Desialylation: A Novel Platelet Clearance Mechanism and a Potential New Therapeutic Target in Anti-GPIb Antibody Mediated Thrombocytopenia

Abstract Abstract 265 Background: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibodies directed at patient's own platelet antigens, primarily glycoprotein (GP)IIbIIIa-integrin (70–80%) and GPIb-complex (20–40%). Current paradigm suggests that clearance of opsonized platelets through the reticuloendothelial system via Fcγ-receptors results in thrombocytopenia and bleeding disorders. However, evidence from others and our group demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP-models (Blood 2006). These observations are consistent with subsequent IVIG studies in human ITP patients. Interestingly, human anti-GPIb-mediated ITP patients seem also resistant to steroid therapy in our recent retrospective study (American Journal of Hematology 2012). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a different mechanism which is currently poorly understood. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa or GPIba deficient mice. Some of the mAbs have cross-reactivity to both mouse and human GPIIbIIIa and GPIba. Flow cytometry was used to evaluate whether these mAbs were able to induce platelet activation, apoptosis and desialylation. GPIbα is heavily glycosylated and the role of desialylation and exposure of underlying galactose and β-N-acetyl-D-glucosamine (βGN) residues on GPIbα in platelet clearance was assessed using the sialidase neuraminidase (NA) and it's inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). Desialyation effects on platelet activation and apoptosis was measured by flow cytometry. We also repeated these experiments with human platelets and plasma from human ITP-patients. We also investigated the effects of anti-GPIbα antibodies on platelet activation, apoptosis and clearance in vivo. Briefly, BALB/c mice were injected with anti-GPIbαor anti-GPIIIa mAbs and 24 hrs later, platelet desialylation, activation and apoptosis were measured by flow cytometry. The effect of desialylation on platelet clearance was assessed with DANA. The possible roles of Ashwell-Morell and MAC-1 receptors in GPIbα-mediated platelet clearance in the liver were examined using immunohistochemistry (anti-CD11b) or blocking of the Ashwell-Morell receptor with asialofetuin. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa mAbs, induced significant P-selectin expression and phosphatidylserine (PS)-exposure, and increased inner membrane mitochondrial depolarization (ΔYm). Interestingly, platelets were desialylated in the presence of anti-GPIbα but not anti-GPIIbIIIa mAbs. Moreover, we found that desialylation of GPIbα lies directly upstream of platelet activation and apoptosis, as prior treatment with DANA diminished PS-exposure, and P-selectin expression. Most importantly, incubations of human platelets with ITP-patient plasma showed similar effects. In vivo, we found significant increases in PS-exposure and ΔYm induced by anti-GPIbα, but not by anti-GPIIIa mAbs, independent of IgG subclass. Interestingly, prior injection with DANA rescued platelets numbers in anti-GPIbα, but not in anti-GPIIIa injected mice. A significant role for the Ashwell-Morell and MAC-1 receptors in the clearance of deglycosylated platelets was observed; blocking of the Ashwell-Morell receptors by asialofetuin, decreased platelet clearance in anti-GPIbα, but not anti-GPIIbIIIa antibody injected mice, there was also increased staining for MAC-1 on Kupffer cells, exclusively in the presence of an anti-GPIbα mAb tested. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce GPIbα desialyation, leading to platelet activation and apoptosis. Therefore, we identified novel Fc-independent platelet clearance pathways, more specifically, via Ashwell-Morell and MAC-1 receptors on hepatocytes and liver macrophages. These findings may lead to novel therapeutic regimens including the potential use of sialidase inhibitors as a solution for anti-GPIb-mediated ITP patients previously refractory to both steroid and IVIG therapies. Disclosures: No relevant conflicts of interest to declare.

