Coagulation profile of human COVID-19 convalescent plasma

Convalescent plasma is used to treat COVID-19. There are theoretical concerns about the impact of pro-coagulant factors in convalescent plasma on the coagulation cascade particularly among patients with severe COVID-19. The aim of this study was to evaluate the coagulation profile of COVID-19 convalescent plasma. Clotting times and coagulation factor assays were compared between fresh frozen plasma, COVID-19 convalescent plasma, and pathogen-reduced COVID-19 convalescent plasma. Measurements included prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, D-dimer, von Willebrand factor activity, von Willebrand factor antigen, coagulation factors II, V, VII–XII, protein S activity, protein C antigen, and alpha-2 plasmin inhibitor. Clotting times and coagulation factor assays were not different between COVID-19 convalescent plasma and fresh frozen plasma, except for protein C antigen. When compared to fresh frozen plasma and regular convalescent plasma, pathogen reduction treatment increased activated partial thromboplastin time and thrombin time, while reducing fibrinogen, coagulation factor II, V, VIII, IX, X, XI, XII, protein S activity, and alpha-2 plasmin inhibitor. The coagulation profiles of human COVID-19 convalescent plasma and standard fresh frozen plasma are not different. Pathogen reduced COVID-19 convalescent plasma is associated with reduction of coagulation factors and a slight prolongation of coagulation times, as anticipated. A key limitation of the study is that the COVID-19 disease course of the convalesced donors was not characterized.

The effects of fructose diphosphate on routine coagulation tests in vitro

To evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0–35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, − 0.990, − 0.983, − 0.989 and − 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied.

Azolo[1,5-a]pyrimidines and Their Condensed Analogs with Anticoagulant Activity

Hypercytokinemia, or cytokine storm, is one of the severe complications of viral and bacterial infections, involving the release of abnormal amounts of cytokines, resulting in a massive inflammatory response. Cytokine storm is associated with COVID-19 and sepsis high mortality rate by developing epithelial dysfunction and coagulopathy, leading to thromboembolism and multiple organ dysfunction syndrome. Anticoagulant therapy is an important tactic to prevent thrombosis in sepsis and COVID-19, but recent data show the incompatibility of modern direct oral anticoagulants and antiviral agents. It seems relevant to develop dual-action drugs with antiviral and anticoagulant properties. At the same time, it was shown that azolo[1,5-a]pyrimidines are heterocycles with a broad spectrum of antiviral activity. We have synthesized a new family of azolo[1,5-a]pyrimidines and their condensed polycyclic analogs by cyclocondensation reactions and direct CH-functionalization and studied their anticoagulant properties. Five compounds among 1,2,4-triazolo[1,5-a]pyrimidin-7-ones and 5-alkyl-1,3,4-thiadiazolo[3,2-a]purin-8-ones demonstrated higher anticoagulant activity than the reference drug, dabigatran etexilate. Antithrombin activity of most active compounds was confirmed using lipopolysaccharide (LPS)-treated blood to mimic the conditions of cytokine release syndrome. The studied compounds affected only the thrombin time value, reliably increasing it 6.5–15.2 times as compared to LPS-treated blood.

Antimicrobial, Anticoagulant and Anticancer Effects of Arum Palaestinum Flowers Extracts

Background: Wild plants are amply utilized in traditional medicine and folkloric food worldwide. Arum palaestinum Boiss. (AP) is one of the wild Palestinian plants which leaves have a long history in the Middle Eastern countries as food and medicine. Herby, the current study aimed to evaluate the antimicrobial, coagulation cascade activities, and anticancer effects of (AP) flowers extract Methods: The aqueous extract of (AP) flowers was screened on its antimicrobial activity using microdilution assay against eight pathogens. While, prothrombin time, activated partial thromboplastin time, and thrombin time tests were measured utilizing standard hematological methods. And Anti cancer effect was assessed by using Parameters of cell cycles and alph feta protein level that were investigated for (AP) flowers fractionated with aqueous, DMSO, and methanol Results: The antimicrobial screening results revealed that the aqueous extract of (AP) has strong antibacterial effects against P. vulgaris and E. faecium compared with Ampicillin with MIC values of 6.25, 6.25 and 18 mg/ml, respectively. The aqueous extract of (AP) showed anticoagulant activity with significant prolonged results in aPTT and TT tests at high concentrations (50 mg/ml and 25 mg/ml) and slightly prolonged results in the PT test at a high concentration (50 mg/ml). The anticancer results indicate a delay in cell cycle through decreased the cell proliferation rate following effects of the AP fractions. The delay in the S phase was in favor of the water fraction. Water and DMSO fractions while maintained the cells in the G2-M phase similar to the DOX, the flower extract in methanol accelerated the cells in the G2-M phase suggesting that (AF) flower extracts have anti-cancer properties. At the same time Aqueous extract decreased HCC aFP to 1.55-fold (P=0.0008). While DMSO and methanolic extract had no significant effects on HCC aFP levels, compared to control untreated cells of 2519.16 ± 198.1 ng/ml. This data show that (AF) aqueous solution is potent inhibitor of alpha-fetoprotein secretion (P-value <0.05), which indicates its anti-carcinogenic effects Conclusion: These results showed that the aqueous extract of (AP) plant possesses bioactive components with antibacterial and anticoagulant properties, which may be exploited in the treatment of infectious diseases and blood coagulation disorders.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro

