Hsp47 Inhibitor Col003 Attenuates Collagen-Induced Platelet Activation and Cerebral Ischemic–Reperfusion Injury in Rats

Ischemic stroke is a major type of stroke worldwide currently without effective treatment, although antiplatelet therapy is an existing option for it. In previous studies, heat shock protein 47 (Hsp47) was found to be expressed on the surface of human and mice platelets and to strengthen the interaction between platelets and collagen. In recent years, Col003 was discovered to inhibit the interaction of Hsp47 with collagen. We evaluated whether the Hsp47 inhibitor Col003 is a promising therapeutic agent for ischemic stroke. Here, we first verified that Hsp47 is also expressed on the surface of rat platelets, and its inhibitor Col003 significantly inhibited thrombus formation in the FeCl3-induced rat carotid arterial thrombus model. Both Col003 and clopidogrel did not alter the bleeding time or coagulation parameters, while aspirin increased the tail-bleeding time (p < 0.05). The low cytotoxicity level of Col003 to rat platelets and human liver cells was similar to those of aspirin and clopidogrel. Col003 inhibited collagen-induced platelet aggregation, adhesion, [Ca2+]i mobilization, P-selectin expression, reactive oxygen species production and the downstream signal pathway of collagen receptors. The results of the middle cerebral artery occlusion model indicated that Col003 has a protective effect against cerebral ischemic–reperfusion injury in rats. The Hsp47 inhibitor Col003 exerted antiplatelet effect and protective effect against brain damage induced by ischemic stroke through the inhibition of glycoprotein VI (GPVI)and mitogen-activated protein kinase (MAPK) signaling events, which might yield a new antiplatelet agent and strategy to treat ischemic stroke.

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity

Introduction: Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. Aim: The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. Materials and methods: Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. Results: SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-induced RBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. Conclusions: This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Antiplatelet and Antithrombotic Effects of Isaridin E Isolated from the Marine-Derived Fungus via Downregulating the PI3K/Akt Signaling Pathway

Isaridin E, a cyclodepsipeptide isolated from the marine-derived fungus Amphichorda felina (syn. Beauveria felina) SYSU-MS7908, has been demonstrated to possess anti-inflammatory and insecticidal activities. Here, we first found that isaridin E concentration-dependently inhibited ADP-induced platelet aggregation, activation, and secretion in vitro, but did not affect collagen- or thrombin-induced platelet aggregation. Furthermore, isaridin E dose-dependently reduced thrombosis formation in an FeCl3-induced mouse carotid model without increasing the bleeding time. Mechanistically, isaridin E significantly decreased the ADP-mediated phosphorylation of PI3K and Akt. In conclusion, these results suggest that isaridin E exerts potent antithrombotic effects in vivo without increasing the risk of bleeding, which may be due to its important role in inhibiting ADP-induced platelet activation, secretion and aggregation via the PI3K/Akt pathways.

Coagulation profile in two nephropathic dogs

Coagulatory abnormalities are common in renal dysfunction in humans. The studies on coagulatory abnormalities in renal failure in dogs are limited. The present paper deals with coagulation profile in acute and chronic kidney disease in dogs. The haemostatic defects observed in acute renal dysfunction included thrombocytopaenia, prolonged capillary bleeding time (CBT), elevated D-Dimer and hypoantithrombinemia which indicated a hypercoagulable state. Prolongation of prothrombin time (PT), activated partial thromboplastin time (aPTT), elevated D-Dimer concentration and hypoantithrombinemia in chronic kidney disease indicated the presence of hypocoagulable state

