Direct thrombin inhibitors fail to reverse the negative effects of heparin on lung growth and function after murine left pneumonectomy.

Neonates with congenital diaphragmatic hernia (CDH) frequently require cardiopulmonary bypass and systemic anticoagulation. We previously demonstrated that even subtherapeutic heparin impairs lung growth and function in a murine model of compensatory lung growth (CLG). The direct thrombin inhibitors (DTIs) bivalirudin and argatroban preserved growth in this model. Although DTIs are increasingly used for systemic anticoagulation clinically, patients with CDH may still receive heparin. In this experiment, lung endothelial cell proliferation was assessed following treatment with heparin-alone or mixed with increasing concentrations of bivalirudin or argatroban. The effects of subtherapeutic heparin with or without DTIs in the CLG model were also investigated. C57BL/6J mice underwent left pneumonectomy and subcutaneous implantation of osmotic pumps. Pumps were preloaded with normal saline, bivalirudin, or argatroban; treated animals received daily intraperitoneal low-dose heparin. In vitro, heparin-alone decreased endothelial cell proliferation and increased apoptosis. The effect of heparin on proliferation, but not apoptosis, was reversed by the addition of bivalirudin and argatroban. In vivo, low-dose heparin decreased lung volume compared with saline-treated controls. All three groups that received heparin demonstrated decreased lung function on pulmonary function testing and impaired exercise performance on treadmill tolerance testing. These findings correlated with decreases in alveolarization, vascularization, angiogenic signaling, and gene expression in the heparin-exposed groups. Together, these data suggest that bivalirudin and argatroban fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of low-dose heparin with DTIs on CDH outcomes are warranted.NEW & NOTEWORTHY Infants with pulmonary hypoplasia frequently require cardiopulmonary bypass and systemic anticoagulation. We investigate the effects of simultaneous exposure to heparin and direct thrombin inhibitors (DTIs) on lung growth and pulmonary function in a murine model of compensatory lung growth (CGL). Our data suggest that DTIs fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of heparin with DTIs on clinical outcomes are thus warranted.

Marine sulfated glycans inhibit the interaction of heparin with S-protein of SARS-CoV-2 Omicron XBB variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS­CoV­2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents.

The mutation of Japanese encephalitis virus envelope protein residue 389 attenuates viral neuroinvasiveness.

The envelope (E) protein of the Japanese encephalitis virus (JEV) is a key protein for virus infection and adsorption of host cells, which determines the virulence of the virus and regulates the intensity of inflammatory response. The mutation of multiple aa residues in the E protein plays a critical role in the attenuated strain of JEV. This study demonstrated that the Asp to Gly, Ser, and His mutation of the E389 site, respectively, the replication ability of the viruses in cells was significantly reduced, and the viral neuroinvasiveness was attenuated to different degrees. Among them, the mutation at E389 site enhanced the E protein flexibility contributed to the attenuation of neuroinvasiveness. In contrast, less flexibility of E protein enhanced the neuroinvasiveness of the strain. Our results indicate that the mechanism of attenuation of E389 aa mutation attenuates neuroinvasiveness is related to increased flexibility of the E protein. In addition, the increased flexibility of E protein enhanced the viral sensitivity to heparin inhibition in vitro, which may lead to a decrease in the viral load entering brain. These results suggest that E389 residue is a potential site affecting JEV virulence, and the flexibility of the E protein of aa at this site plays an important role in the determination of neuroinvasiveness.

In vitro degradation, swelling, and bioactivity performances of in situ forming injectable chitosan-matrixed hydrogels for bone regeneration and drug delivery.

