Akt-mediated platelet apoptosis and its therapeuticimplications in immune thrombocytopeniaMengxing Chena,1, Rong Yana,1,2, Kangxi Zhoua,1, Xiaodong Lia, Yang Zhanga, Chunliang Liua, Mengxiao Jianga,Honglei Yea, Xingjun Menga, Ningbo Panga, Lili Zhaoa, Jun Liua, Weiling Xiaoa, Renping Hua, Qingya Cuia, Wei Zhonga,Yunxiao Zhaoa, Mingqing Zhua, Anning Linb, Changgeng Ruana, and Kesheng Daia,2

aJiangsu Institute of Hematology, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, State Key Laboraotry of RadiationMedicine and Protection, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Suzhou, Jiangsu 215006, China; and bBenMay Department for Cancer Research, The University of Chicago, Chicago, IL 60637

Edited by Barry S. Coller, The Rockefeller University, New York, NY, and approved September 25, 2018 (received for review May 14, 2018)

Immune thrombocytopenia (ITP) is an autoimmune disorder charac-terized by low platelet count which can cause fatal hemorrhage. ITPpatients with antiplatelet glycoprotein (GP) Ib-IX autoantibodiesappear refractory to conventional treatments, and the mechanismremains elusive. Here we show that the platelets undergo apoptosisin ITP patients with anti-GPIbα autoantibodies. Consistent with thesefindings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induceplatelet apoptosis in vitro. We demonstrate that anti-GPIbα antibodybinding activates Akt, which elicits platelet apoptosis through activa-tion of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibi-tion. Genetic ablation or chemical inhibition of Akt or blocking of Aktsignaling abolishes anti-GPIbα antibody-induced platelet apoptosis.We further demonstrate that the antibody-bound platelets are re-moved in vivo through an apoptosis-dependent manner. Phosphati-dylserine (PS) exposure on apoptotic platelets results in phagocytosisof platelets by macrophages in the liver. Notably, inhibition or geneticablation of Akt or Akt-regulated apoptotic signaling or blockageof PS exposure protects the platelets from clearance. Therefore,our findings reveal pathogenic mechanisms of ITP with anti-GPIbαautoantibodies and, more importantly, suggest therapeutic strat-egies for thrombocytopenia caused by autoantibodies or otherpathogenic factors.

immune thrombocytopenia | platelet | apoptosis | Akt |phosphatidylserine exposure

Immune thrombocytopenia (ITP) is an autoimmune disordercharacterized by low platelet count (1, 2) which is caused pri-

marily by autoantibodies against two major receptors of platelets,the fibrinogen receptor glycoprotein (GP) IIb/IIIa and the vonWillebrand factor (VWF) receptor GPIb-IX complex (3–5). Theautoantibody-bound platelets are thought to be removed by Fc-dependent phagocytosis in the spleen (1, 2, 6). Therefore, themain therapeutic strategies for ITP are immune suppression, im-mune modulation, and splenectomy (1, 2, 7). However, ITP patientswith anti–GPIb-IX autoantibodies present more severe decreases inplatelet count (4) and are less responsive to conventional therapiessuch as steroid treatments (8), i.v. IgG (IVIG) (5, 9), and evensplenectomy (10, 11), suggesting that a different pathogenic mech-anism may be involved in anti–GPIb-IX autoantibody-inducedplatelet clearance.Anti-GPIbα monoclonal antibodies were found to activate plate-

lets in vitro (12–16) and induce platelet clearance in vivo (12, 17–20).More recent studies demonstrated that anti-GPIbα antibodiesinduced phagocytosis of platelets in the liver through anFc-independent mechanism (12, 17, 20). Anti-GPIbα antibodiestargeting the N terminus of the receptor cause it to cluster, resultingin phagocytosis of platelets by microphages in the liver (12). On theother hand, GPIbα desialylation was demonstrated to contributeto platelet clearance in an hepatocyte Ashwell–Morell receptor-dependent manner (20). Moreover, shear-induced unfolding of theGPIbα mechanosensory domain by anti-GPIbα monoclonal anti-bodies was found to trigger signaling, leading to platelet clearance(21). Therefore, while increasing evidence suggests that anti-GPIbα

autoantibodies may induce platelet clearance via an Fc-independentmanner, the mechanism for anti-GPIbα antibody-induced thrombo-cytopenia remains elusive.GPIbα, the main subunit of the GPIb-IX complex, contains

binding sites for several important ligands including VWF andthrombin at the N-terminal extracellular domain (16, 22, 23).The interaction of the VWF multimer with GPIbα inducestranslocation and cross-linking of GPIb-IX complexes in lipidrafts (24–27), triggering signaling cascades (28, 29) and leadingto platelet activation and thrombus formation (30, 31). In-terestingly, we found that the GPIbα–VWF interaction could alsoinduce platelet apoptosis, but the mechanism remains unknown(32). We recently reported that protein kinase A (PKA)-mediatedplatelet apoptosis occurs extensively in pathophysiological condi-tions (33). Moreover, accumulating evidence suggests that variouspathological stimuli lead to thrombocytopenia in many commondiseases, such as infection, cancer, diabetes, and heart and circula-tion diseases (34–37). However, little is known about the patho-genesis leading to thrombocytopenia.In this study, we find that anti-GPIbα monoclonal antibodies

induce Akt activation and Akt-mediated platelet apoptosis. Wedemonstrate that platelets undergo apoptosis in ITP patients

Significance

Immune thrombocytopenia (ITP) patients with antiplatelet gly-coprotein (GP) Ib-IX autoantibodies appear refractory to con-ventional treatments; however, the mechanism remains elusive.Here we show that the platelets undergo apoptosis in ITP pa-tients with anti-GPIbα autoantibodies. We demonstrate thatanti-GPIbα antibody binding activates Akt, which elicits plateletapoptosis through activation of phosphodiesterase (PDE3A) andPDE3A-mediated PKA inhibition. Phosphatidylserine (PS) expo-sure results in phagocytosis of anti-GPIbα antibody-boundplatelets by macrophages in the liver. Notably, inhibition orgenetic ablation of Akt or Akt-regulated apoptotic signaling orblockage of PS exposure rescues the platelets from clearance.Therefore, our findings reveal pathogenic mechanisms of ITPwith anti-GPIbα autoantibodies and, more importantly, suggesttherapeutic strategies for thrombocytopenia caused by autoan-tibodies or other pathogenic factors.

Author contributions: M.C., R.Y., K.Z., and K.D. designed research; M.C., R.Y., K.Z., X.L.,Y. Zhang, C.L., M.J., H.Y., X.M., N.P., L.Z., J.L., W.X., R.H., Q.C., W.Z., Y. Zhao, and M.Z.performed research; M.C., R.Y., C.R., and K.D. analyzed data; R.Y. and K.D. wrote thepaper; A.L. provided Bad−/− mice; and K.D. initiated and supervised the project.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1M.C., R.Y., and K.Z. contributed equally to this work.2To whom correspondence may be addressed. Email: yanrongbaobao@163.com or kdai@suda.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1808217115/-/DCSupplemental.

Published online October 18, 2018.

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with anti-GPIbα autoantibodies. The apoptotic platelets arephagocytized by macrophages in the liver in a phosphatidylserine(PS) exposure-dependent manner. Inhibition or genetic ablationof Akt or Akt-regulated apoptotic signaling or blockage of PSexposure rescues the platelets from clearance. Therefore ourfindings reveal pathogenic mechanisms of ITP with anti-GPIbαautoantibodies and, more importantly, suggest therapeuticstrategies for thrombocytopenia caused by autoantibodies orother pathogenic factors.

ResultsAnti-GPIbα Antibodies Induce Platelet Apoptosis. To investigate thepathogenesis of ITP with anti-GPIbα antibodies, we selectedanti-GPIbα monoclonal antibodies (SI Appendix, Table S1) toexamine their effects on platelets. We hypothesized that anti-GPIbα antibodies could induce platelet apoptosis. To test this,we incubated the anti-GPIbα antibodies AN51 and SZ2 withhuman platelets. We found that these antibodies inducedmarked mitochondrial transmembrane potential (ΔΨm) de-polarization (Fig. 1A), which initiates mitochondria-mediatedintrinsic programmed apoptosis in platelets (32–35). Moreover,AN51 and SZ2 significantly elevated caspase-3 activity in plate-lets, as indicated by the appearance of 17-kDa fragments (Fig.1B), and total caspase-3 activity (Fig. 1C). Caspases combinedwith other apoptogenic enzymes disrupt plasma membrane in-tegrity leading to PS externalization during apoptosis (32–35).We found that AN51 and SZ2 indeed induced PS exposure inthe platelets (Fig. 1D). These data indicate that the anti-GPIbα

antibodies induce platelet apoptosis. Moreover, as is consistentwith previous reports (12–16), anti-GPIbα antibodies also in-duced platelet activation, as indicated by P-selectin exposure andPAC-1 binding (SI Appendix, Fig. S1). In contrast, the anti-GPIIb/IIIa antibodies SZ21 and D57 did not induce plateletapoptosis or activation (SI Appendix, Fig. S1). In addition, theanti-GPIbα antibody HIP1 also did not induce obvious plateletapoptosis (Fig. 1 A–D).

