Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.

Abstract Abstract 3003 Poster Board II-980 Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis. Disclosures: No relevant conflicts of interest to declare.

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Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽

10.1182/blood.v99.12.4486 ◽

2002 ◽

Vol 99 (12) ◽

pp. 4486-4493 ◽

Cited By ~ 109

Author(s):

Gregor Theilmeier ◽

Carine Michiels ◽

Erik Spaepen ◽

Ingrid Vreys ◽

Désiré Collen ◽

...

Keyword(s):

Von Willebrand Factor ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Perfusion Studies ◽

Von Willebrand ◽

Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

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The CD47 Pathway Is Deregulated In Human Immune Thrombocytopenia (ITP).

Blood ◽

10.1182/blood.v116.21.3776.3776 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3776-3776

Author(s):

Lucia Catani ◽

Daria Sollazzo ◽

Francesca Ricci ◽

Francesca Palandri ◽

Nicola Polverelli ◽

...

Keyword(s):

Cell Death ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Immunoglobulin Superfamily ◽

Phagocytic Capacity ◽

Platelet Apoptosis ◽

Cell Death Pathway

Abstract Abstract 3776 The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a receptor for Thrombospondin (TSP) and a ligand for signal regulatory protein-alpha (SIRP-alpha) receptor, acting, respectively, as a regulator of apoptosis and as antagonistic to phagocyte activity. Ligation of CD47 with antibodies, its natural physiological ligand TSP or the specific CD47-binding peptide 41NK induces apoptosis in nucleated blood cells. This apoptosis is characterized by mitochondrial damage and the exposure of phosphatydilserine on the outerleaflet of the plasma membrane. Interaction of SIRP-alpha with CD47 is important also for the regulation of phagocytosis. SIRP-alpha is an immunoglobulin superfamily member and is predominantly expressed in neurons, dendritic cells (DCs) and monocytes/macrophages. Phagocytes engulf foreign cells but not “self” in part because “self” cells express CD47 as a ligand for SIRP-alpha, which inhibits phagocytosis. Thus CD47 functions as a “don't eat me” signal. Based on studies in mice, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently suggested in Immune Thrombocytopenia (ITP). Specifically, it has been demonstrated that: 1) platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in immune thrombocytopenia in a mouse model; 2) interaction between platelet CD47 and macrophage SIRP-alpha is important in regulating normal platelet turnover and FcgammaR-mediated clearance of IgG-sensitized platelets; 3) CD47-deficient platelets have a shortened half-life in the circulation of CD47 wild-type mice and are also more sensitive to Fcgamma receptor-mediated clearance, both in vivo and in vitro. However, the role of CD47 pathway in the pathogenesis of human ITP has not yet been studied. Therefore, the main purpose of the present study was to evaluate whether alterations of this system (platelets/phagocytes) might play a pathogenetic role in human ITP. In particular, we investigated whether in ITP: i) platelets are more susceptible to CD47-induced cell death; ii) expression of CD47 on fresh and in vitro aged platelets is reduced; iii) the platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRP-alpha. Phenotypical and functional analysis of the expression of CD47 on platelets and SIRP-αlpha on CD14-derived/circulating DCs and on CD14-derived macrophages was performed in 32 ITP patients. Patients were newly diagnosed (14 cases) or with persistent (15 cases) or chronic (3 cases) ITP. At the time of the study, patients with persistent or chronic ITP were off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 49×109/L (range 14–98). We found that in healthy subjects CD47 expression increased in in vitro aged platelets and ligation of CD47 with anti-CD47 antibody induced a dose-dependent increase of platelet apoptosis. Immature and mature CD14-derived DCs and circulating myeloid DCs were strongly positive for SIRP-α. Conversely, we demonstrated that in ITP: 1) CD47 expression was unchanged in freshly isolated and in vitro aged platelets; 2) increased platelet apoptosis was not due to the activation of the CD47-induced cell death pathway, which instead was shown to be blocked; 3) the blockage of SIRP-αlpha on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs; in contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages. In conclusion, our data demonstrate that in ITP the increased platelet clearance is not due to reduced CD47 expression on platelets. However, platelets from ITP patients are not healthy because 1) apoptosis is increased; 2) platelet apoptosis is independent from CD47 death signal; 3) CD47 expression is not modified by in vitro ageing/apoptosis. In addition, we show that the CD47 pathway plays a role in platelet phagocytosis of macrophages, but not in DCs. We conclude that in ITP patients platelet homeostasis is differentially modulated by the CD47 pathway. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of Protein Kinase a Induces Platelet Apoptosis

