Prolonged coagulation times in severe fever with thrombocytopenia syndrome virus infection, the indicators of heparin-like effect and increased haemorrhagic risk.

Prolonged coagulation times, such as activated partial thromboplastin time (APTT) and thrombin time (TT), are common in patients infected with severe fever with thrombocytopenia syndrome virus (SFTSV) and have been confirmed to be related to patient's poor outcome by previous studies. To find out the reason for prolonged coagulation time in patients with SFTSV infection, and whether it predicts haemorrhagic risk or not. Seventy-eight consecutive patients with confirmed SFTSV infection were enrolled in this prospective, single-centre, observational study. Several global and specific coagulation parameters of these patients on admission were detected, and the haemorrhagic events during hospitalization and their outcomes were recorded. Most of the enrolled patients had prolonged APTT (82.1%) and TT (80.8%), normal prothrombin time (83.3%) and intrinsic coagulation factors above haemostatic levels (97.4%). The heparin-like effect was confirmed by a protamine neutralization test and anti-Xa activity detection in most patients. Interestingly, the APTT and TT results were significantly positively correlated with the levels of endothelial markers and viral load, respectively. The APTT was independently associated with the haemorrhage of patients. The prolonged APTT and TT of SFTS patients may mainly be attributed to endogenous heparinoids and are associated with increased haemorrhagic risk.

Association of Heparin-Like Effect, Factor VII/XIII Deficiency and Fibrinolysis with Rebleeding Risk in Cirrhosis with Acute Variceal Bleeding.

BACKGROUND: Hyperfibrinolysis and coagulation dysfunction may occur in cirrhotic patients with acute variceal bleed (AVB) despite successful endotherapy. AIMS: To prospectively study the association of endogenous heparinoids and coagulation dysfunction with variceal rebleeding and outcome in cirrhosis. METHODS: Consecutive patients were assessed with conventional coagulation tests, SONOCLOT™ [(global(gb) and heparinase(h) treated] and factors VII, VIII, XIII, X, tissue plasminogen activator, and plasminogen activator inhibitor ELISA assays in a university hospital. Heparin-like-effect (HLE) was defined as ≥ 20% difference in paired gb/h-SONOCLOT™ traces for activated clotting time (ACT). RESULTS: Of 143 patients screened, 90 (46.4 ± 11.7 years, males 82.2%, ethanol-related 58.8%) were recruited, who bled from esophageal varices (81,90.0%), gastric varices (6,6.6%), or esophageal varices with portal hypertensive gastropathy (3,3.3%). Twenty (21.7%) had early rebleeding, mainly post-variceal ligation ulcer related (70%). Patients who rebled had low Factor XIII [1.6 (1.2-2.1) vs 2.4 ng/ml (2.0-2.8) P = 0.035] and Factor VII (94.1 ± 46.9 vs. 124.0 ± 50.4, P = 0.023). On receiver operating curve analysis, the gbACT > 252 s (sensitivity 86.8%, specificity 76.9%, P < 0.001), hACT > 215 s (sensitivity 71.1%, specificity 70.3%, P < 0.001), and HLE > 50% (sensitivity 69.5%, specificity 70.3%, P = 0.006) predicted rebleeding. Baseline Factor VIII (HR 1.26; 95% CI 1.17-1.34, P < 0.001), low factor VII (HR 0.89; 95% CI 0.76-0.98, P = 0.035), and lysis (HR 1.25, 95% CI 1.17-1.33, P < 0.001) predicted mortality. Endogenous heparinoids at baseline predicted sepsis (HR 1.8; 95% CI 1.4-6.5; P = 0.022), rebleeding events (HR 1.2; 95% CI 1.1-6.3; P = 0.030), and mortality (HR 1.1; 95% CI 1.0-4.6; P = 0.030). CONCLUSIONS: Hyperfibrinolysis, Factor VII/XIII deficiency, and HLE are associated with rebleeding after AVB. Trial Registration NCT04111120 available from https://clinicaltrials.gov/ct2/show/NCT04111120 .

Significance of heparin induced thrombocytopenia (HIT) in COVID-19: a systematic review and meta-analysis.

