Fcγ Receptors I and III on Splenic Macrophages Mediate GPIIb/IIIa Autoantibody-Dependent Phagocytosis of Platelets in Human Immune Thrombocytopenia

Abstract Autoantibody-opsonized platelets in immune thrombocytopenia (ITP) are thought to be destroyed primarily by macrophage Fc gamma receptor (FcγR)-mediated phagocytosis in the spleen. Blockade of splenic macrophage FcγRs has been proposed as a therapeutic mechanism for ITP intervention. Unfortunately, the contribution of specific FcγRs to disease in ITP remains unknown. Our objective was to determine which FcγRs are responsible for the phagocytosis of ITP autoantibody-opsonized platelets by splenic macrophages. Splenic macrophages were purified by CD14 positive selection from spleens of splenectomized ITP patients, and were treated with blocking antibodies to FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII. Blocking antibodies were deglycosylated to prevent non-specific blocking effects by their Fc region. Two separate ITP sera confirmed positive for anti-GPIIb/IIIa autoantibodies by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay were used to opsonize healthy donor human platelets. Phagocytosis was determined by confocal microscopy and non-phagocytosed (external) platelets were differentiated by an anti-platelet antibody stain following macrophage fixation. Human ITP splenic macrophages were found to express FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII, and expression was not significantly different compared to healthy (trauma) controls (n=5). The two anti-GPIIb/IIIa-positive ITP sera induced a mean 3.7- and 4.2-fold increase of platelet uptake by ITP splenic macrophages relative to normal human serum controls (n=3 each, p<0.001). Blockade of all FcγRs significantly reduced phagocytosis of serum-opsonized platelets for both ITP sera as compared to an IgG control (p<0.001) down to background (non-opsonized) levels. Using single blocking antibodies, inhibition of FcγRI reduced splenic macrophage phagocytosis of platelets by 45% and 37% for the two respective ITP sera as compared to IgG controls (n=3 for each patient sera, p<0.01), while inhibition of FcγRIII reduced phagocytosis of platelets by 43% and 44% (n=3 each, p<0.05). Blockade of FcγRIIa or FcγRIIa/b/c only marginally inhibited splenic macrophage phagocytosis and was not significant. An Fab-like FcγRIII-blocking antibody with a mutant Fc region completely deficient for FcγR binding demonstrated equal FcγRIII blockade compared to the deglycosylated anti-FcγRIII antibody. In comparison to ITP sera-opsonized platelets, FcγRI had a greater involvement in ITP splenic macrophage phagocytosis of anti-D-opsonized human erythrocytes as FcγRI blockade inhibited phagocytosis by 70% (n=5, p<0.001), while FcγRIII blockade inhibited phagocytosis by only 30% (p<0.001) and FcγRIIa or FcγRIIa/b/c blockade had no significant effect. This work demonstrates that FcγRI and FcγRIII are the primary phagocytic receptors on splenic macrophages for anti-GPIIb/IIIa autoantibody-opsonized platelets in human ITP and suggests that FcγRI and FcγRIII are the best targets for FcγR blockade as a potential therapeutic maneuver in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.

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Characterization of the CD47/SIRP-Alpha System in Patients with Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v114.22.1309.1309 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1309-1309

Author(s):

Lucia Catani ◽

Daria Sollazzo ◽

Francesca Ricci ◽

Nicola Polverelli ◽

Francesca Palandri ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Platelet Apoptosis ◽

