Evaluation of Two Structurally Distinct Novel Inhibitors of Apoptosis Signal-Regulating Kinase 1 (MAP3K5), As Potent Anti-Platelet Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3833-3833
Author(s):  
Meghna U. Naik ◽  
Xi Chen ◽  
Brendan Bachman ◽  
Ganesha Rai ◽  
David Maloney ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Receptor Activation ◽  
New Drugs ◽  
Severe Bleeding ◽  
Fibrinogen Receptor

Abstract Platelets play a critical role in both hemostasis and thrombosis. Anti-platelet drugs currently available apart from aspirin are directed against platelet agonist receptors or fibrinogen receptor integrin aIIbb3. These antagonists, although having potent anti-thrombotic activities, cause severe bleeding due to their effect on hemostasis. It is therefore of utmost important to develop new drugs that will protect from thrombosis with minimal effect on hemostasis. Apoptosis signal-regulating kinase (ASK1) is a redox sensitive serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its role in platelets is not known. We found that ASK1 is rapidly activated downstream of all platelet agonists. Ablation of Ask1 gene results in impaired platelet functions such as granule secretion, thromboxane A2 generation, as well as fibrinogen receptor activation, which translates into attenuated platelet aggregation compared to WT littermates. We also found that thrombin failed to activate p38 in Ask1 null platelets, showing that Ask1 is indispensable for p38 activation by thrombin. FeCl3-induced carotid artery injury model of thrombosis showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. These results indicated that ASK1 plays a central role in regulating platelet function, making it a potential target for combating thrombosis. We therefore synthesized two novel and highly specific ASK1 inhibitors based on the published reports, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) and GS-4997. We found that GS-4997 (500nM) and IPTB (5mM) inhibit agonist-induced ASK1 activation in human platelets. They do not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1/2. We also found that IPTB and GS-4997 dose-dependently inhibited activation of p38, a downstream effector kinase, induced by a variety of platelet agonists. Furthermore, these compounds dose-dependently inhibited ADP, collagen, convulxin and PAR4 activating peptide AYPGKF-induced platelet aggregation as well as platelet spreading on immobilized fibrinogen. In-vivo carotid artery thrombosis assay revealed that WT mice injected intraperitoneally with either IPTB (100mg/kg) of GS-4997 (100mg/kg), showed a significantly increased time of occlusion (P=0.028 and P=0.005 respectively) and thrombus formed were unstable as compared to control WT mice treated with saline alone. Furthermore, injection of either IPTB or GS-4997 protected mice against collagen/epinephrine-induced pulmonary thromboembolism. Out of 14 saline-treated mice only two survived whereas, 10 out of 11 mice treated with GS-4997 (100mg/kg) survived (P=0.0002). In case of IPTB 1 out of 12 control mice survived as compared to 9 out of 12 treated mice (P=0.0028). Interestingly, tail-bleeding studies revealed that WT mice treated with either IPTB (1mg/kg) of GS-4997 (1mg/kg), did not affect the average bleeding time (100s) seen in the WT mice treated with saline alone, suggesting that both inhibitors had no effect on in-vivo hemostasis. Moreover, pretreatment of the whole blood with these inhibitors significantly reduced thrombus formation under arterial flow (800s-1) without affecting platelet adhesion to collagen as assessed using a microfluidic device. Our results strongly suggest that both IPTB and GS-4997 protect the mice from thrombosis without affecting hemostasis. Further development of these inhibitors as a potential therapeutic agent to combat thrombotic disorders is highly warranted. Disclosures No relevant conflicts of interest to declare.

Identification Of a Novel Small Molecule Inhibitor Of ASK1 As a Potent Anti-Platelet Agent

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 20-20
Author(s):  
Meghna Ulhas Naik ◽  
Maloney David ◽  
Ramya Turaga ◽  
Hidinori Ichijo ◽  
Ulhas P Naik
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Genetic Ablation ◽  
Fibrinogen Receptor ◽  
Molecule Inhibitor

