2ÞDF


 ; kP 1 hk 2 þ hi DF

(6)

i¼1

where DF is the bin width of the refolding force histogram that starts at Fo, hi is the fraction of refolding in the ith bin normalized by the total count, and i and k are bin numbers. Plots of k(F) versus F were fit with a protein refolding model developed by Evans et al. that accounts for the compliance of the protein (33):

TABLE 1 Fitted Bell-Evans Model Parameters of the MSD Unfolding

Native MSD cSc



 kðFÞ ¼ k0f exp F2 ð2k kB TÞ ;

ku0 (s1)

g (nm)

0.009 5 0.006 0.03 5 0.01

2.1 5 0.3 1.9 5 0.2

Uncertainties are the standard error of the fits.

1962 Biophysical Journal 116, 1960–1969, May 21, 2019

(7)

where k is the compliance of the unfolded protein. Fitting was conducted using linear least squares methods using IGOR Pro software.

Statistical analysis For each pulling speed, 40–200 unfolding and refolding forces were collected. Curve fitting was performed using IGOR Pro or Origin software by minimizing the c-square statistic for the optimal fit. Unless otherwise stated, the data is reported as the mean and the standard error of the estimate. Statistical analyses between groups were performed using an unpaired t-test or analysis of variance, with a p value less than 0.05 considered to be statistically significant.

Binding of antibody 5G6 to the MSD Human platelet-rich plasma (PRP) was prepared from whole blood by standard low-speed centrifugation (12 min, 140
g, no brake, 22 C). The PRP was supplemented with human plasma to a count of 5
105 platelets per mL, mixed gently with 1 mg$mL1 botrocetin, and incubated at room temperature for 10 min. The PRP mixture was then mixed with 0.5 mg$mL1 FITC-conjugated 5G6 Fab or directly transferred to the stationary plate surface of a Cap2000þ cone-plate viscometer (Brookfield Engineering Laboratories, Middleboro, MA) and subjected to a shear stress treatment with the primary shear rate at 25 dyn$cm2 for 60 s at room temperature. Then approximately 50 mL of PRP mixture was collected gently using a microcapillary pipet tip, incubated at room temperature for 10 min, washed with modified Tyrode’s Buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose, 12 mM NaHCO3, 20 mM HEPES (pH 7.35)) when desired, and fixed by the addition of 200 mL 4% paraformaldehyde. If desired, 0.5 mg$mL1 FITC-conjugated 5G6 Fab was added to the PRP mixture after it was collected after the shear treatment and before the incubation. Fixed PRP sample was analyzed on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA) using FlowJo software as described previously (11).

Uniform shear assay Platelets (in PRP) were incubated with the indicated antibodies before shear treatment. The mixture was then sheared for the indicated time periods (0, 0.5, 1, 3, and 5 min) on the Cap2000þ cone-plate viscometer applying 20 dyn$cm2 of shear at 22 C. Shear-treated PRP was labeled with binding proteins for the detection of surface markers (0.08 mM Lact-C2, 20 mg$mL1 anti-P-selectin antibody (BioLegend, San Diego, CA)) for 20 min before fixation in 2% paraformaldehyde. Platelet flow cytometry was performed on a Beckman Coulter CytoFLEX Flow Cytometer (Brea, CA) as previously described (21). Signal strength was quantitated using the height value for the intensity of each fluorophore, and a bisecting gate was drawn based on a negative control to determine the percentage of positive events in each sample. At least 20,000 events were collected for each replicate, and data were analyzed by FlowJo and GraphPad Prism software.

RESULTS WM23 Fab generates stable pulling curves at high forces Recombinant human GPIb-IX was expressed in transfected HEK293 cells. The cytoplasmic tail of GPIX was appended