Injectable, tissue mimetic, bioactive, and biodegradable hydrogels offer less invasive regeneration and repair of tissues. The monitoring swelling and in vitro degradation capacities of hydrogels are highly important for drug delivery and tissue regeneration processes. Bioactivity of bone tissue engineered constructs in terms of mineralized apatite formation capacity is also pivotal. We have previously reported in situ forming chitosan-based injectable hydrogels integrated with hydroxyapatite and heparin for bone regeneration, promoting angiogenesis. These hydrogels were functionalized by glycerol and pH to improve their mechano-structural properties. In the present study, functionalized hybrid hydrogels were investigated for their swelling, in vitro degradation, and bioactivity performances. Hydrogels have degraded gradually in phosphate-buffered saline (PBS) with and without lysozyme enzyme. The percentage weight loss of hydrogels and their morphological and chemical properties, and pH of media were analyzed. The swelling ratio of hydrogels (55%-68%(wt), 6 h of equilibrium) indicated a high degree of cross-linking, can be suitable for controlled drug release. Hydrogels have gradually degraded reaching to 60%-70% (wt%) in 42 days in the presence and absence of lysozyme, respectively. Simulated body fluid (SBF)-treated hydrogels containing hydroxyapatite-induced needle-like carbonated-apatite mineralization was further enhanced by heparin content significantly.

Antithrombin Levels and Heparin Responsiveness during Venoarterial Extracorporeal Membrane Oxygenation: A Prospective Single-center Cohort Study.

BACKGROUND: Unfractionated heparin, administered during venoarterial extracorporeal membrane oxygenation to prevent thromboembolic events, largely depends on plasma antithrombin for its antithrombotic effects. Decreased heparin responsiveness seems frequent on extracorporeal membrane oxygenation; however, its association with acquired antithrombin deficiency is poorly understood. The objective of this study was to describe longitudinal changes in plasma antithrombin levels during extracorporeal membrane oxygenation support and evaluate the association between antithrombin levels and heparin responsiveness. The hypothesis was that extracorporeal membrane oxygenation support would be associated with acquired antithrombin deficiency and related decreased heparin responsiveness. METHODS: Adults receiving venoarterial extracorporeal membrane oxygenation were prospectively included. All patients received continuous intravenous unfractionated heparin using a standardized protocol (target anti-Xa 0.3 to 0.5 IU/ml). For each patient, arterial blood was withdrawn into citrate-containing tubes at 11 time points (from hour 0 up to day 7). Anti-Xa (without dextran or antithrombin added) and antithrombin levels were measured. The primary outcome was the antithrombin plasma level. In the absence of consensus, antithrombin deficiency was defined as a time-weighted average of antithrombin less than or equal to 70%. Data regarding clinical management and heparin dosage were collected. RESULTS: Fifty patients, including 42% postcardiotomy, were included between April 2020 and May 2021, with a total of 447 samples. Median extracorporeal membrane oxygenation duration was 7 (interquartile range, 4 to 12) days. Median antithrombin level was 48% (37 to 60%) at baseline. Antithrombin levels significantly increased throughout the follow-up. Time-weighted average of antithrombin levels was 63% (57 to 73%) and was less than or equal to 70% in 32 (64%) of patients. Overall, 45 (90%) patients had at least one antithrombin value less than 70%, and 35 (70%) had at least one antithrombin value less than 50%. Antithrombin levels were not significantly associated with heparin responsiveness evaluated by anti-Xa assay or heparin dosage. CONCLUSIONS: Venoarterial extracorporeal membrane oxygenation support was associated with a moderate acquired antithrombin deficiency, mainly during the first 72 h, that did not correlate with heparin responsiveness.

Impact of anti-coagulant choice on blood elongational behavior.

Blood is a highly complex fluid with rheological properties that have a significant impact on various flow phenomena. In particular, it exhibits a non-Newtonian elongational viscosity that is comparable to polymer solutions. In this study, we investigate the effect of three different anticoagulants, namely EDTA (ethylene diamine tetraacetic acid), heparin, and citrate, on the elongational properties of both human and swine blood. We observe a unique two stage thinning process and a strong dependency of the characteristic relaxation time on the chosen anticoagulant, with the longest relaxation time and thus the highest elongational viscosity being found for the case of citrate. Our findings for the latter are consistent with the physiological values obtained from a dripping droplet of human blood without any anticoagulant. Furthermore, our study resolves the discrepancy found in the literature regarding the reported range of characteristic relaxation times, confirming that the elongational viscosity must be taken into account for a full rheological characterization of blood. These results have important implications for understanding blood flow in various physiological, pathological and technological conditions.