Platelets Undergo Apoptosis in ITP Patients with Anti-GPIbαAutoantibodies. To verify the observations with anti-GPIbαmonoclonal antibodies in ITP patients, we identified 12 ITPpatients with anti-GPIbα autoantibodies (SI Appendix, TableS2) using the flow cytometric immunobead array (38). Afterhealthy human platelets were incubated with plasma from theITP patients, we found that the plasma obviously inducedΔΨm depolarization (Fig. 1E), PS exposure (Fig. 1F), andcaspase-3 activation in the platelets (Fig. 1G). These datasuggest that the anti-GPIbα autoantibody plasma can induceplatelet apoptosis in vitro. To rule out other factors, such ascytokines and growth factors, that might be present in ITP plasmaand influence the results, we purified IgG fractions from the ITPplasmas containing anti-GPIbα autoantibodies. Compared with IgGfractions from normal plasmas, IgG fractions from ITP plasmasinduced obvious ΔΨm depolarization, PS exposure, and caspase-3 activation in the platelets (SI Appendix, Fig. S2).We also directly examined the platelets from the ITP patients

for apoptotic events. ΔΨm depolarization was detected in the

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Fig. 1. Anti-GPIbα antibodies induce platelet apoptosis. (A–D) Washed human platelets were incubated with 10 μg/mL normal mouse IgG or anti-GPIbαantibody AN51, SZ2, or HIP1 at 37 °C for 8 h. (A) Representative flow cytometric figures of platelet Δψm depolarization. The JC-1 monomers reflect themonomeric form of JC-1 that appeared in the cytosol after mitochondrial Δψm depolarization, and the JC-1 aggregates represent potential-dependentaggregation in the mitochondria. (B) Western blot analysis of caspase 3 with anti–caspase-3 antibody. (C) Analysis of caspase-3 activity with ELISA, n = 6. (D)Representative flow cytometric figures of platelet PS exposure. (E–G) Washed normal human platelets were incubated with plasma from healthy donors(control plasma) or from ITP patients with only anti-GPIbα autoantibodies (ITP plasma) at 37 °C for 8 h (1:1, vol/vol). Platelet Δψm depolarization (E) and PSexposure (F) were detected by flow cytometry. (G, Left) Caspase 3 was analyzed with anti–caspase-3 antibody by Western blot. (Right) Densitometry ofimmunoblot for cleaved caspase 3 from the Western blot data. (H and I) Platelet Δψm depolarization (H) and PS exposure (I) were detected by flow cytometryin PRP isolated from healthy donors (control platelets) and ITP patients with anti-GPIbα autoantibodies (ITP platelets). Data in C and G are expressed asmean ± SD. In E, F, H, and I horizontal lines indicate the median values, and each dot represents one patient. **P < 0.01, ***P < 0.001 compared with controlby one-way ANOVA (C), Mann-Whitney U test (E, F, H, and I), or Student’s t test (G). NS, not significant.

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platelets from ITP patients with anti-GPIbα autoantibodies (Fig.1H). Interestingly, unlike the results with plasma in vitro, therewas no significant difference in PS exposure between plateletsfrom ITP patients and platelets from healthy controls (Fig. 1I).The reason might be that PS-exposed platelets had been re-moved from the circulation in vivo. Moreover, P-selectin exposurewas obviously elevated in healthy human platelets incubated withplasma containing anti-GPIbα autoantibodies (SI Appendix, Fig.S3A) and in platelets from the patients (SI Appendix, Fig. S3B),suggesting the platelets were activated. In addition, we found that,compared with the significant effects of the anti-GPIbα autoanti-body plasma on platelet ΔΨm depolarization and P-selectin expo-sure, plasma from ITP patients with anti-GPIIb/IIIa autoantibodiesinduced only moderate ΔΨm depolarization and P-selectin expo-sure in healthy human platelets (SI Appendix, Fig. S4). Taken to-gether, these data suggest that the platelets undergo apoptosis inITP patients with anti-GPIbα autoantibodies.

Anti-GPIbα Antibody Induces Platelet Apoptosis by Inhibiting PKAActivity. Next, we investigated the mechanism of anti-GPIbαantibody-induced platelet apoptosis. First we detected theamounts of apoptotic proteins in the platelets. Bcl-xL, Bak, andBax did not vary in the antibody-treated (Fig. 2A) and anti-GPIbα autoantibody plasma-treated (SI Appendix, Fig. S5)platelets. We reported recently that PKA plays a key role inregulating platelet apoptosis (33). Reduction of PKA activityresulted in dephosphorylation of Bad at Ser155 leading toplatelet apoptosis (33). Therefore, we detected PKA activity andfound that PKA activity was reduced in AN51- and SZ2-treatedplatelets (Fig. 2B) but not in SZ21- or D57-treated platelets (SIAppendix, Fig. S6), as indicated by dephosphorylation of thePKA substrates GPIbβ Ser166 (39) and vasodilator-associatedstimulated phosphoprotein (VASP) (Fig. 2B) (40). Phosphory-lation of Bad at Ser155, which is mediated by PKA (41), was alsoreduced (Fig. 2B). Similarly, anti-GPIbα autoantibody plasmareduced PKA activity in platelets (SI Appendix, Fig. S7). Wefurther verified the role of PKA in platelet apoptosis with Bad-deficient platelets, which lack PKA substrate, and found thatanti-GPIbα antibody-induced apoptotic events were obviouslyreduced (Fig. 2 C and D) but that the activation events were notobviously different in the Bad-deficient platelets (Fig. 2 E and F).In contrast, activation of PKA with forskolin, as indicated byphosphorylation of VASP at Ser157 (SI Appendix, Fig. S8),markedly reduced anti-GPIbα antibody-induced apoptotic events

(Fig. 2 G and H). These data indicate that anti-GPIbα antibodiesinduce platelet apoptosis through inhibition of PKA activity.

Akt Plays Key Roles in Anti-GPIbα Antibody-Induced Platelet Apoptosis.We further investigated why anti-GPIbα antibody binding inducesapoptosis in PKA-mediated platelets. VWF-induced cross-linking ofmultiple GPIb-IX complexes in lipid rafts triggers signaling cascadesleading to platelet activation (24–27). In light of these findings, wetreated platelets with GM3 and GlcNAc, which inhibit GPIbαclustering (42, 43), and found that both these inhibitors significantlyreduced AN51- and SZ2-induced ΔΨm depolarization and PS ex-posure (Fig. 3 A and B). Moreover, we used MβCD to disrupt lipidrafts (24) and found that MβCD obviously reduced AN51- and SZ2-induced apoptotic events (Fig. 3 A and B). In addition, consistentwith the results of VWF binding (24–27), platelet activation wasdiminished by GM3, GlcNAc, and MβCD in AN51- and SZ2-treated platelets (SI Appendix, Fig. S9). These data suggestthat anti-GPIbα antibodies may initiate platelet apoptosis byinducing GPIbα clustering in lipid rafts.Akt, a downstream effector of PI3K which interacts with the

cytoplasmic domain of GPIbα (44), transduces VWF–GPIbαinteraction signaling leading to platelet activation (28). There-fore, we hypothesized that Akt may be essential for anti-GPIbαantibody-induced apoptotic signaling. In support of this, wefound that Akt was activated in AN51- and SZ2-treated (Fig. 3C)but not in SZ21- or D57-treated (SI Appendix, Fig. S6) platelets.Anti-GPIbα autoantibody plasma also markedly elevated Aktactivity in platelets (SI Appendix, Fig. S10). Inhibitors of PI3K(LY294002 and wortmannin) or Akt (MK 2206 and Akti VIII)obviously reduced anti-GPIbα antibody-induced platelet apo-ptosis (Fig. 3 D and E).There are three isoforms of Akt, all of which are expressed in

both mouse and human platelets (28, 45–47). However, onlyAkt1 and Akt2 play roles in VWF-dependent signaling (28, 47).Therefore, we selected Akt1- and Akt2-knockout mice to in-vestigate the role of Akt in antibody-induced signaling. Wefound that anti-GPIbα antibody-induced apoptotic events andactivation were markedly reduced in both Akt1-deficient (Fig. 3F–I) and Akt2-deficient (SI Appendix, Fig. S11) platelets. Takentogether, these data demonstrate that Akt plays key roles in anti-GPIbα antibody-induced platelet apoptosis.

Akt Regulates PKA Activity Through Regulation of Phosphodiesterase.Akt was reported to directly activate phosphodiesterase (PDE3A)

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Fig. 2. Anti-GPIbα antibody-induced platelet apo-ptosis through inhibition of PKA activity. (A and B,Left) Western blot analysis of protein levels in humanplatelets treated with10 μg/mL IgG, AN51, SZ2, orHIP1 at 37 °C for 8 h with the indicated primary an-tibodies. Data are representative of three separateexperiments. (B, Right) Densitometry of phosphory-lated proteins in the Western blots at Left; n = 3. (C–F) PRP from WT or Bad−/− mice was incubated with5 μg/mL R300 (a mixture of purified rat monoclonalantibodies against mouse GPIbα) at 37 °C for 8 h.Platelet Δψm depolarization (C), PS exposure (D), P-selectin externalization (E), and JON/A binding (F)were analyzed by flow cytometry. (G and H) Washedhuman platelets were pretreated with the indicatedconcentrations of the PKA activator forskolin at RTfor 5 min and then were incubated with 10 μg/mLSZ2 at 37 °C for 8 h. Platelet Δψm depolarization (G)and PS exposure (H) were detected by flow cytom-etry; n = 6. Data are expressed as mean ± SD in B, G,and H. Horizontal lines in C–F indicate the medianvalues, and each dot represents one mouse. *P <0.05, **P < 0.01, ***P < 0.001 compared with controlor WT mice with Student’s t test in B and C–F or one-way ANOVA followed by Dunnett’s post hoc test in Gand H. NS, not significant.

