Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 3965-3970 ◽  
Author(s):  
Cristina P. Vicente ◽  
Li He ◽  
Mauro S. G. Pavão ◽  
Douglas M. Tollefsen
Keyword(s):  
Carotid Artery ◽  
Dermatan Sulfate ◽  
Wild Type ◽  
Antithrombotic Effect ◽  
Porcine Skin ◽  
Heparin Cofactor Ii ◽  
Thrombotic Occlusion ◽  
Antithrombotic Activity ◽  
Occlusion Time ◽  
Deficient Mice

Abstract Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)

scholarly journals Accelerated atherogenesis and neointima formation in heparin cofactor II–deficient mice

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4261-4267 ◽  
Author(s):  
Cristina P. Vicente ◽  
Li He ◽  
Douglas M. Tollefsen
Keyword(s):  
Carotid Artery ◽  
Dermatan Sulfate ◽  
Plasma Lipid ◽  
Atherogenic Diet ◽  
Dependent Manner ◽  
Neointima Formation ◽  
Wild Type ◽  
Heparin Cofactor Ii ◽  
Glucose Levels ◽  
Deficient Mice

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin when bound to dermatan sulfate or heparin. HCII-deficient mice are viable and fertile but rapidly develop thrombosis of the carotid artery after endothelial injury. We now report the effects of HCII deficiency on atherogenesis and neointima formation. HCII-null or wild-type mice, both on an apolipoprotein E–null background, were fed an atherogenic diet for 12 weeks. HCII-null mice developed plaque areas in the aortic arch approximately 64% larger than wild-type mice despite having similar plasma lipid and glucose levels. Neointima formation was induced by mechanical dilation of the common carotid artery. Thrombin activity, determined by hirudin binding or chromogenic substrate hydrolysis within 1 hour after injury, was higher in the arterial walls of HCII-null mice than in wild-type mice. After 3 weeks, the median neointimal area was 2- to 3-fold greater in HCII-null than in wild-type mice. Dermatan sulfate administered intravenously within 48 hours after injury inhibited neointima formation in wild-type mice but had no effect in HCII-null mice. Heparin did not inhibit neointima formation. We conclude that HCII deficiency promotes atherogenesis and neointima formation and that treatment with dermatan sulfate reduces neointima formation in an HCII-dependent manner.

scholarly journals Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4118-4125 ◽  
Author(s):  
Li He ◽  
Tusar K. Giri ◽  
Cristina P. Vicente ◽  
Douglas M. Tollefsen
Keyword(s):  
Carotid Artery ◽  
Dermatan Sulfate ◽  
Thrombus Formation ◽  
Wild Type ◽  
Heparin Cofactor Ii ◽  
Arterial Endothelium ◽  
Carotid Arterial ◽  
Deficient Mice

AbstractHeparin cofactor II (HCII)–deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.

The Venous Antithrombotic Profile of Naroparcil in the Rabbit

1994 ◽  
Vol 72 (06) ◽  
pp. 874-879 ◽  
Author(s):  
Jean Millet ◽  
Jocelyne Theveniaux ◽  
Neil L Brown
Keyword(s):  
Oral Administration ◽  
Bleeding Time ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Bleeding Risk ◽  
Thrombin Time ◽  
Heparin Cofactor Ii ◽  
Antithrombotic Activity ◽  
Thrombin Inhibition ◽  
Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

The Antiaggregating and Antithrombotic Activity of Clopidogrel Is Potentiated by Aspirin in Several Experimental Models in the Rabbit

1998 ◽  
Vol 80 (09) ◽  
pp. 512-518 ◽  
Author(s):  
Frédérique Dol ◽  
André Bernat ◽  
Robert Falotico ◽  
Alain Lalé ◽  
Pierre Savi ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Experimental Models ◽  
Arteriovenous Shunt ◽  
Thrombotic Occlusion ◽  
Antithrombotic Activity ◽  
Rabbit Carotid Artery ◽  
Antithrombotic Efficacy

SummaryIt is unknown whether the addition of aspirin might increase both the efficacy and the potency of clopidogrel, a potent and selective ADP blocker. For that purpose, the efficacy of clopidogrel (1–20 mg/kg, p.o.) administered orally to rabbits alone or in combination with aspirin (0.1–10 mg/kg, p.o.) was determined in several experimental models. A potent synergistic effect of the clopidogrel/aspirin association was demonstrated with regard to collagen-induced platelet aggregation measured ex vivo. Similarly, aspirin potentiated the antithrombotic activity of clopidogrel measured with regard to experimental thrombosis induced by a silk thread or on stents placed in an arteriovenous shunt, thrombus formation following electrical stimulation of the rabbit carotid artery and with regard to 111In-labeled platelet deposition on a stent implanted in an arteriovenous shunt or on the subendothelium following air drying injury of the rabbit carotid artery. A similar potentiating effect of aspirin was obtained with regard to myointimal proliferation (restenosis) in the femoral arteries of atherosclerotic rabbits which occurred as a consequence of stent placement. The clopidogrel/aspirin combination showed only additive-type effects on bleeding time prolongation induced by ear transection in the rabbit, therefore showing that combined inhibition of cyclooxygenase and ADP‘s effects provide a marked enhanced antithrombotic efficacy. Such a combination may provide substantial protection against platelet aggregation leading to thrombotic occlusion at sites of endothelial injuries and coronary artery stenosis in humans.