Bismuth Subgallate as a Local Hemostatic Agent

Background: Patients on clopidogrel increased bleeding risk after surgery. This drug prolonged bleeding time, increased bleeding volume and induced secondary bleeding because its active metabolite inhibited platelets aggregation and interfered with haemostatic plug stabilization. Conventional methods, such as pressing sterile gauze on the surgery site, showed less effective to stop bleeding in patients on clopidogrel. This research aims to prove the haemostatic effect of bismuth subgallate both on normal and delayed platelet aggregation due to clopidogrel.Methods: Twenty-eight Wistar rats were equally and randomly administered with clopidogrel (10 mg/kgBW) or NaCl 0.9% (saline) via oral gavage. After anesthetizing, we amputated transversely their tail 10 mm from the distal tip. Bleeding after amputation was controlled with pressing gauze soaked in saline or bismuth subgallate solution. After 60 seconds, bleeding assays (bleeding time, bleeding volume, and secondary bleeding) have been observed, recorded, and analysed both in normal and clopidogrel groups.Results: Clopidogrel groups had significantly longer bleeding time, greater bleeding volume, and had more secondary bleeding rather than saline groups (p <.05). Using bismuth subgallate as local haemostatic agent decreased bleeding time and bleeding volume significantly (p <.05) both in normal and clopidogrel groups. Conclusions: Bismuth subgallate has a haemostatic effect on both clopidogrel and normal rat tail bleeding models.

EGb 761® Does Not Affect Blood Coagulation and Bleeding Time in Patients with Probable Alzheimer’s Dementia—Secondary Analysis of a Randomized, Double-Blind Placebo-Controlled Trial

Following reports of bleeding upon Ginkgo intake, we assessed whether Ginkgo extract EGb 761® affects coagulation or platelet function or increases the risk of bleeding. In a double-blind, placebo-controlled trial, prothrombin time, activated partial thromboplastin time, international normalized ratio and bleeding time were measured in patients with Alzheimer’s dementia at baseline, weeks 6 and 26. A total of 513 patients were randomized to 120 mg (n = 169) or 240 mg EGb 761® (n = 170) or placebo (n = 174). No relevant changes were found for coagulation parameters and bleeding time. Numbers of bleeding-related adverse events were similar in all groups. Concomitant intake of acetylsalicylic acid was documented for 68 patients in the placebo group and 105 in the EGb 761® groups. Within these groups, the means at baseline and week 26 differed by less than 1 unit for prothrombin time and bleeding time and less than 0.1 unit for international normalized ratio. Data on warfarin treatment in nine patients each taking placebo or EGb 761® did not indicate enhancement of warfarin effects by EGb 761®. No evidence was found that EGb 761® affects hemostasis or increases the bleeding risk. No pharmacodynamic interactions with warfarin or acetylsalicylic acid were found.

Overview of the Effectiveness of Aspirin, Ibuprofen, Acetaminophen On Teeth

The usage of medications like aspirin, ibuprofen and acetaminophen is well-accepted as pain relieving agents worldwide. Aspirin is a medication that is used not only for relieving pain but also for decreasing the risk of heart attack. Acetaminophen and ibuprofen have a significant role in decreasing chronic pain and inflammation. There is a deep connection between the dental procedures and the patients medicine intake. These effects can be known as side effects and damage the natural structure of the tooth or can be any disturbance in dental procedures especially in surgery and tooth extractions. Recognition of common medications and their effects is an important aspect not only for physicians but also for dentists and oral surgeons. The database of this article was derived from valid sources such as PubMed, Google scholar and Web of Science. The search method included the primary keywords like “aspirin”, “ibuprofen” and “acetaminophen” combined with “teeth”, “tooth extraction”, “oral cavity”, “dental procedures'', and/or “bleeding time”. Our results illustrated that a long-term application of aspirin favorably affects the patients who are suffering from angina, stroke or myocardial infarctions. Other findings confirmed that ibuprofen has antipyretic, analgesic and anti – inflammatory features. Effective characteristics of paracetamol as the medicine of choice for the treatment of post-exodontia pain was demonstrated according to several studies. In this review article we provide details about the effects of the three aforementioned medications, their usage in the dental field and any adverse effects on healthy tooth structures.