Blood ◽

10.1182/blood.v124.21.4992.4992 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4992-4992

Author(s):

Lili Zhao ◽

Zhicheng Wang ◽

Weilin Zhang ◽

Mengxing Chen ◽

Kesheng Dai

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Conflicts Of Interest ◽

Cell Types ◽

Surface Expression ◽

Human Platelets ◽

Obvious Effect ◽

Platelet Apoptosis ◽

Apoptosis Protein ◽

Kinase A

Abstract Background: Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelet, or in the development of some platelet-related diseases. However, up to now, little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase A (PKA) is highly expressed and plays central roles in signal transduction in human platelets. The certain level of PKA activity is indispensability for maintaining circulating platelets in resting state. In nucleated cells, PKA stimulated proliferation or promoted cell death via apoptosis in different cell types. However, it is still unclear whether PKA plays a role in platelets apoptosis. The aim of the current study is to investigate the role of PKA in platelets apoptosis. Methods and Results: Apoptotic events were assessed in platelets by flow cytometry, western blotting. PKA inhibitor H89 dose-dependently elucidated depolarization of mitochondrial inner membrane potential (ΔΨm), activation of caspase-3, -9, cleavage of gelsolin, phosphatidylserine (PS) exposure and membrane shrinkage in platelets. Incubation of platelets with PKA catalytic subunit-α (PKACα)-siRNA specific reduced the production PKACα and obviously elucidated platelet apoptosis. Platelet aggregation induced by ADP, botrocetin, or α-thrombin was significantly reduced in platelets treated with H89. However, H89 does not incur P-selectin surface expression and PAC-1 binding in platelets. After intraperitoneal injection of PKA inhibitor (Rp-cAMPS), the circulating platelets were reduced at 8 hour time point and recovered within 24 hours. H89 down-regulated the expression of Bad in platelets, and phosphorylation of Bad at Ser 112 was reduced in platelets. Cyclosporin A and ROS inhibitor (NAC) significantly reduced H89-induced platelet ΔΨm depolarization. However, z-DEVE-fmk, GM6001, MDL28170, forskolin, SB203580, BAPTA had no obvious effect on depolarization of ΔΨm. Conclusions: These data indicate that inhibition of PKA results in ROS-mediated platelet apoptosis. The findings suggest a novel mechanism for PKA in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis. Disclosures No relevant conflicts of interest to declare.

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Kindlin-3 Undergoes Endoproteolysis in Stored Platelets and After Platelet Stimulation

Blood ◽

10.1182/blood.v118.21.1255.1255 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1255-1255

Author(s):

Craig N. Streu ◽

Patrik Nygren ◽

Paul C. Billings ◽

David T. Moore ◽

Kathleen S. Molnar ◽

...

Keyword(s):