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Platelet Desialylation: A Novel Mechanism of Fc-Independent Platelet Clearance and a Potential Diagnostic Biomarker and Therapeutic Target in immune Thrombocytopenia

Blood ◽

10.1182/blood.v124.21.467.467 ◽

2014 ◽

Vol 124 (21) ◽

pp. 467-467

Author(s):

June Li ◽

Dianne Evertdina van der Wal ◽

Guangheng Zhu ◽

Miao Xu ◽

Issaka Yougbare ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Therapeutic Target ◽

Conflicts Of Interest ◽

Ivig Therapy ◽

Human Platelets ◽

Diagnostic Biomarker ◽

Large Patient ◽

Sialidase Inhibitor

Abstract Background:Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa (70-80%) and/or GPIb-complex (20-40%). Current theory suggests antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc-FcγR interactions. However, evidence from us and others demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP models (Blood 2006) and subsequent IVIG studies in human ITP patients, including our recent large patient cohort study (JTH 2014). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a presently unidentified mechanism different than that of anti-GPIIbIIIa. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa-/- or GPIba-/- mice, which also recognize GPIbα and GPIIbIIIa of different species including human. Flow cytometry, immunofluorescence, and western blotting were used to evaluate whether these mAbs induced platelet activation, neuraminidase-1 translocation and desialylation of the heavily glycosylated GPIbα in the presence of sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). These experiments were repeated with human platelets and human ITP patient plasma. We further investigated the effects of anti-GPIbα antibodies on platelet activation, desialylation and clearance in vivo; BALB/c mice were injected with anti-GPIbα or anti-GPIIbIIIa mAbs and following, platelet activation and desialylation were measured by flow cytometry. Hepatocytic Ashwell-Morell receptor (AMR) mediated anti-GPIbα platelet clearance in the liver was examined using immunohistochemistry or blocking the AMR with asialofetuin in both wild-type and macrophage depleted mice. Therapeutic administration of DANA in a murine ITP model assessed the significance of Fc-independent anti-GPIbα mediated platelet clearance in ITP. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induced significant P-selectin expression, JON/A binding, neuraminidase-1 translocation and desialylation in murine platelets. Interestingly, certain human platelets were activated (P-selectin expression) and desialylated in the presence of both anti-GPIbα and anti-GPIIbIIIa mAbs or ITP patient plasma. However, we demonstrate that the anti-GPIIbIIIa antibody mediated platelet effects are dependent on the FcγRIIa present exclusively on human platelets as FcγRII blocker IV.3 completely attenuated the response. In contrast, IV.3 had little effect on anti-GPIbα mediated platelet activation or desialylation. Anti-GPIbα Fab fragments and platelet signal pathway inhibitors demonstrate that anti-GPIbα mediated platelet activation and desialylation are consequences of GPIbα cross linking and are reinforced by a positive feedback loop. In vivo, we found significant increases in P-selectin and desialylation in anti-GPIbα injected mice, independent of IgG subclass. A significant role for the hepatic AMR in the clearance of deglycosylated platelets was observed; particularly in macrophage depleted mice whereby, although anti-GPIIbIIIa mediated platelet clearance was completely attenuated, anti-GPIbα mediated platelets clearance still occurred, but was completely rescued with asialofetuin. Immunohistochemistry revealed significant co-localization of anti-GPIbα opsonized platelets with AMR. These suggest the AMR is the dominant Fc-independent anti-GPIbα mediated platelet clearance pathway in the absence of macrophages. Remarkably, sialidase inhibitor DANA ameliorated anti-GPIbα mediated thrombocytopenia in mice. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce platelet activation leading to GPIbα desialyation and platelet clearance via a novel Fc-independent pathway: the hepatic AMR. Our data also suggested that some anti-GPIIbIIIa autoantibodies in human patients may also induce platelet activation and desialylation via the platelet FcR signaling pathway. These findings may lead to novel therapeutic regimens including designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of both anti-GPIIbIIIa and anti-GPIbα mediated and/or refractory ITP. Disclosures No relevant conflicts of interest to declare.