DMAG, a novel countermeasure for the treatment of thrombocytopenia

Background Thrombocytopenia is one of the most common hematological disease that can be life-threatening caused by bleeding complications. However, the treatment options for thrombocytopenia remain limited. Methods In this study, giemsa staining, phalloidin staining, immunofluorescence and flow cytometry were used to identify the effects of 3,3ʹ-di-O-methylellagic acid 4ʹ-glucoside (DMAG), a natural ellagic acid derived from Sanguisorba officinalis L. (SOL) on megakaryocyte differentiation in HEL cells. Then, thrombocytopenia mice model was constructed by X-ray irradiation to evaluate the therapeutic action of DMAG on thrombocytopenia. Furthermore, the effects of DMAG on platelet function were evaluated by tail bleeding time, platelet aggregation and platelet adhesion assays. Next, network pharmacology approaches were carried out to identify the targets of DMAG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate the underling mechanism of DMAG against thrombocytopenia. Finally, molecular docking simulation, molecular dynamics simulation and western blot analysis were used to explore the relationship between DAMG with its targets. Results DMAG significantly promoted megakaryocyte differentiation of HEL cells. DMAG administration accelerated platelet recovery and megakaryopoiesis, shortened tail bleeding time, strengthened platelet aggregation and adhesion in thrombocytopenia mice. Network pharmacology revealed that ITGA2B, ITGB3, VWF, PLEK, TLR2, BCL2, BCL2L1 and TNF were the core targets of DMAG. GO and KEGG pathway enrichment analyses suggested that the core targets of DMAG were enriched in PI3K–Akt signaling pathway, hematopoietic cell lineage, ECM-receptor interaction and platelet activation. Molecular docking simulation and molecular dynamics simulation further indicated that ITGA2B, ITGB3, PLEK and TLR2 displayed strong binding ability with DMAG. Finally, western blot analysis evidenced that DMAG up-regulated the expression of ITGA2B, ITGB3, VWF, p-Akt and PLEK. Conclusion DMAG plays a critical role in promoting megakaryocytes differentiation and platelets production and might be a promising medicine for the treatment of thrombocytopenia. Graphical Abstract

A Quantitative Systems Pharmacology (QSP) Model to Compare the Non-Clinical Biodistribution and Efficacy between Recombinant Factor IX (rIX) Therapies

