Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives.

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

A low-anticoagulant heparin suppresses metastatic dissemination through the inhibition of tumor cell-platelets association.

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.

Therapeutic implementation in arterial thrombosis with pulmonary administration of fucoidan microparticles containing acetylsalicylic acid.

Several antithrombotic drugs are available to treat cardiovascular diseases due to its high mortality and morbidity worldwide. Despite these, severe adverse effects that can lead to treatment withdrawal have been described, highlighting the importance of new therapies. Thus, this work describes the development of fucoidan microparticles containing acetylsalicylic acid (MP/F4M) for pulmonary delivery and in vitro, ex vivo, and in vivo evaluation. Microparticles were prepared via spray-drying and characterized in vitro (mucoadhesive properties, coagulation time, platelet aggregation, adhesion, and hemolysis) followed by ex vivo platelet aggregation, in vivo arterial thrombosis, and hemorrhagic profile. The formulation physicochemical characterization showed suitable characteristics along with delayed drug release, increased breathable particle fraction, and high washability resistance as well as antiplatelet activity and enhanced platelet adhesion in vitro. In in vivo assays, MP/F4M protected against arterial thrombosis, without changes in the hemorrhagic profile. Finally, no lung changes were observed after prolonged pulmonary administration, whereas isolated ASA led to an inflammatory response. In conclusion, pulmonary administration of fucoidan microparticles with an antiplatelet drug may be an alternative therapy to treat cardiovascular diseases, opening the field for different formulations.

Polysaccharide composition of an anticoagulant fraction from the aqueous extract of Marsypianthes chamaedrys (Lamiaceae).

Marsypianthes chamaedrys (Lamiaceae) is a medicinal plant popularly used against envenomation by snakebite. Pharmacological studies have shown that extracts of M. chamaedrys have antiophidic, anti-inflammatory and anticoagulant properties, supporting the ethnopharmacological use. In this study, an aqueous extract of aerial parts of M. chamaedrys showed anticoagulant activity in the activated partial thromboplastin time assay (0.54 IU/mg). The bioassay-guided fractionation using ethanol precipitation and gel filtration chromatography on Sephadex G-50 and Sephadex G-25 resulted in a water-soluble fraction with increased anticoagulant activity (Fraction F2-A; 2.94 IU/mg). A positive correlation was found between the amount of uronic acids and the anticoagulant potential of the active samples. Chemical and spectroscopic analyses indicated that F2-A contained homogalacturonan, type I rhamnogalacturonan, type II arabinogalactan and α-glucan. UV and FT-IR spectra indicated the possible presence of ferulic acid. Pectic polysaccharides and type II arabinogalactans may be contributing to the anticoagulant activity of the aqueous extract of M. chamaedrys in the APTT assay.

Integrin inhibitors from snake venom: exploring the relationship between the structure and activity of RGD-peptides.

alphaIIbbeta3 is an integrin that is involved in platelet adhesion and aggregation. This receptor may be inhibited by cysteine-rich peptides known as disintegrins. We isolated two disintegrins from Bothrops jararaca venom called jarastatin and jararacin. We evaluated the structural characteristics and the effects on human platelet aggregation of these disintegrins. Inhibitory profiles were compared to six distinct peptides synthesized based on their RGD hairpin loop primary sequences. Both jarastatin and jararacin inhibited ADP and thrombin induction. Conversely, none of the cyclic peptides showed high-quality activity in assays induced by ADP or thrombin. We constructed homology models for all of these molecules, and theoretically evaluated their interaction with the alphaIIbbeta3 crystal structure using a molecular modeling approach. These results support the observations that the cyclic peptides had little effects, and also reinforce the observation that residues outside the disintegrin RGD sequence are required for interactions with receptor.

Synthesis and anti-platelet activity of novel arylsulfonate--acylhydrazone derivatives, designed as antithrombotic candidates.

In this work, we describe a new class of promising anti-platelet drug candidates with significant antithrombotic activity in vivo. This new series of compounds was structurally planned by modification of known thrombin inhibitors based on the use of acylhydrazone subunit, as a nonpeptide scaffold, and variations at P1 moiety. Three different families of arylsulfonate-acylhydrazone derivatives were designed. The bioassays indicated the first class of derivatives represented by 4f (LASSBio-693) and 4j (LASSBio-743), which were active in inhibiting the platelet aggregation induced by thrombin. The second class represented by compounds 4e (LASSBio-774) and 4h (LASSBio-480) that selectively inhibit the platelet aggregation involving TXA(2) formation. Finally, the third class of derivatives was identified acting as a novel symbiotic agent able to inhibit the platelet aggregation induced by collagen or AA and by thrombin, represented by compounds 4b (LASSBio-694) and 4g (LASSBio-770).

The antithrombotic and haemostatic effects of LASSBio-752: a synthetic, orally active compound in an arterial and venous thrombosis model in rats.

OBJECTIVES: In this work, we further investigated the effect of the compound LASSBio-752 in thrombosis models in rats. METHODS: Arterial and venous thrombosis model, ex-vivo recalcification time and aPTT and PT. KEY FINDINGS: In the venous thrombosis model, oral administration of LASSBio-752 [48.2 mg (100 µmol)/kg] one hour before the thrombus induction decreased thrombus weight by 37 ± 0.2%. Interestingly, the antithrombotic action of this compound [48.2 mg (100 µmol)/kg] occurred at 87.5 ± 2.1% of inhibition after 24 h of administration and showed a lasting activity. When tested on the arterial thrombosis model, after a 1-h interval, there was already an increase in time to total occlusion of 34 ± 2.4 min, but the greatest effect was observed at intervals between 6 and 15 h of administration, when no occlusion of the artery was observed. The antithrombotic effect was reduced after 24 h when the occlusion time was 23.8 ± 2.3 min, close to that of the control, 17.6 ± 2.0 min. We also observed that bleeding was not excessive in any of the intervals tested. CONCLUSIONS: Our results indicate that compound LASSBio-752 is a potential candidate for utilization in the treatment of thromboembolic diseases.

