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Cardiovascular diseases caused by blood coagulation system disorders are one of the leading causes of morbidity and mortality in the world. Research shows that blood clotting factors are involved in these thrombotic processes. Among them, factor Xa occupies a key position in the blood coagulation cascade. Another coagulation factor, XIa, is also a promising target because its inhibition can suppress thrombosis with a limited contribution to normal hemostasis. In this regard, the development of dual inhibitors as new generation anticoagulants is an urgent problem. Here we report the synthesis and evaluation of novel potential dual inhibitors of coagulation factors Xa and XIa. Based on the principles of molecular design, we selected a series of compounds that combine in their structure fragments of pyrrolo[3,2,1-ij]quinolin-2-one and thiazole, connected through a hydrazine linker. The production of new hybrid molecules was carried out using a two-stage method. The reaction of 5,6-dihydropyrrolo[3,2,1-ij]quinoline-1,2-diones with thiosemicarbazide gave the corresponding hydrazinocarbothioamides. The reaction of the latter with DMAD led to the target methyl 2-(4-oxo-2-(2-(2-oxo-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1(2H)-ylidene)hydrazineyl)thiazol-5(4H)-ylidene)acetates in high yields. In vitro testing of the synthesized molecules revealed that ten of them showed high inhibition values for both the coagulation factors Xa and XIa, and the IC[sub.50] value for some compounds was also assessed. The resulting structures were also tested for their ability to inhibit thrombin.

Abstract Objective To compare the impact on thrombin generation of the new combined oral contraceptive containing 15 mg estetrol and 3 mg drospirenone with ethinylestradiol (30 or 20 mcg) associated either with 150 mcg levonorgestrel or with 3 mg drospirenone. Methods Data were collected from the “E4/DRSP Endocrine Function, Metabolic Control and Hemostasis Study” (NCT02957630). Overall, the per-protocol set population included 24 subjects in the ethinylestradiol/levonorgestrel arm, 28 subjects in the ethinylestradiol/drospirenone arm, and 34 subjects in the estetrol/drospirenone arm. Thrombograms and thrombin generation parameters (lag time, peak, time to peak, endogenous thrombin potential, and mean velocity rate index) were extracted for each subject at baseline and after 6 cycles of treatment. Results After 6 cycles of treatment, ethinylestradiol-containing products arms show a mean thrombogram outside the upper limit of the reference range, that is the 97.5th percentile of all baseline thrombograms. On the other hand, the mean thrombogram of estetrol/drospirenone is within this reference interval. After 6 cycles of treatment, all thrombin generation parameters are statistically less affected by estetrol/drospirenone than ethinylestradiol-containing products. Conclusions In conclusion, an association of 15 mg estetrol with 3 mg drospirenone does not have an impact on thrombin generation compared with ethinylestradiol-containing products that, either associated with levonorgestrel or drospirenone, are able to increase the production of procoagulant factors and decrease the production of anticoagulant ones, shifting the patient to a prothrombotic state. Ethinylestradiol-containing products thus generate prothrombotic environments contrary to estetrol which demonstrates a neutral profile on hemostasis.

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than −8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.

DNA origami-based nanorobot presents thrombin to cause tumor infarction after specific recognition of a tumor vessel marker. Nanoscale robots have potential as intelligent drug delivery systems that respond to molecular triggers 1 , 2 , 3 , 4 . Using DNA origami we constructed an autonomous DNA robot programmed to transport payloads and present them specifically in tumors. Our nanorobot is functionalized on the outside with a DNA aptamer that binds nucleolin, a protein specifically expressed on tumor-associated endothelial cells 5 , and the blood coagulation protease thrombin within its inner cavity. The nucleolin-targeting aptamer serves both as a targeting domain and as a molecular trigger for the mechanical opening of the DNA nanorobot. The thrombin inside is thus exposed and activates coagulation at the tumor site. Using tumor-bearing mouse models, we demonstrate that intravenously injected DNA nanorobots deliver thrombin specifically to tumor-associated blood vessels and induce intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth. The nanorobot proved safe and immunologically inert in mice and Bama miniature pigs. Our data show that DNA nanorobots represent a promising strategy for precise drug delivery in cancer therapy.