Cell Death Pathway ◽

Macrophage Phagocytosis

Abstract Abstract 1309 Poster Board I-331 Introduction The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a ligand for SIRP-alpha (signal regulatory protein-alpha) receptor and as a receptor for Thrombospondin, acting, respectively, as antagonistic to phagocyte activity and as regulator of apoptosis. Based on mouse studies, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently proposed in immune thrombocytopenia. This mechanism suggests that platelet homeostasis is regulated by platelet expression of CD47 and that interaction between platelet CD47 and macrophage SIRP-alpha receptor is important in regulating platelet macrophage phagocytosis. However, the role of this system in platelet uptake/phagocytosis by dendritic cells (DCs) has never been investigated in immune thrombocytopenia (ITP) in humans. Therefore, our purpose was to evaluate whether alterations of the CD47/SIRP-alpha system may have a role in the pathogenesis of ITP in humans. Patients and methods Twenty five ITP patients were studied. We phenotypically characterized apoptosis (Annexin-V FITC staining) and the expression of CD47 on platelets and SIRP-alpha on CD14-derived and circulating DCs by flow cytometry. To determine whether platelet apoptosis was due to activation of CD47-cell death pathway, in parallel experiments, we assessed the in vitro sensitivity of platelets to antibody CD47 ligation. In addition, to investigate the role of CD47/SIRP-alpha system on platelet phagocytic capacity of CD14-derived DCs, immature DCs were coincubated with PKH26-labelled platelets in the presence or absence of antibodies against CD47 and SIRP-alpha. The percentage of ingested platelets was then evaluated by flow cytometry. Results We demonstrate that in ITP: 1) CD47 expression is not altered in freshly isolated platelets; 2) after in vitro aging, platelet apoptosis is increased as compared with the normal counterparts; 3) CD47 expression is unchanged in apoptotic platelets; by contrast, it increases in normal platelets; 4) the increased platelet apoptosis is not due to the activation of the CD47-induced cell death pathway; 5) despite low level expression of SIRP-alpha in CD14-derived DCs and in circulating DCs, the CD47/ SIRP-alpha system does not play a central role for in vitro platelet phagocytosis of DCs, since blockage of SIRP-alpha on DCs or CD47 on platelets by specific antibodies failed to modify phagocytosis. Conclusions In conclusion, we demonstrate that in ITP platelet CD47 expression does not play a role in the pathogenesis of the disease. We also show that, in ITP patients, higher platelet apoptosis is not due to different CD47-induced cell death susceptibility. Whether this is due to the fact that CD47 expressed on apoptotic platelets from ITP patients may have a peculiar conformation, avoiding the delivery of cell death signal, remains open question. Furthermore, the platelet uptake/phagocytosis by DCs is not significantly influenced by the CD47/ SIRP-alpha system. Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section) Disclosures No relevant conflicts of interest to declare.

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How Does CD47-SIRPα ‘Don’t Eat Me Signal’ Physically Signal Self

Blood ◽

10.1182/blood.v122.21.953.953 ◽

2013 ◽

Vol 122 (21) ◽

pp. 953-953

Author(s):

Sosale Nisha ◽

Dennis E. Discher

Keyword(s):

Blood Cells ◽

Conflicts Of Interest ◽

Myosin Ii ◽

Time Lapse ◽

Splenic Macrophage ◽

Macrophage Phagocytosis ◽

Self Recognition ◽

Protein Functions

Abstract Resident macrophages in spleen and liver are particularly adept at recognizing foreign pathogens through recognition of ‘non-self’ proteins on the pathogen surface but also through the absence of ‘self’ proteins that are highly displayed on circulating blood cells. Red blood cells display a ‘marker of self’ protein CD47 which increases the in vivo half-life and decreases red-pulp splenic macrophage uptake of mouse RBC (Oldenborg et al, Science 2000) and also of particles displaying human-CD47 in recent studies by our group (Rodriguez et al, Science 2013). CD47 signals self through its counter receptor SIRPa, which is highly expressed on the surfaces of myeloid cells but also highly polymorphic. The CD47 protein functions in vitro as a marker of self toward human SIRPa on human macrophages and monocytes, inhibiting accumulation of myosin II motor protein to the phagocytic synapse (Tsai 2008). The work here aims to clarify when and how CD47-SIRPa inhibition physically signals ‘self’ during macrophage phagocytosis uptake. While it is clear that CD47 reduces the number of uptake events, here we use time-lapse and confocal microscopy to examine the forces of distortion imparted by phagocytes on opsonized red blood cell targets during uptake. Glutaraldehyde-fixed RBC are also used as a model to assess the affects of cell rigidity in this self-recognition process, since rigidity is relevant to processes as diverse as RBC aging and sickle RBC to malaria but also because adhesion (by macrophages) is expected to activate the myosin-II contractility system and oppose CD47 signaling. Through blocking and pharmacological approaches, we parse the pathways between foreign, self, and rigidity sensing. Disclosures: No relevant conflicts of interest to declare.