Abstract Apoptosis signal-regulating kinase (ASK1) is a serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its presence and role in platelets are not known. We found that ASK1 is expressed in platelets and is rapidly activated during platelet stimulation by various agonists in a dose-dependent manner. In addition, we found that TRAF2/6, known endogenous activators of ASK1, are expressed in platelets and associate with ASK1 upon platelet activation with agonists. Furthermore, genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2x10-9). While WT mice showed an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by a mixture of collagen and epinephrine as determined by increased survival and lack of large occlusive thrombi in the lung. We also found that ADP- and AYPGKF (PAR4 receptor peptide) -induced platelet aggregation was diminished in Ask1 null mice compared to WT mice. Furthermore, PAR4 peptide-induced alpha- and dense-granular secretion was also reduced in Ask1 null platelets compared to WT. Interestingly, we also found that Ask1 null platelets bind less FITC-fibrinogen compared to the WT upon activation by PAR4 peptide. Furthermore, thrombin failed to activate MKK6 and p38 in Ask1 knockout platelets, showing that Ask1 is indispensable for p38 activation by thrombin. These results indicated that ASK1 regulates platelet function by augmenting platelet secretion as well as fibrinogen receptor activation, making it an important target for combating thrombosis. We therefore synthesized a novel and highly specific ASK1 inhibitor, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) based on the published report. IPTB has been found to be a very potent inhibitor that inhibits ASK1 activity at nM concentrations. IPTB is also highly specific to ASK1 and does not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1. We found that in human platelets, IPTB dose-dependently inhibits p38 activation induced by a variety of platelet agonists. Furthermore, IPTB dose-dependently inhibited ADP and PAR4 peptide-induced platelet aggregation. Interestingly, IPTB also dose-dependently inhibited platelet spreading on immobilized fibrinogen. Our results strongly suggest that the dose of IPTB could be adjusted so that it attenuates thrombosis without affecting hemostasis. This development would make IPTB a novel potential therapeutic agent to be used to combat thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

Local delivery of soluble platelet collagen receptor glycoprotein VI inhibits thrombus formation in vivo

2006 ◽  
Vol 95 (05) ◽  
pp. 763-766 ◽  
Author(s):  
Andreas Bültmann ◽  
Christian Herdeg ◽  
Zhongmin Li ◽  
Götz Münch ◽  
Christine Baumgartner ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Vascular Injury ◽  
Carotid Arteries ◽  
Thrombus Formation ◽  
Critical Role ◽  
Local Delivery ◽  
Computer Assisted ◽  
Glycoprotein Vi ◽  
Collagen Receptor

SummaryPlatelet-mediated thrombus formation at the site of vascular injury isa major trigger for thrombo-ischemic complications after coronary interventions. The platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in the initiation of arterial thrombus formation. Endothelial denudation of the right carotid artery in rabbits was induced through balloon injury. Subsequently, local delivery of soluble, dimeric fusion protein of GPVI (GPVI-Fc) (n=7) or control Fc (n=7) at the site of vascular injury was performed with a modified double-balloon drugdelivery catheter.Thrombus area within the injured carotid artery was quantified using a computer-assisted image analysis and was used as index of thrombus formation.The extent of thrombus formation was significantly reduced in GPVI-Fc- compared with control Fc-treated carotid arteries (relative thrombus area, GPVI-Fc vs. Fc: 9.3 ± 4.2 vs. 2.3 ± 1.7, p<0.001). Local delivery of soluble GPVI resulted in reduced thrombus formation after catheter-induced vascular injury.These data suggest a selective pharmacological modulation of GPVI-collagen interactions to be important for controlling onset and progression of pathological arterial thrombosis, predominantly or even exclusively at sites of injured carotid arteries in the absence of systemic platelet therapy.

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.

Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2168-2168
Author(s):  
Lu Wang ◽  
Yi Wu ◽  
Junsong Zhou ◽  
Syed S. Ahmad ◽  
Bulent Mutus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Abstract 2168 Several members of the protein disulfide isomerase family of enzymes are important in platelet function and in thrombosis. Platelet protein disulfide isomerase (PDI) has been shown to have an important role in platelet function but is reported to not be required for thrombus formation in vivo. A novel platelet PDI called ERp57 mediates platelet aggregation but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis we generated a megakaryocyte/platelet specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times, and defective activation of the αIIbβ3 integrin and platelet aggregation. The aggregation defect was corrected by addition of exogenous ERp57 implicating surface ERp57 in platelet aggregation. Platelet surface ERp57 protein and activity increased substantially with platelet activation. We conclude that platelet-derived ERp57 is required for hemostasis and thrombosis and platelet function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Impaired Platelet Responses to Thromboxane a2 and Thrombin in Mice Lacking Receptor-Interacting Protein Kinase 3

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2762-2762
Author(s):  
Yiwen Zhang ◽  
Jian Zhang ◽  
Rong Yan ◽  
Jie Zhang ◽  
Mengxing Chen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Protein Kinase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Thromboxane A2 ◽  
Dense Granule ◽  
In Vivo Model ◽  
Interacting Protein ◽  
Granule Secretion