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Platelet Tissue Factor: Fast and Specific Clotting Activation Pathway Mediated by VWF-GPIba Interaction and Platelet Membrane FVII. Human Platelets Contain All the Components for Assembling the Prothrombinase Complex and Their Procoagulant Function Is Independent of Aggregation/Secretion and GPVI Function.

Blood ◽

10.1182/blood.v114.22.3000.3000 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3000-3000

Author(s):

Olga Panes ◽

Valeria Matus ◽

Claudia G. Sáaez ◽

Jaime Pereira ◽

Diego Mezzano

Keyword(s):

Flow Cytometry ◽

Tissue Factor ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Human Platelets ◽

Platelet Membrane ◽

Prothrombinase Complex ◽

Serotonin Secretion ◽

Trap Activation

Abstract Abstract 3000 Poster Board II-977 Human platelets synthesize and store functionally silent tissue factor (TF) which expresses procoagulant activity (PCA) after platelet activation. Fast activation of TF was elicited by VWF-Ristocetin (VWF-R) through GPIbαa activation and Src-Lyn transduction pathway (Blood, Nov 2008; 112:113). Given that GPVI, along with GPIb and TF have been found in “lipid rafts”, and the activated form of GPVI signals through Fyn, another member of the Src family, we tested if GPVI was involved in TF-initiated PCA. We also studied the time-course and pathway specificity of TF activation and the role of platelet FVII in PCA. Weak TF immunofluorescence and co-localization with GPIba were observed in non stimulated washed platelets. A mild increase of TF fluorescence was detected 2 min after TRAP activation, which augmented when the stimulus was VWF-R. Furthermore, striking enhancement of TF fluorescence occurred 2 min after depositing platelets over a VWF-coated surface, but not over fibrinogen or albumin. Platelets adherent to VWF matrix showed GPIb clustering and loss of co-localization with TF. Externalization of TF was confirmed by immunoprecipitation (Ip) of biotinylated membranes before and after platelet activation. Concomitantly, TF-dependent FXa generation increased 5-10-fold shortly after VWF stimulus. Washed platelets stimulated with VWF-R agglutinated normally when stirred in an aggregometer, and the fraction of platelets exposing anionic phospholipids (annexin V binding) was similar to parallel samples stimulated with TRAP. However, VWF-R induced null 14C-serotonin secretion and P-selectin exposure (flow cytometry) in washed platelets. In contrast, TRAP, collagen, ADP and convulxin induced full platelet aggregation, 14C-serotonin and P-selectin secretion at 2-5 min, but with no increase in FXa generation. Platelet PCA was inhibited by antibodies against TF, GPIba, FVIIa, as well as by SU6656 and PP2 (Src pathway inhibitors), but not by Gö6850 (a PKC inhibitor) or exogenous TFPI. p85, a subunit of PI-3K constitutively associated with GPIb complex, becomes strongly associated with TF after stimulation with VWF-R, though only weakly after TRAP activation, confirming the coordinate activation of GPIb and TF. FVII and FX were revealed in platelet membrane fractions by immunoblotting and both co-precipitate with TF in non-stimulated platelets. Two min after activation with VWF-R striking co-precipitations of TF with FVII and FX light chains were evidenced, denoting activation of platelet FVII and FX. When exogenous FX was added to the assay, the amount of FXa generated after 1 and 2 min stimulation was similar whether or not exogenous FVIIa was added. Platelets from four non-related patients with bleeding related to hereditary defect of GPVI had null aggregation and secretion with convulxin and collagen, less than 7% labeling of GPVI by flow cytometry and an immunoreactive membrane GPVI of Mr≈40kDa (native GPVI Mr=62kDa). All of them had normal agglutination with VWF-R and normal FXa generation. In summary, GPIb activation by VWF constitutes a unique and fast inducer of platelet TF-dependent PCA. This process requires anionic phospholipid exposure, but is independent of platelet GPIIb/IIIa and GPVI function. Platelet FVII can initiate FXa generation without need of plasma FVII. The associations of platelet FVII and FX with TF on membrane fractions, together with the large amount of FV in platelets, indicate that human platelets provide not just TF and a PCA phospholipid platform, but also all the components of the prothrombinase complex to trigger the clotting process. Taken together, our results underline the central role of platelets in the whole hemostatic process, unifying primary and secondary hemostasis and circumscribing thrombin generation and fibrin deposition where platelet plug is being formed. Platelet PCA should become a pharmacological target for preventing or managing bleeding and thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