Immune-stealth VP28-conjugated heparin nanoparticles for enhanced and reversible anticoagulation.

Heparins are a family of sulfated linear negatively charged polysaccharides that have been widely used for their anticoagulant, antithrombotic, antitumor, anti-inflammatory, and antiviral properties. Additionally, it has been used for acute cerebral infarction relief as well as other pharmacological actions. However, heparin's self-aggregated macrocomplex may reduce blood circulation time and induce life-threatening thrombocytopenia (HIT) complicating the use of heparins. Nonetheless, the conjugation of heparin to immuno-stealth biomolecules may overcome these obstacles. An immunostealth recombinant viral capsid protein (VP28) was expressed and conjugated with heparin to form a novel nanoparticle (VP28-heparin). VP28-heparin was characterized and tested to determine its immunogenicity, anticoagulation properties, effects on total platelet count, and risk of inducing HIT in animal models. The synthesized VP28-heparin trimeric nanoparticle was non-immunogenic, possessed an average hydrodynamic size (8.81 ± 0.58 nm) optimal for the evasion renal filtration and reticuloendothelial system uptake (hence prolonging circulating half-life). Additionally, VP28-heparin did not induce mouse death or reduce blood platelet count when administered at a high dosein vivo(hence reducing HIT risks). The VP28-heparin nanoparticle also exhibited superior anticoagulation properties (2.2× higher prothrombin time) and comparable activated partial thromboplastin time, but longer anticoagulation period when compared to unfractionated heparin. The anticoagulative effects of the VP28-heparin can also be reversed using protamine sulfate. Thus, VP28-heparin may be an effective and safe heparin derivative for therapeutic use.

Taurolidine and Heparin as Catheter Lock Solution for Central Venous Catheters in Hemodialysis.

BACKGROUND: Chronic kidney disease can lead to end-stage renal disease, and the prevalence is increasing. Many patients starting hemodialysis require central venous catheters (CVCs). Catheter-related bloodstream infections (CRBSIs) are a common complication and lead to significant morbidity and mortality. Interventions to prevent CRBSI include antimicrobial lock therapy but concern for the development of antimicrobial resistance and adverse effects. Nonantimicrobial antiseptics as catheter lock solutions have also been used. Taurolidine and heparin catheter lock solution is first approved by the Food and Drug Administration for the prevention of CRBSI in patients on hemodialysis. Taurolidine has a unique mechanism of action and favorable safety profile. MECHANISM OF ACTION, PHARMACODYNAMICS, AND PHARMACOKINETICS: Taurolidine and heparin catheter lock solution have both antimicrobial and anticoagulant properties. Taurolidine is derivative of the amino acid taurine, and heparin is derived from porcine intestinal mucosa. Taurolidine not only damages microbial cell walls but also prevents the adherence of microorganisms to biological surfaces, preventing biofilm formation. Taurolidine and heparin catheter lock solution is intended to be used intraluminally within the catheter and should be aspirated. Because it is used locally, limited pharmacokinetic and pharmacodynamic data are available. CLINICAL TRIALS: The LOCK-IT-100 trial is a randomized, double-blind, phase 3 study, which included 795 end-stage renal disease patients on hemodialysis with CVC. Taurolidine and heparin was compared with the control heparin alone. The results of the study showed a 71% risk reduction in CRBSI for taurolidine and heparin arm (95% confident interval, 38%-86%, P = 0.0006). Other studies have also shown that taurolidine lock solution leads to decreased CRBSI episodes. Several systematic reviews and meta-analysis consisted of taurolidine in adult, and pediatric populations also showed reduction in the incidence of CRBSIs. THERAPEUTIC ADVANCE: Taurolidine and heparin lock solution represents a novel preventive strategy for those undergoing hemodialysis through a CVC by reducing the risk of CRBSI. This is significant progress because there are no other similar options available for patients for whom catheters are the only options for their life-saving treatment.

Antithrombin supplementation attenuates heparin resistance in plasma spiked with Gla-domainless factor Xa S195A in vitro.

BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.

Multianticoagulant Pseudothrombocytopenia in a Patient with Primary Carcinoma of the Liver with Hypersplenism.

