scholarly journals Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice

2019 ◽  
Vol 119 (08) ◽  
pp. 1321-1331
Author(s):  
Vaishali V. Inamdar ◽  
John C. Kostyak ◽  
Rachit Badolia ◽  
Carol A. Dangelmaier ◽  
Bhanu Kanth Manne ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Extracellular Region ◽  
C Terminus

Background and Objective CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. Methods and Results We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. Conclusion We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

scholarly journals Impaired GPVI Mediated Platelet Functional Responses and Signaling in CD45 Knockout Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1355-1355
Author(s):  
Vaishali Inamdar ◽  
John Kostyak ◽  
Rachit Badolia ◽  
Carol Dangelmaier ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Dense Granule ◽  
Receptor Protein ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Terminal Domain ◽  
Deficient Mice

Abstract Background: CD45 is a Receptor Protein Tyrosine Phosphatase C (PTPRC) and regulates Src Family Kinases activation in Lymphocytes. Although it is known to be absent from the platelet surface, proteomics studies prove that the CD45 c-terminal catalytic domain is present in platelets. Thus the aim of this study is to identify presence of CD45 c-terminal domain in platelets and characterize the functional implications of CD45 deficiency in platelets using a global CD45 knockout mouse. Results: Platelets from CD45-deficient mice displayed a selective impairment of aggregation and dense granule secretion mediated by the collagen receptor Glycoprotein VI. CD45 deficient mice show increased bleeding times, indicating an important role for CD45 in hemostasis. However, there was no difference observed in thrombus generation and thrombus stability using the ferric chloride-induced carotid artery injury model. Signaling downstream of the GPVI receptor, indicated by Src Family Kianse (SFK), Syk and Phospholipase C_2 (PLCg2) tyrosine phosphorylation, was also impaired. In order to establish the presence of CD45 in platelets we used an established primary antibody that recognizes the c-terminal domain of CD45. We observed that this antibody recognized a protein of approximately 65 kDa, which is the expected size of the c-terminal 1 and 2 domains of CD45, in wild type (WT) mice but not in knockout (KO)mice. Conclusion: Thus we conclude that CD45 is expressed in platelets as a truncated form, possibly generated by proteolytic cleavage, and regulates GPVI signaling, through regulation of Src Family Kinase activation. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 648-657 ◽  
Author(s):  
Ilaria Canobbio ◽  
Lina Cipolla ◽  
Alessandra Consonni ◽  
Stefania Momi ◽  
Gianni Guidetti ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Focal Adhesion Kinase ◽  
Platelet Activation ◽  
Focal Adhesion ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
In Vivo Model ◽  
Glycoprotein Vi

Abstract In the present study, we used a knockout murine model to analyze the contribution of the Ca2+-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin αIIbβ3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA2 phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein–coupled receptors in supporting platelet aggregation and thrombus formation.

scholarly journals The Endothelial Tyrosine Phosphatase SHP-1 Plays an Important Role for Vascular Haemostasis in TNFα-Induced InflammationIn Vivo

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Elisabeth Koch ◽  
Joachim Pircher ◽  
Thomas Czermak ◽  
Erik Gaitzsch ◽  
Stefan Alig ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Surface Expression ◽  
Negative Regulator ◽  
Sodium Stibogluconate ◽  
Dorsal Skinfold Chamber ◽  
Endothelial Inflammation

Introduction. Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα-induced endothelial inflammationin vivo.Methods. Arteriolar thrombosis and platelet rollingin vivowere investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model.Results. Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interactionin vivounder physiological conditions but led to an augmented fraction of rolling platelets in TNFα-induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα-induced inflammation. Platelet aggregationin vitroas well asex vivowas not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα-induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFαincreased SHP-1 activity and protein expression.Conclusions. The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasisin vivo,which is crucial in TNFα-induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

1994 ◽  
Vol 71 (01) ◽  
pp. 095-102 ◽  
Author(s):  
Désiré Collen ◽  
Hua Rong Lu ◽  
Jean-Marie Stassen ◽  
Ingrid Vreys ◽  
Tsunehiro Yasuda ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Bolus Injection ◽  
Cell Injury ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Femoral Vein ◽  
Thrombotic Occlusion ◽  
Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

RPR120844, a Novel, Specific Inhibitor of Coagulation Factor Xa Inhibits Venous Thrombosis in the Rabbit

1999 ◽  
Vol 81 (01) ◽  
pp. 157-160 ◽  
Author(s):  
Ross Bentley ◽  
Suzanne Morgan ◽  
Karen Brown ◽  
Valeria Chu ◽  
Richard Ewing ◽  
...  
Keyword(s):  
Jugular Vein ◽  
Drug Effect ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Antithrombotic Activity ◽  
Fxa Inhibitor

SummaryThe in vivo antithrombotic activity of RPR120844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p <0.05 vs saline-treated animals) by RPR120844 at 30 and 100 μg/kg/min. At doses of 10, 30 and 100 μg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8 ± 0.3, 1.5 ± 0.9 and 2.4 ± 0.6 μM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.

scholarly journals Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets

2017 ◽  
Vol 37 (5) ◽  
pp. 823-835 ◽  
Author(s):  
Christopher W. Smith ◽  
Steven G. Thomas ◽  
Zaher Raslan ◽  
Pushpa Patel ◽  
Maxwell Byrne ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Thrombus Formation ◽  
Physiological Role ◽  
Src Family Kinase ◽  
Glycoprotein Vi ◽  
Collagen Receptor ◽  
Deficient Mice

Objective— Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif–containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif–containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1–deficient mice. Approach and Results— Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1–deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI–specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI–FcRγ-chain and integrin αIIbβ3 in megakaryocytes because of enhanced Src family kinase activity. Conclusions— Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein–coupled receptors.

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.

Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Reheman Adili ◽  
Katherine Mast ◽  
Michael Holinstat
Keyword(s):  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Human Platelets ◽  
Mass Spectrometry Analysis ◽  
Vessel Occlusion ◽  
Spectrometry Analysis ◽  
Thrombosis Model

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

scholarly journals Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1858
Author(s):  
Justine Habault ◽  
Claire Fraser ◽  
Ewa Pasquereau-Kotula ◽  
Maëlys Born-Bony ◽  
Anne Marie-Cardine ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Clinical Settings ◽  
Aspartic Acid Residue ◽  
Circulating Cells ◽  
C Terminus ◽  
Cell Penetrating ◽  
Endocytosis Pathway ◽  
Cancerous Cells

In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377–399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients’ circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.

scholarly journals A High-Throughput Screen for Receptor Protein Tyrosine Phosphatase–γ Selective Inhibitors

2011 ◽  
Vol 16 (5) ◽  
pp. 476-485 ◽  
Author(s):  
Kingsley K. Appiah ◽  
Walter A. Kostich ◽  
Samuel W. Gerritz ◽  
Yanling Huang ◽  
Brian D. Hamman ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Protein ◽  
Protein Tyrosine ◽  
Compound Collection ◽  
Ptp 1B

Protein tyrosine phosphatase–γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z′-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC50 determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC50 value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.

Sign in / Sign up

Export Citation Format

Share Document