Heparin-induced thrombocytopenia (HIT) occurs in approximately 3% of patients receiving heparinoids. About 30-75% of patients with type 2 of HIT develop thrombosis as a result of platelet activation. The most important clinical symptom is thrombocytopenia. Patients with severe COVID-19 are among those receiving heparinoids. This meta-analysis performed to picture the current knowledge and results of published studies in this field. Three search engines were searched and 575 papers were found. After evaluation, 37 articles were finally selected of which 13 studies were quantitatively analyzed. The pooled frequency rate of suspected cases with HIT in 13 studies with 11,241 patients was 1.7%. The frequency of HIT was 8.2% in the extracorporeal membrane oxygenation subgroup with 268 patients and 0.8% in the hospitalization subgroup with 10,887 patients. The coincidence of these two conditions may increase the risk of thrombosis. Of the 37 patients with COVID-19 and confirmed HIT, 30 patients (81%) were treated in the intensive care unit or had severe COVID-19. The most commonly used anticoagulants were UFH in 22 cases (59.4%). The median platelet count before treatment was 237 (176-290) x 103/µl and the median nadir platelet count was 52 (31-90.5) x 103/µl.

What is the impact of circulating histones in COVID-19: a systematic review.

The infectious respiratory condition COVID-19 manifests a clinical course ranging from mild/moderate up-to critical systemic dysfunction and death linked to thromboinflammation. During COVID-19 infection, neutrophil extracellular traps participating in cytokine storm and coagulation dysfunction have emerged as diagnostic/prognostic markers. The characterization of NET identified that mainly histones, have the potential to initiate and propagate inflammatory storm and thrombosis, leading to increased disease severity and decreased patient survival. Baseline assessment and serial monitoring of blood histone concentration may be conceivably useful in COVID-19. We performed a literature review to explore the association among increased circulating levels of histones, disease severity/mortality in COVID-19 patients, and comparison of histone values between COVID-19 and non-COVID-19 patients. We carried out an electronic search in Medline and Scopus, using the keywords "COVID-19" OR "SARS-CoV-2" AND "histone" OR "citrullinated histones" OR "hyperhistonemia", between 2019 and present time (i.e., June 07th, 2022), which allowed to select 17 studies, totaling 1,846 subjects. We found that substantially elevated histone values were consistently present in all COVID-19 patients who developed unfavorable clinical outcomes. These findings suggest that blood histone monitoring upon admission and throughout hospitalization may be useful for early identification of higher risk of unfavorable COVID-19 progression. Therapeutic decisions in patients with SARS-CoV-2 based on the use of histone cut-off values may be driven by drugs engaging histones, finally leading to the limitation of cytotoxic, inflammatory, and thrombotic effects of circulating histones in viral sepsis.

Marine glycan-based antiviral agents in clinical or preclinical trials.

The constant outbreak of diseases caused by viral infections has caused serious harm to human health all over the world. Although many antiviral drugs have been approved for clinical use during the past decade, important issues, such as unsatisfactory efficacy, toxicity, and high cost of drugs, remain unresolved. Glycans are major components of the surfaces of both host cells and most viruses and play critical roles in the steps of viral infection. Marine glycans have more structural diversities than those found in humans. Most importantly, low toxicity and low-cost marine glycans have demonstrated potent antiviral activities through multiple molecular mechanisms. As a result, a series of marine glycan-derived agents are undergoing preclinical and clinical trials. This review discusses the recent progress in research on the marine glycan-based antiviral agents in clinical trials, relating to their structural features and clinical applications. In addition, molecular mechanisms of marine glycans involved in viral infection and novel strategies used in glycan-based drug development are critically reviewed and discussed.

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion.

Sevuparin is a novel drug candidate in phase II development as a treatment for vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS-RBCs) to stimulated cultured endothelial cells in vitro. Importantly, sevuparin prevents vaso-occlusion and normalizes blood flow in an in vivo mouse model of SCD vaso-occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P- and L-selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso-occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti-adhesive efficacy similar to the low molecular weight heparin tinzaparin both in vitro and in vivo. These results suggest that the anti-adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso-occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

New insight into the effects of heparinoids on complement inhibition by C1-inhibitor.

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa(®) -kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases.

Structural elucidation of Sulodexide with multidimensional chromatography and online in-source acid-induced dissociation mass spectrometry.