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Pathological Shear Regulates ADAM10 Activity on Circulating Platelets

Blood ◽

10.1182/blood.v118.21.2194.2194 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2194-2194

Author(s):

Elizabeth E Gardiner ◽

Jianlin Qiao ◽

Cheewee Tan ◽

Jane Frances Arthur ◽

Mohammad Al-Tamimi ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Platelet Reactivity ◽

Factor Xa ◽

Fold Increase ◽

Human Platelets ◽

Calcium Flux ◽

Short Exposure ◽

Protective Mechanism

Abstract Abstract 2194 Metalloproteinase-mediated ectodomain shedding of platelet-specific receptors for collagen (GPVI) and von Willebrand factor (GPIbα of the GPIb-IX-V complex) is triggered by either ligand-induced platelet activation-dependent pathways, or by activation-independent pathways mediated by Factor Xa or induced by the thiol-modifying agent, N-ethylmaleimide. We recently reported that shed soluble GPVI (sGPVI) was elevated in plasma of 159 ischaemic stroke patients compared with 159 community-based controls (P=0.0168), and in 29 patients with disseminated intravascular coagulation compared with healthy donors (n=25, P=0.002), consistent with a pathophysiological role for GPVI shedding from human platelets. Our new studies now show that transient exposure of human platelets to arterial or pathological shear rates of 3000–10,000 s−1 for 1–5 min ex vivo in a cone-plate viscometer, in the absence of GPVI ligand or platelet activation, activated sheddases producing a 2- to 3-fold increase in plasma sGPVI and a corresponding loss of surface GPVI from sheared platelets. Shear-induced GPVI shedding was blocked by GM6001 or GI254023, a selective inhibitor of ADAM10. In contrast to shear-induced platelet aggregation, shedding was unaffected by inhibitors of aggregation (VWF-blocking anti-GPIbα mAb, AK2, or the αIIbβ3 antagonist, RGD peptide) or by the absence of VWF in a patient with von Willebrand's disease Type III (VWF antigen levels <1%). Further, shedding of GPVI increased for up to 10 min after cessation of a short exposure of platelets to shear even when signalling, secretion and aggregation was blocked by inhibiting intracellular kinases (PP2, piceatannol), thromboxane generation (aspirin), ADP (apyrase) and calcium flux (BAPTA). Together, the combined results provide the first evidence that receptor sheddase activity can be regulated by hydrodynamic shear stress independent of cellular activation. This may represent a novel protective mechanism for down-regulating platelet reactivity as a response to pathological shear. Disclosures: No relevant conflicts of interest to declare.

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Platelet Tissue Factor Activity In Patients with Hypercholesterolemia: Modulation of Procoagulant Activity by Statins

Blood ◽

10.1182/blood.v116.21.156.156 ◽

2010 ◽

Vol 116 (21) ◽

pp. 156-156 ◽

Cited By ~ 1

Author(s):

Olga Panes ◽

Mónica Acevedo ◽

Valeria Matus ◽

Claudia G. Sáez ◽

Jaime Pereira ◽

...