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(48, 49). PDE3A hydrolyzes intracellular cAMP, leading to re-duction of PKA activity (50). Therefore, it is possible that Aktregulates PKA-mediated platelet apoptosis through activation ofPDE3A. To test this, platelets were treated with PI3K or Akt in-hibitors. We found that inhibitors of PI3K (LY294002 and wort-mannin) or Akt (MK 2206 and Akti VIII) markedly elevated PKAactivity in SZ2-treated platelets (Fig. 4A). Correspondingly, theantibody-induced apoptotic events were markedly reduced by PI3Kor Akt inhibitors (Fig. 3 D and E). Moreover, PDE3A inhibitors(milrinone and cilostazol) directly elevated PKA activity (Fig. 4B)and reduced apoptotic events in SZ2-treated platelets (Fig. 4 C andD). These data indicate that Akt reduces PKA activity and regulatesPKA-mediated platelet apoptosis through activation of PDE3A.

Anti-GPIbα Antibody-Induced Platelet Apoptosis and Activation OccurIndependently Through Two Separate Signaling Cascades. Deletionof Akt reduced anti-GPIbα antibody-induced apoptosis and ac-

tivation (Fig. 3 F–I). However, Bad deficiency only impaired theantibody-induced apoptosis (Fig. 2 C–F), suggesting that theantibody-induced apoptosis and activation occur independentlydownstream of Akt. To further verify this, we used BAPTA [1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid] to de-plete intracellular calcium, which is essential for Akt-dependentplatelet activation signaling (46, 51). BAPTA reduced P-selectin (Fig.5A) but did not affect ΔΨm depolarization (Fig. 5B), suggesting thatCa2+ mobilization induced by anti-GPIbα antibodies only contributesto platelet activation. We incubated platelets with the caspase in-hibitor Q-VD-OPh (quinoline-Val-Asp-difluorophenoxymethyl ke-tone) to block the apoptotic process. Q-VD-OPh only reduced PSexternalization (Fig. 5C) but did not affect P-selectin exposure (Fig.5A). Since ΔΨm depolarization is up-stream of caspase activation,we found that Q-VD-OPh did not affect ΔΨm depolarization (Fig.5B). These findings indicate that anti-GPIbα antibody-inducedplatelet apoptosis and activation occur independently through twoseparate signaling cascades. Similar effects were observed in mouseplatelets stimulated with anti-mouse GPIbα antibody R300 (SIAppendix, Fig. S12).Platelet apoptosis and activation alone may lead to platelet

clearance in vivo. We therefore investigated the chronologicalorder of anti-GPIbα antibody-induced apoptosis and activation.The results showed that apoptotic and activated events occurredalmost simultaneously (Fig. 5 D–G). Similar effects of anti-mouse GPIbα polyclonal antibodies on mouse platelets wereobserved with R300 (SI Appendix, Fig. S13).

Anti-GPIbα Antibodies Induce Apoptosis-Dependent Platelet Clearance.ITP patients with anti-GPIbα autoantibodies appear refractory toFc-dependent therapy (5, 9–11). It has been known that anti-GPIbαantibodies induce thrombocytopenia in an Fc-independent manner(12, 17, 20). However, how the anti-GPIbα antibody-bound plateletsare removed remained unclear. Therefore, we investigated whetherplatelet apoptosis contributed to platelet clearance in vivo. First, wedemonstrated that both intact and F(ab′)2 fragments of anti-mouseGPIbα antibodies (R300) could induce platelet apoptosis in vitro

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Fig. 3. Akt plays key roles in anti-GPIbα antibody-induced platelet apo-ptosis. (A and B) Washed human platelets were pretreated with GM3(100 μM), GlcNAc (100 mM), MβCD (1 mM), or vehicle control at 37 °C for30 min and were further incubated with 10 μg/mL AN51 or SZ2 at 37 °C for8 h. Platelet Δψm depolarization (A) and PS exposure (B) were detected byflow cytometry; n = 5. (C) Western blot analysis of Akt phosphorylation withanti-pAkt Ser-473 and Thr-308 antibodies. Data are representative of threeseparate experiments. (D and E) Washed human platelets were pretreatedwith the PI3K inhibitors LY294002 (20 μM) or wortmannin (100 μM), the Aktinhibitors MK2206 (6 μM) or Akti VIII (2 μM), or vehicle at RT for 5 min andwere incubated with 10 μg/mL SZ2 at 37 °C for 8 h. Platelet Δψm de-polarization (D) and PS exposure (E) were detected by flow cytometry; n = 7.(F–I) PRP from WT or Akt1−/− mice was incubated with 5 μg/mL R300 at 37 °Cfor 8 h. Δψm depolarization (F), PS exposure (G), P-selectin externalization(H), and JON/A binding (I) were analyzed by flow cytometry. Data in A, B, D,and E are expressed as mean ± SD. Horizontal lines in F–I indicate the medianvalues, and each dot represents one mouse. *P < 0.05, **P < 0.01, ***P <0.001, compared with vehicle control or WT mice by Student’s t test.

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Fig. 4. Akt regulates PKA activity through regulation of PDE3A. (A) Washedhuman platelets were pretreated with PI3K inhibitors LY294002 (20 μM) andwortmannin (100 nM), Akt inhibitors MK2206 (6 μM) and Akti VIII (2 μM), orvehicle control at RT for 5 min and then were incubated with 10 μg/mL SZ2 at37 °C for 8 h. VASP phosphorylation was analyzed with anti-pVASPSer157 antibody by Western blot; data are representative of three sepa-rate experiments. (B–D) Washed human platelets were pretreated withPDE3A inhibitors milrinone (10 μM) and cilostazol (10 μM) or vehicle controlat RT for 5 min and then were incubated with 10 μg/mL SZ2 at 37 °C for 8 h.VASP phosphorylation was analyzed with anti-pVASP Ser157 antibody byWestern blot (B), and platelet Δψm depolarization (C) and PS exposure (D)were detected by flow cytometry; n = 7. Data are expressed as mean ± SD;**P < 0.01, ***P < 0.001, compared with vehicle control with Student’s t test.

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(SI Appendix, Fig. S14 A and B). Second, we injected these anti-bodies into mice and found that, consistently with a previous report(17), both intact and F(ab′)2 fragments of R300 dose-dependentlyinduced platelets clearance (SI Appendix, Fig. S14 C and D), furtherdemonstrating that the Fc is not required for the anti-GPIbαantibody-induced platelet clearance. Third, apoptotic events weredetected in the platelets from R300-treated mice. We found thatthe circulating platelet counts were negatively correlated with ap-optotic events, Δψm depolarization, and PS exposure (Fig. 6 A andB), suggesting that platelet apoptosis may be essential for plateletclearance.Therefore, we next tried to investigate the role of platelet

apoptosis in anti-GPIbα antibody-induced platelet clearance.Fig. 1A shows that anti-GPIbα antibody SZ2, but not HIP1, in-duced platelet apoptosis. Because these monoclonal antibodiesare all antibodies against human platelet GPIbα, we set up ahumanized GPIbα murine model by using the human GPIbαgene to replace the mouse GPIbα gene (Fig. 6C and SI Appendix,Fig. S15). The expression of humanized GPIbα in the mouseplatelets was verified by flow cytometry (SI Appendix, Fig. S16).The homozygous humanized GPIbα mice (hGPIbα) did notdiffer fromWT mice in the numbers of platelets or red and whiteblood cells or in hemoglobin concentration (SI Appendix, TableS3). Consistent with the observation in human platelets, SZ2, butnot HIP1, resulted in humanized GPIbα platelet apoptosis (Fig.6 D and E). Then, we injected SZ2 and HIP1 into hGPIbα miceand found that SZ2, but not HIP1, induced platelet clearance(Fig. 6F). These data demonstrate that only the anti-GPIbα an-tibody, which induces platelet apoptosis, can initiate plateletclearance in vivo.To further verify the apoptosis-dependent platelet clearance

and evaluate the clinical relevance of these findings, we preparedF(ab′)2 fragments from the IgG fractions of ITP patients withonly anti-GPIbα autoantibodies. The F(ab′)2 fragments inducedthe humanized GPIbα mouse platelet apoptosis in vitro (SI Ap-pendix, Fig. S17). When injected into hGPIbα mice, the F(ab′)2fragments indeed induced platelet clearance (Fig. 6G).