Platelet GPIb-IX Biomechanics

with a biotag and thereby biotinylated (20). The biotinylated GPIb-IX was immobilized on a streptavidin bead for the single-molecule force measurement (Fig. 1 b). When A1 was used to capture GPIb-IX in our previous experiments, the tether rupture forces in the pulling curves were typically 5–30 pN, and only 20% of the pulling traces showed MSD unfolding (20). This was because of the limited mechanical strength of the A1-GPIba bond. Using this approach, tethers usually broke after one or two pulling-relaxation cycles. This limitation made the measurement less efficient than what was required for a thorough examination of the mechanical properties of the MSD (11). Our current experimental setup is illustrated in Fig. 1 b. To thoroughly examine mechanical properties of the MSD, WM23, a monoclonal antibody recognizing the sialomucin region of GPIba (34), was utilized as a ligand to pull on GPIb-IX. Because the epitope of WM23 is located between the LBD and the MSD of GPIba (34), a pulling force via WM23 will not be applied to, nor unfold, the LBD (35). To maximize the events of single-molecule binding and pulling in our experiments, the monovalent Fab fragment of WM23 was prepared and tethered to the DNA handle (Fig. 1 b). Given that WM23 binds to GPIba with a high affinity (34), the WM23-GPIba linkage is likely to withstand strong pulling forces. Indeed, as shown in Fig. 1 c, the strong

a

b

tethered bond between WM23 and GPIba enabled multiple cycles of stretching and relaxation without the detachment of WM23 from GPIb-IX. Once a single tether was caught between the two beads, confirmed by DNA overstretching at 67 pN, 85% of the tethers showed MSD unfolding. Fig. 2 a illustrates repetitive pulling and relaxation cycles, accompanied by recorded consecutive unfolding and refolding events. For the tethers displaying MSD unfolding, we applied a pulling protocol in which force was ramped up from 0.5 to 80 pN under varying loading rates and then decreased back to 0.5 pN using the same unloading rate. The tether was relaxed at 0.5 pN for 10 s before the next pulling cycle begins. Four different loading rates, which were determined by a linear fitting of the force versus time trace before each unfolding event occurred, were used in this study: 2.9, 6.9, 14.3, and 24.8 pN/s. The unfolding force versus unfolding extension was plotted, and the curve was fitted to the worm-like chain (WLC) model (Fig. 2 b). The WLC fit of the MSD unfolding force and extension yielded a contour length of 25.0 5 0.9 nm and a persistence length of 0.81 5 0.11 nm. These two numbers are very close to the MLC parameters in our previous study using A1 to pulling GPIb-IX (Fig. 2 b), in which the contour and persistence lengths were 25.1 5 0.3 and 0.74 5 0.04 nm, respectively (20). These results indicate that

FIGURE 2 Unfolding properties of MSD. (a) Shown is an overlay of 53 consecutive pullingrelaxation traces of stretching a GPIb-IX complex from one single tether by mAb WM23. Inset: a zoomed-in force-trap position traces show the force-induced unfolding and refolding of MSD from pulling at 150 nm/s. (b) Superimposed plots of unfolding force versus unfolding extension data and their fits to the WLC model is shown
2 FðxÞ,Lp 1 x (dashed line):  14 þ Lxc kB T ¼ 4 1  Lc

where F(x) is the applied force on the polymer, x is the end-to-end distance, Lc is the contour length, c d and Lp is the persistence length of the polymer. The unfolding extension is defined as the increase in end-to-end length of MSD when it unfolds. A histogram of unfolding extension (inset) was used to find peak extension. The fit and data plot of A1 pulling on GPIb-IX was from an earlier study (20). Error bars are one SD for force and half width of the Gaussian fit for extension. (c) Shown are plots of the most probable unfolding force of the MSD in GPIb-IX and of a recombinant isolated MSD (cSc) as a function of loading rates. Unfolding forces at different loading rates were plotted as histograms (Fig. S1). The most probable unfolding forces were identified from these histograms and plotted against their corresponding loading rates. The error bars are the half bin width. The solid line is a linear fit of the data to the Bell-Evans model (Eq. 3). (d) Shown are plots of unfolding force versus unfolding extension data for the MSD and cSc and their fits to the WLC model (dashed line). Error bars are one SD for force and half width of the Gaussian fit for extension. MSD unfolding data was obtained from 32 tethers, which generated 636 unfolding forces. cSc unfolding data was obtained from 57 tethers and a total of 410 unfolding forces.

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Zhang et al.