Background and Aims: Replacement FIX therapy (rIX) is an effective treatment for hemophilia B even with undetectable levels in the blood 1. However, the mechanistic reason for hemostasis with low plasma levels is not well understood. There is growing evidence that FIX interactions with one or multiple binding partners (BP), may play a significant role in the exposure and hemostatic efficacy of rIX 2,3. The aim of this study is to explore this hypothesis by comparing the plasma PK, tissue biodistribution, and in vivo endpoints of different rIX variants using a mouse QSP model. Method: in vitro and in vivo FIX-KO mice studies and mathematical models were used to build a QSP model consisting of 8 tissue compartments , with each tissue divided into vascular, endothelial and interstitial spaces 4,5. The model simulates endogenous mouse IgG (mIgG), mouse serum albumin (MSA), and rIX dynamics including key clearance and distribution mechanisms. Competition for the endothelial FcRn receptor between Fc, albumin, mIgG, and MSA is explicitly modeled 6,7,8. The model was calibrated using mouse studies of radiolabeled rIX-Fc (Alprolix®), rIX-WT (BeneFIX®), and rIX-FP (Idelvion®). Tail-clip experiments following administration of rIX-Fc, rIX-WT, and rIX-FP were used to correlate the predicted exposures with the observed effects on bleeding time and total blood loss. Results: Preliminary simulations proved that having at least one BP best explains the rapid distribution of rIX-Fc and rIX-WT into the tissues, and the long plasma T 1/2 of rIX-Fc and rIX-FP. Visual predictive checks of the full PBPK model showed good agreement with the PK in the tissues. The best fit was achieved using a specific arrangement of four distinct binding partners: Shared BP (SBP) between all compounds (e.g. N-terminal binder) located within the vasculature with estimated K D of 470/600/4100 nM, for rIX-WT/rIX-FP/rIX-Fc, respectively. BP binding specific to rIX-WT (e.g. C-terminal binder) located in the interstitium of the tissue (varying densities) with estimated K D of 23 nM BP binding only for rIX-FP (e.g. albumin binder) located in both; the vasculature and interstitium of the tissue with estimated K D 20/0.05 μM (vascular/interstitial) BP binding only for rIX-Fc (e.g. Fc binder) located in the interstitium of tissue (varying densities) with estimated K D 3 μM The high degree of extravasation of rIX-Fc (and rIX-WT to a lesser degree) results in rapid distribution and sequestration in the tissues. The limited extravasation of rIX-FP and its high affinity to the SBP, results in increased recovery and a greater pool of bound rIX available in the tissue vasculature. Additionally, strong inverse correlation between the bound rIX in the vasculature and bleeding time/total blood loss suggests that the vascular pool plays a more significant role in FIX pharmacology, as compared to the pool in the extravascular space. Conclusion: The mouse QSP model demonstrated that the plasma and tissue biodistribution of rIX-Fc, rIX-FP, and rIX-WT cannot be explained without a BP, and that it is plausible to assume that different binding partners, both intra- and extravascular, for different rFIX variants exist. The correlation between the levels of bound rIX and the coagulation endpoints suggests that the vascular bound rIX may be the pharmacologically active pool or reservoir for haemostasis. The extravasation and sequestration of rIX-WT and rIX-Fc into the tissues may explain the decreased vascular exposure, and hence, the reduced efficacy (increased bleeding time/total blood loss) at later time points. Although the exact identity of the BP's remains to be further elucidated, the model estimates of their affinity, density and location provide guidance for further experimental investigations. Expansion of the QSP model with additional data and coagulation kinetics will further our understanding of the role of BPs in rIX pharmacology. References 1Srivastava A et al (2013) Haemophilia 19(1), e1-47 2Feng D et al (2013) JTH, Vol. 11 (12), 2176-2178 3Cheung WF et al (1996) PNAS USA, 93(20), 11068-11073 4Li L et al (2014) AAPS Journal 16(5), 1097-1109 5Shah DK & Betts AM (2012) J Pharmacokinet Pharmacodyn 39(1), 67-86 6Chia J et al (2018) J Biol Chem 293(17), 6363-6373 7Andersen JT et al (2010) J Biol Chem 285(7), 4826-4836 8Andersen JT et al (2013) J Biol Chem 288(33), 24277-24285 Disclosures Pestel: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Rezvani-Sharif: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Muir: CSL Behring Ltd: Current Employment, Current holder of stock options in a privately-held company. Krupa: CSL Behring LLC: Current Employment, Current equity holder in publicly-traded company. Brechmann: CSL Behring Innovation GmbH, Ended employment in the past 24 months: Bayer Ag (Bayer Pharmaceuticals),: Current Employment, Ended employment in the past 24 months, Patents & Royalties: Bayer. Verhagen: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Dower: CSL Behring Ltd: Current Employment, Current equity holder in publicly-traded company. Herzog: CSL Behring GmbH: Current Employment, Current equity holder in publicly-traded company.

Pharmacokinetics and Pharmacodynamics of Recombinant FIX Variants in the FIX Knockout Mouse Tail Clip Bleeding Model