PS Exposure Is Responsible for Apoptotic Platelet Clearance. To ex-plore the mechanism of apoptosis-dependent platelet clearance,we noticed that PS exposure is a late event of both platelet ap-optosis and activation. As known, PS exposure is an “eat-me”signal leading to phagocytosis of apoptotic cells in vivo (52).Therefore, we investigated the role of PS in platelet phagocy-tosis. We found that the PS blocker annexin V effectively pro-tected platelets from anti-GPIbα antibody (R300)-inducedclearance (Fig. 7A). Moreover, annexin V rescued anti-human

GPIbα antibody SZ2-induced platelet clearance in hGPIbα mice(SI Appendix, Fig. S18). In contrast, lactadherin, which enhancesthe interaction between PS and PS receptor (53), increasedplatelet clearance (Fig. 7A). These data suggest the role of PSexposure in anti-GPIbα antibody-induced platelet clearance.Since apoptosis and activation both contribute to PS exposure

in anti-GPIbα antibody-treated platelets, both these two signal-ing pathways should contribute to anti-GPIbα antibody-inducedplatelet clearance in vivo as well. To investigate this, we injectedBAPTA and Q-VD-OPh into mice. We found that, consistentwith the in vitro data, BAPTA and Q-VD-OPh both obviouslyprotected platelets from anti-GPIbα antibody-induced clear-ance. Moreover, the rescuing effects of Q-VD-OPh plus BAPTAwere almost double those of Q-VD-OPh or BAPTA alone(Fig. 7B).PS externalization during the activating process requires

TMEM16F (54). To further confirm the role of PS exposure inplatelet clearance, we generated TMEM16F-knockout mice (Fig.7C). Because very few homozygotes of TMEM16F-knockout micewere born, we had to use heterozygotes of TMEM16F-knockoutmice (TMEM16F+/−) for the experiment. The deficiency ofTMEM16F in the TMEM16F+/− platelets was examined byWesternblot (Fig. 7D). We found TMEM16F+/− and WT mice did not differin the number of platelets and red and white blood cells or in he-moglobin concentration (SI Appendix, Table S4). Anti-GPIbαantibody-induced PS exposure was obviously reduced in theplatelets from TMEM16F+/− mice (SI Appendix, Fig. S19). Wei.v. transfused antibody-treated platelets from TMEM16F+/− miceand WT littermates into WT mice and found that the plateletsfrom TMEM16F+/− mice were obviously less cleared than theplatelets of WT littermates (Fig. 7E). These data demonstrate thatPS exposure is essential for anti-GPIbα antibody-induced plateletclearance.

The Apoptotic Platelets Are Phagocytosed by Macrophages in theLiver. Next, we investigated where the PS-exposed platelets areremoved. Endothelial cells were reported to engulf bacterial-activated platelets (55); therefore, endothelial cells from anti-GPIbα antibody-treated mice were examined. No platelet wasfound to be colocalized with endothelial cells (SI Appendix, Fig.S20A). Neutrophils phagocytose activated platelets during acutemyocardial infarction (56). However, we found that depletionof neutrophils could not rescue anti-GPIbα antibody-inducedplatelet clearance (SI Appendix, Fig. S20 B and C). These datasuggest that endothelial cells or neutrophils may not contributeto anti-GPIbα antibody-induced platelet clearance. Then, weused Clophosome-N to deplete macrophages in vivo. After

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Fig. 5. Anti-GPIbα antibody-induced platelet apoptosis and activation occur independently through two separate signaling cascades. (A–C) Washed humanplatelets were pretreated with the Ca2+ chelator BAPTA (10 μM), the pan-caspase inhibitor Q-VD-OPh (100 μM), or vehicle at RT for 5 min and were incubated with10 μg/mL AN51 or SZ2 at 37 °C for 8 h. Platelet P-selectin externalization (A), Δψm depolarization (B), and PS exposure (C) were detected by flow cytometry; n =7. (D–G) Time course of apoptosis and activation occurring in human platelets treated with 10 μg/mL IgG, AN51, SZ2, or HIP1. Data are expressed asmean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, compared with vehicle control or normal IgG control by Student’s t test in A–C and by two-way ANOVAfollowed by Dunnett’s post hoc test in D–G.

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injection of R300 into the mice, we found that depletion ofmacrophages markedly rescued platelets from clearance (Fig. 7F).Macrophages contain Fc receptors, which can engulf platelets inan Fc-dependent manner. To investigate this possibility, we in-jected R300 F(ab′)2 into macrophage-depleted mice. Depletionof macrophages still protected platelets from clearance com-pared with results in undepleted mice (Fig. 7G), furtherindicating that Fc is not required for phagocytosis of antibody-bound platelets.Macrophages present in the reticuloendothelial system through-

out the whole body. We reported previously that anti-GPIbαantibody-bound platelets were removed by macrophages inthe liver (12). To determine where the apoptotic plateletswere removed, we labeled R300 and R300 F(ab′)2 with Alexa-

Fluor 488 and injected the antibodies into mice. The fluo-rescence images indicated that the Alexa-Fluor 488 signal wasprimarily detected in the liver (SI Appendix, Fig. S21A). Wefurther examined the liver (SI Appendix, Fig. S21B) and spleen(SI Appendix, Fig. S21C) by immunofluorescent staining andfound that platelets were primarily colocalized with macro-phages in the liver. Taken together, these data suggest thatPS-exposed platelets are removed by macrophages in the liver.

Inhibition of Akt and Akt-Mediated Apoptosis Protects the Anti-GPIbα Antibody-Bound Platelets from Clearance in Vivo. As shownin Fig. 3, GlcNAc and GM3 inhibited anti-GPIbα antibody-induced platelet apoptosis in vitro. Therefore, GlcNAc andGM3 should rescue the antibody-induced platelet clearance invivo. To verify this, GlcNAc or GM3 was i.v. injected into mice,

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Fig. 6. Anti-GPIbα antibodies induce apoptosis-dependent platelet clearance.(A and B) C57BL/6J mice were i.v. injected with 0.1 μg/g R300. Platelet countand Δψm depolarization (A) and platelet count and PS exposure (B) weremeasured at the indicated time points. n = 5 mice per group. (C) Schematicoverview of the strategy to generate the hGPIbα-knockin allele. The human-ized GPIbα coding sequence (hGPIbα cDNA) was fused to the signal peptide ofthe mouse GPIbα gene. The scissors indicate the Cas9 nuclease and its cuttingsites at the GPIbα locus. The homologous arms of the donor vector are in-dicated as “L-HA” (1.7 kb) and “R-HA” (1.7 kb). Regions of homology betweenthe WT locus and the donor vector are depicted by thick black lines, UTRs areshown as open bars, and the translated region is represented by solid bars. Therestriction enzyme used for Southern blot analysis is shown. The purple boxesindicate the probe used for Southern blotting. The arrowheads indicate thelocations of primers 1, 2, 3, and 4 used for genotyping and sequencing. (D andE) PRP from WT and hGPIbα mice was incubated with 10 μg/mL SZ2, HIP1, ornormal mouse IgG at 37 °C for 8 h. Platelet Δψm depolarization (D) and PSexposure (E) were measured by flow cytometry. (F) hGPIbα mice were i.p. in-jected with 0.4 μg/g normal mouse IgG, SZ2, or HIP1. Platelet counts were de-termined at the indicated time points; n = 5 mice per group. (G) hGPIbα micewere i.v. injected with 200 μL IgG F(ab′)2 fragments from four healthy donors orfrom five ITP patients with only anti-GPIbα autoantibodies. Platelet counts weredetermined at the indicated time points. Baseline is defined as the plateletnumber before antibody injection. Data are expressed as mean ± SD in A, B, F,and G. Horizontal lines indicate the median values, and each dot represents onemouse in D and E. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA in Dand E, by two-way ANOVA followed by Tukey’s post hoc test in F, and by two-way ANOVA followed by Bonferroni’s post hoc test in G. NS, not significant.

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Fig. 7. PS exposure-dependent phagocytosis of platelets by macrophage. (A)C57BL/6J mice were i.v. preinjected with 0.5 μg/g annexin V, 0.025 μg/g lac-tadherin, or vehicle control and then were transfused with calcein-labeledR300-treated platelets. (B) Calcein-labeled mouse platelets pretreated with10 μM BAPTA, 100 μM Q-VD-OPh, or vehicle control at RT for 5 min were in-cubated with R300 and then were transfused to C57BL/6J mice. The percent-age of calcein-positive platelets remaining in circulation was assessed at theindicated time points by flow cytometry; n = 6–11 mice per group. Baseline isdefined as the percentage of calcein-positive platelets within 1 min afterplatelet transfusion. (C) Generation of TMEM16F frameshift mutant mice us-ing CRISPR/Cas9 genome editing. Exon 2 of the mouse TMEM16F gene wastargeted with two CRISPR gRNAs indicated by bold letters and underlined,respectively (protospacer ajacent motif is shown in red). Founder alleles aredepicted with deleted bases represented by dashes and inserted bases shownin green. (D) Western blot analysis of the TMEM16F level in WT andTMEM16F+/− mice. (E) C57BL/6J mice were i.v. transfused with calcein-labeledR300-treated platelets from TMEM16F+/− or WT littermates, and the percent-age of calcein-positive platelets remaining in circulation was assessed at theindicated time points by flow cytometry; n = 5 mice per group. Baseline isdefined as in A. (F and G) Effect of macrophage depletion on platelet clear-ance induced by R300 (F) and R300 F(ab′)2 (G) fragments. C57BL/6J mice wereinjected with control and clodronate liposomes 24 h before R300 (0.1 μg/g) orR300 F(ab′)2 fragments (0.3 μg/g) were injected i.p. into mice, and plateletnumber was determined at the indicated time points; n = 5 or 6 mice pergroup. Baseline is defined as the platelet number before anti-GPIbα antibodyinjection. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P <0.001, by two-way ANOVA followed by Bonferroni’s post hoc test.