Abstract Objective: Receptor-interacting protein 3 (RIP3) is a member of RIP family with a Ser/Thr protein kinase domain in its amino-terminus which is essential for kinase activity and autophosphorylation. The roles of RIP3 in embryonic development and different disease pathologies, such as inflammation and infections, have been reported in recent years. However, the role of RIP3 in thrombosis and hemostasis remains unknown. Methods: Hematologic analysis was performed and tail bleeding time was monitored. Mouse platelets were isolated from anti-coagulated whole blood. Platelet aggregation and secretion were recorded at real time. Platelet P-selectin exposure and specific fibrinogen binding were detected by flow cytometry. TXA2 generation was measured with enzyme immunoassay (EIA) kit. Protein phosphorylations were detected by western blotting. Result: RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited delayed thrombus formation, fewer accumulated platelets, smaller thrombi, and prolonged occlusion times. RIP3 was expressed in both human and mouse platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619. The defect in ADP secretion appears responsible for the impaired platelet aggregation, because addition of exogenous ADP rescued the reduced platelet aggregation. Although TXA2 generation and α-granule secretion were not impaired, integrin αIIbβ3 activation was attenuated in RIP3-/- platelets. Moreover, phosphorylation of Akt induced by U46619 or thrombin was markedly reduced in the absence of RIP3. Activation of Akt signaling restored the impaired aggregation of RIP3-/- platelets. ERK and p38 phosphorylation elicited by either U46619 or thrombin was attenuated in RIP3-/- platelets. In contrast, U46619- and thrombin-induced activation of PTEN, PDK1, or Src was not impaired in RIP3-/- platelets. Conclusion: Our data demonstrate a novel role for RIP3 in amplifying U46619- and thrombin-induced platelet activation by mediating Akt-dependent ADP secretion, and in supporting hemostasis and thrombus formation in vivo. RIP3 may represent a novel target to modulate PARs and TP signaling and a potential new target for antithrombotic strategy. Disclosures No relevant conflicts of interest to declare.

Apolipoprotein AIV (ApoA-IV) Is a Novel Ligand of Platelet β3 Integrin That Negatively Regulates Platelet Adhesion, Aggregation, and Thrombosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 156-156
Author(s):  
Christopher M. Spring ◽  
Wuxun Jin ◽  
Hong Yang ◽  
Adili Reheman ◽  
Guangheng Zhu ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Protein S ◽  
Vessel Occlusion ◽  
Platelet Adhesion And Aggregation ◽  
Β3 Integrin

Abstract Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

Cadherin-6 Forms a Functional Adhesion Complex with Alpha-Catenin and Beta-Catenin in Platelets and Is Required for Platelet Aggregation and Thrombus Formation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2232-2232 ◽  
Author(s):  
Michele Mumaw ◽  
Maria de la Fuente ◽  
Carolyn Aldana ◽  
Wei Li ◽  
Marvin T Nieman
Keyword(s):  
Platelet Aggregation ◽  
Adhesion Molecules ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Beta Catenin ◽  
Adhesion Complex

Abstract The regulation of hemostasis and thrombus formation is a tightly controlled event that has catastrophic consequences when it is deregulated. One of the hallmarks of the thrombus is aggregated platelets. Upon platelet stimulation, adhesion molecules become activated and mediate multiple cell-cell interactions. Therapeutically, blocking platelet adhesion is a proven method for preventing pathological arterial thrombus formation. However, targeting the primary adhesion receptor, integrin αIIbβ3, results in severe bleeding complications. Therefore, identifying novel proteins or uncovering novel functions for known proteins in platelets is a necessary first step to facilitate the development of safer anti-platelet therapeutics. We have identified that the cell adhesion molecule cadherin-6 forms a functional adhesion complex with α-catenin and β-catenin in platelets. The goal of our project was to determine the mechanism of cadherin-6 mediated adhesion in platelets. Our initial experiments demonstated that cadherin-6 and β-catenin co-localize at the plasma membrane in platelets using confocal immunofluorescence microscopy. We determined that α-catenin and β-catenin co-immunoprecipitate with cadherin-6 from platelet lysates. To examine the functional role of cadherin-6 on platelet aggregation we used a cadherin-6 blocking antibody (10 μg/ml). Blocking cadherin-6 inhibited mouse platelet aggregation induced by PAR4 peptide. We next determined the role of cadherin-6 in vivo by examining carotid artery thrombosis after 7.5% FeCl3 treatment. C57Bl6 mice were injected with cadherin-6 antibody IV and labeled with rhodamine 6G by jugular vein injection. Thrombus formation was imaged in real time by fluorescent intravital microscopy. Blocking cadherin-6 prevented thrombosis for the duration of the experiment (30 min). To verify that the effects that we observed were specific to cadherin-6 expressed on platelets, we isolated platelets from donor mice and treated with cadherin-6 antibody or control IgG ex vivo. The treated platelets were perfused into recipient mice that were irradiated with 11 Gy to make the animals thrombocytopenic. The cadherin-6 antibody treated platelets formed an occlusion at 26.4 ± 3.6 min vs. 13.7 ± 2.0 min for the IgG (p=0.03). Importantly, the cadherin-6 antibody did not affect platelet counts compared to IgG controls 2.97 ± 0.40 (×108) vs. 3.02 ± 0.20 (×108). These combined studies show that caderhin-6 forms a complex with the necessary proteins required to mediate adhesion in platelets. Our results demonstrate that platelet cadherin-6 has a physiologically important role during platelet activation and thrombus formation in vivo. In summary, we have identified a novel adhesion complex in platelets that may provide a mechanism to limit platelet aggregation therapeutically. On going studies will determine the regulation of the cadherin-6/catenin complex and how cadherin-6 cooperates with other platelet adhesion molecules. Disclosures No relevant conflicts of interest to declare.