Conflicts Of Interest ◽

Dissociation Constants ◽

Leukocyte Adhesion ◽

Human Platelets ◽

Experimental Conditions ◽

Chip Surface ◽

Ferm Domain ◽

Platelet Storage

Abstract Abstract 1255 Kindlins are a family of FERM domain proteins that are essential for inside-out integrin activation. In particular, kindlin-3 is required for the conversion of the major platelet integrin αIIbβ3 from its resting conformation to its active ligand binding conformation. Moreover, naturally-occurring kindlin-3 mutations result in the inherited disorder leukocyte adhesion deficiency III, one component of which is defective platelet function that mimics Glanzmann thrombasthenia. Despite the importance of kindlin-3 in initiating αIIbβ3 function in platelets, little is known about its regulation in resting platelets or its fate in activated platelets. To address these questions, we purified full-length kindlin-3 from outdated human platelets where it is present in substantial amounts and also developed a procedure to synthesize substantial amounts of recombinant kindlin-3 in SF9 cells. We found that in stored human platelets, kindlin-3 is cleaved into two fragments as a function of the time of storage. We also found that kindlin-3 is cleaved into identical fragments when fresh human platelets are stimulated with the thrombin receptor activating peptide (TRAP) for 5 minutes. To identify the site of kindlin-3 cleavage, as well as the responsible protease, we used a proteomics method in which an engineered peptide ligase, subtiligase, was used to selectively biotinylate the unblocked α amines of proteins obtained from platelet lysates (Mahrus et al, Cell 134:866–76, 2008). Biotinylated proteins were digested with trypsin and the resulting biotinylated peptide fragments were then captured using avidin agarose and identified using tandem mass spectrometry. Using this method, we identified the kindlin-3 peptide (G)SAPTDVLDSLTTIPELKDHL in lysates of stored platelets, thereby mapping the cleavage site to residues 335–336. We obtained identical results using proteins isolated from TRAP-stimulated platelets. Further, we were able to recapitulate these results in vitro using purified kindlin-3 and the calcium-activated protease calpain, implying that calpain is the responsible protease in vivo. Kindlin-3 is thought to initiate αIIbβ3 function by binding to the distal NITY motif in the β3 cytosolic tail in an interaction that also involves S752. Previously, we reported a model for αIIbβ3 regulation based on an NMR structure of the β3 cytosolic tail (Metcalf et al, PNAS 107:24775–83, 2010). In this structure, the NITY motif is located in a distal dynamic amphiphilic helix where the motif is transiently masked by interacting with the membrane. To validate this model, we have studied the interaction of both purified and recombinant kindlin-3 with the β3 tail using surface plasmon resonance (SPR). A peptide corresponding to β3 residues 719–762, encompassing the complete β3 tail, was immobilized on a CM5 chip and kindlin-3 was flowed over the chip surface. The resulting sensorgrams could then be fit to two binding events with dissociation constants of 2.2 nM and 2.8 μM. The biphasic behavior could have resulted from heterogeneity of the β3 tail on the chip surface or heterogeneity of the interaction between kindlin-3 and the carboxymethylated dextran. Similar SPR experiments measuring binding of the talin-1 FERM domain to the β3 tail also could be fit to two binding events with dissociation constants of 155 nM and 3.5 μM. Thus, under these experimental conditions, kindlin-3 binds approximately 70-fold more tightly to the β3 tail than does the talin-1 FERM domain. In summary, these studies demonstrate that kindlin-3 undergoes calpain-mediated endoproteolysis during platelet storage, an event that may contribute to the development of the platelet storage lesion. Kindlin-3 also undergoes an identical cleavage following platelet stimulation by agonists such as thrombin. Since high affinity binding of kindlin-3 to the β3 cytosolic tail is required for physiologic αIIbβ3 activation, it is possible that kindlin-3 cleavage attenuates αIIbβ3 activity. Disclosures: No relevant conflicts of interest to declare.

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Lenalidomide Reduces Survival of Chronic Lymphocytic Leukemia Cells in Primary Co-Cultures by Altering the Myeloid Microenvironment

Blood ◽

10.1182/blood.v120.21.3894.3894 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3894-3894

Author(s):

Angela Schulz ◽

Claudia Dürr ◽

Thorsten Zenz ◽

Stephan Stilgenbauer ◽

Peter Lichter ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Drug Effects ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

Cell Surface Expression ◽

Clinical Activity ◽

Altered Expression

Abstract Abstract 3894 Chronic lymphocytic leukemia (CLL) cells are highly dependent on their microenvironment. External stimuli provided by bone marrow stromal cells or non-malignant leukocytes are required for their survival and proliferation. Interestingly, peripheral blood-derived monocytes differentiate in the presence of CLL cells to so-called Nurse-like cells (NLCs), which are round or fibroblast-shaped adherent cells that were shown to promote survival of CLL cells in vitro and to exist in lymph nodes of CLL patients. In search of new therapeutic options for patients with CLL, the immunomodulatory drug lenalidomide turned out to have significant clinical activity in CLL. Lenalidomide does not induce apoptosis in CLL cells directly, but is rather believed to act via the microenvironment. Several studies described that it alters cytokine levels and the activation status of the cells. Further, a CLL-specific T-cell defect was shown to be repaired by lenalidomide, which might represent a major activity of this drug in CLL. However, its mechanism of action seems to be complex and is not well understood. As monocytes as well as NLCs are very effective in maintaining survival of CLL cells, we aimed to investigate whether lenalidomide interferes with these supportive cell-cell interactions. To do this, we established primary co-cultures of monocytes and CLL cells in the presence or absence of lenalidomide and observed a significantly decreased viability of CLL cells after 14 days of treatment, suggesting an impact of this drug on the survival support of NLCs. Therefore, we analyzed the immunophenotype of NLCs by flow cytometry, as well as the secretion of cytokines in the co-cultures by ELISA and antibody-coupled bead arrays. Among the effects induced by lenalidomide, we observed reduced cell surface expression of the MHC II protein HLA-DR on NLCs as well as lower levels of the chemokine CCL2, but higher levels of IL-10 in the culture supernatant, indicating an altered inflammatory milieu in the co-cultures. The enhanced IL-10 levels resulted in an increase in STAT1 phosphorylation in CLL cells as measured by Western blot analysis. As a consequence, enhanced expression of the adhesion molecule ICAM-1 (CD54) and an altered expression of cytoskeletal genes (e.g. RHOC and CORO1B) were observed in CLL cells after lenalidomide treatment. Chemotaxis assays using transwell culture dishes and SDF1-α as chemoattractant revealed an impaired migratory potential of lenalidomide-treated CLL cells, which was not due to reduced expression of the SDF1-α receptor CXCR4. In summary, our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug effects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide impairs the migratory potential of CLL cells which may affect circulation and homing of CLL cells in vivo. Disclosures: No relevant conflicts of interest to declare.