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and then the mice were injected with calcein-labeled R300-treated platelets. As expected, GlcNAc and GM3 rescued anti-GPIbα antibody-induced platelet clearance (Fig. 8A). It wasreported that GPIbα clustering might enhance the affinity ofGPIbα with the CR3 receptor on macrophages, leading toplatelet clearance (57). However, CR3 deficiency did not protectanti-GPIbα antibody-treated platelets from clearance (SI Ap-pendix, Fig. S22). The reason may be that anti-GPIbα antibodybinding has steric hindrance to block the interaction of GPIbαwith CR3 receptors.PI3K-Akt activation is essential for anti-GPIbα antibody-induced

platelet apoptosis. Thus, it is conceivable that inhibition of PI3K-Akt activation should rescue anti-GPIbα antibody-induced plateletclearance in vivo. Indeed, Fig. 8B shows that PI3K and Akt in-hibitors markedly reduced anti-GPIbα antibody-induced plate-let clearance. We further verified the role of Akt in anti-GPIbαantibody-induced platelet clearance with Akt-deficient mice.Since only a very few Akt1−/− mice were generated, we had to useAkt1+/− mice to perform the experiment. After injection of anti-GPIbα antibodies into the Akt1+/− mice, platelet clearance wassignificantly prevented (Fig. 8C).We further demonstrated the role of anti-GPIbα antibody-

induced apoptosis in platelet clearance with Bad−/− mice. Wefound that after injection of R300 the platelets were less cleared

in Bad−/− mice than in WT mice (Fig. 8D). Taken together, thesedata demonstrate that inhibition of Akt or Akt-mediated plateletapoptosis rescues anti-GPIbα antibody-induced platelet clear-ance in vivo.

DiscussionThe data described in this study indicate that (i) anti-GPIbαantibodies induce Akt activation and Akt-mediated plateletapoptosis; (ii) Akt regulates platelet apoptosis, independent ofactivation signaling, through PDE3A-mediated PKA activity;(iii) PS exposure on apoptotic platelets rather than Fc results inphagocytosis of anti-GPIbα antibody-bound platelets in the liver;and (iv) agents that block Akt signaling or PS externalizationprotect the antibody-bound platelets from clearance and thussuggest therapeutic strategies for thrombocytopenia caused byautoantibodies or other pathogenic factors.We demonstrate that anti-GPIbα antibodies induce Akt acti-

vation, which results in platelet apoptosis and activation. PI3Kassociated with the cytoplasmic domain of GPIbα transduces theVWF-binding signaling, leading to Akt activation (28, 45). Wefound that Akt, the downstream effector of PI3K, was activatedafter anti-GPIbα antibody binding. Inhibition of PI3K or Akt orgenetic ablation of Akt disrupted the antibody-induced apoptosisand activation. Moreover, Akt activated platelets through Ca2+elevation. These findings suggest that anti-GPIbα antibody ac-tivates platelets via signaling cascades similar to that of VWFbinding (28). Distinct from the activation signaling, we foundthat Akt elicited platelet apoptosis via activation of PDE3A. Wereported recently that inhibition of PKA caused intrinsicallyprogrammed platelet apoptosis (33). Consistent with this report,PKA activity was markedly reduced after anti-GPIbα antibodybinding. We demonstrate that Akt activates PDE3A, which canreduce cAMP concentration, leading to PKA inhibition. Insupport of this finding, we reported previously that VWF-bindingalso induced platelet apoptosis (23). The current study providesa theoretical explanation for this finding. Therefore, our studyidentifies the dual roles of Akt in regulating both apoptosis andactivation in platelets (Fig. 8E). Because Akt is abundant inplatelets and eukaryotes and is involved in many functionalsignaling cascades (29, 46–48), the current findings may havegeneral implications.To explore the mechanism for anti-GPIbα antibody-induced

Akt activation, we found that inhibitors of GPIbα clustering andthe reagent that disrupts lipid rafts markedly reduced anti-GPIbαantibody-dependent platelet activation and apoptosis. Thesedata suggest that anti-GPIbα antibodies may, like the VWFmultimer (24–27), induce Akt activation by cross-linking GPIbαin lipid rafts. This possibility is supported by previous evidence.First, antibody-mediated cross-linking of target proteins has beendemonstrated to associate with lipid rafts, rather than simpledimerization, to initiate signaling (58, 59). Second, two GPIbαmolecules were in close proximity upon anti-GPIbα antibodybinding, leading to platelet activation (12). Third, disrupting theassociation of GPIbα with lipid rafts abolished GPIbα-mediatedplatelet aggregation (24, 25). However, it is intriguing that, un-like AN51 and SZ2, the anti-GPIbα antibody HIP1 could notactivate Akt and induce Akt-mediated platelet activation andapoptosis. Similar effects on platelets have been reported withother anti-GPIbα antibodies (12, 21). During the current sub-mission, a report suggested that some anti-GPIbα antibodies, butnot others, can exert a pulling force on GPIbα by cross-linkingplatelets under shear flow, which unfolds their mechanosensorydomain, leading to platelet activation (21). This model providesan explanation for the different effects of anti-GPIbα antibodieson platelet activation under shear-flow conditions. However, inour current and previous (12) in vitro experiments and in similarexperiments by others (24–26), the pretreated platelets wereincubated under static conditions. There was no shear stress topull the antibodies, but the antibodies still induced platelet ac-tivation. Interestingly, we find that the anti-GPIbα antibodiesAK2 and HIP1 with epitopes located in the leucine-rich repeat

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Fig. 8. Inhibition of Akt activation and Akt-mediated apoptosis rescuesanti-GPIbα antibody-induced platelet clearance in vivo. (A) C57BL/6J micewere i.v. preinjected with GM3 (6 μg/g), GlcNAc (110 μg/g), or vehicle controland then were transfused with calcein-labeled R300-treated platelets. (B)Mouse platelets were pretreated with 100 nM wortmannin, 6 μM MK2206,or vehicle control at RT for 5 min and then were transfused to C57BL/6J mice.The percentage of calcein-positive platelets remaining in circulation wasassessed at the indicated time points by flow cytometry; n = 6–9 mice pergroup. Baseline is defined as the percentage of calcein-positive plateletswithin 1 min after platelet transfusion. (C and D) WT and Akt1+/− (C) andBad−/− (D) mice were i.p. injected with R300 (0.1 μg/g). Platelet counts weredetermined at the indicated time points; n = 6–8 mice per group. Baseline isdefined as the platelet number before anti-GPIbα antibody injection. Dataare expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 compared bytwo-way ANOVA followed by Bonferroni post hoc test. (E) The dual roles ofAkt in regulating activation and apoptosis in platelets. Akt, the downstreameffector of PI3K, is activated after anti-GPIbα antibody binding. Akt activatesplatelets through Ca2+ elevation. Meanwhile, Akt activates PDE3A, leadingto PKA inhibition and platelet apoptosis. The Akt-regulated activation andapoptosis signaling independently lead to PS externalization.

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region could not activate platelets (12, 21); on the other hand,epitopes of some of the reported anti-GPIbα antibodies which(except for VM16d) could activate platelets are located in the N-or C-terminal flanks or anionic sulfated sequence of GPIbα (12,21). This finding suggests that epitope location or conformation-related affinity of the antibody may, as in VWF (60), play a rolein the initiation of activation signaling. Future work is needed tosolve this mystery.We and others reported previously that Fc was not required

for anti-GPIbα antibody-induced platelet clearance (12, 17, 20,21, 61). Consistent with these findings, although binding toplatelets, HIP1 did not induce platelet clearance in vivo. Incontrast, SZ2, which induces platelet apoptosis, initiated plateletclearance. Blocking or genetic ablation of PS externalization,induced by platelet activation and apoptosis independently,prevented the platelets from clearance. We further demonstratedthat the PS-exposed platelets were engulfed by macrophages in theliver. These findings demonstrate that the anti-GPIbα antibody-induced platelet clearance is through a PS-dependent rather thanan Fc-dependent manner. Thus, our findings explain the long-standing puzzle that ITP patients with anti-GPIb-IX autoanti-bodies appear less responsive to conventional therapies, suchas IVIG and even splenectomy (9–11).Notably, GPIbα desialylation was demonstrated to contribute to

anti-GPIbα antibody-induced platelet clearance (20). We verifiedthat an inhibitor of sialidase rescued anti-GPIbα antibody-inducedplatelet clearance in our experimental condition (SI Appendix, Fig.S23). Anti-GPIbα antibody-induced platelet activation was foundto be a prerequisite for GPIbα desialylation (20). The removal ofsialic residues facilitates GPIbα clustering and accelerates plateletactivation (20, 42). Therefore, it is likely that GPIbα desialylationcontributes to platelet clearance by enhancing platelet activationand PS-dependent platelet phagocytosis. In support of this, plate-lets lacking sialylation of O-glycans were also primarily phagocy-tosed by macrophages in the liver (62). This explanation might alsoapply to GPIbα desialylation-mediated chilled platelet clearance(43, 63).We demonstrate that anti-GPIbα antibodies induce platelet

clearance through Akt activation and Akt-mediated platelet apo-ptosis and activation. Therefore, it is reasonable that inhibitionof GPIbα clustering in lipid rafts, PI3K and Akt activation, or Akt-mediated activation and apoptosis signaling all significantly re-duced anti-GPIbα antibody-induced platelet clearance. Thus, ourfindings provide various potential therapeutic strategies for thetreatment of ITP with anti-GPIbα autoantibodies.More importantly, we demonstrate that PS exposure is re-

sponsible for the phagocytosis of apoptotic platelets in the liver.PS externalization is a common and later-stage event for bothapoptosis and activation (64). We and others reported thatplatelet apoptosis occurs extensively in physiologic conditions,blood bank platelets, and many common diseases (33–35).Platelet activation has long been demonstrated in many commondiseases, such as infection, cancer, and heart diseases (36–38,65). A large amount of evidence indicates that thrombocytope-nia, which can result in fatal bleeding, occurs in many commondiseases in which platelets are activated or undergo apoptosis(31–35). However, the means by which the apoptotic or activatedplatelets are removed from the circulation and result in throm-bocytopenia remains elusive. Therefore, our findings provide apathogenetic explanation for thrombocytopenia during variousdiseases in which the platelets are activated or undergo apo-ptosis. Inhibition of PS exposure-dependent platelet clearancerepresents a general therapeutic strategy for thrombocytopeniaoccurring in various common diseases.In conclusion, we demonstrate that anti-GPIbα antibodies

induce Akt activation and Akt-mediated platelet apoptosis. Theapoptotic platelets are phagocytosed by macrophages in the liverin a PS exposure-dependent manner. Inhibition or genetic ab-lation of Akt or Akt-regulated apoptotic signaling or blocking ofPS externalization protects the platelets from clearance. There-fore, our findings reveal the pathogenic mechanisms of anti-