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Modulation of Thrombin-Induced Platelet Activation By Defibrotide

Blood ◽

10.1182/blood-2019-125945 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3614-3614

Author(s):

Kristin N. Maher ◽

Xu Han ◽

Keith B. Neeves ◽

Marvin T. Nieman ◽

Jorge Di Paola

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Cleavage Site ◽

Conflicts Of Interest ◽

Geometric Mean ◽

Human Platelets ◽

Antibody Binding ◽

Activation Markers ◽

Terminal Domain ◽

Significant Difference

Defibrotide is a mixture of single-stranded phosphodiester oligonucleotides 9-80 bases in length derived from mammalian tissue and it is the only medication that is FDA approved for the prevention and treatment of post-transplant veno-occlusive disease, also known as sinusoidal obstructive syndrome (VOD/SOS). The mechanism of action of defibrotide is not well understood. A better understanding of the molecular mechanism by which defibrotide prevents and reverses the microvascular occlusion seen in VOD/SOS will be critical in the development of novel therapeutics for VOD/SOS and other related disorders. Our central hypothesis is that single stranded phosphodiester oligonucleotide mixtures such as defibrotide alter thrombin-induced platelet and endothelial cell activation primarily via altered signaling through protease activated receptors (PARs). To determine the effect of defibrotide on thrombin-induced platelet activation we performed flow cytometry on washed platelets from humans and mice. Washed platelets were incubated with agonist (1 nM thrombin, 100 μM synthetic PAR1 agonist TRAP6, or 100 μM synthetic PAR4 agonist PAR4AP) in the presence or absence of 1 mg/mL of defibrotide. Fluorescent antibodies against P-selectin (anti-CD62P antibody) and against the high affinity conformation of the αIIβ3 receptor (PAC1 antibody for human samples and JON/A antibody for murine samples) were added to assess for inhibition of platelet activation by defibrotide. Fluorescent anti-PAR1 antibodies SPAN12 (which spans the canonical thrombin cleavage site) and WEDE15 (which binds at the hirudin-like domain) were also added to separate samples to assess whether defibrotide inhibits cleavage of the PAR1 N-terminal domain by thrombin. After twenty minutes of incubation at room temperature the samples were analyzed by flow cytometry. We calculated the geometric mean of the fluorescence intensity (MFI) for three replicate samples and data are shown as the fold change compared to the washed platelet control without addition of agonist or defibrotide. For thrombin-stimulated human platelets, both anti-CD62P binding (MFI fold over control 5.7 vs. 1.2) and PAC1 antibody binding (MFI fold over control 7.1 vs. 1.7) are significantly reduced in the presence of defibrotide (See Figure 1a). For thrombin-stimulated murine platelets there is a small decrease in anti-CD62P antibody binding (MFI fold over control 8.4 vs. 6.6), but no significant change in JON/A antibody binding (See Figure 1b). Binding of both anti-CD62P and PAC1 antibodies to human platelets stimulated with synthetic PAR agonists was unaffected by the presence of defibrotide (See Figure 1c). There is no significant difference in SPAN12 or WEDE15 binding in thrombin-stimulated platelets in the absence or presence of defibrotide. However, defibrotide alone does appear to interfere with WEDE15 binding in unstimulated platelets (See Figure 1d). In conclusion, we showed that defibrotide inhibits thrombin-induced human platelet activation at low concentrations of thrombin, as shown by a decrease in surface platelet activation markers measured by flow cytometry. This effect is much less pronounced under the same conditions if mouse platelets are used, supporting the hypothesis that defibrotide inhibits platelet activation by alteration of PAR1 receptor signaling, since PAR1 receptors are not present on mouse platelets. Defibrotide does not interfere with TRAP6-induced platelet activation, evidence against interference with the intracellular portion of the signaling pathway, since TRAP6 is a synthetic PAR1 agonist that binds directly into the ligand-binding pocket of the receptor, eliminating the need for cleavage of the PAR1 extracellular domain. The decrease in WEDE15 antibody binding to washed platelets seen with defibrotide alone suggests that the mechanism may involve alterations in the hirudin-like domain of the PAR1 receptor which is crucial for thrombin binding and cleavage site specificity. Ongoing studies including investigating the effect of defibrotide on peptide mimics of the extracellular N-terminal domain of the PAR1 receptor and the effect of defibrotide on the activation of endothelial cells will likely shed light on the exact mechanism of platelet response modulation by defibrotide and may provide new targets for treatment of the microvascular occlusion that characterizes VOD/SOS. Disclosures No relevant conflicts of interest to declare.