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N-Glycosylation Of Human ITP Autoantibodies Affects Complement Activation, Platelet Phagocytosis, and Platelet Survival In a Murine Model

Blood ◽

10.1182/blood.v122.21.569.569 ◽

2013 ◽

Vol 122 (21) ◽

pp. 569-569

Author(s):

Ulrich J. Sachs ◽

Kathrin Walek ◽

Annika Krautwurst ◽

Mathias J. Rummel ◽

Gregor Bein ◽

...

Keyword(s):

Complement Activation ◽

Laser Scanning ◽

Conflicts Of Interest ◽

Human Platelets ◽

Laser Scanning Microscopy ◽

Platelet Survival ◽

Scid Mouse Model ◽

Rapid Clearance

Abstract Introduction Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets. The IgG effector functions of autoantibodies depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. Material and Methods AAbs were purified from ITP patients (n=15) and controls (n=10) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs (deAAbs) were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. The capability of AAbs and deAAbs to eliminate human platelets in vivo was studied in a NOD/SCID mouse model in presence and absence of a complement source. Results AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs. 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and induced complement consumption. N-glycan cleavage significantly reduced C1q binding (MFI 16.4 vs. 4.9, p=0.017) and complement consumption. In vivo survival of human PLTs was assessed after cotransfusion with native or deAAbs in NOD/SCID mice. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL5h) 75% vs. 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL5h 82% vs. 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL5h42%, p<0.001). Conclusion Removal of N-glycan from AAbs interferes with Fc-mediated phagocytosis and complement activation and thereby prolongs platelet survival in vivo. Our study provides tools for better characterizing ITP AAbs and sheds light on the heterogeneity of AAbs in ITP. Clinical studies should aim to assess such additional characteristics, since this could lead to the identification of ITP patient subgroups with increased responses to specific or new interventions such as, targetting complement factors. Disclosures: No relevant conflicts of interest to declare.

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POL5551, a Novel and Potent CXCR4 Antagonist, Enhances Sensitivity to Chemotherapy in an in Vivo Model of High-Risk (HR) Pediatric Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood.v124.21.3707.3707 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3707-3707

Author(s):

Edward Allan R. Sison ◽

Daniel Magoon ◽

Li Li ◽

Barbara Romagnoli ◽

Garry Douglas ◽

...

Keyword(s):