GPIbα autoantibodies in ITP and, more importantly, suggesttherapeutic strategies for thrombocytopenia caused by autoan-tibodies or other pathogenic factors.

Materials and MethodsMice.Generation of human GPIbα-knockin mice. For GPIbα targeting, exon 2 wastargeted by two gRNAs designed to cut both ends of the exon to replace themature mouse GPIbα chain-coding region with the human sequence (Fig.6C). CRISPR single-guide RNAs (sgRNAs) were designed and screened for on-target activity. The T7 promoter sequence was added to the Cas9 and sgRNAtemplates by PCR amplification. T7-Cas9 and T7-sgRNA PCR products were pu-rified and used as the template for in vitro transcription. Both the Cas9 mRNAand the sgRNA were purified. A circular donor vector was used to minimizerandom integrations. The donor plasmid containing human GPIbα cDNA andthe WPRE-pA cassette was flanked by 1.7-kb and 1.7-kb homolog arms. C57BL/6female mice and Kunming (KM) mouse strains were used as embryo donorsand pseudopregnant foster mothers, respectively. Cas9 mRNA, sgRNAs, anddonor vector were mixed at different concentrations and coinjected into thecytoplasm of fertilized zygotes at the one-cell stage. After injection, survivingzygotes were transferred into the oviducts of KM pseudopregnant females.Genomic DNAwas extracted from the tail of the 7-d-old mice. The genotype forthe human GPIbα-knockin allele was confirmed by PCR amplification, Southernblotting (SI Appendix, Fig. S15), and direct sequencing.Generation of TMEM16F-knockout mice. The TMEM16F-mutant mouse modelwas established by CRISPR/Cas9 genome editing technology to inducedouble-stranded breaks in exon 2 of TMEM16F. Cas9 mRNA was in vitrotranscribed. Two independent guide RNAs targeting exon 2 of the TMEM16Fgene were designed and transcribed in vitro using the MEGAshortscriptKit (Thermo Scientific). The sequences of sgRNAs were gRNA1 5’-ACAATTGTCTGCCCCACCTTTGG-3’ and gRNA2 5’-CTGATTCTCCAGTGATC-CAAAGG-3’. The in vitro-transcribed Cas9 mRNA and sgRNAs were injectedinto the cytoplasm of C57BL/6J fertilized eggs, transferred to pseudopreg-nant recipients, and allowed to develop to term. Founders were screened forinsertions/deletions (indels) by PCR amplification across the targeted region (F:5′-TTTGACCTCTGGCTCATCTATTC-3′, R: 5′-CCT-AGTCCTTCTGGGGTTGC-3′). PCRproducts from indel-carrying founders were Sanger sequenced to identify spe-cific mutations. The founder mice were bred to WT C57BL/6J mice to generateheterozygous TMEM16F-mutant mice and then were intercrossed to generatehomozygous TMEM16F-mutant mice.Other mouse strains. Bad−/− (66), Akt1−/− (67), and Akt2−/− (68) mice weregenerated as described previously. CR3−/− mice (003991) were purchasedfrom The Jackson Laboratory. C57BL/6J WT mice were purchased from JOINNLaboratories. All mutations had been backcrossed onto the C57BL/6J back-ground for at least 10 generations before this study. Mice were 6–12 wk old,and experiments included balanced groups of male and female mice unlessotherwise stated. All animal experiments complied with the regulatorystandards of and were approved by the Ethics Committee of The First Af-filiated Hospital of Soochow University.

Patients and Healthy Volunteers. Approval for obtaining whole-blood samplesfrom healthy volunteers and patients was obtained from the Ethics Committeeof The First AffiliatedHospital of SoochowUniversity, and informed consentwasobtained from all subjects according to the Declaration of Helsinki. Twelve ITPpatients with anti-GPIbα autoantibodies and 30 ITP patients with anti-GPIIb/IIIaautoantibodies as characterized by flow cytometric immunobead array (38)were enrolled from The First Affiliated Hospital of Soochow University betweenMay 8, 2016 and August 30, 2017, and age- and gender-matched healthycontrol subjects were recruited simultaneously for the studies.

Antibodies and Reagents. Antibodies and reagents are provided in SI Ap-pendix, Supplementary Materials.

Platelet Counts and Preparation. Platelet and blood cell counts were per-formedwith Sysmex XP-100 Hematologic Analyzer (Sysmex Corporation). Theplatelets from healthy volunteers were prepared as previously described (30–33). Briefly, whole blood was drawn from the inferior vena cava and anti-coagulated with a 1:7 volume of acid–citrate–dextrose (ACD: 2.5% trisodiumcitrate, 2.0% D-glucose, 1.5% citric acid). Platelet-rich plasma (PRP) was col-lected from the whole blood by centrifugation at 200 × g for 11 min.Platelets were washed twice with CGS buffer (0.123 M NaCl, 0.033 MD-glucose, 0.013 M trisodium citrate, pH 6.5), resuspended in modifiedTyrode’s buffer (MTB) (2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mMNaHCO3, 5.5 mM D-glucose, 1 mM CaCl2, 1 mM MgCl2, pH 7.4) to a final

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concentration of 5 × 108/mL, and allowed to incubate at 22 °C for 1–2 h. Forthe preparation of murine platelets, whole blood from mice was collectedfrom the postorbital vein using a 1:7 volume of ACD as anticoagulant.Platelets were washed with CGS buffer, resuspended in MTB to a concen-tration of 5 × 108/mL, and allowed to incubate at 22 °C for 1–2 h. For thepreparation of platelets from patients, whole blood was drawn from the cubitalvein and anticoagulated with ACD. For preparation of PRP, whole blood wasanti-coagulated with a 1:9 volume of 3.8% trisodium citrate. Human and murinePRP was obtained by centrifugation at 200 × g and 100 × g, respectively.

In Vitro Platelet Antibody Assays. Washed human platelets were treated with10 μg/mL anti-human platelet monoclonal antibodies at 37 °C for 2–10 h. Arelatively long time of incubation was selected to compensate for the no-shear-stress condition in vitro. Murine PRP was treated with 5 μg/mL anti-GPIbα poly-clonal antibody R300 at 37 °C for 2–10 h. In inhibition experiments, humanplatelets or murine PRP were preincubated with various inhibitors and theircorresponding vehicle controls at for 5 min at RT or at for 30 min at 37 °C andthen were treated with AN51, SZ2, or R300 at 37 °C for 8 h.

Flow Cytometry. P-selectin expression was detected with FITC-labeled anti-human (10 μg/mL) or anti-murine P-selectin antibody (1:5). GPIIb/IIIa activa-tion was detected by FITC-labeled PAC-1 (25 μg/mL) binding to humanplatelets and PE-labeled JON/A (1:5) binding to murine platelets. Mito-chondrial inner transmembrane potential (ΔΨm) depolarization in human ormurine platelets was measured by JC-1 (2 μg/mL). PS exposure of human ormurine platelets was detected by FITC-labeled lactadherin (10 μg/mL).Platelets were measured by a flow cytometer (FC 500; Beckman-Coulter).

Western Blot. Washed human platelets (5 × 108/mL) were incubated with anti-GPIbα antibodies at 37 °C for 8 h or were preincubated with various inhibitors atRT for 5 min before anti-GPIbα antibody treatment and then were lysed with anequal volume of lysis buffer on ice for 30 min. For detection of the mouseTMEM16F level, washed murine platelets (5 × 108/mL) were lysed with an equalvolume of lysis buffer on ice for 30 min. Proteins were separated and visualizedby the ECL system. Quantification was performed with ImageJ software (NIH).

Caspase-3 Activity Assay. Washed human platelets (5 × 108/mL) were in-cubated with 10 μg/mL AN51, SZ2, HIP1, or normal mouse IgG at 37 °C for 8 hand were lysed with an equal volume of lysis buffer on ice for 30 min. Thecaspase-3 activity assay was performed according to the manufacturer’sprotocol. Briefly, 10 μL of platelet lysate per sample was mixed with 80 μL ofreaction buffer and 10 μL of caspase-3 substrate (Ac-DEVD-pNA, 2 μM).Samples were further incubated at 37 °C for 4 h and activity was determinedby an ELISA reader at an absorbance of 450 nm. The specific activity ofcaspase 3 was normalized for the total protein of sample.