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Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

Blood ◽

10.1182/blood-2019-128250 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4883-4883

Author(s):

Stephanie Renna ◽

Leonard C. Edelstein ◽

Steven Edward McKenzie

Keyword(s):

Amino Acid ◽

Platelet Activation ◽

Knockout Mice ◽

Transmembrane Domain ◽

Conflicts Of Interest ◽

Wild Type Mouse ◽

Human Platelets ◽

Amino Acid Sequences ◽

Wild Type

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

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The Bcl-2 Family Protein Inhibitor, ABT-263, Promotes Apoptotic-Like Response in Isolated Platelets.

Blood ◽

10.1182/blood.v108.11.1122.1122 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1122-1122

Author(s):

Chris Tse ◽

Haichao Zhang ◽

Jun Chen ◽

Ryan M. Fryer ◽

Richard Nelson ◽

...

Keyword(s):

Platelet Activation ◽

Reticuloendothelial System ◽

Human Platelets ◽

Dense Granule ◽

Protein Inhibitor ◽

Cytochrome C Release ◽

Family Protein ◽

Granule Release

Abstract Platelets are relatively short-lived, anucleated cells derived from megakaryocytes that are essential for proper hemostasis. Although they lack a nucleus, platelets possess a highly organized structure with multiple cellular organelles including mitochondria and a number of specific granules. Upon recruitment to sites of endothelial injury, platelet activation stimulates their adhesion, aggregation and release of granule components leading to coagulation. Many of the cellular changes observed during platelet activation and platelet senescence resemble those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, cell shrinkage and microparticle formation (analogous to membrane blebbing). ABT-263 is an orally bioavailable Bcl-2 family protein inhibitor currently in clinical development. This compound is a potent antagonist of the anti-apoptotic proteins Bcl-xL, Bcl-2 and Bcl-w and induces apoptosis in cancer cells dependent on these proteins for survival. ABT-263 induces a unique thrombocytopenia in multiple species that is characterized by a rapid clearance of circulating platelets with recovery to normal platelet counts within several days after treatment cessation. To elucidate the mechanisms underlying this thrombocytopenia, a series of in vitro experiments were performed using freshly isolated platelets comparing ABT-263, its enantiomer (which has significantly lower activity against Bcl-2 family proteins) and known platelet activators. ABT-263, but not the enantiomer control, reduced the viability of dog and human platelets and activated key apoptotic processes, including cytochrome c release, caspase 3 activation, and PS externalization. ABT-263 did not induce aggregation of isolated platelets and did not induce surface expression of GPIIb/IIIa that is essential for platelet adhesion to fibrinogen during clot formation. While ABT-263 had no effect on markers of lysosomal- or alpha-granule release, increases in a marker of dense granule release were apparent at high concentrations. Taken together these data suggest that ABT-263 induces an apoptotic-like response in platelets that is distinct from platelet activation. Furthermore, the lack of effect of the enantiomer control compound suggests this effect is mediated through inhibition of antiapoptotic Bcl-2 family members. Loss of membrane asymmetry and PS externalization has been suggested to be the primary signal for the recognition and clearance of senescent platelets by phagocytes of the reticuloendothelial system. Induction of apoptosis in platelets by Bcl-2 family protein inhibitors such as ABT-263 may coopt this process inducing a premature platelet senescence resulting in a novel type of thrombocytopenia, in vivo.