WM23 mediates the same extent of MSD unfolding as VWF-A1 does (11,21). The unglycosylated MSD exhibits a weaker unfolding force than the MSD in full-length GPIb-IX In addition to biotinylated GPIb-IX, a recombinant MSD in isolation, designated cSc, was characterized by force. The cSc protein contains human GPIba residues 417–483, with a number of serine and threonine residues that are O-glycosylated in the native form. Because cSc was expressed in E. coli, it is not glycosylated (20). The addition of flanking cysteines in cSc does not significantly affect the structure of the MSD in isolation, although it modestly stabilizes the domain (20). The flanking cysteines also enabled the coupling of cSc to two pieces of DNA handles through disulfide bonds as described previously (20,27). Carboxyl polystyrene beads of 2.0-mm diameter were covalently coupled with streptavidin, anti-digoxigenin Fab, and were used to pull cSc in repetitive pulling and relaxation cycles. To obtain the unfolding kinetic rates, the most probable unfolding forces were identified from the mode (peak) of each histogram (Fig. S1), and these forces were then plotted against the logarithm of the loading rates. Unfolding forces for both native MSD in GPIb-IX and the isolated cSc were grouped by different loading rates. As shown in Fig. 2 c, the most probable forces increase exponentially with the loading rate, similar to what has been found in many other ligand-receptor systems (36). However, the unfolding forces of native MSD are greater than those of the cSc at all the tested loading rates, suggesting that the native MSD is more stable than its recombinant unglycosylated form. The unfolding kinetic rates were extracted using the BellEvans model (29). As predicted by the model, the most probable unbinding force f* is a linear function of the logarithm of the loading rate (Eq. 3). These kinetics parameters were characterized by fitting Eq. 3 to the f* versus the loading rate curve shown in Fig. 2 c. The fitted curves are overlaid on Fig. 2 c, and the best-fit parameters ku0 and g are tabulated in Table 1. Fitting Fig. 2 c to the Bell-Evans’ model yielded an unstressed unfolding rate k0 of 0.009 5 0.006 s1. The energy barrier position parameter g describes the sensitivity of the MSD unfolding rate as a function of force. The unfolding rate would rise exponentially when a force scale of kBT/g is applied. For the MSD, this force scale kBT/g is 2.0 pN. In comparison, the unstressed unfolding rate of recombinant cSc is 0.03 5 0.01 s1, which is 3-fold greater than that of the native MSD, suggesting that the cSc is less mechanically stable than the native MSD. In addition to using the Bell-Evans model, we also fitted the unfolding data to the Dudko-Hummer-Szabo model

1964 Biophysical Journal 116, 1960–1969, May 21, 2019

(37). The distributions of unfolding forces at different loading rates (Fig. S1) were first fitted to a statistical model (37) to obtain the force-dependent lifetimes (Fig. S2). The average lifetimes for MSD and cSc unfolding were then fitted to the Dudko-Hummer-Szabo model (Fig. S2, A and B). The fitted results were summarized in Fig. S2 C. The Dudko-Hummer-Szabo model fit yielded comparable zero-force unfolding rates as obtained from the Bell-Evans model. Despite some differences in the trend of force dependency, both models yielded the conclusion that, when exposed to a pulling force, the native MSD exhibits greater force resistance (i.e., longer folded lifetimes) compared to the cSc (Fig. S2 D). To further compare the differences in unfolding between the MSD and cSc, we plotted the unfolding force versus unfolding extension and fit the curves to a WLC model (Fig. 2 d). The WLC fit to the cSc data yielded a contour length of 21.6 5 1.3 nm, significantly shorter than that of the MSD, 25.0 5 0.9 nm (Fig. 2 d). The contour length reflects the difference between the fully unfolded structure and the end-to-end distance of the folded structure. The contour length difference (3.4 nm) between native MSD and cSc suggests that there might be a structural difference involving longer end-to-end distance in the folded cSc. Unfolded MSD can refold under force In pulling-relaxation traces of native full-length GPIb-IX, MSD refolding was evident, manifested by an abrupt shortening of end-to-end distance in the relaxation trace (Fig. 1 c). Overall, refolding of the MSD was observed in 70% of the relaxation traces. When refolding cannot be clearly observed at the low force region (i.e., either between 0.5 and 1 pN or during the 10-s force-clamped period at 0.5 pN), MSD unfolding was observed after 10 s of relaxation, suggesting that refolding still occurred during the relaxation period. Refolding forces were first grouped by eight different unloading rates and plotted in histograms (Fig. S3). The most probable refolding forces were then obtained from the histograms and plotted against the logarithm of the unloading rates (Fig. 3 a). It is evident from Fig. 3 a that the most probable refolding force is inversely proportional to the logarithm of the unloading rates. The data was fitted to a modified Bell-Evans model (30) described in Methods. The fit yielded an unstressed refolding rate of 17 5 3 s1 and a distance to transition state 1.1 5 0.1 nm. Hence, the force scale on which MSD refolding depends is kBT/1.1 nm or 3.7 pN. In addition, we extracted the refolding rates at various forces from the refolding force distributions (Fig. S3) using a statistical method (Eq. 6) described in Methods. The force dependence of these refolding rates was fitted to a model developed by Evans et al. (33) that takes into account the compliance of the unfolded protein (Eq. 7) (Fig. 3 b). This