Human IgG Purification. EDTA-anticoagulated human plasma was diluted withPB (0.1 M Na2HPO4, 0.1 M NaH2PO4, pH 7.5) and applied to a protein G affinitycolumn (GenScript). The column was washed thoroughly with PB, and the boundIgG was eluted with elution buffer (0.1 M glycine, pH 2.8). Purified IgG was di-alyzed against PBS and concentrated to the original volume of the plasma samples.

IgG F(ab′)2 Fragmentation. R300 F(ab′)2 fragments and human IgG F(ab′)2fragments were generated using the Thermo Scientific IgG F(ab′)2 kit accordingto the manufacturer’s protocol. Briefly, 0.5 mg/mL of R300 or purified humanIgG (<5 mg/mL) was desalted and digested with immobilized Pepsin in digestionbuffer. Digest products were purified with Protein A beads and dialyzed. HumanIgG F(ab′)2 fragments cleaved from 500 μL intact IgG were further concentratedto 300 μL. Purified F(ab′)2 was proved by SDS/PAGE and Coomassie blue staining.

Immunofluorescence Microscopy. C57BL/6J mice were i.p. injected with normalrat IgG (0.1 μg/mL), R300 (0.1 μg/mL), or R300 F(ab′)2 fragments (0.4 μg/mL).Animals were killed after 4 h, and organs were immediately excised. Liverand spleen cryosections were fixed in ice-cold acetone and subsequentlywere blocked in 5% BSA/PBS at RT for 1 h. Mouse anti-mouse F4/80 (10 μg/mL)or rabbit anti-mouse CD31 (10 μg/mL) was incubated overnight at 4 °C,and Alexa-Fluor 488-conjugated goat anti-mouse (5 μg/mL) or Alexa-Fluor488-conjugated goat anti-rabbit (5 μg/mL) antibody was incubated at RT

for 1 h. Subsequently, R300 (10 μg/mL) and Alexa-Fluor 555-conjugated goatanti-rat antibody (5 μg/mL) were incubated at RT for 1 h. Cell nuclei werestained with 5 μg/mL DAPI.

Southern Blot Analysis. Genomic DNA extracted from mouse tails wasdigested and electrophoresed in 1% agarose gel. The DNA in gel wastransferred to a positively charged nylon membrane. The membranewas hybridized and detected using the DIG Luminescent Detection kit (RocheGroup). For probe labeling, 3′ externally and internally DIG-labeled probeswere prepared by PCR using Taq DNA polymerase and incorporating DIG-11-dUTP according to the manufacturer’s instructions.

Macrophage Depletion. C57BL/6J mice (6- to 8-wk-old) were i.p. injected withcontrol liposomes (700 μg per mouse) or clodronate liposomes (700 μg permouse), and the liver was removed from mice after 24 h. Macrophage de-pletion was confirmed by immunofluorescence.

In Vivo Imaging Systems. C57BL/6J mice (6- to 8-wk-old) were i.p. injected withrat IgG (0.1 μg/g), R300 (0.1 μg/g), or R300 F(ab′)2 fragments (0.4 μg/g) premixedwith Alexa-Fluor 488-conjugated goat anti-rat antibody. The mice were killed5 min after injection. The liver, spleen, lung, heart, and kidney were excised andimaged immediately using an in vivo imaging system (Caliper Life Sciences).

Platelet Clearance in Vivo. C57BL/6J mice (6–8 wk old) i.p. received normal ratIgG, R300, or R300 F(ab′)2 in 100 μL of sterile PBS. Transgenic mice expressinghuman GPIbα (6- to 8-wk-old) were i.p. injected with normal mouse IgG, SZ2,or HIP1 or were i.v. injected with 200 μL IgG F(ab′)2 fragments from normalcontrols or ITP patients. The blood was collected from the post-glomus ve-nous plexus and was anticoagulated with 3.8% trisodium citrate. Plateletswere counted by a Sysmex XP-100 hematologic analyzer.

Posttransfusion Experiment. Washed murine platelets labeled with 5 μg/mLcalcein were incubated with 2 μg/mL R300 at RT for 1 h and were transfusedto acceptor mice through the post-glomus venous plexus (1 × 108 platelets in100 μL of MTB). Blood was collected from the post-glomus venous plexus at1 min (baseline), 15 min, and 30 min, and total platelets were labeled withPE-conjugated anti-CD41 antibody. The percentage of calcein-labeledplatelets remaining in circulation was assessed by flow cytometry. Forblocking of PS and GPIbα clustering, mice were preinjected through the post-glomus venous plexus with annexin V (0.5 μg/g), lactadherin (0.025 μg/g),GM3 (6 μg/g), GlcNAc (110 μg/g), or vehicle control before platelet transfusion.For inhibition of calcium mobilization and caspase, PI3K, and Akt activity,calcein-labeled platelets were pretreated with BAPTA (10 μM), Q-VD-OPh(100 μM), wortmannin (100 nM), MK 2206 (6 μM), or vehicle control DMSOat RT for 5 min before incubation with 2 μg/mL R300.

Statistical Analysis. All data are expressed as mean ± SD. Numeric data wereanalyzed using one-way (for a single variant) or two-way (for multiple vari-ants) ANOVA. Two groups were compared by the two-tailed Student’s t test.The significance of data was assessed using GraphPad Prism 5 software. Dif-ferences were considered significant at P < 0.05. Different levels of significanceare indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. All experiments re-quiring the use of animals were subject to randomization based on litter. Noanimals or samples were excluded from the study. Sample size was pre-determined based on the variability observed in prior experiments and onpreliminary data. Investigators were not blinded to outcome assessment.

ACKNOWLEDGMENTS. We thank Zhongzhou Yang (Nanjing University) andYulong He (Soochow University) for providing Akt1−/− and Akt2−/− mice. Thiswork was supported by Key Program of the National Natural Science Foun-dation of China Grant 81130008 (to K.D.), National Natural Science Founda-tion of China Grants 81570102 and 81770117 (to K.D.), National Key BasicResearch Program of China Grant 2012CB526600 (to K.D.), National NaturalScience Foundation of China Grant 81770113 (to R.Y.), the Priority AcademicProgram Development of Jiangsu Higher Education Institutions, Jiangsu Pro-vincial Special Program of Medical Science Grant BL2012005, Jiangsu ProvinceKey Medical Center Grant ZX201102, and Jiangsu Province’s Outstanding Med-ical Academic Leader Program Award (to K.D.).

1. Neunert C, et al.; American Society of Hematology (2011) The American Society of

Hematology 2011 evidence-based practice guideline for immune thrombocytopenia.

Blood 117:4190–4207.2. Stasi R, et al. (2008) Idiopathic thrombocytopenic purpura: Current concepts in

pathophysiology and management. Thromb Haemost 99:4–13.

3. McMillan R (2000) Autoantibodies and autoantigens in chronic immune thrombocy-

topenic purpura. Semin Hematol 37:239–248.4. Hou M, Stockelberg D, Kutti J, Wadenvik H (1995) Antibodies against platelet GPIb/IX,

GPIIb/IIIa, and other platelet antigens in chronic idiopathic thrombocytopenic pur-

pura. Eur J Haematol 55:307–314.

E10690 | www.pnas.org/cgi/doi/10.1073/pnas.1808217115 Chen et al.

Dow

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0, 2

020

5. Go RS, Johnston KL, Bruden KC (2007) The association between platelet autoantibodyspecificity and response to intravenous immunoglobulin G in the treatment of pa-tients with immune thrombocytopenia. Haematologica 92:283–284.

6. McMillan R (2007) The pathogenesis of chronic immune thrombocytopenic purpura.Semin Hematol 44(4 Suppl 5):S3–S11.

7. Zufferey A, Kapur R, Semple JW (2017) Pathogenesis and therapeutic mechanisms inimmune thrombocytopenia (ITP). J Clin Med 6:E16.

8. Zeng Q, et al. (2012) Relative efficacy of steroid therapy in immune thrombocytopeniamediated by anti-platelet GPIIbIIIa versus GPIbα antibodies. Am J Hematol 87:206–208.

9. Peng J, et al. (2014) Association of autoantibody specificity and response to in-travenous immunoglobulin G therapy in immune thrombocytopenia: A multicentercohort study. J Thromb Haemost 12:497–504.

10. McMillan R, Wang L, Tani P (2003) Prospective evaluation of the immunobead assayfor the diagnosis of adult chronic immune thrombocytopenic purpura (ITP). J ThrombHaemost 1:485–491.

11. Kim M, et al. (2017) Platelet count evolution as a predictor of outcome after sple-nectomy for immune thrombocytopenic purpura. Int J Hematol 105:433–439.

12. Yan R, et al. (2015) Glycoprotein Ibα clustering induces macrophage-mediatedplatelet clearance in the liver. Thromb Haemost 113:107–117.

13. Yanabu M, et al. (1997) Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody. Blood 89:1590–1598.

14. Cauwenberghs N, et al. (2001) Fc-receptor dependent platelet aggregation inducedby monoclonal antibodies against platelet glycoprotein Ib or von Willebrand factor.Thromb Haemost 85:679–685.

15. Bergmeier W, Rackebrandt K, Schröder W, Zirngibl H, Nieswandt B (2000) Structuraland functional characterization of the mouse von Willebrand factor receptor GPIb-IXwith novel monoclonal antibodies. Blood 95:886–893.

