Antithrombotic Activity of Heparinoid G2 and Its Derivatives from the Clam Coelomactra antiquata

Clam heparinoid G2 (60.25 kDa) and its depolymerized derivatives DG1 (24.48 kDa) and DG2 (6.75 kDa) prepared from Coelomactra antiquata have been documented to have excellent fibrinolytic and anticoagulant activity. In this study, to further explore the antithrombotic activity of G2, DG1 and DG2, azure A, sheep plasma, and clot lytic rate assays were used to determine their anticoagulant and thrombolytic activity in vitro. The results indicated that the anticoagulant titer of G2 was approximately 70% that of heparin and the thrombolytic activity of DG2 was greater than G2, DG1, and heparin activities. Moreover, in a carrageenan-induced venous thrombosis model, oral administration of G2 and DG1 each at 20 mg/kg and 40 mg/kg for 7 days significantly reduced blacktail thrombus formation, increased tissue-type plasminogen activator, fibrin degradation products, and D-dimer levels, decreased von Willebrand factor and thromboxane B2 levels, and restored phylum and genus abundance changes of intestinal bacteria. DG2 had no antithrombotic effect. At 20 mg/kg, G2, DG1, and heparin had comparable antithrombotic activities, and DG1 at 40 mg/kg had more muscular antithrombotic activity than G2. Thus, DG1 could be an antithrombotic oral agent owing to its more robust antithrombotic activity and lower molecular weight.

The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

Screening of Potential Anti-Thrombotic Ingredients from Salvia miltiorrhiza in Zebrafish and by Molecular Docking

Background: Danshen (DS), the dry root of Salvia miltiorrhiza Bge., has been used in traditional Chinese medicine (TCM) for many years to promote blood circulation and to inhibit thrombosis. However, the active ingredients responsible for the anti-thrombotic effect and the underlying mechanisms are yet to be fully elucidated. Methods: Molecular docking was used to predict the active ingredients in DS and their potential targets by calculating the scores of docking between DS ingredients and thrombosis-related proteins. Then, a chemical-induced zebrafish thrombosis model was applied to confirm their anti-thrombotic effects. Result: The molecular docking results indicated that compared to the control ligand, higher docking scores were observed for several compounds in DS, among which salvianolic acid B (SAB), lithospermic acid (LA), rosmarinic acid (MA), and luteolin-7-O-β-d-glucoside (LG) could attenuate zebrafish caudal vein thrombosis and recover the decrease in heart red blood cells (RBCs) in a dose-dependent manner. Conclusions: Our study showed that it is possible to screen the potential active components in natural products by combining the molecular docking method and zebrafish in vivo model.

The Resolution Mediator Annexin A1 Affords Protection Against Thromboinflammation

Stroke is a leading cause of death and disability worldwide, with the majority (~85 %) being ischemic in origin. Age is the most important non-modifiable risk factor for acute ischemic stroke (AIS). While inflammation with ageing is a well-known complication of AIS, a new model is emerging in which ageing-associated thrombosis is being viewed as a multi-step, multi-cellular process driven by inflammatory stimuli and recruitment/activation of leukocytes. The ideal outcome of inflammation is resolution, an active process involving specific endogenous mediators (e.g. annexin A1 [AnxA1]) and related pathways (e.g. formyl peptide receptor-2 [Fpr2/ALX] pathway).[1,2] The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of thromboinflammation associated with AIS. We have shown that the AnxA1 mimetic peptide AnxA1 Ac2-26 ameliorates thrombotic responses in thromboinflammatory conditions such as Sickle Cell Disease,[3] however, the role that AnxA1 plays in age-related thrombosis is currently unknown. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in age-related thrombosis. Initially, to evaluate the role of AnxA1, thrombosis in cerebral vessels was induced using the light/dye thrombosis model.[2] Male and female adult (10-14 weeks) and ageing (18-24 months) wild type (WT, C57/BL6) or AnxA1 knock-out (AnxA1 -/-) mice were used. WT mice received AnxA1 (1 µg/mouse), or saline vehicle injected 20 min before the onset of thrombus formation in cerebral pial vessels. Thrombogenesis and blood flow cessation times were quantified. AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, an effect which was more pronounced in ageing mice (p<0.05) via regulation of the FPR2/ALX-pathway. Next, to investigate the mechanism of action of AnxA1 in an inflammatory backdrop (i.e. lipopolysaccharide [LPS]), the effect of AnxA1 on platelet P-selectin and αIIbβ3 receptor expression, following stimulation with the GPVI collagen receptor agonist convulxin (CVX), was performed. CVX treatment increased platelet activation, which was suppressed by AnxA1 co-administration (100 ng. p<0.05). CVX+LPS increased platelet αIIbβ3 or P-selectin levels, which were inhibited by the administration of AnxA1. Finally, to determine whether a deletion of AnxA1 impacts thrombosis, we performed the light/dye thrombosis model in AnxA1 −/− mice. These mice displayed accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) compared to WT mice in both arterioles and venules (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p<0.05). In conclusion, these results demonstrate the ability of AnxA1 to modify the thromboinflammatory environment, including reducing platelet activation under inflammatory conditions via GPVI. Collectively, these data show the importance of the AnxA1/Fpr2/ALX system in effecting the resolution of cerebral thromboinflammation in ageing and may provide a novel therapeutic strategy for AIS and other thromboinflammatory conditions. Disclosures No relevant conflicts of interest to declare.