scholarly journals JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3352-3360 ◽  
Author(s):  
Meghna U. Naik ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
Ulhas P. Naik
Keyword(s):  
Platelet Activation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Genetic Ablation ◽  
Fibrinogen Receptor ◽  
Inside Out

Abstract Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

scholarly journals Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1131-1131
Author(s):  
Jasna Marjanovic ◽  
Brad Rumancik ◽  
Luke Weber ◽  
Felix Wangmang ◽  
Dane Fickes ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Glycogen Synthase ◽  
Thrombus Formation ◽  
Type I ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

Platelets Derived From Patients with Myelodysplastic Syndromes (MDS) Show Defective Platelet Aggregation and Integrin aIIbβ3 Receptor (GPIIb/IIIa) Signaling Caused by a Reduced Expression of Proteins Required for Inside-out Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 598-598
Author(s):  
Akos G. Czibere ◽  
Julia Fröbel ◽  
Sonja Hartwig ◽  
Ron-Patrick Cadeddu ◽  
Matthias Wilk ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Clinical Problem ◽  
Receptor Activation ◽  
In Vivo Model ◽  
Inside Out ◽  
Platelet Transfusions

Abstract Abstract 598 Thrombocytopenia is prevalent in up to 65% of patients with myelodysplastic syndrome (MDS) at the time of diagnosis and thrombocytopenic hemorrhage is a significant clinical problem that is often complicated by platelet aggregation defects. Little is known about the pathophysiology of this insufficient platelet function. Here, we delineate a reduced expression of critical platelet aggregation-related proteins by analyzing the platelet proteome of 7 patients with MDS and 7 normal donors. Patients' median platelet count was 60 × 10E9/L (range 37–109 × 109/L) and none of the patients examined had received prior anticoagulant treatment, chemotherapy or platelet transfusions. Differential 2-dimensional in-gel electrophoresis coupled with a time-of-flight Ultraflex-Tof/Tof mass spectrometer enabled the discovery of 120 differential protein spots. Among these, we identified a total of 35 proteins including 26 proteins that are integral part of the integrin aIIbβ3 receptor (GPIIb/IIIa, Fibrinogen receptor) signaling such as Talin-1 and Vinculin. In resting platelets the integrin aIIbβ3 receptor exhibits a low-affinity (inactive) state which is shifted to a high-affinity (active) state following inside-out activation. Talin-1 expression has been shown to be essential for this inside-out activation of the integrin aIIbβ3 receptor and consecutive platelet aggregation in an in-vivo model. We hypothesized that the reduced expression of Talin-1 and its co-factor Vinculin may inhibit activation of the integrin aIIbβ3 receptor and thereby contribute to the platelet aggregation defect seen in patients with MDS. Therefore, we performed further functional studies on integrin aIIbβ3 receptor activation and platelet spreading/aggregation with platelets derived from an independent cohort of 7 patients with MDS and 7 normal donors. In this new cohort, patients' median platelet count was 94 × 109/L (range 60–120 × 109/L) and again all patients had never received prior platelet transfusions or anti-coagulant treatment. When we looked at the surface expression of the integrin aIIbβ3 receptor on resting platelets by means of flow-cytometry, we did not detect any differences between platelets from patients with MDS and normal donors. Then, we activated platelets from normal donors and patients with MDS with 0.01U/μl and 0.001U/μl thrombin and measured binding of PAC-1, which is highly specific for detection of the active form of the integrin aIIbβ3 receptor. Here, we found a significantly lower shift from the inactive to the active form in platelets derived from patients with MDS dropping from 92.15% and 91.46% in normal donors to 41.97% and 48.45% (p = 0.01 and p = 0.006), respectively. We confirmed this suggested lack of adhesion and aggregation capacities in MDS platelets by confocal microscopy and single platelet imaging of washed platelets stimulated with 0.01U/μl thrombin which were adhered to immobilized fibrinogen. Consecutive platelet aggregation assays also revealed an insufficient response to stimuli like Collagen and ADP. Our findings provide for the first-time insight into the molecular pathology of defective platelet aggregation in MDS and suggest a mechanism of defective inside-out signaling caused by a reduced expression of proteins required for integrin aIIbβ3 receptor activation. Disclosures: No relevant conflicts of interest to declare.

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