Ankaferd hemostat (ABS) as a potential mucosal topical agent for the management of COVID-19 syndrome based on its PAR-1 inhibitory effect and oestrogen content.

COVID-19 due to the SARS-CoV-2 infection is a multi-systemic immune syndrome affecting mainly the lungs, oropharyngeal region, and other vascular endothelial beds. There are tremendous ongoing efforts for the aim of developing drugs against the COVID-19 syndrome-associated inflammation. However, currently no specific medicine is present for the absolute pharmacological cure of COVID-19 mucositis. The re-purposing/re-positioning of already existing drugs is a very important strategy for the management of ongoing pandemy since the development of a new drug needs decades. Apart from altering angiotensin signaling pathways, novel drug candidates for re-purposing comprise medications shall target COVID-19 pathobiology, including pharmaceutical formulations that antagonize proteinase-activated receptors (PARs), mainly PAR-1. Activation of the PAR-1, mediators and hormones impact on the hemostasis, endothelial activation, alveolar epithelial cells and mucosal inflammatory responses which are the essentials of the COVID-19 pathophysiology. In this context, Ankaferd hemostat (Ankaferd Blood Stopper, ABS) which is an already approved hemostatic agent affecting via vital erythroid aggregation and fibrinogen gamma could be a potential topical remedy for the mucosal management of COVID-19. ABS is a clinically safe and effective topical hemostatic agent of plant origin capable of exerting pleiotropic effects on the endothelial cells, angiogenesis, cell proliferation and vascular dynamics. ABS had been approved as a topically applied hemostatic agent for the management of post-surgical/dental bleedings and healing of infected inflammatory mucosal wounds. The anti-inflammatory and proteinase-activated receptor axis properties of ABS with a considerable amount of oestrogenic hormone presence highlight this unique topical hemostatic drug regarding the clinical re-positioning for COVID-19-associated mucositis. Topical ABS as a biological response modifier may lessen SARS-CoV-2 associated microthrombosis, endothelial dysfunction, oropharyngeal inflammation and mucosal lung damage. Moreover, PAR-1 inhibition ability of ABS might be helpful for reducing the initial virus propagation and mocasal spread of COVID-19.

A novel protease-activated receptor 1 inhibitor from the leech Whitmania pigra.

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.

Designing Natural Product Hybrids Bearing Triple Antiplatelet Profile and Evaluating Their Human Plasma Stability.

Cardiovascular diseases (CVDs) are becoming major contributors to the burden of disease due to genetic and environmental factors. Despite current standard oral care, cardiovascular risk remains relatively high. A triple antiplatelet therapy with a cyclooxygenase-1 (COX-1) inhibitor, a P2Y12 receptor antagonist, and a protease-activated receptor-1 (PAR-1) antagonist has been established in the secondary prevention of atherothrombosis in patients with acute myocardial infraction and in those with peripheral artery disease. However, due to the combinatorial use of three different drugs, patients receiving this triple therapy are exposed to enhanced risk of bleeding. Conforming to polypharmacology principles, the discovery of a single compound that can simultaneously block the three platelet activation pathways (PAR-1, P2Y12, and COX-1) is of importance. Natural products have served as an inexhaustible source of bioactive compounds presenting a diverse pharmaceutical profile, including anti-inflammatory, antioxidant, anticancer, and antithrombotic activity. Indeed, principal component analysis indicated that natural products have the potential to inhibit the three aforementioned pathways, though existed reports refer to single inhibition mechanism on specific receptor(s) implicated in platelet activation. We thus set out to explore possibilities that take advantage of this potential of natural products and shape the basis to produce novel compounds that could simultaneously target PAR-1, P2Y12, and COX-1 platelet activation pathways. Polyunsaturated fatty acids (PUFAs) have multiple effects leading to improvements in blood pressure and cardiac function and arterial compliance. A promising approach to achieve the desirable goal is the bioconjugation of natural products with PUFAs. Herein, we describe the principles that should be followed to develop molecular hybrids bearing triple antiplatelet activity profile.

Neuroprotective effect of ginsenoside-Rg1 on cerebral ischemia/reperfusion injury in rats by downregulating protease-activated receptor-1 expression.

AIMS: Ginsenoside-Rg1 (G-Rg1), a saponin that is a primary component of ginseng, is very useful and important in traditional Chinese medicine for stroke. The objective of this study was to explore the mechanisms underlying the neuroprotective effect of G-Rg1 on focal cerebral ischemia/reperfusion. MAIN METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longa's 5-point scale. The brain infarct volume was determined by the 2,3,5-triphenyltetrazolium chloride staining. The permeability of the blood-brain barrier (BBB) was evaluated by Evans blue dye. Western blot and quantitative RT-PCR were used to assess protease-activated receptor-1 (PAR-1) expression. KEY FINDINGS: After G-Rg1 treatment, there was a significant decrease in the neurobehavioral function score compared with normal saline (NS) treatment after ischemia/reperfusion (P<0.05). G-Rg1 significantly reduced the infarct volume compared with NS treatment after ischemia/reperfusion (P<0.001). The permeability of the BBB was significantly decreased in the G-Rg1 group compared with the NS group (P<0.05 or P<0.01). Western blot and quantitative real time RT-PCR indicated that G-Rg1 administration down-regulated the expression of PAR-1 in the ischemic hemisphere compared with NS administration (P<0.01 and P<0.05, respectively). The level of PAR-1 expression strongly correlated with BBB permeability in both the G-Rg1- and NS-treated rats (r=0.856 and r=0.908, respectively, P<0.01). SIGNIFICANCE: G-Rg1 may ameliorate the neurological injury, the brain infarct volume and the BBB permeability induced by focal cerebral ischemia in rats and its neuroprotective mechanism is related to the down-regulation of PAR-1 expression.