16. Li C, et al. (2011) The maternal immune response to fetal platelet GPIbα causes fre-quent miscarriage in mice that can be prevented by intravenous IgG and anti-FcRntherapies. J Clin Invest 121:4537–4547.

17. Nieswandt B, Bergmeier W, Rackebrandt K, Gessner JE, Zirngibl H (2000) Identifica-tion of critical antigen-specific mechanisms in the development of immune throm-bocytopenic purpura in mice. Blood 96:2520–2527.

18. Becker BH, Miller JL (1989) Effects of an antiplatelet glycoprotein Ib antibody onhemostatic function in the guinea pig. Blood 74:690–694.

19. Cadroy Y, et al. (1994) Relative antithrombotic effects of monoclonal antibodiestargeting different platelet glycoprotein-adhesive molecule interactions in non-human primates. Blood 83:3218–3224.

20. Li J, et al. (2015) Desialylation is a mechanism of Fc-independent platelet clearanceand a therapeutic target in immune thrombocytopenia. Nat Commun 6:7737.

21. Quach ME, et al. (2018) Fc-independent immune thrombocytopenia via mechano-molecular signaling in platelets. Blood 131:787–796.

22. Li R, Emsley J (2013) The organizing principle of the platelet glycoprotein Ib-IX-Vcomplex. J Thromb Haemost 11:605–614.

23. Li Z, Delaney MK, O’Brien KA, Du X (2010) Signaling during platelet adhesion andactivation. Arterioscler Thromb Vasc Biol 30:2341–2349.

24. Shrimpton CN, et al. (2002) Localization of the adhesion receptor glycoprotein Ib-IX-Vcomplex to lipid rafts is required for platelet adhesion and activation. J Exp Med 196:1057–1066.

25. Sullam PM, et al. (1998) Physical proximity and functional interplay of the glycopro-tein Ib-IX-V complex and the Fc receptor FcgammaRIIA on the platelet plasmamembrane. J Biol Chem 273:5331–5336.

26. Kasirer-Friede A, et al. (2002) Lateral clustering of platelet GP Ib-IX complexes leads toup-regulation of the adhesive function of integrin alpha IIbbeta 3. J Biol Chem 277:11949–11956.

27. Gitz E, et al. (2013) Platelet interaction with von Willebrand factor is enhanced byshear-induced clustering of glycoprotein Ibα. Haematologica 98:1810–1818.

28. Yin H, Stojanovic A, Hay N, Du X (2008) The role of Akt in the signaling pathway ofthe glycoprotein Ib-IX induced platelet activation. Blood 111:658–665.

29. Liu J, Fitzgerald ME, Berndt MC, Jackson CW, Gartner TK (2006) Bruton tyrosine ki-nase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombusformation in vivo. Blood 108:2596–2603.

30. Zhang Y, et al. (2017) Receptor-interacting protein kinase 3 promotes platelet acti-vation and thrombosis. Proc Natl Acad Sci USA 114:2964–2969.

31. Dai K, Bodnar R, Berndt MC, Du X (2005) A critical role for 14-3-3zeta protein inregulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeuticimplications. Blood 106:1975–1981.

32. Li S, et al. (2010) The glycoprotein Ibalpha-von Willebrand factor interaction inducesplatelet apoptosis. J Thromb Haemost 8:341–350.

33. Zhao L, et al. (2017) Protein kinase A determines platelet life span and survival byregulating apoptosis. J Clin Invest 127:4338–4351.

34. Leytin V (2012) Apoptosis in the anucleate platelet. Blood Rev 26:51–63.35. Tang WH, et al. (2014) Aldose reductase-mediated phosphorylation of p53 leads to

mitochondrial dysfunction and damage in diabetic platelets. Circulation 129:1598–1609.

36. Ojha A, et al. (2017) Platelet activation determines the severity of thrombocytopeniain dengue infection. Sci Rep 7:41697.

37. van Es N, Sturk A, Middeldorp S, Nieuwland R (2014) Effects of cancer on platelets.Semin Oncol 41:311–318.

38. Zhao Y, et al. (2015) An improved flow cytometric immunobead array to detect au-toantibodies in plasma from patients with immune thrombocytopenic purpura. ClinChim Acta 438:396–400.

39. Wardell MR, Reynolds CC, Berndt MC, Wallace RW, Fox JE (1989) Platelet glycoproteinIb beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase.J Biol Chem 264:15656–15661.

40. Smolenski A, et al. (1998) Analysis and regulation of vasodilator-stimulated phos-phoprotein serine 239 phosphorylation in vitro and in intact cells using a phospho-specific monoclonal antibody. J Biol Chem 273:20029–20035.

41. Lizcano JM, Morrice N, Cohen P (2000) Regulation of BAD by cAMP-dependent pro-tein kinase is mediated via phosphorylation of a novel site, Ser155. Biochem J 349:547–557.

42. Gitz E, et al. (2012) Improved platelet survival after cold storage by prevention ofglycoprotein Ibα clustering in lipid rafts. Haematologica 97:1873–1881.

43. Hoffmeister KM, et al. (2003) Glycosylation restores survival of chilled blood platelets.Science 301:1531–1534.

44. Mu FT, et al. (2008) A functional 14-3-3zeta-independent association of PI3-kinasewith glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glyco-protein Ib-IX-V complex. Blood 111:4580–4587.

45. Woulfe D, et al. (2004) Defects in secretion, aggregation, and thrombus formation inplatelets from mice lacking Akt2. J Clin Invest 113:441–450.

46. Chen J, et al. (2004) Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood 104:1703–1710.

47. O’Brien KA, Stojanovic-Terpo A, Hay N, Du X (2011) An important role for Akt3 inplatelet activation and thrombosis. Blood 118:4215–4223.

48. Zhang W, Colman RW (2007) Thrombin regulates intracellular cyclic AMP concen-tration in human platelets through phosphorylation/activation of phosphodiesterase3A. Blood 110:1475–1482.

49. Han SJ, et al. (2006) Protein kinase B/Akt phosphorylation of PDE3A and its role inmammalian oocyte maturation. EMBO J 25:5716–5725.

50. Hunter RW, Mackintosh C, Hers I (2009) Protein kinase C-mediated phosphorylationand activation of PDE3A regulate cAMP levels in human platelets. J Biol Chem 284:12339–12348.

51. Chow TW, Hellums JD, Moake JL, Kroll MH (1992) Shear stress-induced von Wille-brand factor binding to platelet glycoprotein Ib initiates calcium influx associatedwith aggregation. Blood 80:113–120.

52. Savill J, Gregory C (2007) Apoptotic PS to phagocyte TIM-4: Eat me. Immunity 27:830–832.

53. Hanayama R, et al. (2002) Identification of a factor that links apoptotic cells tophagocytes. Nature 417:182–187.

54. Fujii T, Sakata A, Nishimura S, Eto K, Nagata S (2015) TMEM16F is required forphosphatidylserine exposure and microparticle release in activated mouse platelets.Proc Natl Acad Sci USA 112:12800–12805.

55. Ma R, et al. (2017) Phosphatidylserine-mediated platelet clearance by endotheliumdecreases platelet aggregates and procoagulant activity in sepsis. Sci Rep 7:4978.

56. Maugeri N, et al. (2009) Neutrophils phagocytose activated platelets in vivo: Aphosphatidylserine, P-selectin, and beta2 integrin-dependent cell clearance program.Blood 113:5254–5265.

57. Hoffmeister KM, et al. (2003) The clearance mechanism of chilled blood platelets. Cell112:87–97.

58. Janes PW, Ley SC, Magee AI (1999) Aggregation of lipid rafts accompanies signalingvia the T cell antigen receptor. J Cell Biol 147:447–461.

59. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572.60. Peng Y, Shrimpton CN, Dong JF, López JA (2005) Gain of von Willebrand factor-

binding function by mutagenesis of a species-conserved residue within the leucine-rich repeat region of platelet glycoprotein Ibalpha. Blood 106:1982–1987.

61. Li J, et al. (2013) Fc-independent phagocytosis: Implications for IVIG and other ther-apies in immune-mediated thrombocytopenia. Cardiovasc Hematol Disord DrugTargets 13:50–58.

62. Li Y, et al. (2017) Sialylation on O-glycans protects platelets from clearance by liverKupffer cells. Proc Natl Acad Sci USA 114:8360–8365.

63. Rumjantseva V, et al. (2009) Dual roles for hepatic lectin receptors in the clearance ofchilled platelets. Nat Med 15:1273–1280.

64. Schoenwaelder SM, et al. (2009) Two distinct pathways regulate platelet phosphati-dylserine exposure and procoagulant function. Blood 114:663–666.

65. Linden MD, et al. (2007) Indices of platelet activation and the stability of coronaryartery disease. J Thromb Haemost 5:761–765.

66. Ranger AM, et al. (2003) Bad-deficient mice develop diffuse large B cell lymphoma.Proc Natl Acad Sci USA 100:9324–9329.

67. Yang ZZ, et al. (2003) Protein kinase B alpha/Akt1 regulates placental developmentand fetal growth. J Biol Chem 278:32124–32131.

68. Dummler B, et al. (2006) Life with a single isoform of Akt: Mice lacking Akt2 andAkt3 are viable but display impaired glucose homeostasis and growth deficiencies.Mol Cell Biol 26:8042–8051.

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