Platelet GPIb-IX Biomechanics

a

b

FIGURE 3 Refolding properties of MSD. (a) The plot of most probable refolding forces as a function of unloading rates is shown. The most probable refolding forces were obtained from refolding force histograms of eight different unloading rates (Fig. S3). The error bars are the half bin width. The solid line is a linear fit of the data to the modified Bell-Evans model to describe protein refolding (Eq. 4). (b) The plot of refolding rates kf as a function of pulling force is shown. The kf values were calculated using Eq. 6 from the refolding force distributions (Fig. S3). The force dependence of refolding was fitted to a protein refolding model developed by Evans et al. (33) that takes into account the soft compliance of the unfolded state. The refolding data was generated from 38 tethers and 867 refolding curves. To see this figure in color, go online.

model has been previously used to describe the refolding of the A2 domain of VWF (27). The fitting yielded an unstressed refolding rate of 18 5 1 s1, and k, the compliance parameter was 2.1 5 0.3 pN/nm. The unstressed refolding rate is in good agreement with the value obtained from the Bell-Evans model. The k value is approximately one order of magnitude higher than that of the A2 domain (27), suggesting that the unfolded MSD is much stiffer than the A2. This could be because of possible interactions between the MSD and the surrounding GPIbb and GPIX subunits as well as to the O-linked glycosylation on the MSD. Using the unstressed folding and unfolding rates obtained from the Bell-Evans model analysis, we estimated the free energy difference between the two states: DG ¼ –kBT∙ln (ku0/kf0) y 7.5 kBT. Because most folded proteins have a free energy of >10 kBT (38), our data suggest that the MSD is metastable, making it suitable to function as a mechanical sensor. Refolding of MSD weakens platelet mechanical signals The monoclonal antibody 5G6 specifically recognizes a linear epitope in the MSD, exhibiting very similar nanomo-

lar binding affinities for the epitope peptide and the fulllength GPIb-IX (26,39). Although 5G6 can bind to GPIb-IX on the surface of resting platelets, its binding is significantly increased when platelets are stimulated by GPIba ligands under shear stress, in which the MSD becomes unfolded and the 5G6 epitope more exposed (11,21,40). Similar to 5G6, the Fab fragment of 5G6 exhibits higher binding when the MSD is unfolded and has overall better sensitivity, likely because of its smaller size. An increase in 5G6 Fab binding, as detected by flow cytometry, was observed when it was premixed with platelets before shear stress was applied (Fig. 4 a). Interestingly, when 5G6 Fab was added to platelets shortly (10 s) after the shear stress treatment, its binding only marginally increased compared to that with no shear treatment and was significantly less than that of the premixed sample (Fig. 4 a). This observation suggests that the MSD may refold once the shear stress was removed, consistent with the relatively fast unstressed refolding rate (17 s1) observed in our single-molecule experiments. In our recent study of immune thrombocytopenia, we showed that antibodies targeting the LBD of GPIba can cross-link platelets and apply tensile force to the MSD, leading to MSD unfolding and platelet activation (21). This cross-linking-dependent activation is contingent upon a high unbinding force between the antibody and LBD. 11A8, an antibody with an average unbinding force of 30 pN, is sufficient to sustain the 15 pN of force required to unfold the MSD, unlike AK2, with an unbinding force <15 pN (21). Here, the two antibodies of differing unbinding forces were incubated with PRP during shear. When incubated with 11A8, platelet activation increased with shear duration, an effect not observed with AK2, which did not trigger activation at any shear duration tested. The extent of platelet activation, as measured by the surface expression of P-selectin and PS, after brief shear exposure of 30 s was markedly less than after longer exposures of >3 min (67 and 45% less for P-selectin and PS, respectively) (Fig. 4, b–d). Notably, the increase in activation signals with respect to time appeared to taper off after 3 min of shear exposure. Increasing the shear time to 5 min did not lead to a significant increase of activation signals (Fig. 4, b–d). These results indicate that activation of GPIb-IX in platelets was dependent on the duration of shear exposure. They are consistent with a recent observation in which pulling on the LBD via engaged VWF for at least 2 s is required to trigger a sustained calcium influx in platelets (25) and support the idea that applying tension on GPIb-IX for a period of time may be needed to sustain unfolding of the MSD and to activate GPIb-IX. In the shear flow assays shown in Fig. 4, platelets were incubated with either human plasma (with a typical VWF concentration of 10 mg/mL) with botrocetin (Fig. 4 a) or with one of two antibodies (11A8 and AK2) recognizing the N-terminal LBD of GPIba (Fig. 4, b–d). During