TNFα causes thrombin-dependent vagal neuron apoptosis in inflammatory bowel disease.

BACKGROUND: The role of peripheral tumor necrosis factor alpha (TNFα) in inflammatory bowel disease (IBD) is well established, but its central nervous system (CNS) effects are not understood. Thrombin, another mediator of inflammation in IBD, has been implicated in CNS vagal neuron apoptosis in the dorsal motor nucleus of the vagus (DMV). This study evaluates DMV TNFα exposure, characterizes effects of TNFα on DMV neurons, and identifies a relationship between DMV TNFα and thrombin in IBD. METHODS: 2,4,6-Trinitrobenzene sulfonic acid was administered via enema to induce colonic inflammation in rats. TNFα in serum, cerebrospinal fluid (CSF), and DMV tissues were determined by ELISA and DMV TNFα expression by quantitative reverse transcription PCR (RT-PCR). TNFα was administered into the fourth intracerebral ventricle (4 V) adjacent to the DMV, with and without blockade of TNF receptor 1 (TNFR1) and the thrombin receptor proteinase-activated receptor 1 (PAR1). Immunofluorescence was used to evaluate microglial activation (Cd11b) and prothrombin presence in DMV sections. Apoptosis was examined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) and activated caspase-3 immunofluorescence. RESULTS: IBD is associated with increased TNFα protein in serum, CSF, and DMV tissue; DMV TNFα transcription is also increased. TNFα (4 V) caused a 54 % increase in microglial activation, a 27 % increase in DMV prothrombin protein, and a 31 % increase in vagal neuron apoptosis by TUNEL. There was a 52 % increase in activated caspase-3 immunofluorescence in TNFα-treated animals (p < 0.05). All effects of 4 V TNFα were prevented by TNFR1 blockade. TNFα-induced apoptosis was prevented by PAR1 blockade. CONCLUSIONS: IBD is associated with DMV exposure to TNFα, causing excess DMV prothrombin and vagal apoptosis.

Baicalin attenuates focal cerebral ischemic reperfusion injury by inhibition of protease-activated receptor-1 and apoptosis.

OBJECTIVE: To investigate the neuro-protective effects of baicalin in Wistar rats with focal cerebral ischemic reperfusion injury. METHODS: Ninety adult male Wistar rats weighing 320-350 g were randomly divided into the following groups (n=5): (a) sham control group; (b) vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; (c-e) baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 (PAR-1), PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction (RTPCR) analysis and immunohistochemistry, respectively. RESULTS: Significant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group (P<0.01). Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group (P<0.01). Compared with the low-dose baicalin group (25 mg/kg), remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group (P<0.05). CONCLUSION: Baicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis.

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical signalling pathways in pathology and physiology. Bleomycin-induced lung fibrosis has been shown to be diminished in PAR-1-deficient mice. The purpose of this study is to investigate whether pharmacological PAR-1 inhibition is a potential therapeutic option to combat pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type mice with or without a specific PAR-1 antagonist (ie, P1pal-12, a pepducin that blocks the PAR-1/G-protein interaction). Fibrosis was assessed by hydroxyproline analysis, immunohistochemistry, quantitative PCR and western blot for fibrotic markers expression. RESULTS: We first show that P1pal-12 effectively inhibits PAR-1-induced profibrotic responses in fibroblasts. Next, we show that once daily treatment with 0.5, 2.5 or 10 mg/kg P1pal-12 reduced the severity and extent of fibrotic lesions in a dose-dependent manner. These findings correlated with significant decreases in fibronectin, collagen and α smooth muscle actin expression at the mRNA and protein level in treated mice. To further establish the potential clinical applicability of PAR-1 inhibition, we analysed fibrosis in mice treated with P1pal-12 1 or 7 days after bleomycin instillation. Interestingly, when administered 7 days after the induction of fibrosis, P1pal-12 was as effective in limiting the development of pulmonary fibrosis as when administration was started before bleomycin instillation. CONCLUSIONS: Overall, targeting PAR-1 may be a promising treatment for pulmonary fibrosis.

Inhibitory effects of bromelain, a cysteine protease derived from pineapple stem (Ananas comosus), on intestinal motility in mice.