Sulodexide, a heparinoid medicine, is wildly used in clinic for prophylaxis and treatment of thromboembolic diseases and diabetic nephropathy. Despite its widespread use, the structure of Sulodexide remains poorly understood. It consists of various polysaccharides characterized by differing sugar compositions, linkages, and sulfonation patterns, yet they share common features such as strong hydrophilicity, high native charges, and considerable polydispersity, posing significant challenges for conventional chromatographic and online mass spectrometry (MS) characterization. In this work, a novel analytical method combining multiple-heart cut 2D-LC and in-source acid-induced dissociation (inAID) MS was developed. Three polysaccharides in Sulodexide were separated by high efficient strong-anion-exchange chromatography, followed by desalting with the second dimensional size-exclusion chromatography before MS. A novel MS strategy employing inAID technique was utilized for online analysis, leading to the initial identification of Sulodexide polysaccharide components. The results were validated through disaccharide composition analysis of those three polysaccharide components after offline preparation. This advanced strategy, merging various techniques, enable a comprehensive structural elucidation of such complex drugs and provides a viable tool for potential routine analysis of complex biomolecules.

Do Circulating Histones Represent the Missing Link among COVID-19 Infection and Multiorgan Injuries, Microvascular Coagulopathy and Systemic Hyperinflammation?

Several studies shed light on the interplay among inflammation, thrombosis, multi-organ failures and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Increasing levels of both free and/or circulating histones have been associated to coronavirus disease 2019 (COVID-19), enhancing the risk of heart attack and stroke with coagulopathy and systemic hyperinflammation. In this view, by considering both the biological and clinical rationale, circulating histones may be relevant as diagnostic biomarkers for stratifying COVID-19 patients at higher risk for viral sepsis, and as predictive laboratory medicine tool for targeted therapies.

A Case of Livedoid Vasculopathy Successfully Treated with Sulodexide.

We report a 29-year-old female with a one-month history of non-healing multiple erythematous to violaceous plaques with crusts over both legs and feet. Tender, scarring ulcers with surrounding erythema were present. The clinical manifestation, together with histopathologic findings of fibrinoid plugs within vascular lumens and walls, as well as red blood cell extravasation, led to diagnosis of livedoid vasculopathy. The patient experienced recurrent painful violaceous plaques with ulcerations during the two years of treatment with oral pentoxifylline 400 mg three times daily. The cutaneous lesions and symptoms dramatically improved after the treatment regimen changed to oral sulodexide (250 lipasemic units) three times daily. Sulodexide, a highly purified mixture of glycosaminoglycans including dermatan sulfate and low-molecular weight heparin, could be an effective therapy for recalcitrant livedoid vasculopathy. Herein, we report a case of livedoid vasculopathy treated with sulodexide, which has not previously been reported.

Fine structural characterization of sulodexide.

Sulodexide is a heparinoid which combines the properties of its components heparin and dermatan sulfate and is used not only for the prophylaxis and treatment of thromboembolic diseases but also for the treatment of diabetic nephropathy. Despite many clinical studies have been conducted to investigate its activity and safety, no data are available on the fine chemical characterization of its components. In this work, the in-depth investigation on the structural features of both the whole mixture and the isolated components was accomplished, involving the analysis of molecular weight distribution and of their mono, di and oligosaccharide composition by HP-SEC/TDA, 2D-NMR and HPLC-MS techniques. Moreover, also the separation of fractions endowed of graded affinity to antithrombin was achieved followed again by detailed structural analysis. The combination of different techniques permits to profile in depth the structural features of such a drug and offers a useful tool for possible analysis of batch production.

Natural heparinoids isolated from Halymenia sp. (Rhodophyceaes) delivery on the Ceará coast - doi: 10.4025/actascibiolsci.v32i3.6282 / Heparinoides naturais isolados de rodofíceas (Halymenia sp.) arribadas na costa cearense - doi: 10.4025/actascibiolsci.v32i3.6282

