STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 485-485
Author(s):  
Firdos Ahmad ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Teshell K Greene ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Phosphatidyl Serine ◽  
Flow Chamber ◽  
Procoagulant Activity ◽  
Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

Agonist-Induced Binding of the Regulatory Scaffold Protein Spinophilin to Protein Phosphatase-1 In Platelets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2029-2029
Author(s):  
Andrew Sinnamon ◽  
Peisong Ma ◽  
Lawrence F. Brass
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Scaffold Protein ◽  
Western Blots ◽  
Co Precipitation

Abstract Abstract 2029 Platelet regulation plays a critical role in hemostasis. Underactivation can result in failure to stop bleeding, whereas inappropriate platelet activation can cause thrombus formation. The 130-kDa scaffold protein spinophilin (SPL) has recently been shown to play a role in preventing platelet overactivation by forming a complex with the proteins RGS10, RGS18, and the tyrosine phosphatase SHP-1. This complex dissociates when platelet are activated by thrombin or thromboxane A2 and evidence from spinophilin knockout mice suggests that this regulates platelet activation in vitro and in vivo. Spinophilin was originally isolated as a binding partner for the serine/threonine phosphatase, PP-1, in neurons. Here we asked whether PP-1 forms a complex with spinophilin in human platelets and, if so, whether the complex is affected by platelet activation. The approaches that we used to answer this question included Western blotting with antibodies to PP-1 and spinophilin, and co-precipitation studies looking for an association between spinophilin and PP-1. The results of the Western blots confirm the presence of PP-1 in platelets. The initial co-precipitation studies show that little, if any, PP-1 is associated with spinophilin in resting platelets, but there is a time-dependent increase in the SPL/PP-1 complex when platelets are activated with the PAR1 (thrombin receptor) activating peptide, SFLLRN. Thus it appears that within approximately the same time frame that the SPL/RGS/SHP-1 complex is decaying in activated platelets, the SPL/PP-1 complex is forming. Targets for PP1 have not been fully identified in platelets, but it is known that spinophilin localizes to the plasma membrane upon platelet activation. Since spinophilin is thought to direct PP1 targeting in neurons, it is reasonable to propose that it may be directing PP1 to targets in platelets in a similar manner. The studies described in this abstract were supported in part by a 2010 ASH Trainee Research Award to Andrew Sinnamon, who is a first year medical student at the University of Pennsylvania. Disclosures: No relevant conflicts of interest to declare.

Phosphatidylserine-Mediated Platelet Clearance By Endothelium Decreases Platelet Aggregates and Procoagulant Activity in Sepsis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2538-2538
Author(s):  
Ruishuang Ma ◽  
Xiaoming Wu ◽  
Lixiu Wang ◽  
Lu Zhao ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Disease Process ◽  
Procoagulant Activity ◽  
Activation Markers ◽  
Fibrin Formation ◽  
Activated Platelets

