ANTITHROMBOTIC ACTIONS OF A SULFOMUCOPOLYSACCHARIDE MIXTURE (ATERIOD) IN ANIMAL MODELS

Ateriod obtained from beef mucosal lining is a sulfomuco-polysaccharide mixture of various glycosaminoglycans which contains derma tans, heparatans and traces of heparin. It has been used in the treatment ofatherosclerosis and related vaso-oclusive disorders. Ateriod is standardized in terms of its lipoprotein lipase activation actions. Ateriod contains signfi-cant in vitro anticoagulant and antiprotease (anti-factor Xa and anti-factor Ila) activities as measured by clot-based and chromr ogenic substrate methods. However, this in vitro activity is 7-10 times lesser than heparin. In order to study the antithrombotic actions of this agent in subcutaneous, intravenous and oral routes, we utilized a rabbit stasis thrombosis model with a prothrombin complex concentrate/Russell's viper venom thrombogenic challenge and prolonged stasis. The apparent ED50 for the antithrombotic action were found to be: IV (75-100 ug/ kg), SC (0.8-1.3 mg/kg) and oral (20-30 mg/kg). In both the IV- and SC studies, sustained anticoagulant and antiprotease actions were evident. The observed antithrombotic actions did not relate to the anti-factor IIa or anti-factor Xa actions. Pretreatment of Ateriod with equigravimetric amounts of protamine and platelet factor 4 did not neutralize the antithrombotic actions of this agent in the rabbit model. In a primate (Macaca mulatta) model of pharmacokinetics, ex vivo analysis following subcutaneously administered Ateriod showed sustained anticoagulant and antiprotease effects. The time course of the subcutaneously administered Ateriod was markedly different than heparin and a low molecular weight heparin. Treated animals were shown to resist induced hypercoagulability following injection of homologous serum as measured by FPA generation for extended periods. These studies suggest that Ateriod produces a strong antithrombotic action and that it has highly sustained pharmacokinetics. The antithrombotic activity appears to be primarily mediated via non-antithrombin - HI dependent events which may be related to heparin cofactor II and vascular/ cellular modifications.

Download Full-text

ANTITHROMBOTIC ACTIVITY OF A SYNTHETIC HEPARIN PENTASACCHARIDE IN A RABBIT STASIS THROMBOSIS MODEL USING DIFFERENT THROMBOGENIC CHALLENGES

10.1055/s-0038-1644161 ◽

1987 ◽

Author(s):

J M Walenga ◽

J Fareed ◽

M Petitou ◽

J C Lormeau ◽

M Samama ◽

...

Keyword(s):

Prothrombin Complex Concentrate ◽

Ex Vivo ◽

Factor Xa ◽

Antithrombotic Effect ◽

Antithrombotic Activity ◽

Thrombosis Model ◽

Activated Prothrombin Complex Concentrate ◽

Dose Dependent

The synthetic pentasccharide, representing the critical sequence required in heparin for binding to antithrombin III, provides a unique tool to study the question of whether an agent solely capable of inhibiting factor Xa but devoid of anti-factor Ila activity in vitro, has the capacity to produce an antithrombotic effect in vivo. We have previously demonstrated in a rabbit stasis thrombosis model using a human serum challenge, a significant antithrombotic effect of the pentasaccharide (Walenga et al., Thromb Res 43:243, 1986). To extend and confirm these studies, four modifications of the stasis thrombosis model were developed using more specified induction sites of thrombosis. The following thrombogenic challenges were selected: monkey brain thromboplastin, an activated prothrombin complex concentrate, a non-activated prothrombin complex concentrate administered simultaneously with Russell's viper venom, and factor Xa. Dose-dependent antithrombotic responses were obtained in all four systems with ED50 values between 25-43 ug/kg for pentasaccharide as compared to 16-47 ug/kg for heparin. Complete inhibition of induced thrombosis was obtained in all four systems for pentasaccharide. Ex vivo analysis revealed expected anti-factor Xa levels but no anti-factor IIa activity. It was also shown that pentasaccharide in the rabbit was capable of inhibiting the generation of thrombin without directly inhibiting formed thrombin. It is concluded that an oligosaccharide with high anti-factor Xa activity, devoid of anti-factor Ila activity, is capable of inhibiting thrombosis induced in rabbit stasis models, but that higher dosages than heparin are required for this effect-in terms of anti-factor Xa activity.