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a

c

b

d

incubation, platelets may be cross-linked by VWF (facilitated by botrocetin) or antibodies and form a dumbbelllike structure. Shankaran and Neelamegham (41) have estimated the tensile force exerted on platelet dumbbells under shear flow. The maximal tensile force can be expressed as Ft ¼ an m G, where G is the shear rate, m is viscosity, and an is a coefficient dependent on platelet radius as well as the length of the linker between the two platelets. Using the an computed by Shankaran et al., the 1250 s1 shear rate used in our shear flow assay, and assuming the linker length being 60 and 190 nm for antibody- and dimeric VWF-cross-linking cases, respectively, we found the tensile forces applied to platelets to be 25 pN. Note that if the platelets were cross-linked by VWF multimers larger than a dimer, the tensile forces would be even greater. Therefore, the tensile forces experienced by GPIb-IX in the shear flow assays were 25 pN or higher and should be sufficient to unfold the MSD during the assay. DISCUSSION The GPIb-IX complex has long been recognized as a major shear sensor and shear receptor on platelets as the activation of GPIb-IX by its ligands requires shear stress (14). Sitespecific biotinylation of GPIb-IX enabled single-molecule force measurement on the intact receptor complex and the initial identification of the MSD therein (20). Follow-up studies have provided evidence linking the unfolding of the MSD to GPIb-IX activation and platelet signaling (11,21,25,40,42). In this study, utilizing the anti-GPIba monoclonal antibody WM23 with a strong unbinding force,

1966 Biophysical Journal 116, 1960–1969, May 21, 2019

FIGURE 4 MSD refolding weakens platelet activation signals. (a) Shown is the overlaid flow cytometry histograms comparing the binding of 5G6 Fab to platelets under the indicated conditions, in the presence of botrocetin (1 mg$mL1). (b–d) Shown is the percentage of platelets positive for (b) P-selectin exposure, (c) phosphatidylserine (PS) exposure, or (d) both on human platelets treated with control IgG, 11A8, or AK2 under 20 dyn/cm2 of shear force at the indicated time points. n ¼ 3; significance determined by oneway analysis of variance with Tukey test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. To see this figure in color, go online.

we were able to achieve efficient, consecutive cycles of pulling and relaxation in single-molecule force measurements. This has led to the first direct detection of refolding of the MSD in GPIb-IX. Fitting the refolding data to two independent protein refolding models produced an unstressed refolding rate of the MSD at 17 s1 (Fig. 3). Under applied tension, MSD refolding slows down exponentially over a force scale of 3.7 pN. The consecutive cycles of pulling and relaxation also facilitated a systematic characterization of MSD unfolding under various loading rates. The unfolding and refolding properties make the MSD particularly suitable for sensing blood forces. In the vasculature, shear rates range from tens of s1 in venules and veins up to a few thousand s1 in arterioles and arteries. Under pathological conditions, such as in hemorrhage or thrombosis, shear rates may increase significantly to the order of 10,000 s1. Elevated shear may stretch and activate circulating multimeric VWF, which may cross-link platelets via its A1 domains. A shear rate of 1000 s1 is sufficient to generate a tensile force of 20 pN on cross-linked platelet dumbbells (41). Forces at this magnitude are expected to unfold MSD and further activate platelets. Upon vascular damage, VWF can tether onto subendothelial collagen via its A3 domain. In a recent pivotal study by Fu and colleagues (43), the tension developed on tethered VWF under shear has been shown to activate A1 domain. The activated A1 on VWF may capture platelets from the flowing blood (4). Under shear flow, the significant tensile forces may be applied to the bonded pair of VWF and GPIba (4,44,45). The tensile force applied to a platelet tethering to a wall at a shear rate of 1000 s1 has