Introduction: The recessive X-linked bleeding disorder Hemophilia B is caused by a mutation in the coagulation factor (F) IX gene leading to partial or total loss of its function. Preventive treatment with replacement long-acting FIX is an attractive option for patients to reduce administration frequency and prevent bleeding. New recombinant FIX therapeutics like the albumin-fused FIX (rFIX-FP) or the Fc-fused FIX (rFIX-Fc) enable longer half-life in circulation and thus less frequent administration, as compared to non-fused FIX (rFIX). Studies in FIX knockout (KO) mice were conducted to characterize the effect of the modifications on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the different recombinant FIX products. Methods: Pharmacokinetics: Recombinant FIXs were administered intravenously at doses of 25 nmol/kg (corresponding to ~175-400 IU/kg FIX clotting activity) to FIX KO mice. Blood samples were collected starting at 5 min, and up to 336 h. FIX plasma levels were measured with an ELISA-based assay with anti-human FIX paired antibodies. PK was evaluated by non-compartmental analysis. Biodistribution: 3H-labeled recombinant FIXs were administered intravenously at doses of 200 IU/kg to FIX KO mice. Plasma levels and organ distribution were quantified starting at 15 min, and up to 240 h. Pharmacodynamics: FIX KO mice were treated intravenously with 50 IU/kg FIX clotting activity (nominal or labeled potency) of different rFIX products at 24, 72, 120 168 and 336 h prior to determination of bleeding time and total blood loss in a tail clip bleeding model. Immediately upon lesion, the tail tip was submerged in isotonic saline (0.9 %), kept at the mice physiological body temperature. Time to hemostasis is quantified as the time until bleeding stops for a minimum of 2 min. The volume of total blood loss was calculated by measuring the hemoglobin present in the isotonic saline solution at the end of the experiment. Results: Distinct PK profiles were observed for the three FIX molecules, where rFIX and rFIX-Fc exhibit an initial rapid distribution phase from plasma, while rFIX-FP showed a monophasic elimination profile up to 120 h post administration (p.a.). In the terminal phase, rFIX levels were quantifiable for up to 48 h p.a., while both; rFIX-FP and rFIX-Fc were measurable in plasma up to 240 h p.a. In line with this, the overall exposure AUC 0-inf is ranked in the following order: rFIX-FP &gt; rFIX-Fc &gt; rFIX. In the biodistribution study, a similar plasma PK profile was determined. Given the sensitivity of the radioactive method, an exposure plateau was observed for rFIX-Fc, and at lower levels for rFIX, whereas rFIX-FP continued to exhibit monophasic plasma clearance behavior. rFIX-FP exposure in the extravascular space (EVS) was lower than for the other FIX products. This is in line with volumes of distribution (Vss and Vz) which were highest for rFIX-Fc (AUC ranking rFIX-Fc &gt; rFIX &gt; rFIX-FP). FIX hemostatic efficacy in tail clip model was comparable for all 3 FIXs at the early time points but diverged at later time points post dosing. The blood loss and bleeding time measurements returned to baseline within 168 h for rFIX and rFIX-Fc, while the rFIX-FP group maintained robust hemostatic activity for up to 336 h. In contrast to lowest tissue exposure of rFIX-FP, the plasma AUC for rFIX-FP was highest, compared to FIX-Fc or FIX. In line, efficacy over time was also highest for rFIX-FP, suggesting that tissue exposure might not be the main driver for hemostatic activity. Conclusion: Different FIX products exhibit divergent PK and PD behaviors. rFIX-FP plasma PK profile suggests somewhat lower tissue distribution in comparison to rFIX-Fc and rFIX, which was confirmed in the tissue biodistribution study. Despite its limited extravasation into tissue, rFIX-FP exhibits superior and prolonged hemostatic activity in the FIX KO mouse tail clip model. rFIX and rFIX-Fc show comparable tissue biodistribution behavior, with robust extravasation into the EVS. Despite having the longest half-life and overall (plasma and tissue) exposure in the mouse, rFIX-Fc lost hemostatic activity in the tail clip model significantly faster than rFIX-FP. As a result, hemostatic efficacy was highest for the FIX-FP, the product with the lowest distribution volumes. The results therefore suggest that EVS is not the main determining factor for FIX efficacy in vivo. Disclosures Pestel: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Peil: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Knoll Machado: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Claar: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Raquet: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Ponnuswamy: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Mischnik: CSL Behring Innovation GmbH: Current Employment, Current equity holder in publicly-traded company. Herzog: CSL Behring GmbH: Current Employment, Current equity holder in publicly-traded company.