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.

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.

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.

scholarly journals Lipolysis generates platelet dysfunction after in vivo heparin administration

2002 ◽  
Vol 103 (4) ◽  
pp. 433-440 ◽  
Author(s):  
Elijah W. MURIITHI ◽  
Philip R. BELCHER ◽  
Stephen P. DAY ◽  
Mubarak A. CHAUDHRY ◽  
Muriel J. CASLAKE ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Cardiopulmonary Bypass ◽  
Lipoprotein Lipase ◽  
Whole Blood ◽  
Ex Vivo ◽  
Hepatic Lipase ◽  
Platelet Factor 4 ◽  
Platelet Factor

Heparin, when administered to patients undergoing operations using cardiopulmonary bypass, induces plasma changes that gradually impair platelet macroaggregation, but heparinization of whole blood in vitro does not have this effect. The plasma changes induced by heparin in vivo continue to progress in whole blood ex vivo. Heparin releases several endothelial proteins, including lipoprotein lipase, hepatic lipase, platelet factor-4 and superoxide dismutase. These enzymes, which remain active in plasma ex vivo, may impair platelet macroaggregation after in vivo heparinization and during cardiopulmonary bypass. In the present study, proteins were added in vitro to hirudin (200units·ml-1)-anticoagulated blood from healthy volunteers, and the platelet macroaggregatory responses to ex vivo stimulation with collagen (0.6μg·ml-1) were assessed by whole-blood impedance aggregometry. Over a 4h period, human lipoprotein lipase and human hepatic lipase reduced the platelet macroaggregatory response from 17.0±2.3 to 1.5±1.3 and 1.2±0.6Ω respectively (means±S.D.) (both P<0.01; n = 6). Other lipoprotein lipases also impaired platelet macroaggregation, but platelet factor-4 and superoxide dismutase did not. Platelet macroaggregation showed an inverse linear correlation with plasma concentrations of non-esterified fatty acids (r2 = 0.69; two-sided P<0.0001; n = 8), suggesting that heparin-induced lipolysis inhibits platelet macroaggregation. Lipoprotein degradation products may cause this inhibition by interfering with eicosanoids and other lipid mediators of metabolism.

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.

Evaluation of anti-platelet and anti-thrombotic effects of cilostazol with PFA-100® and Multiplate® whole blood aggregometer in vitro, ex vivo and FeCl3-induced thrombosis models in vivo

2011 ◽  
Vol 127 (6) ◽  
pp. 565-570 ◽  
Author(s):  
Chae-Wook Kim ◽  
Jun-Won Yun ◽  
Il-Hong Bae ◽  
Yang-Hui Park ◽  
Yeon Su Jeong ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo

scholarly journals Targeted Deletion of the Lipopolysaccharide (LPS)-binding Protein Gene Leads to Profound Suppression of LPS Responses Ex Vivo, whereas In Vivo Responses Remain Intact

1997 ◽  
Vol 186 (12) ◽  
pp. 2051-2056 ◽  
Author(s):  
Mark M. Wurfel ◽  
Brian G. Monks ◽  
Robin R. Ingalls ◽  
Russell L. Dedrick ◽  
Russell Delude ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Ex Vivo ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Cellular Responses ◽  
Tnf Α ◽  
Deficient Mice ◽  
Lps Binding

Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-α. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-α levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.

Comparison Of The Effects Of Aspirin In Humans Ex Vivo In Platelet-Rich Plasma And Whole Blood

1981 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Trisodium Citrate ◽  
Blood Platelet ◽  
Maximal Response ◽  
Blood Samples ◽  
Platelet Concentration

The effect of aspirin on human platelet function is usually assessed using platelet-rich plasma (PRP). Some preliminary results in vitro suggested that the effect of aspirin appears to be greater in PRP than whole blood. To explore this possibility further, a comparison of the effect of aspirin in humans ex vivo has been made taking measurements simultaneously in whole blood and PRP at 2 platelet concentrations. Blood samples (36ml) were drawn from 7 male volunteers after a light breakfast. Each took 300mg soluble aspirin and blood samples were drawn again 2 hours later. Blood was mixed with 0.1 volumes 129nM trisodium citrate. Some (30ml) was then centrifuged to prepare PRP and platelet -poor plasma (PPP) by standard techniques. Platelet concentration of some PRP was adjusted with PPP to equal that of the corresponding blood sample; the rest was adjusted to 350,000 per μl. Aggregation in response to collagen (Horm, Munich) was measured photometrically at 37°. Aggregation in 0.5ml aliquots of whole blood was measured after 4 min stirring with 154mM NaCl (control) or collagen at 37° as the fall in single platelet count determined using an Ultraflo- 100 whole blood platelet counter (Clay Adams). The concentrations of collagen producing a 50% maximal response (EC50) in PRP and blood were determined. Dose-ratios for each volunteer were calculated by dividing the EC50 obtained after aspirin by the corresponding value obtained before aspirin.The effect of aspirin was significantly (p<0.001) less in blood than PRP. Whether or not the results in whole blood more closely reflect the effect of aspirin in vivo remains to be determined.

scholarly journals The Influence of Methylsulfonylmethane on Inflammation-Associated Cytokine Release before and following Strenuous Exercise

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Mariè van der Merwe ◽  
Richard J. Bloomer
Keyword(s):  
Whole Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Knee Extension ◽  
Strenuous Exercise ◽  
Peripheral Blood Mononuclear ◽  
Physically Active ◽  
Inflammatory Molecules

Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties.Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise.Ex vivoandin vitrotesting consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, whilein vivotesting included the evaluation of cytokines before and through 72 hours after exercise.Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM.Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise.

scholarly journals Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice

2018 ◽  
Vol 148 (9) ◽  
pp. 1415-1420 ◽  
Author(s):  
Xiaoying Wang ◽  
Yang Yuan ◽  
Inka C Didelija ◽  
Mahmoud A Mohammad ◽  
Juan C Marini
Keyword(s):  
Ex Vivo ◽  
Transcript Abundance ◽  
Mutant Mice ◽  
Ex Vivo Model ◽  
Endogenous Production ◽  
Gene Coding ◽  
Cycle Disorders ◽  
Deficient Mice

Abstract Background The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. Objective The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. Methods Six-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Results Liver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg−1 · h−1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA−1 · d−1; P < 0.001). Conclusions Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

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.

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