Download Full-text

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645198 ◽

1990 ◽

Vol 63 (02) ◽

pp. 220-223 ◽

Cited By ~ 19

Author(s):

J Hauptmann ◽

B Kaiser ◽

G Nowak ◽

J Stürzebecher ◽

F Markwardt

Keyword(s):

Factor Xa ◽

Thrombin Inhibitors ◽

Factor Xa Inhibitors ◽

Venous Stasis ◽

Clotting Time ◽

Thrombosis Model ◽

Activity Factor ◽

Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Download Full-text

The Effect of Pentosan Polysulphate (SP54) on the Fibrinolytic Enzyme System - A Human Volunteer and Experimental Animal Study

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660143 ◽

1985 ◽

Vol 54 (04) ◽

pp. 833-837 ◽

Cited By ~ 7

Author(s):

N A Marsh ◽

P M Peyser ◽

L J Creighton ◽

M Mahmoud ◽

P J Gaffney

Keyword(s):

Plasminogen Activator ◽

Surface Effect ◽

Ex Vivo ◽

Vena Cava ◽

Tissue Type ◽

Similar Response ◽

Small Contribution ◽

Thrombosis Model ◽

Pentosan Polysulphate

SummaryPentosan polysulphate causes an increase in plasminogen activator activity in plasma both after oral ingestion and after subcutaneous injection. The effect is greatest after 3 h and has disappeared by 6 h. Repeat doses by mouth over 5 days elicit a similar response. The recorded increase in activity is due largely to the release of tissue-type plasminogen activator (tPA) from the endothelium according to the antigen assay although there could be a small contribution from Factor XH-related “intrinsic” fibrinolysis induced in vitro. SP54 enhances activity ex vivo by a non-specific surface effect, and this phenomenon may contribute the increased levels of activity seen in vitro. Administration of SP54 to animals elicits a similar increase in activator activity, the intramuscular route being slightly more effective. Results with an inferior vena cava thrombosis model in the rat suggest that pentosan polysulphate may induce a thrombolytic effect.

Download Full-text

Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽

10.3390/cells10040730 ◽

2021 ◽

Vol 10 (4) ◽

pp. 730

Author(s):

Biji Mathew ◽

Leianne A. Torres ◽

Lorea Gamboa Gamboa Acha ◽

Sophie Tran ◽

Alice Liu ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Extracellular Vesicles ◽

Time Course ◽

Ganglion Cells ◽

Ex Vivo ◽

Dependent Manner ◽

Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

Download Full-text

Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide

Blood ◽

10.1182/blood.v91.11.4197 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4197-4205 ◽

Cited By ~ 151

Author(s):

J.M. Herbert ◽

J.P. Hérault ◽

A. Bernat ◽

R.G.M. van Amsterdam ◽

J.C. Lormeau ◽

...

Keyword(s):

Thrombin Generation ◽

Ex Vivo ◽

Thrombus Formation ◽

Factor Xa ◽

Therapeutic Index ◽

Inhibitory Effect ◽

Experimental Models ◽

Treatment And Prevention ◽

Long Acting

Abstract SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.

Download Full-text

STUDIES ON THE ANTITHROMBOTIC ACTION OF A LOW MOLECULARWEIGHT HEPARIN OBTAINED FROM BEEF MUCOSAL ORIGIN AND PEROXIDATIVE CLEAVAGE

10.1055/s-0038-1644164 ◽

1987 ◽

Author(s):

P Bianchini ◽

R Nonn ◽

J Fareed ◽

J M Walenga ◽

A Kumar

Keyword(s):

