ABSTRACT
Violations of the hemostasis (blood aggregation control, BAC) system in patients with COVID-19 in the acute period and at the stage of convalescence have been studied and methods of targeted correction of the identified disorders are considered. Prevention of serious complications related to COVID-19 infection requires complex assessment of the hemostasis system and prompt correction of disorders. Methods of clinical hemostasiograms and low-frequency piezothromboelastography (LPTEG) provide comprehensive and informative assessment of functional state of the BAC system and monitoring of the effectiveness of therapy, both in hospital and on outpatient basis. It was established that hemostasis system disorders had unspecified character with hyper- or hypocoagulation in the acute period and structural or chronometric hypercoagulation in the recovery period. Under LPTEG monitoring in hospital, the identified disorders were corrected by low-molecular-weight (LMW) heparins, blood-based preparations, and fibrinolysis inhibitors;at the outpatient stage, the therapy was supplemented with sulodexide and anticoagulants. Personalized correction of the hemostatic potential was based on the following LPTEG parameters. Prescription of the anti-aggregant and vasoprotective therapy required that the response time (t1) would be reduced below 0.9 min and thrombin activity (TA) constant increased above 40 relative units. The anticoagulant therapy was prescribed when the gelation point (t3) decreased to 4.7 min and the coagulation drive intensity (CDI) index was above 50 relative units. The fibrinolytic activity was corrected when the clot polymerization intensity (CPI) index was above 20 relative units, the cross-linked fibrin formation time (t5) decreased to 27 min, and the clot retraction and lysis intensity (CRLI) index exceeded 15%. The boundary values of these LPTEG parameters were adjusted at the levels of moderate hypercoagulation or reference normal coagulation. The LPTEG monitoring and personalization of the prescribed antithrombotic therapy allowed the risk of thrombo-hemorrhagic complications to be reduced at all stages of COVID-19 treatment.Copyright © 2021 Authors. All rights reserved.
ABSTRACT
Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3“-galloyl-4”,6“-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh, inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. [Formula presented] Disclosures: No relevant conflicts of interest to declare.