BACKGROUND: Bromelain (BR) is a cysteine protease with inhibitory effects on intestinal secretion and inflammation. However, its effects on intestinal motility are largely unexplored. Thus, we investigated the effect of this plant-derived compound on intestinal contractility and transit in mice. METHODS: Contractility in vitro was evaluated by stimulating the mouse isolated ileum, in an organ bath, with acetylcholine, barium chloride, or electrical field stimulation. Motility in vivo was measured by evaluating the distribution of an orally administered fluorescent marker along the small intestine. Transit was also evaluated in pathophysiologic states induced by the pro-inflammatory compound croton oil or by the diabetogenic agent streptozotocin. KEY RESULTS: Bromelain inhibited the contractions induced by different spasmogenic compounds in the mouse ileum with similar potency. The antispasmodic effect was reduced or counteracted by the proteolytic enzyme inhibitor, gabexate (15 × 10(-6) mol L(-1) ), protease-activated receptor-2 (PAR-2) antagonist, N(1) -3-methylbutyryl-N(4) -6-aminohexanoyl-piperazine (10(-4) mol L(-1) ), phospholipase C (PLC) inhibitor, neomycin (3 × 10(-3) mol L(-1) ), and phosphodiesterase 4 (PDE4) inhibitor, rolipram (10(-6) mol L(-1) ). In vivo, BR preferentially inhibited motility in pathophysiologic states in a PAR-2-antagonist-sensitive manner. CONCLUSIONS & INFERENCES: Our data suggest that BR inhibits intestinal motility - preferentially in pathophysiologic conditions - with a mechanism possibly involving membrane PAR-2 and PLC and PDE4 as intracellular signals. Bromelain could be a lead compound for the development of new drugs, able to normalize the intestinal motility in inflammation and diabetes.

Green tea polyphenol epigallocatechin-3-gallate inhibits thrombin-induced hepatocellular carcinoma cell invasion and p42/p44-MAPKinase activation.

Thrombin has been recently demonstrated to promote hepatocellular carcinoma (HCC) cell migration by activation of the proteinase-activated receptor (PAR) subtypes PAR1 and PAR4 suggesting a role of these proteinase-receptor systems in HCC progression. In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic compound of green tea on thrombin-PAR1/PAR4-mediated hepatocellular carcinoma cell invasion and p42/p44 MAPKinase activation. In this study we used the permanent liver carcinoma cell line HEP-3B and two primary cultures established from surgically resected HCCs. We found that stimulation of HCC cells with thrombin, the PAR1-selective activating peptide, TFLLRN-NH2, and the PAR4-selective activating peptide, AYPGKF-NH2, increased cell invasion across a Matrigel-coated membrane barrier and stimulated activation of p42/p44 MAPKinase phosphorylation. Both the effects on p42/p44 MAPKinases, and on cell invasiveness induced by thrombin and the PAR1/4 subtype-selective agonist peptides were effectively blocked by EGCG. The results clearly identify EGCG as a potent inhibitor of the thrombin-PAR1/PAR4-p42/p44 MAPKinase invasive signaling axis in hepatocellular carcinoma cells as a previously unrecognized mode of action for EGCG in cancer cells. Moreover, the results suggest that (-)-epigal-locatechin-3-gallate might have therapeutic potential for hepatocellular carcinoma.

Inhibition of PAR4 signaling mediates ethanol-induced attenuation of platelet function in vitro.

BACKGROUND: Reduction in coronary heart disease morbidity in response to moderate consumption of alcoholic beverages may be partly mediated by ethanol-induced inhibition of platelet function. However, the precise mechanisms by which ethanol modulates platelet activation induced by thrombin, which plays a central role in hemostasis, remain unclear. The goal of this study was to investigate ethanol-induced changes in platelet function and clarify the underlying mechanisms including PAR1 and PAR4 activity and [Ca2+]i dynamics in vitro. METHODS: Platelet aggregation, increase in intracellular calcium ([Ca2+]i), and release of platelet factor 4 and beta-thromboglobulin induced by alpha-thrombin, PAR1-agonist peptide (AP), or PAR4-AP were assessed in the presence or absence of ethanol. RESULTS: Ethanol exposure inhibited low-dose thrombin (0.5 nM)-induced aggregation but not an increase in [Ca2+]i. In contrast, ethanol had no effect on high-dose thrombin (10 nM)-induced aggregation or the [Ca2+]i increase. Ethanol did not significantly inhibit thrombin-induced release of platelet factor 4 and beta-thromboglobulin. Ethanol reduced PAR1-AP-induced aggregation, but did not affect the spike form of [Ca2+]i increase. In contrast, ethanol inhibited the increase in [Ca2+]i as well as the aggregation in response to PAR4-AP and resulted in delayed [Ca2+]i peak time. Furthermore, ethanol inhibited both PAR1-AP- and PAR4-AP-induced platelet factor 4 and beta-thromboglobulin release. CONCLUSIONS: These data suggest that ethanol inhibits platelet aggregation via inhibition of PAR4 signaling and subsequent inhibition of Ca2+ influx and granule release. This phenomenon may contribute to the reduction in coronary heart disease morbidity in response to consumption of alcoholic beverages.