Platelet GPIb-IX Biomechanics

been estimated to be 97 pN (46). This force may also unfold the MSD and transduce mechanical signals into platelets. Under physiological conditions, the force applied to unfold MSD likely stems from stretching the LBD domain of GPIba via the engagement of A1. Hence, the mechanical properties of the A1-LBD linkage are tightly connected to whether or not the MSD can be unfolded. It is reasonable to assume that the lifetime of the A1-LBD bond must be longer than that of the folded MSD, so that a sufficient duration of force can be applied to unfold the MSD. If not, the A1-LBD linkage would likely break before any MSD unfolding could take place. Fig. 5 a shows a comparison of mechanical properties of the MSD and the reported A1-LBD interactions, including our previous study (20) using optical tweezers (OT), an earlier study by Kim et al. using OT, and those from Ju et al. (35) determined by the biomembrane a

b

FIGURE 5 Comparison of lifetimes and MSD-unfolded fraction as a function of force. (a) Shown is a comparison of bond/folded lifetime as a function of the force of folded MSD, A1-GPIba interactions, and interactions between GPIba and activated A1 (e.g., by botrocetin) or plasma autoantibodies found in immune thrombocytopenia against Iba. The force-dependent lifetimes (i.e., the reciprocal dissociation rate constant) were determined by Eq. 1, using the Bell-Evans model parameters determined for MSD unfolding from the current study or from literature using optical tweezers (OT) (20,47) or a biomembrane force probe (BFP) (35). (b) Shown is the unfolding fraction of MSD at equilibrium as a function of force. The unfolded fraction is estimated using kunfold/krefold/(1 þ kunfold/krefold).

force probe. An examination of the kinetic lifetime profiles revealed that when the force was lower than 12 pN, the lifetime of the A1-LBD bond is either shorter (biomembrane force probe data) than or comparable (OT data) to the MSD unfolding time. Under these conditions, the A1LBD linkage may not be able to trigger MSD unfolding. However, at higher forces (>14 pN), the A1-LBD bond becomes more stable (46,47) than the folded MSD, suggesting that only under higher forces can the A1-LBD interaction trigger MSD unfolding. Significantly, in comparison to the lifetime of the botrocetin-enhanced A1-LBD bond (11), or the lifetimes of the bonds between strong-binding antibodies (e.g., 11A8 and 6B4) and the LBD (21), the MSDunfolding time is at least one order of magnitude shorter (Fig. 5 a). These differences can explain our earlier observations that VWF with botrocetin and some anti-LBD antibodies can trigger MSD unfolding and GPIb-IX signaling in platelets and GPIb-IX-transfected cells (11,21). There exist some discrepancies on the force dependence within the lower force regime (<14 pN) among the cited A1LBD lifetime studies (Fig. 5 a). This difference is likely due to the actual A1 and GPIba constructs being studied, such as their activation states, how the proteins were immobilized on the force probes, and whether they contained proper O-linked glycosylation. Moreover, using the force-dependent kinetic rates, kunfold(f) and krefold(f), calculated using the Bell-Evans model, the predicted unfolded fraction of the MSD at equilibrium as a function of force can be estimated (Fig. 5 b). Our results indicate that the MSD unfolded fraction starts to increase when the force rises to 5 pN, and the MSD unfolding is saturated at around 15 pN. Together with the observations reported in Figs. 4 and 5 a, these results suggest a threshold of force over time (>15 pN) for the activation of GPIb-IX on the platelet surface (11,21,25). It should be noted that neither the botrocetin-enhanced A1-LBD bond nor the 6B4-LBD bond exhibit the feature of a catch bond, yet both botrocetin and 6B4 can readily activate GPIb-IX and induce fast platelet clearance (11,21). Thus, a catch bond is not required for the activation of GPIb-IX. The force-regulated unfolding/refolding parameters of the MSD in the full-length GPIb-IX complex were also compared to those of the recombinant cSc that mimics an isolated MSD. The unfolding forces of native MSD are greater than those of the cSc at all loading rates, and the contour length of the native MSD is 3–4 nm longer than that of the cSc (Figs. 2 and 3). These results indicate that although the cSc is mechanosensitive, it differs from the native MSD in several aspects. One possibility is that the native MSD contains more residues than the cSc. The MSD has been mapped to a juxtamembrane location in GPIba by analysis of deletion mutations (20), and its C-terminal boundary is likely delimited by the transmembrane domain of GPIba (unpublished data). However, its N-terminal boundary has not been precisely determined. It was estimated based on