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Surface Ifitms on Megakaryocytes and Platelets Regulate Fibrinogen Endocytosis Under Inflammatory Conditions

Blood ◽

10.1182/blood.v126.23.1034.1034 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1034-1034

Author(s):

Robert A. Campbell ◽

Jesse W Rowley ◽

Andrew S. Weyrich ◽

Matthew T. Rondina

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Surface Protein ◽

Human Platelets ◽

Inflammatory Conditions ◽

Inflammatory Stimuli ◽

And Gender

Abstract Background IFITM proteins (IFITM-1, -2, and -3) mediate cellular resistance to influenza, dengue, and other viruses. IFITM expression on human platelets has not been previously recognized. Our laboratory recently demonstrated that IFITMs are robustly expressed by human platelets and megakaryocytes after stimulation by pathogens and inflammatory mediators and restrict viral infection. IFITMs, which are interferon inducible, also mediate clathrin localization and associated protein endocytosis. Nevertheless, whether IFITMs regulate protein endocytosis by platelets and megakaryocytes remains unknown. Aims We investigated IFITM expression on murine megakaryocytes and platelets and determined whether IFITMs regulate fibrinogen endocytosis under basal and inflammatory conditions. Methods We examined the expression of IFITMs and clathrin in bone-marrow derived murine megakaryocytes and platelets under basal conditions and following interferon-beta (IFN-β) stimulation. To determine whether upregulation of IFITM causes increased fibrinogen endocytosis, megakaryocytes were stimulated ex vivo with IFN-β and treated with labeled fibrinogen. Endocytosis of labeled fibrinogen was then measured by immunocytochemistry and flow cytometry. To determine whether this response also occurred in vivo, C57Bl/6 mice were injected intraperitoneally (IP) with 50,000 units of IFN-β over four days. On the fourth day, 100 μg of labeled fibrinogen was injected into the tail vein and the amount of endocytosed, labeled fibrinogen in platelets was determined the next day via flow cytometry. Parallel experiments were performed in age and gender matched IFITM-/- mice. Results Bone-marrow derived murine megakaryocytes and platelets basally express IFITMs. Upon IFN-β stimulation, IFITM and clathrin expression significantly increased (p<0.05). Fibrinogen endocytosis by murine megakaryocytes occurred under resting conditions and appeared to be punctate and granular in nature. Upon IFN-b stimulation, fibrinogen endocytosis in megakaryocytes significantly increased compared to unstimulated conditions (p<0.004). The increase in endocytosis appeared independent of changes in αIIbβ3 expression as IFN-β stimulation did not change αIIbβ3 surface protein. Fibrinogen endocytosis after IFN-β stimulation did not increase in megakaryocytes from IFITM-/- mice, suggesting that IFITMs regulate fibrinogen uptake under these conditions. We next determined if fibrinogen endocytosis occurred in platelets isolated from IFITM-/- mice. Platelet counts and activation indices (assessed by JonA staining) were similar in C57Bl/6 mice (WT) and IFITM-/- mice. Nevertheless, the injection of IFN-β IP results in significant increases in fibrinogen endocytosis by platelets in vivo in WT but not IFITM-/- mice (p<0.02). Summary/Conclusions These findings suggest IFITMs, in addition to their anti-viral roles, mediate fibrinogen endocytosis. Further, in settings where inflammatory stimuli such as interferons are increased, enhanced IFITM expression may promote upregulation of fibrinogen endocytosis by platelets and megakaryocytes. Disclosures No relevant conflicts of interest to declare.