The success in treatment of venous thromboembolism and acute coronary syndromes using direct thrombin inhibitors has stimulated research aimed at finding a new anticoagulant from haematophagous organisms. This study deals with the comparison between hirudin-1 from Hirudomedicinalis(desirudin), being the first-known and most well-studied natural anticoagulant, along with recombinant analogs of haemadin from the leech Haemadipsa sylvestris, variegin from the tick Amblyomma variegatum, and anophelin from Anopheles albimanus. These polypeptides were chosen due to their high specificity and affinity for thrombin, as well as their distinctive inhibitory mechanisms. We have developed a universal scheme for the biotechnological production of these recombinant peptides as pharmaceutical substances. The anticoagulant activities of these peptides were compared using the thrombin amidolytic activity assay and prolongation of coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time) in mouse and human plasma. The preliminary results obtained suggest haemadin as the closest analog of recombinant hirudin-1, the active substance of the medicinal product Iprivask (Aventis Pharmaceuticals, USA) for the prevention of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. In contrast, variegin can be regarded as a natural analog of bivalirudin (Angiomax, The Medicines Company), a synthetic hirudin-1 derivative certified for the treatment of patients undergoing percutaneous coronary intervention and of patients with unstable angina pectoris after percutaneous transluminal coronary angioplasty.

rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick Hyalomma dromedarii

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 μM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

Clot Waveform Analysis Demonstrates Low Blood Coagulation Ability in Patients with Idiopathic Thrombocytopenic Purpura

Background: Although platelets, which contain large amounts of phospholipids, play an important role in blood coagulation, there is still no routine assay to examine the effects of platelets in blood coagulation. Methods: Hemostatic abnormalities in patients with thrombocytopenia, including those with idiopathic thrombocytopenic purpura (ITP), were examined using clot wave analysis (CWA)–small-amount tissue-factor-induced FIX activation (sTF/FIXa) and thrombin time (TT). Results: Although there were no marked differences in the three parameters of activated partial thromboplastin time (APTT) between normal healthy volunteers and typical patients with ITP, the peak heights of the CWA-sTF/FIXa were markedly low in patients with ITP. The three peak times of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly longer than those in patients with a platelet count > 8.0 × 1010/L and the peak heights of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly lower than those in patients with >8.0 × 1010/L. The peak heights of the CWA-APTT in patients with ITP were significantly lower than in patients with other types of thrombocytopenia. The three peak heights of the CWA-sTF/FIXa in ITP patients were significantly lower than those in patients with other types of thrombocytopenia. The CWA-TT showed lower peak heights and longer peak times in patients with ITP in comparison to patients with other types of thrombocytopenia. Conclusions: The CWA-sTF/FIXa and CWA-TT results showed that blood coagulation is enhanced by platelets and that the blood coagulation ability in ITP patients was low in comparison to healthy volunteers and patients with other types of thrombocytopenia.

Extended Coagulation Profiling in Isolated Traumatic Brain Injury: A CENTER-TBI Analysis