Cell Lines ◽

Xenograft Model ◽

Surface Expression ◽

Vehicle Control ◽

Surface Adhesion ◽

Time Points ◽

Pediatric All ◽

Potent Antagonist

Abstract Background: Interaction between surface receptor CXCR4 (s-CXCR4) and chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow (BM) microenvironment. We previously demonstrated: 1) chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) and ALL enhances stromal protection from chemotherapy-induced apoptosis; 2) the FDA-approved CXCR4 inhibitor plerixafor reverses stromal protection and chemotherapy resistance both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged (MLL-R) ALL; 3) the novel Protein Epitope Mimetic POL5551, a selective and potent antagonist of CXCR4, blocks the SDF-1-binding site of CXCR4, inhibits SDF-1-induced chemotaxis, and reverses stromal-mediated protection from chemotherapy in vitro in pre-B and T ALL cell lines. Here, we further characterize the effects of POL5551 (POL) on surface adhesion molecule expression in ALL, and its in vivo effects in a xenograft model of HR pediatric ALL. In Vitro Methods/Results: We have previously shown that POL inhibits 12G5 antibody binding to s-CXCR4 in ALL cell lines, suggesting an overlapping of the two binding sites. We sought to verify these results using primary samples of pediatric ALL. We treated 3 pre B and 3 T cell ALL primary samples with a dose range of POL and measured s-CXCR4 by FACS at multiple time points. POL inhibition of 12G5 binding was potent (average IC50 at 2 hours pre B 8.3 nM, T 1.4 nM), rapid (<1 hour) and sustained (>24 hours). Further, POL was significantly more potent at inhibiting 12G5 binding than plerixafor (average IC50 at 2 hours pre B 18.4 nM, T 8.4 nM). To further characterize POL’s effects in ALL, we treated 2 pre B and 2 T cell ALL cell lines with POL or vehicle control and then treated with SDF-1α or vehicle control. Treatment with POL inhibited SDF-1α-induced phosphorylation of ERK1/2 in a dose-dependent manner. In parallel, we measured POL-induced compensatory upregulation of the alternative surface adhesion molecules CXCR7 and VLA-4 (CD49d), and found that POL led to increased CXCR7 expression at early time points that began to decrease after 24 hours. We did not find a consistent effect of POL on CD49d surface expression. Xenograft Methods/Results: Infant MLL-R ALL primary samples (n=4) were transplanted into sublethally irradiated NSG mice. After 2 weeks, mice were treated on days 1-3 of 2 consecutive weeks with 1) vehicle control (C), 2) POL (5 mg/kg SC), 3) AraC (200 mg/kg IP), or 4) POL followed by AraC 4 hours later (POL+AraC). One week after treatment, cells were harvested from BM, spleen, and peripheral blood (PB). Leukemic blasts were defined as human CD19+ and CD45+. Overall leukemic burden (average % blasts in BM+spleen+PB) did not differ between mice treated with either C (56.2%) or POL (49.5%, p=0.12). However, treatment with AraC (36.7%, p=3E-07) or POL+AraC (26.3%, p=4E-15) significantly decreased total leukemic burden compared to C. Notably, POL+AraC significantly decreased total leukemic burden compared to AraC alone (p=0.001), demonstrating that POL increased overall sensitivity to AraC. When analyzed by organ-specific leukemic burden, POL+AraC resulted in decreased leukemic burden compared to AraC alone in BM (42.8 vs. 49.8%, p=0.27), spleen (16.9 vs. 30.8%, p=0.002), and PB (19.3 vs. 29.6%, p=0.008). Interestingly, AraC and POL+AraC led to significantly increased CXCR7 expression (blasts from BM p<0.01, spleen p<0.04, and PB p<0.03 vs. C) as well as a trend toward increased CD49d expression compared to C. Conclusions: POL5551 is a potent antagonist of s-CXCR4 in pediatric ALL cell lines and primary samples. Surface expression of adhesion molecules in ALL blasts is dynamic and affected by anti-leukemic therapy. Importantly, POL5551 enhances sensitivity to AraC in a xenograft model of infant MLL-R ALL, a HR subtype of pediatric ALL. Our findings support the continued development of BM microenvironment-targeted agents as a therapeutic strategy for pediatric ALL. Disclosures Romagnoli: Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Tuffin:Polyphor Ltd.: Employment. Zimmermann:Polyphor Ltd.: Employment.

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The Role of Ca2+I in Procoagulant Surface Expression and Apoptosis in Human Platelets.

Blood ◽

10.1182/blood.v106.11.3569.3569 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3569-3569

Author(s):

Adam M. Gwozdz ◽

Hong Wang ◽

K.W. Annie Bang ◽

Marian A. Packham ◽

John Freedman ◽

...

Keyword(s):