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Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648166 ◽

1991 ◽

Vol 65 (04) ◽

pp. 432-437 ◽

Cited By ~ 9

Author(s):

A W J Stuttle ◽

M J Powling ◽

J M Ritter ◽

R M Hardisty

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Aggregation ◽

Calcium Ions ◽

Human Platelet ◽

Human Platelets ◽

In Vivo Detection ◽

Fibrinogen Binding ◽

Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648952 ◽

1994 ◽

Vol 72 (05) ◽

pp. 745-749 ◽

Cited By ~ 7

Author(s):

Elza Chignier ◽

Maud Parise ◽

Lilian McGregor ◽

Caroline Delabre ◽

Sylvie Faucompret ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Peripheral Blood ◽

Vascular Trauma ◽

Activated Platelets ◽

Group I ◽

Group Ii ◽

Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

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Abstract 489: Differential Phosphoproteomic Analysis of Platelet Activation by Specific Oxidized Phospholipids and Thrombin Reveals Mechanisms of Platelet Activation in Hyperlipidemia

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a489 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Alejandro Zimman ◽

Bjoern Titz ◽

Evangelia Komisopoulou ◽

Thomas G Graeber ◽

Eugene A Podrez

Keyword(s):

Signaling Pathways ◽

Signaling Pathway ◽

Platelet Activation ◽

Scavenger Receptor ◽

Human Platelets ◽

Oxidized Phospholipids ◽

Systematic Analysis ◽

Bioinformatic Tools ◽

Induced Changes

We previously showed that specific oxidized phospholipids (oxPC CD36 ) activate platelets via the scavenger receptor CD36 and promote platelet hyper-reactivity in hyperlipidemia, however the signaling pathway(s) induced in platelets by oxPC CD36 are not defined. We employed mass spectrometry-based phosphoproteomics for the unbiased analysis of changes in protein phosphorylation induced by oxPC CD36 and thrombin, a strong platelet agonist, in human platelets. oxPC CD36 induced changes in phosphorylation of 148 unique phosphorylation sites (116 proteins) while thrombin induced changes of 297 unique sites (181 proteins). Most of the changes in phosphorylation induced by oxPC CD36 and thrombin identified in our study have never been reported before in platelets and include high- and low-abundant proteins with diverse molecular functions located in the plasma membrane, cytosol, or cytoskeleton. Analysis using multiple bioinformatic tools identified protein interaction networks, signaling pathways, activated kinases, and enriched phosphorylation motifs. Comparison between platelet agonists revealed multiple differences including the specific activation of a signaling pathway involving Src-family kinases (SFK), SYK kinase, and PLCγ2 by oxPC CD36 . Subsequent biochemical studies in human platelets demonstrated that this pathway is critical for platelet activation by oxPC CD36 and is downstream of CD36. In conclusion, systematic analysis of platelet activation pathways provided novel insights into the mechanism of platelet activation and specific signaling pathways induced by oxidized phospholipids that modulate platelet function in vivo in hyperlipidemia.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

2020 ◽

Vol 4 (4) ◽

pp. 638-643

Author(s):

Manuel Salzmann ◽

Sonja Bleichert ◽

Bernhard Moser ◽

Marion Mussbacher ◽

Mildred Haase ◽

...

Keyword(s):

Platelet Activation ◽

Active Site ◽

Bleeding Time ◽

Thrombus Formation ◽

Human Platelets ◽

Iκb Kinase ◽

Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

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