Keyword(s):

Statin Therapy ◽

Conflicts Of Interest ◽

Plasma Cholesterol ◽

Fold Increase ◽

Cholesterol Content ◽

Human Platelets ◽

Procoagulant Activity ◽

Specific Stimulus ◽

Activated Platelets ◽

Platelet Membranes

Abstract Abstract 156 Human platelets store TF that expresses procoagulant activity (PCA) associated with platelet activation (Panes O. et al, Blood 2007;109:5242-50). We have also observed that platelet TF-induced PCA is specifically and rapidly induced by activation of GPIb complex by VWF-Ristocetin (Panes O. et al. Blood 2008;112:A113). Hypercholesterolemia has been associated with increased platelet function and hypercoagulability, but the mechanisms are still unknown. The cholesterol content in cell membranes is directly related with the plasma concentration of the sterol. Inhibition of HMG-CoA reductase by statins reduces cardiovascular risk by decreases in plasma cholesterol and by other independent (“pleiotropic”) effects. We studied the relationship between total and LDL plasma cholesterol with platelet TF content and TF-dependent PCA of washed platelets activated for 5 min with VWF-Ristocetin (VWF-R). We also examined these variables after atorvastatin or rosuvastatin intake (80 and 20 mg/day × 1 month, respectively) in 25 subjects with hypercholesterolemia. TF was measured by ELISA and PCA by FXa generation (chromogenic assay) in washed platelet suspensions before and after activation. We found no significant differences between both statins regarding the decrease in plasma LDL-cholesterol levels and platelet TF-dependent PCA; both statins had any significant effects on the levels of blood inflammatory markers (usCRP, fibrinogen and VWF). Thus, we analyzed the pooled data of patients receiving either one or the other statin. Total and LDL-Chol dropped from 271±41 and 185±40 to 167±30 and 87±23 mg dL-1, respectively. This was associated with decrease in basal PCA (non-stimulated platelets) from 34±27 to 22±14 nmol of FXa/2*107platelets (p=0.02). After VWF-R stimulation, a mean 3.48-fold increase in platelet PCA was observed in hypercholesterolemic patients before taking statins. Surprisingly, a mean 20-fold increase in FXa generation in activated platelets was observed after 1 month on statin therapy, and this difference was statistically significant (p=0.018). These changes in PCA were not associated with significant changes in TF content of platelet membranes (621±481 to 555±300 pg/mg prot). We also found that plasma LDL-Chol was negatively correlated with platelet PCA induced by VWF-R activation (r = −0.239, p = 0.026) and with the ratio of PCA between activated and non-activated platelets (r = −0.27, p = 0.011). The striking post-statin increase of platelet-PCA without change in the total platelet TF protein suggests that PCA depends more on the fraction of platelet TF available for activation than on the total mass of platelet TF. The decrease in basal PCA of non-stimulated platelets after statin therapy suggests that lower concentration of cholesterol in platelet membranes makes the platelets more stable during the isolation procedure. The higher increase after stimulation would reflect a better hemostatic response to a specific stimulus. Thus, changes in plasma cholesterol, possibly through decrease in platelet membrane cholesterol, modulates the PCA of platelets, either by making platelets more stable under resting conditions and more responsive after specific activation. Disclosures: No relevant conflicts of interest to declare.

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Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647928 ◽

1976 ◽

Vol 35 (02) ◽

pp. 350-357 ◽

Cited By ~ 8

Author(s):

Hana Bessler ◽

Galila Agam ◽

Meir Djaldetti

Keyword(s):

Protein Synthesis ◽

Fold Increase ◽

Human Platelets ◽

Polystyrene Latex ◽

Latex Particles ◽

The Third ◽

Platelet Protein ◽

Dose Dependent ◽

Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

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Macrophage Inhibition of Collagen-Induced Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657016 ◽

1979 ◽

Vol 42 (04) ◽

pp. 1207-1216 ◽

Cited By ~ 1

Author(s):

Berit Mørland

Keyword(s):