Molecular Weight ◽

Structural Integrity ◽

Factor Xa ◽

Inhibitory Effects ◽

Heparin Cofactor Ii ◽

At Iii ◽

Antithrombotic Effects ◽

In Vitro Systems ◽

Mean Molecular Weight

We have studied a low molecular weight heparin (LMWH) obtained by acontrolled peroxidative depolymerization of beef mucosal heparin (OP 2123, Opocrin, Corlo, Italy). This product was found to be significantly different from other LMWHs in that it exhibits the same COO-/SO2- ratios as unfractionated heparin, contains reducing end groups composed of 2-sulfated iduronic acid or 6-disulfated glucosamine and retains an identical structural integrity as that of native heparin. As opposed to most other LMWHs the oligosaccharide components of OP 2123 consist of homogeneous progressive units. In addition, the relative amount of AT-IIIaffinity components in OP 2123 were 20-30% less than other LMWHs. OP 2123 has a mean molecular weight of 6200 daltons with a potency of 90 anti-factor Xa U/mg and 68 USP U/mg. This agent produced strong antithrombotic actions in a rabbit stasis model against both an activated prothrombin complex and a prothrombin complex concentrate/Russell's viper venomcombination (ED50:(IV) 30-70 ug/kg;(SC) 0.6-1.5 mg/kg). The antithrombotic effects were comparable to other LMWHs in normal rabbits: however, in AT III depleted rabbits (immunodepleted and y thrombin depleted), OP 2123 produced stronger antithrombotic effects than most other LMWHs. The in vitro systems in contrast to other LMWHs, CP 2123 produced stronger inhibitory effects in AT III depleted plasma as measured by fibrinopeptide A generation and amidolytic anti-factor Xa and anti-factor Ila methods. The relative heparin cofactor II activity as measured by amidolytic method was also found to be higher than with most LMWHs. These results suggest that OP 2123, unlike most LMWHs, non AT III mediated actions play a major role in themendiation of the antithrombotic actions.

Download Full-text

Suitability of a Progenitor Cell-Enriching Device for In Vitro Applications

Coatings ◽

10.3390/coatings11020146 ◽

2021 ◽

Vol 11 (2) ◽

pp. 146

Author(s):

Antonio Celentano ◽

Tami Yap ◽

Giuseppe Pantaleo ◽

Rita Paolini ◽

Michael McCullough ◽

...

Keyword(s):

Cell Viability ◽

Time Course ◽

Ex Vivo ◽

Trypan Blue Exclusion ◽

Trypan Blue Exclusion Test ◽

Initial Reduction ◽

Metal Wear ◽

Class 1 ◽

Electron Energy Loss

Rigenera® is a novel class-1 medical device that produces micro-grafts enriched of progenitors cells without ex vivo manipulation of donor tissues. The manufacturer’s protocol has been supported for a wide variety of clinical uses in the field of regenerative medicine. This study aimed to evaluate its potential use for in vitro cell models. Human primary oral fibroblasts were cultured under standard conditions and processed through Rigenera® over a time course of up to 5 min. Cell viability was assessed using a Trypan Blue exclusion test. It is possible to process fibroblasts through Rigenera® although an initial reduction of cell viability was observed. Additionally, debris was evident in the cell suspension of the processed samples. Scanning electron microscopy (SEM) microanalysis of the debris and electron energy-loss spectroscopy confirmed the presence of metal wear possibly due to the processing conditions used in this study. Interestingly, pore sizes within Rigeneracons® grids were found to range between 250–400 μm. This is the first report assessing the suitability of Rigenera® and Rigeneracons® for in vitro applications. Whilst Rigenera® workflow was found to be amenable to laboratory uses, our results strongly suggest that further research and development is necessary to support the utilization of this technology for enrichment of micro-graft derived cells and cell sorting in vitro.

Download Full-text

ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i4.22474 ◽

2018 ◽

Vol 10 (4) ◽

pp. 44

Author(s):

Mihir K Patel ◽

Kiranj K. Chaudagar ◽

Anita A. Mehta

Keyword(s):

Platelet Aggregation ◽

Arterial Thrombosis ◽

Ex Vivo ◽

Platelet Activity ◽

Aggregation Assay ◽

Thrombosis Model ◽

Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

Download Full-text

Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies

Blood ◽

10.1182/blood-2011-05-353391 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1248-1255 ◽

Cited By ~ 69

Author(s):

Krystin Krauel ◽

Christine Hackbarth ◽

Birgitt Fürll ◽

Andreas Greinacher

Keyword(s):

Factor Xa ◽

Platelet Factor 4 ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Alternative Anticoagulation ◽

Heparin Complexes ◽

History Of

Abstract Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.

Download Full-text