Thrombin generation (TG) is reduced after cardiac surgery using cardiopulmonary bypass (CPB), contributing to coagulopathy and bleeding. Plasma transfusion or four-factor prothrombin complex concentrate (PCC) are commonly used to treat coagulopathic bleeding after CPB without knowledge of how each may restore TG. To determine the effect of PCC infusion on restoration of thrombin generation compared with plasma transfusion, we performed a laboratory-based secondary analysis of a randomized, controlled trial of adult patients undergoing cardiac surgery to assess efficacy and safety of 4 F-PCC versus plasma for treatment of perioperative coagulopathic bleeding after CPB. Participants were randomized to receive either PCC (15 IU/kg) or plasma (10–15 ml/kg) after separation from CPB. Participant blood samples were obtained at pre-specified serial timepoints, with laboratory assays for TG and factor levels subsequently performed. The primary outcome was change in thrombin generation (TG) parameters after each randomized treatment through postoperative day 5. Secondary outcomes included serially derived clotting factor levels. Of 100 randomized participants, 99 were included in this laboratory analysis (PCC group, N  = 51; plasma group, N  = 48). After treatment, participants in the PCC group compared with those in the plasma group showed higher endogenous thrombin potential (ETP, Median, Interquartile range, IQR: 688 [371–1069] vs. 1088 [550–1691] nM minutes, P  = 0.01), a greater increase din ETP ( P  = 0.002) and peak TG ( P  = 0.01) in the timepoints between heparin reversal and after treatment administration. Both groups demonstrated similar values in all TG assays by postoperative day 1 ( P  > 0.05). The PCC group also demonstrated higher levels of proteins C, S, and Factors II, VII, IX and X, early after treatment ( P  < 0.001 for all comparisons). Antithrombin levels were initially higher in the plasma group after treatment (Median, IQR: 66% [61-71%] vs. 56% [51-65%], P  = 0.002) but differences did not persist beyond postoperative day 3. In this laboratory analysis from a recent randomized trial in adult cardiac surgery, PCC administration restored thrombin generation more rapidly than plasma in the early postoperative period without laboratory evidence of hypercoagulability. ClinicalTrials.gov identifier: NCT02557672 [1]. Key points Question How does prothrombin complex concentrate (PCC) compare with plasma to restore thrombin generation (TG) in patients with coagulopathic bleeding after cardiopulmonary bypass? Findings In this subanalysis of a randomized clinical trial in 99 adult cardiac surgical patients, TG was restored more rapidly in the PCC group compared with the plasma group after treatment: endogenous thrombin potential (PCC, 688 [371–1069] vs. plasma, 1088 [550–1691], P  = 0.01nM minutes). Both groups displayed similar post-treatment TG by postoperative day one. Meaning These results support PCC administration to restore TG while avoiding acquired hypercoagulability in the early period after cardiac surgery.

Background/Objectives: Thrombin, a serine protease central to coagulation and platelet activation, remains an important target for the development of safer and more effective antithrombotic agents. Naturally derived pentacyclic triterpenoids, such as betulinic acid and its acetylated derivatives, 3β-acetoxybetulinic acid, exhibit promising antiplatelet aggregation activity in validated in vitro and ex vivo assays; however, the molecular determinants underlying their direct thrombin inhibition remain unexplored. Results: Docking and MM/GBSA analyses revealed that Baa exhibits the strongest binding affinity (ΔG = −29.58 ± 2.97 kcal/mol), exceeding those of Ba (−20.94 ± 5.81 kcal/mol) and Asp (−18.87 ± 4.18 kcal/mol). Baa forms a highly persistent hydrogen bond with Trp96 (95.5% occupancy) and extensive hydrophobic contacts with Trp215, Leu99, Ile174, and Tyr60A residues defining thrombin’s aryl-binding pocket. MD trajectories demonstrated that Baa binding reduced solvent-accessible surface area (SASA) and residue fluctuations, indicating enhanced structural compaction and stability. In contrast, Ba exhibited weaker, transient hydrogen bonding, while Asp bound primarily near the catalytic triad. The triterpenes exhibit limited oral bioavailability, free PAINS alerts, favourable permeability and metabolic stability. Conclusions: Acetylation at C-3 (acetoxy substitution) substantially enhances thrombin binding via cooperative hydrogen bonding and van der Waals stabilisation, explaining the superior experimental inhibitory potency of Baa. These findings provide a mechanistic framework for structure-guided optimisation of triterpenoid-based thrombin inhibitors and support their further experimental development. Methods: In this study, molecular docking, molecular dynamics (MD) simulations (400 ns), and MM/GBSA free energy analyses were employed to elucidate the binding mechanisms of 3β-acetoxybetulinic acid (Baa), betulinic acid (Ba), and aspirin (Asp) within the thrombin receptor active site. The simulations were explicitly grounded in previously reported chromogenic antithrombin assays and platelet aggregation studies and were designed to mechanistically rationalise the experimentally observed inhibitory potency.

Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as with biological drugs, most cyclic peptides cannot be applied orally because they are rapidly digested and/or display low absorption in the gastrointestinal tract, hampering their development as therapeutics. In this study, we developed a combinatorial synthesis and screening approach based on sequential cyclization and one-pot peptide acylation and screening, with the possibility of simultaneously interrogating activity and permeability. In a proof of concept, we synthesized a library of 8,448 cyclic peptides and screened them against the disease target thrombin. Our workflow allowed multiple iterative cycles of library synthesis and yielded cyclic peptides with nanomolar affinities, high stabilities and an oral bioavailability (%F) as high as 18% in rats. This method for generating orally available peptides is general and provides a promising push toward unlocking the full potential of peptides as therapeutics. Cyclic peptides show promise for modulating difficult disease targets; however, they often cannot be administered orally. The authors developed a method to synthesize and screen large libraries of small cyclic peptides while enabling the simultaneous interrogation of activity and permeability. This approach was applied to the disease target thrombin to discover peptides with high affinity, stability and oral bioavailability of up to 18% in rats.