Enzyme Activity ◽

Platelet Aggregation ◽

Peritoneal Macrophage ◽

Cultured Cells ◽

Culture Media ◽

Human Platelets ◽

Mouse Peritoneal Macrophage ◽

Collagenase Activity ◽

Partial Inhibition ◽

Macrophage Phagocytosis

SummaryCollagen was incubated with cells or media fractions of mouse peritoneal macrophage cultures, and its aggregating effect on human platelets was tested. Incubation with lysates of cultured cells completely abolished the normal collagen-induced platelet aggregation, while incubation with media fractions only caused partial inhibition. The latter inhibition was more pronounced after macrophage phagocytosis of latex particles, while endocytosis of endotoxin had no effect.Corresponding macrophage cultures were also tested for specific collagenase activity, using 14C-glycine labelled collagen as substrate. Collagenase activity was found in the culture media fractions only, and the enzyme activity could be enhanced by endocytosis of latex as well as endotoxin.It appears that the effect of macrophage lysates and media on collagen-platelet interaction cannot be ascribed only to secretion of collagenase from macrophages.

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Emerging Therapies in Immune Thrombocytopenia

Journal of Clinical Medicine ◽

10.3390/jcm10051004 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1004

Author(s):

Sylvain Audia ◽

Bernard Bonnotte

Keyword(s):

Tyrosine Kinase ◽

Immune Thrombocytopenia ◽

Plasma Cells ◽

Autoimmune Disorder ◽

Intravenous Immunoglobulins ◽

Therapeutic Interventions ◽

Autoimmune Response ◽

Platelet Destruction ◽

Classical Complement Pathway ◽

Splenic Macrophages

Immune thrombocytopenia (ITP) is a rare autoimmune disorder caused by peripheral platelet destruction and inappropriate bone marrow production. The management of ITP is based on the utilization of steroids, intravenous immunoglobulins, rituximab, thrombopoietin receptor agonists (TPO-RAs), immunosuppressants and splenectomy. Recent advances in the understanding of its pathogenesis have opened new fields of therapeutic interventions. The phagocytosis of platelets by splenic macrophages could be inhibited by spleen tyrosine kinase (Syk) or Bruton tyrosine kinase (BTK) inhibitors. The clearance of antiplatelet antibodies could be accelerated by blocking the neonatal Fc receptor (FcRn), while new strategies targeting B cells and/or plasma cells could improve the reduction of pathogenic autoantibodies. The inhibition of the classical complement pathway that participates in platelet destruction also represents a new target. Platelet desialylation has emerged as a new mechanism of platelet destruction in ITP, and the inhibition of neuraminidase could dampen this phenomenon. T cells that support the autoimmune B cell response also represent an interesting target. Beyond the inhibition of the autoimmune response, new TPO-RAs that stimulate platelet production have been developed. The upcoming challenges will be the determination of predictive factors of response to treatments at a patient scale to optimize their management.

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Physiological regulation of total tubulin and polymerized tubulin in tissues.

The Journal of Cell Biology ◽

10.1083/jcb.74.2.351 ◽

1977 ◽

Vol 74 (2) ◽

pp. 351-357 ◽

Cited By ~ 29

Author(s):

D G Pipeleers ◽

M A Pipeleers-Marichal ◽

D M Kipnis

Keyword(s):

Mouse Liver ◽

Marked Decrease ◽

Human Lymphocytes ◽

Fold Increase ◽

Human Platelets ◽

Degree Of Polymerization ◽

Polymerization Process ◽

Physiological Factors ◽

Physiological Regulation ◽

Tubulin Content

Polymerized and depolymerized forms of tubulin were measured in rat and mouse liver, rat islets, human lymphocytes, and platelets. The percent of the total tubulin present in the polymerized form varied from 30.3 +/- 1.5% in the liver of the fed rat to 89.2 +/- 0.2% in human platelets. Fasting decreased the total tubulin and to a greater extent the polymerized form of tubulin in both rat and mouse liver. Glucose feeding increased the polymerized tubulin without affecting the total tubulin content in rat liver. Phytohemagglutinin-stimulated lymphocytes exhibited at least a three-fold increase in total tubulin (expressed in terms of DNA content), which during the initial 48 h of incubation was accounted for in toto by an increase in polymerized tubulin. It is suggested that the lectin not only accelerates tubulin synthesis but also stimulated the polymerization process. Storage of platelets at 4 degrees C for 6 days resulted in a marked decrease in total tubulin and an even greater reduction in the polymerized form. It is concluded that both the total tubulin content and its degree of polymerization can be modulated independently by a wide variety of physiological factors.