Abstract Introduction: Disorders of coagulation are common in sepsis, with disseminated intravascular coagulation (DIC) occurring in approximately 35 % of severe cases, contributing to microvascular dysfunction and death. Intensive platelet activation in sepsis facilitates platelet aggregation, leading to the formation of microthrombi and platelet depletion. This results in the development of DIC and sepsis-associated thrombocytopenia. Therefore, platelets must be cleared locally and quickly in the early phase of activation. Previous studies mainly focused on the clearance of activated cold-stored and aging platelets as well as platelets in immune-mediated thrombocytopenia. However, platelet activation and their clearance in sepsis are poorly understood. Platelets can form aggregates with leukocytes resulting in leukocyte death, the release of extracellular traps (ETs), increased endothelial permeability, and aggravated thrombosis. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Methods: The subjects were septic patients (n=48) and healthy controls (n=48). Platelet engulfment by ECs was observed by electron microscopy, immunofluorescence, or immunochemistry both in vitro and in animal models. The PCA of platelets was measured by clotting time, purified coagulation complex assays, and fibrin formation. Results: Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered to and were ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or integrin on ECs attenuated platelet clearance, resulting in increased platelet count in a mouse model of sepsis (p<0.05). Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets (p<0.01). Pretreatment with lactadherin increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. A large decline in PS exposure on platelets, associated platelet PCA, and PLA formation is seen in patients in remission, which could be attributed to the elimination of abnormal platelets. Conclusions: Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contributes to maintaining platelet homeostasis during acute inflammation. Antiplatelet treatment has been suggested as a novel strategy in sepsis, and we speculate that promoting efficient removal of activated and apoptotic platelets could further improve patient outcomes. Therefore, clearance of activated platelets earlier in the disease process could hasten recovery of homeostasis in circulation by eliminating catalytic platforms for the coagulation pathway, protecting blood cells from excessive activation, and restoring their normal function. Endothelium, at least in part, contributes to platelet disposal and may further improve the hypercoagulable status in inflammation. It is noteworthy that PS-mediated and lactadherin-strengthened platelet engulfment may modify coagulopathy, and thus provide a new modality for treatment of septic clotting disorders. Figure 1 Phagocytosis of platelets by endothelial cells in vitro. Figure 1. Phagocytosis of platelets by endothelial cells in vitro. Figure 1 Effect of lactadherin-mediated phagocytosis on procoagulant activity and fibrin formation. Figure 1. Effect of lactadherin-mediated phagocytosis on procoagulant activity and fibrin formation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 91-91
Author(s):  
Wolfgang Bergmeier ◽  
David S Paul ◽  
Lucia Stefanini ◽  
Raymond F. Robledo ◽  
E. Ricky Chan ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
P2y12 Inhibitors ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. Disclosures No relevant conflicts of interest to declare.

Abstract 41: Platelet Dream Plays a Critical Role During Thrombogenesis in Mice

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Kyungho Kim ◽  
Alan Tseng ◽  
Andrew Barazia ◽  
Joseph E Italiano ◽  
Jaehyung Cho
Keyword(s):  
Thrombus Formation ◽  
Regulatory Element ◽  
Critical Role ◽  
Flow Chamber ◽  
Regulatory Function ◽  
Pi3k Activation ◽  
Granule Secretion ◽  
Phosphoinositide 3 Kinase

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain. Using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that hematopoietic and endothelial cell DREAM are required for platelet thrombus formation following arteriolar injury. DREAM deletion also prolonged tail bleeding times. In vitro flow chamber assays and in vivo adoptive transfer experiments indicated the importance of platelet DREAM in thrombogenesis. We found that deletion of platelet DREAM does not alter ultrastructural features but significantly impaired aggregation and ATP secretion induced by collagen-related peptide (CRP), ADP, or A23187, but not thrombin or U46619. Biochemical studies showed that platelet DREAM is required for phosphoinositide 3-kinase (PI3K) activation induced by GPVI-, A23187-, or integrin-mediated signaling. Studies using DREAM-null platelets and isoform-specific PI3K inhibitors revealed that platelet DREAM positively regulates granule secretion, Ca2+ mobilization, and aggregation induced by CRP or A23187 through PI3K class Iβ (PI3K-Iβ) activity. Genetic and pharmacological studies in megakaryoblastic MEG-01 cells showed that DREAM regulates A23187-induced Ca2+ mobilization and that the regulatory function of DREAM requires Ca2+ binding and PI3K-Iβ activity. Taken together, we have identified platelet DREAM as a novel regulator of PI3K-Iβ activity during thrombus formation.