The increasing demand for heparin (HEP) has led to a search for alternative sources of natural anticoagulants. This study aimed to evaluate the anticoagulant activity of sulfated polysaccharides (SP) isolated from a Halymenia rhodophyceae genus native to the coast of Ceará, Brazil. Total SP were obtained by consecutive digestions with papain in 0.1 M sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by ion-exchange chromatography on DEAE-cellulose column. The obtained fractions were concentrated by lyophilization and submitted to 0.5% agarose gel electrophoresis. Anticoagulant activity was evaluated by the activated partial thromboplastin time (APTT) using plasma from rabbits and a standard HEP (100 IU mg-1) curve. The extractions (53.96%) showed marked differences during the fractionation and in the degree of purification of SP. The species SP showed higher activity anticoagulant than that of HEP. However, the APTT of the fractions changed sharply among the extractions, expressing itself in a dose-dependent manner and increasing from 110.40 (1st extraction) to 143.10 IU mg-1 (3rd extraction). The results suggest that the anticoagulant activity of SP isolated from Halymenia sp. was promoted by inhibition of the intrinsic and/or common pathway of the coagulation cascade. The changes on APTT possibly will be elucidated through the mechanisms of actio

A crescente carência de heparina (HEP) motiva a busca por fontes alternativas de novos anticoagulantes naturais. Objetivou-se avaliar a atividade anticoagulante dos polissacarídeos sulfatados (PS) isolados de uma rodofícea do gênero Halymenia, nativa do litoral cearense, Brasil. Os PS totais foram obtidos por digestões consecutivas com papaína em tampão acetato de sódio 0,1 M (pH 5,0), contendo cisteína 5 mM e EDTA 5 mM, seguidas por cromatografia de troca iônica em coluna de DEAE-celulose. As frações obtidas foram concentradas por liofilização e submetidas à eletroforese em gel de agarose a 0,5%. Os ensaios anticoagulantes foram realizados pelo tempo de tromboplastina parcial ativada (TTPA), usando-se plasma de coelho e uma curva padrão de HEP (100 UI MG-1). As extrações (53,96%) mostraram diferenças marcantes durante o fracionamento e no grau de resolução dos PS. A espécie apresentou PS com atividade anticoagulante superior a HEP. O TTPA das frações modificou-se acentuadamente entre as extrações, expressando-se de maneira dose-dependente e sofrendo um acréscimo de 110,40 (1ª extração) para 143,10 UI MG-1 (3ª extração). Os resultados sugerem que a atividade anticoagulante dos PS isolados de Halymenia sp. foi promovida pela inibição da via intrínseca e/ou comum da cascata de coagulação. As modificações no TTPA possivelmente serão elucidadas pelos mecanismos de

Management of Heparin-Induced Thrombocytopenia During Renal Replacement Therapy.

Awareness is increasing concerning the development of antibodies to heparin-platelet factor 4 complex in both regular hemodialysis patients and those treated with continuous forms of renal replacement therapy. Although the development of antibodies does not result in thrombocytopenia or thrombosis in some patients, most patients present with thrombocytopenia, premature platelet activation, and clotting of the extracorporeal circuit. When systemic anticoagulation is also required to treat venous thrombosis, then synthetic heparinoids or recombinant hirudin will be the agents of choice. However, neither the synthetic heparinoids nor hirudin are without problems. A few patients may have cross-reacting antibodies against the currently available heparinoids. Similarly, antibodies may develop against recombinant hirudin, leading to a potentiation of anticoagulant activity and increased risk of hemorrhage. In the future, thrombin inhibitors such as recombinant hirudin and the arginine derivative argatroban will probably be the agents most widely used to prevent thromboembolic complications. However, anti-platelet agents used alone or in combination with hirudin or synthetic heparinoids may provide adequate treatment by inhibiting both platelet and clotting cascade activation.

Anticoagulation in Patients With Acute Renal Failure Treated With Continuous Renal Replacement Therapies.

Although continuous renal replacement therapy (CRRT) provides greater cardiovascular and cerebrovascular stability compared to standard intermittent hemodialysis and/ or hemofiltration, to provide adequate solute removal, the CRRT circuit must function continuously. Patients with acute renal failure are usually prothrombotic, with activation of the contact coagulation cascade and reduction in the natural anticoagulants. Thus clotting within the extracorporeal circuit can be problematic. Coagulation requires activation of the coagulation protein enzymes, calcium, platelets, and contact with phospholipid cell surface or, in the case of the CRRT circuit, plastic tubing and the dialyzer membrane. This results in a platelet plug stabilized by cross-linking strands of fibrin. Anticoagulation regimes are either directed at trying to prevent contact activation of clotting factors and platelets or the administration of agents designed to prevent coagulation by blocking the coagulation cascade or platelet activation.