Background Trauma-induced coagulopathy in traumatic brain injury (TBI) remains associated with high rates of complications, unfavorable outcomes, and mortality. The underlying mechanisms are largely unknown. Embedded in the prospective multinational Collaborative European Neurotrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study, coagulation profiles beyond standard conventional coagulation assays were assessed in patients with isolated TBI within the very early hours of injury. Methods Results from blood samples (citrate/EDTA) obtained on hospital admission were matched with clinical and routine laboratory data of patients with TBI captured in the CENTER-TBI central database. To minimize confounding factors, patients with strictly isolated TBI (iTBI) (n = 88) were selected and stratified for coagulopathy by routine international normalized ratio (INR): (1) INR < 1.2 and (2) INR ≥ 1.2. An INR > 1.2 has been well adopted over time as a threshold to define trauma-related coagulopathy in general trauma populations. The following parameters were evaluated: quick’s value, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin, coagulation factor activity of factors V, VIII, IX, and XIII, protein C and S, plasminogen, D-dimer, fibrinolysis-regulating parameters (thrombin activatable fibrinolysis inhibitor, plasminogen activator inhibitor 1, antiplasmin), thrombin generation, and fibrin monomers. Results Patients with iTBI with INR ≥ 1.2 (n = 16) had a high incidence of progressive intracranial hemorrhage associated with increased mortality and unfavorable outcome compared with patients with INR < 1.2 (n = 72). Activity of coagulation factors V, VIII, IX, and XIII dropped on average by 15–20% between the groups whereas protein C and S levels dropped by 20%. With an elevated INR, thrombin generation decreased, as reflected by lower peak height and endogenous thrombin potential (ETP), whereas the amount of fibrin monomers increased. Plasminogen activity significantly decreased from 89% in patients with INR < 1.2 to 76% in patients with INR ≥ 1.2. Moreover, D-dimer levels significantly increased from a mean of 943 mg/L in patients with INR < 1.2 to 1,301 mg/L in patients with INR ≥ 1.2. Conclusions This more in-depth analysis beyond routine conventional coagulation assays suggests a counterbalanced regulation of coagulation and fibrinolysis in patients with iTBI with hemostatic abnormalities. We observed distinct patterns involving key pathways of the highly complex and dynamic coagulation system that offer windows of opportunity for further research. Whether the changes observed on factor levels may be relevant and explain the worse outcome or the more severe brain injuries by themselves remains speculative.

THE USE OF THROMBODYNAMICS TEST IN DIAGNOSTICS OF HEMOSTASIS DISORDERS IN PATIENTS WITH COVID-19 OF VARYING SEVERITY

Background: At the moment, an urgent and unresolved problem is the search for a diagnostic method for disorders of the hemostasis system in patients against the background of the course of a new coronavirus infection. Presumably, integral tests, in particular, the thrombodynamics test, will make it possible to monitor changes in blood clotting, predict the course of the disease in patients with COVID-19. Aims: to comparative assessment of plasma hemostasis parameters and thrombodynamics test in patients with COVID-19 viral infection of varying severity. Methods: The study included 96 patients with a confirmed diagnosis of COVID-19, hospitalized in an infectious diseases hospital on the basis of National Medical Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov in the period from 04.23.2020 to 06.20.2020 and discharged at the end of treatment. SARS-CoV-2 was identified by PCR. Patients were stratified by severity into 3 groups: mild course (n = 25), moderate course (n = 54), severe course (n = 17). Diagnostics and treatment of patients was carried out in accordance with the Temporary Methodological Recommendations of the Ministry of Health of the Russian Federation for the prevention, diagnosis and treatment of new coronavirus infection, versions 5, 6, 7. In the dynamics of treatment, patients were assessed APTT, prothrombin %, prothrombin time and thrombin time, fibrinogen, D-dimer, platelet count and thrombodynamic test (V / Vi / Vst, Tlag, Cs, D). Results: It was found that significant differences before admission and a week after the start of hospital treatment were observed for the thrombin time, D-dimer, platelet count, and thrombodynamic parameters: V / Vst, Cs, D. PT, APTT, TD (Tlag, D)) with the duration of hospital stay. There was a positive relationship between the content of fibrinogen and D (r = 0.6307, p 0.0001) and a strong positive relationship between PT and Tlag (r = 0.7499, p 0.0001). Conclusions: The thrombodynamics test can be recommended as a potential tool for a personalized approach to monitoring the hemostasis system and treating patients with COVID-19.

Azolo[1,5-a]pyrimidines and Their Condensed Analogs with Anticoagulant Activity

Hypercytokinemia, or cytokine storm, is one of the severe complications of viral and bacterial infections, involving the release of abnormal amounts of cytokines, resulting in a massive inflammatory response. Cytokine storm is associated with COVID-19 and sepsis high mortality rate by developing epithelial dysfunction and coagulopathy, leading to thromboembolism and multiple organ dysfunction syndrome. The anticoagulant therapy is an important tactic to prevent thrombosis in sepsis and COVID-19, but recent data show the incompatibility of modern direct oral anticoagulants and antiviral agents. It seems relevant to develop dual-action drugs with antiviral and anticoagulant properties. At the same time it was shown that azolo[1,5-a]pyrimidines are heterocycles with a broad spectrum of antiviral activity. We have synthesized a new family of azolo[1,5-a]pyrimidines and their condensed polycyclic analogs by cyclocondensation reactions and direct CH-functionalization and studied their anticoagulant properties. Five compounds among 1,2,4-triazolo[1,5-a]pyrimidin-7-ones and 5-alkyl-1,3,4-thiadiazolo[3,2-a]purin-8-ones demonstrated higher anticoagulant activity than the reference drug, dabigatran etexilate. Antithrombin activity of lead compounds was confirmed using lipopolysaccharide (LPS) treated blood to mimic conditions of cytokine release syndrome. The studied compounds affected only the thrombin time value, reliably increasing it 6.5&ndash;15.2 times as compared to LPS-treated blood.