Wdr1-Mediated Actin Reorganization Is Essential for Integrin αIIbβ3 Activation in Platelets

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2231-2231
Author(s):  
Swapan Kumar Dasgupta ◽  
Qi Da ◽  
Anhquyen Le ◽  
Miguel A. Cruz ◽  
Perumal Thiagarajan
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Carotid Artery ◽  
Bleeding Time ◽  
Wild Type ◽  
Integrin Αiibβ3 ◽  
Thrombosis Model ◽  
Actin Turnover ◽  
Deficient Mice

Abstract In resting platelets, the heterodimeric integrin αIIbβ3 is present in a low-affinity state. During platelet activation, the intracytoplasmic signals induce conformational changes that results in a swung-out conformation of the extracellular domain competent to bind ligands such as fibrinogen with high affinity to mediate platelet aggregation. Actin turnover is essential for this process and dynamic assembly and disassembly of actin filaments regulate it. We have identified Wdr1, a cofilin and actin binding protein containing WD40 repeats, as an essential component of the machinery that orchestrates actin fiber reorganization that leads to integrin αIIbβ3 activation. Methods: Wdr1-deficient mouse strain, Wdr1rd/rd. was obtained through an N-ethyl-N-nitrosourea mutagenesis screen in Baylor College of Medicine. The mutant mouse has a T>A transversion in the second dinucleotide of the intron 9 splice donor site and it produces a mutant transcript containing a 6-bp in-frame deletion that results in a incorrectly folded, nonfunctional protein. Normal splicing produces a small amount of Wdr1 protein (~2%) resulting in a hypomorphic allele. Wdr1-deficient mice are moderately thrombocytopenic (85± 11 x 106 ml Wdr1 deficient versus 427± 52 x 106/ml for wild-type). Platelets were isolated from Wdr1-deficient and control mice. Platelet aggregation was carried out by standard turbidometric methods. Calcium mobilization was measured by incubating Wdr1-deficient and WT (wild-type) platelets with Fura 2 AM and measuring the Fura 2 fluorescence after collagen treatment. Conformational change in αIIbβ3 was determined by flow cytometry with a conformation-specific anti-αIIbβ3 antibody JON/A. In vivo hemostasis was assessed by tail bleeding time and FeCl3-induced endothelial carotid injury/thrombosis model was used to assess the occlusion in carotid artery of mice. Results: Aggregation response of Wdr1-deficient platelets to different doses of collagen was significantly impaired compared to WT platelets. Under similar conditions, the calcium response was similar to the WT. In a parallel-plate flow chamber assay, WT platelets stably adhered to collagen surface and formed stable thrombus. On the other hand, significantly less number of Wdr1 deficient platelets were stably attach to the collagen surface and it did not form stable thrombus. As expected the tail bleeding time of Wdr1 deficient mice is significantly prolonged (> 10 minutes) compared to WT mice (<2 min). In vivo, in FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1 deficient was prolonged significantly compared wild type mice (15.8 ± 12.6 minutes versus 9.0 ± 10.5 minutes (p=0.041, Mann-Whitney non parametric comparison). To examine directly the activation of αIIbβ3, we used JON/A antibody, which selectively binds to activated αIIbβ3 integrins on mouse platelets. Binding of collagen treated Wdr1-deficient platelets to JON/A as determined by flow cytometry, is significantly less compared to WT platelets (6.1±0.3 fluorescence units (FU) versus 17.4±0.6 FU, p≤0.05) indicating impaired inside-out activation of αIIbβ3. Since, Wdr1 promotes actin disassembly, which is essential for the rearrangement of the actin fibers that occurs during platelet activation, we measured actin turn over by measuring F-actin and G-actin ratios of collagen treated platelets at various time points. Actin turnover is highly impaired in Wdr1 deficient platelets compared to WT platelets. Furthermore, integrin αIIbβ3 association with actin cytoskeleton was markedly impaired in Wdr1 deficient mice compared to their WT controls. These studies show that Wdr1 mediated actin cytoskeleton reorganization is essential for integrin αIIbβ3 activation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Selatogrel, the reversible and specific P2Y12 receptor antagonist, does not interfere with haemostasis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M.A Riederer ◽  
L Crescence ◽  
E Caroff ◽  
F Hubler ◽  
L Panicot-Dubois ◽  
...  
Keyword(s):  
Blood Loss ◽  
P2y12 Receptor ◽  
Funding Source ◽  
Wild Type ◽  
Antithrombotic Effect ◽  
Pronounced Increase ◽  
Coronary Syndrome ◽  
Dosing Regimens ◽  
The Stability ◽  
Deficient Mice

