Recombinant-activated factorⅦand neuronal apoptosis in a rat model of intracerebral hemorrhage

BACKGROUND： Activated clotting factor VII has been demonstrated to exhibit obvious anti-apoptosis effects. OBJECTIVE： To observe the effect of activated clotting factor VII on neuronal apoptosis at different time points following rat intracerebral hemorrhage （ICH）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Neurobiological Laboratory of Second Military Medical University from October 2005 to April 2006. MATERIALS： Recombinant-activated clotting factor Vlla （rFVtla） was purchased from Danish Novo Nordisk, Denmark. In situ cell death detection kit-POD kit was purchased from Roche, Switzerland. Caspase-3 activity determination kit from Biovision, USA. METHODS： A total of 72 healthy, male, Sprague Dawley rats, aged 5-8 months, were randomly assigned to three groups （n = 24）： sham-operated, ICH model, and rFVIla. In the ICH model and rFVIla groups, 80.0μL autologous non-clotting blood from rat tails was injected into the right caudate putamen to establish the ICH. The empty microinjector was inserted into the caudate putamen in the sham-operated group. The ICH model and rFVIla groups were subdivided into four subsets separately： 6, 24, 72 hours and 7 days following ICH. The rats in the rFVIla group were injected with 160 μg/kg rFVIla via the dorsal vein of the penis. MAIN OUTCOME MEASURES： Apoptotic cells were detected in the right caudate putamen by TUNEL; caspase-3 activity by spectrophotometry; and rat neurological function was evaluated by neurological functional impairment scales. RESULTS： Rat neurological function was deteriorated at 24, 72 hours, and 7 days following ICH. The TUNEL-positive cells and caspase-3 activity in the right caudate putamen was significantly increased in the ICH rats （P 〈 0.05）; rFVlla treatment reduced the number of TUNEL-positive cells and caspase-3 activity in the right caudate putamen （P 〈 0.05）, and neurological function was significantly improved （P 〈 0.05）. CONCLUSION： rFVIla was applied within 72 hours after tCH, which reduced the amount of neuronal apoptosis and promoted neurological function restoration by possibly inhibiting caspase-3 activity.

In silico evidence of Remdesivir action in blood coagulation cascade modulation in COVID-19 treatment

BACKGROUND Coronavirus disease 2019(COVID-19)has demonstrated several clinical manifestations which include not only respiratory system issues but also liver,kidney,and other organ injuries.One of these abnormalities is coagulopathies,including thrombosis and disseminated intravascular coagulation.Because of this,the administration of low molecular weight heparin is required for patients that need to be hospitalized.In addition,Remdesivir is an antiviral that was used against Middle East Acute Respiratory Syndrome,Ebola,Acute Respiratory Syndrome,and other diseases,showing satisfactory results on recovery.Besides,there is evidence suggesting that this medication can provide a better prognosis for patients with COVID-19.AIM To investigate in silico the interaction between Remdesivir and clotting factors,pursuing a possibility of using it as medicine.METHODS In this in silico study,the 3D structures of angiotensin-converting enzyme 2(ACE2),Factor I(fibrinogen),Factor II(prothrombin),Factor III(thromboplastin),Factor V(proaccelerin),Factor VII(proconvertin),Factor VIII(antihemophilic factor A),Factor IX(antihemophilic factor B),Factor X(Stuart-Prower factor),and Factor XI(precursor of thromboplastin(these structures are technically called receptors)were selected from the Protein Data Bank.The structures of the antivirals Remdesivir and Osetalmivir(these structures are called ligands)were selected from the PubChem database,while the structure of Atazanavir was selected from the ZINC database.The software AutoDock Tools(ADT)was used to prepare the receptors for molecular docking.Ions,peptides,water molecules,and other ones were removed from each ligand,and then,hydrogen atoms were added to the structures.The grid box was delimited and calculated using the same software ADT.A physiological environment with pH 7.4 is needed to make the ligands interact with the receptors,and still the software Marvin sketch®(ChemAxon®)was used to forecast the protonation state.To perform molecular docking,ADT and Vina software was connected.Using PyMol®software and Discovery studio®software from BIOVIA,it was possible to analyze the amino acid residues from receptors that were involved in the interactions with the ligands.Ligand tortions,atoms that participated in the interactions,and the type,strength,and duration of the interactions were also analyzed using those software.RESULTS Molecular docking analysis showed that Remdesivir and ACE2 had an affinity energy of-8.8 kcal/moL,forming a complex with eight hydrogen bonds involving seven atoms of Remdesivir and five amino acid residues of ACE2.Remdesivir and prothrombin had an interaction with six hydrogen bonds involving atoms of the drug and five amino acid residues of the clotting factor.Similar to that,Remdesivir and thromboplastin presented interactions via seven hydrogen bonds involving five atoms of the drug and four residues of the clotting factor.While Remdesivir and Factor V established a complex with seven hydrogen bonds between six antiviral atoms and six amino acid residues from the factor,and Factor VII connected with the drug by four hydrogen bonds,which involved three atoms of the drug and three residues of amino acids of the factor.The complex between Remdesivir and Factor IX formed an interaction via 11 hydrophilic bonds with seven atoms of the drug and seven residues of the clotting factor,plus one electrostatic bond and three hydrophobic interactions.Factor X and Remdesivir had an affinity energy of-9.6 kcal/moL,and the complex presented 10 hydrogen bonds and 14 different hydrophobic interactions which involved nine atoms of the drug and 16 amino acid residues of the clotting factor.The interaction between Remdesivir and Factor XI formed five hydrogen bonds involving five amino acid residues of the clotting factor and five of the antiviral atoms.CONCLUSION Because of the in silico significant affinity,Remdesivir possibly could act in the severe acute respiratory syndrome coronavirus 2 infection blockade by interacting with ACE2 and concomitantly act in the modulation of the coagulation cascade preventing the hypercoagulable state.

Role of platelet-derived extracellular vesicles in traumatic brain injury-induced coagulopathy and inflammation

Extracellular vesicles are composed of fragments of exfoliated plasma membrane,organelles or nuclei and are released after cell activation,apoptosis or destruction.Platelet-derived extracellular vesicles are the most abundant type of extracellular vesicle in the blood of patients with traumatic brain injury.Accumulated laboratory and clinical evidence shows that platelet-derived extracellular vesicles play an important role in coagulopathy and inflammation after traumatic brain injury.This review discusses the recent progress of research on platelet-derived extracellular vesicles in coagulopathy and inflammation and the potential of platelet-derived extracellular vesicles as therapeutic targets for traumatic brain injury.

Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.