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.

scholarly journals Catestatin prevents endothelial inflammation and promotes thrombus resolution in acute pulmonary embolism in mice

2019 ◽  
Vol 39 (11) ◽  
Author(s):  
Hua Chen ◽  
Dongxia Liu ◽  
Lan Ge ◽  
Tao Wang ◽  
Zhenzhen Ma ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Acute Pulmonary Embolism ◽  
Thrombus Formation ◽  
Vascular Dysfunction ◽  
In Vivo Model ◽  
Protective Effects ◽  
Inhibitory Peptide ◽  
Endothelial Inflammation

AbstractCatestatin (CTS), a catecholamine-release inhibitory peptide, exerts pleiotropic cardiac protective effects. Pulmonary embolism caused by deep vein thrombosis involving vascular dysfunction. The present study aims to investigate the effects of CTS on thrombus formation that may inhibit the development of pulmonary embolism and its potential pathway. Acute pulmonary embolism (APE) model was developed as an in vivo model. The effects of CTS on mice with APE were examined. Human pulmonary artery endothelial cells (HPAECs) were pretreated with CTS before thrombin stimulation, and endothelial inflammation and underlying mechanisms were evaluated in vitro. That plasma CTS level was decreased in APE mice, while the number of platelets was significantly increased. The decreased circulating CTS level negatively associated with the number of platelets. CTS administration increased the survival rate of APE mice and protected against microvascular thrombosis in lung. APE-induced the increase in platelets number and plasma von Willebrand factor (VWF) were inhibited by CTS. Platelets from CTS-treated APE mice showed impaired agonist-induced platelets aggregation and spreading. CTS also ameliorated APE-induced the systemic inflammatory response. In in vivo study, thrombin-induced the increase in inflammation, TLR-4 expression and p38 phosphorylation were abrogated by CTS in HPAECs. Furthermore, TLR-4 overexpression inhibited the effect of CTS on VWF release and inflammation in HPAECs. Collectively, CTS increases thrombus resolution by attenuating endothelial inflammation at partially via inhibiting TLR-4-p38 pathway. The present study may provide a novel approach for anti-thrombosis.

Abstract 1144: Successful Surface-Aminated Nanofiber Expansion of Human Umbilical Cord-Derived CD133+ Cells Leads to Augmentation of Angiogenic Functionality In Vitro and In Vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hiranmoy Das ◽  
Matthew Joseph ◽  
Nasreen Abdulhameed ◽  
Hai-Quan Mao ◽  
Vincent J Pompili
Keyword(s):  
Umbilical Cord ◽  
Hind Limb ◽  
Ex Vivo ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Capillary Density ◽  
Therapeutic Angiogenesis ◽  
Human Umbilical Cord

Background: Umbilical cord blood (UCB) and marrow-derived CD133+ cells have been shown to mediate encouraging effects on therapeutic angiogenesis in both animal models and early clinical trials. Low numbers of CD133+ cells derived from a single donor have been a limitation of use of these cells in cardiovascular therapy. We hypothesized that an ex vivo aminated nanofiber system combined with cytokine supplementation would provide optimized topographical and biochemical signals to allow the expansion and potential functional augmentation of CD133+ cells without promoting terminal differentiation. Methods and Results: Human UCB derived CD133+ progenitor cells were isolated by MACS sorting and ex vivo expanded on aminated nanofiber plates with cytokine rich media. Cells harvested 10 days after expansion demonstrated a 225X increase in total number. Flow cytometric analysis demonstrated CD133–24%, CD34–93%, CXCR4–97%, LFA-97% surface expression. The expanded cells can uptake AcLDL efficiently and demonstrate a 2.3X increase in transwell migration to SDF-1 as compared to fresh UCB CD133+ cells. In vitro analysis revealed that expanded cells have potential to differentiate into endothelial or smooth muscle phenotype as demonstrated with CD31, vWF, VCAM-1 and F-pholloidin, α-actin, and SM myosin heavy chain immunocytochemistry when re-cultured for 14d in EGM2 or SMBM respectively. RT-QPCR analysis of 1% O 2 exposed (hypoxic) cells demonstrated a 2X increase in VEGF and 3X increase in IL-8 gene expression compared to normoxic control. In vivo functionality in a NOD/SCID mouse hind limb ischemic model demonstrated that mice treated with 5 x 10 6 expanded cells (n=7) augmented blood flow ratio (ischemic/control limb) as compared to mice treated with CD133+ cells (n=7) and control (n=7) at 28d. (control 0.32±.02 vs. UCB133+ 0.37±.02 vs. expanded cells 0.50±.04 p<0.01) Capillary density in ischemic hind-limb was increased at 28d (control 62.5±5.4 vs. expanded cell 97.6±2.5 p< 0.01) Conclusions: These studies demonstrate successful high level expansion of UCB derived CD133+ cells into functionally potent stem cells which have the capacity to differentiate into vascular cells and promote in vivo neovascularization.

Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3442-3442 ◽  
Author(s):  
Reheman Adili ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Platelet ADP receptors contribute to the initiation of intravascular coagulation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 594-600 ◽  
Author(s):  
Catherine Leon ◽  
Meike Alex ◽  
Antje Klocke ◽  
Eberhard Morgenstern ◽  
Christine Moosbauer ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Intravascular Coagulation ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Deficient Mice ◽  
Storage Pools

Abstract While the adenosine 5′-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

1995 ◽  
Vol 73 (02) ◽  
pp. 318-323 ◽  
Author(s):  
K Azzam ◽  
L I Garfinkel ◽  
C Bal dit Sollier ◽  
M Cisse Thiam ◽  
L Drouet
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Mesenteric Arteries ◽  
Antithrombotic Effects ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model

1998 ◽  
Vol 79 (01) ◽  
pp. 222-227 ◽  
Author(s):  
F. Stockmans ◽  
W. Deberdt ◽  
Å. Nyström ◽  
E. Nyström ◽  
J. M. Stassen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Peak Plasma ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Cell Deformability

SummaryIntravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.

scholarly journals TREM-like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice

2018 ◽  
Vol 2 (16) ◽  
pp. 2072-2078 ◽  
Author(s):  
Christopher W. Smith ◽  
Zaher Raslan ◽  
Lola Parfitt ◽  
Abdullah O. Khan ◽  
Pushpa Patel ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Surface Expression ◽  
Activated Platelets ◽  
Key Points ◽  
Sensitive Marker

Key Points Platelet activation in vitro results in a more rapid and greater upregulation of TLT-1 surface expression compared with P-selectin. TLT-1 is more rapidly translocated to the surface of activated platelets than P-selectin during thrombus formation in vivo.

Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation

2005 ◽  
Vol 93 (06) ◽  
pp. 1161-1167 ◽  
Author(s):  
Burcin Özüyaman ◽  
Susanne Küsters ◽  
Elisabeth Kirchhoff ◽  
Rüdiger Scharf ◽  
Jürgen Schrader ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Surface Expression ◽  
No Synthase ◽  
Minor Role ◽  
Endothelial No Synthase ◽  
Tail Tip ◽  
A Minor

SummaryEndothelial NO synthase (eNOS) expressed in the vascular en-dothelium or formed within platelets was postulated to inhibit platelet activation and aggregation. We have assessed the role of eNOS in platelet aggregation in vitro and in vivo by comparison of WT and eNOS-/- mice. Aggregometer studies revealed that collagen over a concentration range of 0.36–10 µg aggregated WT and eNOS-/- platelets to the same extent (10 µg: WT 86.7±4.7%, eNOS-/- 91±12%, n=6). Collagen treatment did not result in a significant increase in cGMP formation and VASP phosphorylation. Thrombin-induced P-selectin surface expression was unchanged in eNOS-/- platelets. In line with these findings no eNOS protein was detectable within the platelets of WT mice. In vivo, bleeding time after tail tip resection tended to be shorter in eNOS/- mice (WT: 116±35 s; eNOS-/- 109±37 s, n.s). Similarly, time to occlusion of the A.carotis after focal induction of thrombosis was 501±76 s (WT) and 457±95 s (eNOS-/-) (n.s.). These data demonstrate that eNOS-deficiency minimally affects platelet aggregation and is not associated with accelerated arterial thrombosis in vivo. Thus, in the mouse endothelial NO synthase does not play a major role in the autocrine modulation of platelet function and in thrombosis of conduit vessels in vivo.

Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Reheman Adili ◽  
Katherine Mast ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
High Shear ◽  
Vessel Occlusion ◽  
12 Lipoxygenase ◽  
Induced Aggregation

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

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