Abstract Background Combination of a P2Y12 receptor (P2Y12R) antagonist (clopidogrel, prasugrel, ticagrelor) with aspirin is the recommended standard of care for patients with acute coronary syndrome. Selatogrel is a reversible and potent antagonist of P2Y12R. Interestingly, in an experimental thrombosis model in rat, at equivalent antithrombotic effect, blood loss was lower in the presence of selatogrel, compared with clopidogrel or ticagrelor. Purpose To characterise the lower risk of bleeding previously observed with selatogrel Methods Mechanistic studies were performed to profile laser-induced thrombosis in wild-type and P2Y12 deficient mice with real-time intravital microscopy. Ticagrelor and clopidogrel were used as selatogrel comparators. Results Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to the haemostatic seals, were present. The phenotype of these haemostatic seals depended on the P2Y12R antagonist used. In the presence of clopidogrel or ticagrelor, the stability of haemostatic seals was reduced. In contrast, in the presence of selatogrel, the apparent stability was not disturbed. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel interfered with laser-induced calcium mobilisation in the endothelium, restricted subsequent neutrophil adhesion and thus reduced fibrin-mediated stabilisation of the haemostatic seals in wild type mice. The effects of ticagrelor were also observed in P2Y12R-deficient mice, indicating that the effects are P2Y12R independent and off-target. In contrast, an equivalent antithrombotic dosing regimen of selatogrel did not interfere with the process of haemostasis in wild-type or P2Y12R-deficient mice. The degree of interference with the stability of the haemostatic seals correlated with the blood loss profile. The dosing regimens of clopidogrel and ticagrelor, corresponding to the equivalent antithrombotic effects, induced a more pronounced increase in blood loss than that observed with selatogrel. Conclusion Our data offer a novel mechanistic explanation for the differences in bleeding risk of clopidogrel, ticagrelor and selatogrel. Clopidogrel and ticagrelor were found to interfere with haemostasis due to off-target activities. In contrast, selatogrel did not interfere with haemostasis in wild-type and P2Y12-deficient mice, inferring that the process of haemostasis, as defined by formation of haemostatic seals, is independent of P2Y12R. In addition, our data emphasize that the absence of interference with haemostasis is paramount to preserve the advantage of P2Y12R antagonism. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Idorsia Pharmaceuticals Ltd. Allschwil, Switzerland

scholarly journals Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

2009 ◽  
Vol 29 (20) ◽  
pp. 5517-5528 ◽  
Author(s):  
Marco Maccarana ◽  
Sebastian Kalamajski ◽  
Mads Kongsgaard ◽  
S. Peter Magnusson ◽  
Åke Oldberg ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tensile Strength ◽  
Collagen Fibril ◽  
Glucuronic Acid ◽  
Dermatan Sulfate ◽  
Collagen Structure ◽  
Wild Type ◽  
Iduronic Acid ◽  
Skin Collagen ◽  
Deficient Mice

ABSTRACT Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.

KINETICS OF EXPERIMENTAL ANTITHROMBOTIC ACTIVITY OF MF 701 DERMATAN SULFATE

1987 ◽  
Author(s):  
A Morani ◽  
F Gianese ◽  
P Bianchini
Keyword(s):  
Dermatan Sulfate ◽  
Vena Cava ◽  
Maximum Effect ◽  
Antithrombotic Effect ◽  
Antithrombotic Activity ◽  
Total Inhibition ◽  
Long Duration ◽  
Antithrombotic Efficacy ◽  
Kinetics Of ◽  
Dose Dependent

In order to evaluate the intensity and duration of the prophylactic activity of dermatan sulfate against thrombosis induced by ligature of the inferior vena cava (Reyers'model), we treated 6 groups of rats at increasing intervals of time before ligature (1 min, 30 min, 45 min, 2 h, 16 h). Within each group the rats were injected subcutaneously with saline solution (controls) or increasing doses (2.5, 5, 10 and 20 mg/kg) of MF 701, dermatan sulfate (Mediolanum Farmaceutici, Milan, Italy). Two hours after ligature the presence of thrombi and their weight were recorded.A significant, dose-dependent antithrombotic effect was observed for all the treatment times. The maximum effect occured when the compound was administered 45 min before ligature (total abolition of thrombosis at the dose of 5 or more mg/kg). Total inhibition was also observed at 2 h (with 10 mg/kg) and at 4 h (with the same dose). A significant antithrombotic effect, over 50% com pared with the controls, was still found at 16 h at the dose of 20 mg/kg.These results confirm the experimental antithrombotic efficacy of dermatan sulfate already reported in a different animal model by F. Fernandez et al. (Br J. Haematol. 64, 309, 1986) and demonstrate the long duration of the prophylactic effect of the compound.

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