Counteracting effect of glycyrrhizin on the hemostatic abnormalities induced by Bothrops jararaca snake venom.

1. Envenomation by the snake Bothrops jararaca is typically associated with hemostatic abnormalities including pro- and anticoagulant disturbances. Glycyrrhizin (GL) is a plant-derived thrombin inhibitor that also exhibits in vivo antithrombotic properties. Here, we evaluated the ability of GL to counteract the hemostatic abnormalities promoted by B. jararaca venom. 2. GL inhibited the human fibrinogen clotting (IC50 = approximately 1.0 mg ml(-1); 1.2 mM), H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride hydrolysis (IC50 = approximately 0.4 mg ml(-1); 0.47 mM) and platelet aggregation (IC50 = approximately 0.28 mg ml(-1); 0.33 mM) induced by B. jararaca venom, in vitro. 3. The in vivo effect of GL was tested in rats using a model of venous thrombosis in which intravenous (i.v.) administration of B. jararaca venom (100 microg kg(-1)) produced in all animals a thrombus with a mean weight of 10.6+/-1.7 mg. 4. Prior administration of GL (180 mg kg(-1)) or antibothropic serum (27 microl kg(-1)) inhibited thrombus formation by 86 and 67%, respectively. Remarkably, co-administration of ineffective doses of GL and antibothropic serum markedly decreased thrombus weight, suggesting a synergistic effect. 5. Co-administration of GL with antibothropic serum abolished venom-induced bleeding. Ex vivo clotting times showed that rat plasma was non-clotting after i.v. administration of B. jararaca venom. Treatment with GL, antibothropic serum or both before venom administration efficiently prevented this abnormality. 6. Altogether, we demonstrate here that GL prevents both in vitro and in vivo venom-induced changes in hemostasis, suggesting a potential antiophidic activity.

Exploiting the antithrombotic effect of the (pro)thrombin inhibitor bothrojaracin.

Bothrojaracin is a 27 kDa C-type lectin-like protein from Bothrops jararaca snake venom. It behaves as a potent thrombin inhibitor upon high-affinity binding to thrombin exosites. Bothrojaracin also forms a stable complex with prothrombin that can be detected in human plasma. Formation of the zymogen-inhibitor complex severely decreases prothrombin activation and contributes to the anticoagulant activity of bothrojaracin. In the present study, we employed two rodent models to evaluate the antithrombotic effect of bothrojaracin in vivo: stasis-induced thrombosis and thrombin-induced pulmonary thromboembolism. It was observed that bothrojaracin interacts with rat prothrombin in plasma. Ex-vivo assays showed stable complex formation even after 24 h of a single bothrojaracin dose. As a result, bothrojaracin showed significant antithrombotic activity in a rat venous thrombosis model elicited by thromboplastin combined with stasis. The antithrombotic activity of bothrojaracin (1 mg/kg) persisted for up to 24 h and it was associated with moderate bleeding as assessed by a tail transection method. Formation of bothrojaracin-prothrombin complex has been also observed following intravenous administration of the inhibitor into mice. As a result, bothrojaracin effectively protected mice from thrombin-induced fatal thromboembolism. We conclude that bothrojaracin is a potent antithrombotic agent in vivo and may serve as a prototype for the development of new zymogen-directed drugs that could result in prolonged half-life and possible decreased hemorrhagic risk.

Phenolic chemical composition of Petroselinum crispum extract and its effect on haemostasis.

From the aqueous extract (Pc) of Petroselinum crispum (Mill) flat leaves specimens were isolated and identified the flavonoids apigenin (1), apigenin-7-O-glucoside or cosmosiin (2), apigenin-7-O-apiosyl-(1 --> 2)-O-glucoside or apiin (3) and the coumarin 2",3"-dihydroxyfuranocoumarin or oxypeucedanin hydrate (4). The inhibitory activity toward clotting formation and platelet aggregation was assessed for Pc flavonoids (1) and (2), and the coumarin (4). Pc showed no inhibition on clotting activity when compared with the control. On the other hand, a strong antiplatelet aggregation activity was observed for Pc (IC50 = 1.81 mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin (IC50 = 0.18 mg/mL). In all cases ADP was used as inductor of platelet aggregation. Our results showed that Pc, apigenin and cosmosiin interfere on haemostasis inhibiting platelet aggregation. To the best of our knowledge this is the first report for the cosmosiin antiplatelet aggregation in vitro activity.

Bothrojaracin, a Bothrops jararaca snake venom-derived (pro)thrombin inhibitor, as an anti-thrombotic molecule.

Bothrojaracin (BJC) is a selective and potent thrombin inhibitor (KD = 0.6 nM) which also binds to prothrombin on proexosite I (KD = 175 nM). Incubation of BJC with human or rat plasma produced a band that co-migrates with purified prothrombin-BJC complex. We further analyzed the in vivo anti-thrombotic effect of BJC on a venous thrombosis model in rats that combines stasis and hypercoagulability. The administration of 1 mg/kg (i.v.) doses of BJC decreased thrombus weight by approximately 95%. Evaluation of the in vivo effect of BJC in mice using a pulmonary thromboembolism model induced by thrombin showed that BJC protects 100% of mice from death. Altogether, our data show that BJC is a potent anti-thrombotic agent that could further help the development of new prothrombin-directed drugs.