In Vitro Study ◽

Fold Increase ◽

Surface Expression ◽

Human Platelets ◽

Apoptotic Pathway ◽

Outer Leaflet ◽

Activated Platelets

Abstract Asymmetry of phospholipids across the plasma membrane bilayer is a feature of all eukaryotic cells. When platelets are stimulated with certain agonsists, phospholipids are randomized by the action of a Ca2+-dependent scramblase enzyme, resulting in exposure of the anionic aminophospholipid phosphatidylserine (PS) on the outer leaflet that provides a procoagulant surface, catalyzing thrombin formation. We have previously demonstrated that the procoagulant surface of activated platelets persists in vitro for at least 4 hrs (Blood100:63b, 2002). Such persistence may propagate thrombosis in vivo when activated procoagulant platelets re-enter the circulation after fibrinolysis. There is currently little information concerning the mechanisms by which the procoagulant surface persists on activated platelets. In this in vitro study, the Ca2+-chelator BAPTA (0.1 μmol/109 platelets) was used to investigate the role of intracellular Ca2+ (Ca2+i) in procoagulant surface expression and persistence; PS expression was determined flow cytometrically by the binding of annexin A5-FITC. Unexpectedly, chelation of Ca2+i resulted in a 2–2.5x-fold increase in PS expression on the surface of platelets 5 min after activation with thrombin or thrombin+collagen (T+C), and this persisted for up to 4 hrs (last time point tested). Since PS expression is a hallmark of apoptosis in nucleated cells, we also examined another platelet apoptosis marker, the collapse of the mitochondrial inner membrane potential (ΔΨm), by flow cytometry using the potential-sensitive dye TMRM; PS expression was measured concurrently. This allowed us to distinguish between activated platelets expressing PS with an intact ΔΨm and apoptotic platelets expressing PS with a dissipated ΔΨm. 70–85% of the thrombin- or T+C-activated platelets expressing PS had an intact ΔΨm, which persisted for up to 4 hrs after activation. Thus, PS expression can occur independently of ΔΨm loss. However, chelation of Ca2+i with BAPTA resulted in 60–70% of the thrombin- or T+C-activated platelets persistently expressing PS to also have a collapsed ΔΨm, indicating that apoptotic pathways similar to those found in nucleated cells may modulate PS expression in platelets and may depend on Ca2+i concentrations. Caspases and calpain are centrally involved in apoptotic signaling and execution in nucleated cells. Caspases-9 and -3 have been identified in human platelets and may be responsible for downstream activation of calpain. We examined the effects of Ca2+i chelation in thrombin- and T+C- activated platelets on the activation of procaspases and calpain by Western blotting. In keeping with our observations of increased PS expression with concurrent ΔΨm loss in activated platelets with Ca2+i chelation, we observed cleavage of both procaspase-9, procaspase-3 and calpain, which did not occur in activated platelets without Ca2+i chelation. Taken together, our results indicate that Ca2+i levels in activated platelets may serve as a decisional checkpoint for the apoptotic pathway in human platelets, where procaspase-9 and procaspase-3 along with downstream calpain may function in a Ca2+-sensitive manner to protect platelets against PS exposure and ΔΨm collapse.

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Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v118.21.2323.2323 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2323-2323

Author(s):

Shinji Hirata ◽

Ryoko Jono-Ohnishi ◽

Satoshi Nishimura ◽

Naoya Takayama ◽

Sou Nakamura ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Imaging System ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Thrombus Formation ◽

Human Platelets ◽

P38 Map Kinase

Abstract Abstract 2323 Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs. Disclosures: No relevant conflicts of interest to declare.

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Filamin A Deficiency Leads to Premature Platelet Formation and Increased Clearance

Blood ◽

10.1182/blood.v116.21.2027.2027 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2027-2027

Author(s):

Antonija Jurak Begonja ◽

Sarah E. Weber ◽

Karin M. Hoffmeister ◽

John H. Hartwig ◽

Herve Falet

Keyword(s):

Conflicts Of Interest ◽

Surface Expression ◽

Signaling Proteins ◽

Filamin A ◽

Membrane Glycoproteins ◽

Filament Networks ◽

Von Willebrand ◽

Willebrand Factor ◽

Platelet Formation

Abstract Abstract 2027 Filamin A (FlnA) is a large cytoplasmic protein that crosslinks and stabilizes actin filament networks and links membrane glycoproteins and signaling proteins to the underlying cytoskeleton. FlnA loxP-PF4-Cre mice that specifically lack FlnA in their platelets and megakaryocytes (MKs) have a macrothrombocytopenia with increased tail bleeding time. FlnA KO platelets have decreased surface expression of the von Willebrand factor receptor (vWfR) and are cleared rapidly from the circulation of WT mice, indicating an inherent surface defect that leads to detection and removal. FlnA loxP-PF4-Cre mice have a marked increase in MK numbers in bone marrow and spleen. Detailed analysis of platelet production by FlnA KO MKs in vitro reveals an altered maturation program and defects in vWfR stability, compared to WT. Surprisingly, the surface expression of vWfR components (Ibα, Ibβ, IX and V) on FlnA KO MKs is normal or increased compared to WT MKs, although the total amount of GPIbα is decreased and GPIbα is not associated with the actin cytoskeleton. Analysis of the GPIbβ subunit shows increased degradation in FlnA KO MKs and platelets. Although FlnA KO and WT MK cultures contain comparable cell numbers, FlnA KO MKs more rapidly convert their proplatelets into large CD61+ platelet-sized particles. These findings suggest that aberrant platelet maturation by FlnA KO MKs results in enlarged platelets that are cleared rapidly because of altered vWfR expression. Disclosures: No relevant conflicts of interest to declare.

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