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Zhang et al.

the contour length of the MSD and the assumption that the polymorphic 13-residue variable number tandem repeat sequence in GPIba is not a part of the MSD (20), and the cSc was produced accordingly. Another possibility is that the end-to-end distance of the folded cSc is significantly different from that of the native MSD in the GPIb-IX complex. Earlier and current studies suggest that the MSD is not very stable. It is thus conceivable that the conformation of the cSc differs somewhat from that of the native MSD. Moreover, cSc is produced from E. coli and therefore unglycosylated. In contrast, the MSD contains several serine and threonine residues and is heavily glycosylated (20). Such difference in glycosylation may also contribute to their structural and stability difference. Although it is potentially critical to delineate the molecular basis for the aforementioned difference, our results suggest that the cSc may not be an ideal model for the native MSD. The structure of the MSD, in the context of the full-length GPIb-IX complex, remains to be determined. SUPPORTING MATERIAL Supporting Material can be found online at https://doi.org/10.1016/j.bpj. 2019.03.037.

6. Huizinga, E. G., S. Tsuji, ., P. Gros. 2002. Structures of glycoprotein Ibalpha and its complex with von Willebrand factor A1 domain. Science. 297:1176–1179. 7. Deng, W., Y. Wang, ., R. Li. 2017. A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor. J. Thromb. Haemost. 15:1867–1877. 8. De Marco, L., A. Girolami, ., Z. M. Ruggeri. 1985. Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation. Proc. Natl. Acad. Sci. USA. 82:7424–7428. 9. Goto, S., D. R. Salomon, ., Z. M. Ruggeri. 1995. Characterization of the unique mechanism mediating the shear-dependent binding of soluble von Willebrand factor to platelets. J. Biol. Chem. 270:23352– 23361. 10. Savage, B., F. Almus-Jacobs, and Z. M. Ruggeri. 1998. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell. 94:657–666. 11. Deng, W., Y. Xu, ., R. Li. 2016. Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor. Nat. Commun. 7:12863. 12. Quach, M. E., W. Chen, and R. Li. 2018. Mechanisms of platelet clearance and translation to improve platelet storage. Blood. 131:1512– 1521. 13. Sanders, W. E., Jr., R. L. Reddick, ., M. S. Read. 1995. Thrombotic thrombocytopenia induced in dogs and pigs. The role of plasma and platelet vWF in animal models of thrombotic thrombocytopenic purpura. Arterioscler. Thromb. Vasc. Biol. 15:793–800. 14. Kroll, M. H., J. D. Hellums, ., J. L. Moake. 1996. Platelets and shear stress. Blood. 88:1525–1541.

AUTHOR CONTRIBUTIONS

15. Luo, S. Z., X. Mo, ., R. Li. 2007. Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib-IX complex. J. Thromb. Haemost. 5:2494–2502.

X.F.Z. designed study, performed experiments, analyzed results, and wrote the manuscript. W.Z., M.E.Q., and W.D. performed experiments, analyzed data, prepared figures, and edited manuscript. R.L. designed study and wrote the manuscript.

16. Luo, S. Z., and R. Li. 2008. Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex. J. Mol. Biol. 382:448–457.

ACKNOWLEDGMENTS

18. McEwan, P. A., W. Yang, ., J. Emsley. 2011. Quaternary organization of GPIb-IX complex and insights into Bernard-Soulier syndrome revealed by the structures of GPIbb and a GPIbb/GPIX chimera. Blood. 118:5292–5301.

We acknowledge the technical assistance from the Emory Pediatrics flow cytometry core. R.L. is the inventor of a U.S. patent on the 5G6 monoclonal antibody (US10081678). This work was supported in part by National Institutes of Health grants HL082808, HL123984 (R.L.) and HL134241 (M.E.Q.) and a Faculty Research Grant from Lehigh University (X.F.Z.).

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