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Cross-Reactivity of Drug-Dependent Antibodies in Patients with Immune Thrombocytopenia Induced By Beta-Lactam Antibiotics

Blood ◽

10.1182/blood-2019-131375 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2350-2350

Author(s):

Matthew John Slaught ◽

Daniel W. Bougie ◽

Richard H. Aster

Keyword(s):

Bacterial Infections ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Cross Reactivity ◽

Beta Lactam ◽

Cross Reactions ◽

Beta Lactam Antibiotics ◽

Wide Range ◽

Group 2 ◽

Group 1

More than 50 beta lactam (BL) antibiotics are now in active use for treatment of a wide range of bacterial infections. BL antibiotics are among the most common drugs capable of inducing antibodies (DDAbs) that cause drug-induced immune thrombocytopenia (DITP). Most DDAbs are highly specific for the sensitizing drug but beta lactams all have a common core structure and many similarities among side groups that are added to augment potency and modify specificity, raising the possibility that a DDAb specific for one BL may cross-react with another. We studied DDAbs from 33 patients with DITP induced by 9 commonly used BL drugs to determine whether patterns of cross-reactivity exist that might influence the choice of an alternative antibiotic in a patient with BL-induced DITP. DDAbs were demonstrated in a flow cytometric assay considered to be "positive" when immunoglobulins in patient serum but not normal serum react with normal platelets in the presence, but not in the absence of drug (Blood 2018;131:1486). DDAbs detected in the 33 patients were specific for 9 different BL drugs that were divided into two groups, "penicillins" (Group 1) and cephalosporins (Group 2) on the basis of structural similarities (Figure 1). In Group 1 were 19 DDAbs specific for amoxicillin (2), nafcillin (4) and piperacillin (13). Structurally similar ampicillin and penicillin were also tested with these abs. In Group 2 were 14 DDAbs specific for cefadroxil (1), cefepime (2), ceftazidime (2), ceftizoxime (1), ceftriaxone (7) and cephalexin 1). Cross-reactions identified within these groups of DDAbs are shown in Tables 1 and 2. Cross-reactions, many quite strong (S) were observed among DDAbs specific for drugs in both structural groups (Tables 1 and 2). Particularly noteworthy were cross-reactions of the 19 Group 1 DDAbs with ampicillin (6) and penicillin (6) (Table 1) and of the 14 Group 2 DDAbs with cefepime (6), ceftizoxazole (6) and ceftriaxone (3) (Table 2). The findings show that platelet-specific DDAbs induced by beta lactam antibiotics, in contrast with those induced by medications like quinine, sulfamethoxazole and vancomycin, commonly cross-react with other antibiotics of this class. In patients with immune thrombocytopenia induced by a beta lactam antibiotic, it may be prudent to avoid switching to another beta lactam or, if this is necessary, to monitor platelet counts carefully. Disclosures No relevant conflicts of interest to declare.

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Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα

Blood Advances ◽

10.1182/bloodadvances.2018024950 ◽

2019 ◽

Vol 3 (3) ◽

pp. 275-287 ◽

Cited By ~ 5

Author(s):

Caroline Kardeby ◽

Knut Fälker ◽

Elizabeth J. Haining ◽

Maarten Criel ◽

Madelene Lindkvist ◽

...

Keyword(s):

Platelet Aggregation ◽

Physiological Role ◽

Human Platelets ◽

Minor Role ◽

Phosphatidylinositol 3 Kinase ◽

Akt Phosphorylation ◽

Therapeutic Value ◽

Direct Binding ◽

A Minor ◽

Blocking Antibodies

Abstract Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)–dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)–like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

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