Tetrahydrocurcumin Downregulates MAPKs/cPLA2 Signaling and Attenuates Platelet Thromboxane A2 Generation, Granule Secretion, and Thrombus Growth

Platelet granule secretion plays a key role in atherothrombosis. Curcumin, a natural polyphenol compound derived from turmeric, exerts multiple biological activities. The current study sought to investigate the efficacy of tetrahydrocurcumin (THC, the major active metabolite of curcumin) on platelet granule secretion in vitro and thrombus formation in vivo. We found that THC significantly attenuated agonist-induced granule secretion in human gel-filtered platelets in vitro, including CD62P and CD63 expression and platelet factor 4, CCL5, and adenosine triphosphate release. These inhibitory effects of THC were partially mediated by the attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation, leading to a decrease in thromboxane A2 (TxA2) generation. Moreover, the MAPK (Erk1/2, JNK1/2, and p38 MAPK) signaling pathways were downregulated by THC treatment, resulting in reduced cPLA2 activation, TxA2 generation, and granule secretion. Additionally, THC and curcumin attenuated murine thrombus growth in a FeCl3-induced mesenteric arteriole thrombosis model in C57BL/6J mice without prolonging the tail bleeding time. THC exerted more potent inhibitory effects on thrombosis formation than curcumin. Through blocking cyclooxygenase-1 activity and thus inhibiting platelet TxA2 synthesis and granule secretion with aspirin, we found that THC did not further decrease the inhibitory effects of aspirin on thrombosis formation. Thus, through inhibiting MAPKs/cPLA2 signaling, and attenuating platelet TxA2 generation, granule secretion, and thrombus formation, THC may be a potent cardioprotective agent.

Effect of Yiqi Huoxue Granules on Platelet Activation Induced by Thrombin

Objective. To study the effects of Yiqi Huoxue (YQHX) granules on platelet activation and aggregation induced by thrombin. Methods. The effect of YQHX on platelet aggregation rate was detected by platelet aggregation instrument; the effect of YQHX on thrombosis time was observed by the mouse mesentery thrombosis model. DAMI cells were induced to transform into platelet-like granules using PMA, and the effects of SCH (PAR-1 inhibitor) on thrombin-induced changes in platelet intracellular calcium concentration, PAR-1 protein expression, and phosphorylation of MAPK were examined. Results. Compared with the control group, the platelet aggregation rate, PAR-1 protein expression, phosphorylation of ERK1/2, and p38 protein in the YQHX group decreased ( P < 0.05 ), and there was no significant difference between the YQHX + SCH group and YQHX group ( P > 0.05 ). Conclusion. YQHX suppresses the platelet activation induced by thrombin by inhibiting PAR-1 expression.

The recombinant plant Bauhinia bauhinioides elastase inhibitor reduces rat thrombus without alterations in hemostatic parameters

The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His(6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His(6), heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His(6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His(6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His(6) (1.40 mg/kg), with the rBbCI-His(6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His(6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His(6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.