Critical Role of CalDAG-GEFI In FCγRIIa-Dependent Platelet Activation and Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3196-3196
Author(s):  
Moritz Stolla ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Firdos Ahmad ◽  
Michael P Reilly ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Nucleotide Exchange ◽  
Pulmonary Thrombosis ◽  
Nucleotide Exchange Factor ◽  
Immune Mediated

Abstract Abstract 3196 Immune-mediated thrombosis is a major cause of death in autoimmune diseases and contributes to complications in drug treatments (e.g. Heparin Induced Thrombocytopenia). The major receptor on platelets for immunoglobulin-mediated activation is FcγRIIA. FcγRIIA signals through an immunoreceptor tyrosine-based activation motif (ITAM) that leads to phospholipase C activation, which induces the release of calcium and diacylglycerol (DAG). In our previous work, we identified CalDAG-GEFI (calcium and DAG regulated guanine nucleotide exchange factor I) as a key component of collagen/ITAM-mediated platelet activation. In the current study, we evaluated if CalDAG-GEFI is a potential target for the intervention with FcγRIIA receptor dependent, immune-mediated thrombosis. Mice transgenic for the human FcγRIIA (hFCR) and deficient for CalDAG-GEFI-/- (hFCR/CDGFI-/-) were generated. In vitro, aggregation of hFCR/CDGFI-/- platelets required 50–100-fold higher concentrations of anti-CD9 antibodies than hFCR/WT controls. In comparison, inhibition of P2Y12, the target of clopidogrel, shifted the dose response curve for anti-CD9 in hFCR/WT platelets by only ∼2-fold. In addition to their aggregation defect, hFCR/CDGFI-/- platelets were characterized by markedly impaired Rap1 activation. To assess the role of CalDAG-GEFI in FcγRIIA -mediated thrombosis in vivo, we developed a model of antibody-mediated thrombosis, were we injected mice with an Alexa-750 labeled antibody against GPIX and extracted the lungs to visualize pulmonary thrombosis on a LICOR scanner. Anti-GPIX induced pulmonary thrombosis in hFCR mice but not in WT animals. Pretreatment with clopidogrel did not provide a substantial protection from thrombosis in hFCR mice. In contrast, no pulmonary thrombi were observed in hFCR/CDGFI-/- mice. Together, our studies are the first to highlight the importance of CalDAG-GEFI downstream of platelet Fc-receptor/ITAM signaling and suggest CalDAG-GEFI as a powerful new target in the intervention of immune-mediated thrombosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IκB kinase 2 is not essential for platelet activation

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.

Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation

2005 ◽  
Vol 146 (4) ◽  
pp. 216-226 ◽  
Author(s):  
George Hsiao ◽  
Ying Wang ◽  
Nien-Hsuan Tzu ◽  
Tsorng-Hang Fong ◽  
Ming-Yi Shen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Inhibitory Effects ◽  
In Vitro Platelet Activation

A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3526-3526 ◽  
Author(s):  
Laurence Panicot-Dubois ◽  
Christophe Dubois ◽  
Barbara C. Furie ◽  
Bruce Furie ◽  
Dominique Lombardo
Keyword(s):  
Bile Salt ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Pancreatic Acinar Cells ◽  
Cremaster Muscle ◽  
Terminal Domain ◽  
Laser Injury ◽  
Phosphatidylserine Exposure

Abstract Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.

Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1508-1508 ◽  
Author(s):  
Shawn M. Jobe ◽  
Katina M. Wilson ◽  
Lori Leo ◽  
Jeffery D. Molkentin ◽  
Steven R. Lentz ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Mitochondrial Permeability Transition ◽  
Critical Role ◽  
In Vitro Tests ◽  
Cyclophilin D ◽  
Wild Type ◽  
Glycoprotein Vi ◽  
Stimulation Of

Abstract Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation includeincreased surface retention of procoagulant alpha granule proteins,high-level phosphatidylserine (PS) externalization, andmodulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A. Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨm), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H2O2 (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05). Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses. We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3376-3376
Author(s):  
Romain Gioia ◽  
Cedric Leroy ◽  
Claire Drullion ◽  
Valérie Lagarde ◽  
Serge Roche ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Dependent Manner ◽  
Stable Isotope Labelling ◽  
Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

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