BACKGROUND: Pseudothrombocytopenia (PTCP) is a relatively rare phenomenon in vitro, the mechanism is not completely clear, and there is no unified solution for it. How to identify and solve PTCP accurately is a challenge for laboratory personnel. METHODS: According to the patient's clinical manifestations, thrombocytopenia caused by hypersplenism was excluded. PTCP was confirmed by platelet volume histograms, scattergrams and platelet clumps on the blood smears. Commonly used alternative anticoagulants such as sodium citrate or heparin were used for platelet counting. The corrective effect of the platelet count was not good, so non-anticoagulant blood was collected and tested immediately, and blood smears were used to count platelets manually. RESULTS: The PTCP of the patient could not be solved using sodium citrate and heparin anticoagulation. By collecting non-anticoagulant blood and testing immediately, the platelet count returned to normal (180 x 109/L), which is consistent with the results of manual counting on the patient's blood smears (175 x 109/L). CONCLUSIONS: When PTCP is confirmed, commonly used alternative anticoagulants can be used. If these do not work, non-anticoagulant blood can be collected and tested immediately, and blood smears can be used to count platelets manually.

The Effects of Different Blood Sample and Anticoagulant Types on Quantitative Tests of B-Type Natriuretic Peptide.

BACKGROUND: BNP is a sensitive and widely used biomarker for an early diagnosis of heart failure. Currently, most commercial BNP detection products use EDTA plasma samples. The aim of this study was to evaluate the clinical performance of the BNP test by using whole blood samples compared to plasma samples, and to evaluate the effect of the anticoagulant type on the BNP test result. METHODS: In total, 106 patients with different BNP levels from the Dahua Hospital volunteered for this study. Clinically homogenous samples, including EDTA anticoagulant plasma, EDTA whole blood, and heparin anticoagulant plasma, were collected and analyzed by using i-Reader S automatic immuno-analyzer and its supporting reagent kits. Pearson's correlation and weighted least squares linear regression analysis, Bland-Altman plotting, and Kappa test were used for statistical analysis. RESULTS: Correlation analysis showed that BNP concentrations, measured from EDTA anticoagulated plasma samples, had a good linear regression relationship with BNP from whole blood samples, with a slope of 0.9477, r = 0.9978, p < 0.05. A similar correlation was observed between EDTA anticoagulated plasma samples and heparin anticoagulant plasma, with a slope of 0.8413, r = 0.9793, p < 0.05. The BNP concentration measured from the heparin plasma samples were lower than of the EDTA plasma samples. Bland-Altman analysis for assessing BNP concentration agreement showed there was no outlier ratio between EDTA whole blood and EDTA plasma within the range of the detection system, as well as no outlier between EDTA anticoagulated and heparin anticoagulant plasma. Kappa coefficient of BNP concentration between homologous EDTA anticoagulated and heparin anticoagulant plasma was 0.8553 (p < 0.001), and for EDTA anticoagulated plasma and homologous whole blood it was 0.8941 (p < 0.001). CONCLUSIONS: The diagnostic performance of EDTA anticoagulated whole blood samples did not differ significantly from EDTA anticoagulated plasma samples for the BNP test. This study showed no big significant difference between EDTA anticoagulated and heparin anticoagulated plasma measurements within 2 hours. The type of anticoagulant should be carefully chosen when performing the BNP test if BNP samples were in vitro for a long time.

Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Pretreatment with a dual antiplatelet and anticoagulant (APAC) reduces ischemia-reperfusion injury in a mouse model of temporary middle cerebral artery occlusion-implications for neurovascular procedures.

BACKGROUND: Several neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia-reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia-reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites. METHODS: Brain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified. RESULTS: Both APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either. CONCLUSIONS: Pretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.

Agreement between the thromboelastography reaction time parameter using fresh and citrated whole blood during extracorporeal membrane oxygenation with Teg®5000 and Teg®6s.

PURPOSE: We aimed at assessing the correlation between TEG reaction time (TEG-R) in citrated and fresh blood samples with TEG5000 and TEG 6S during heparin administration in patients with and without ECMO support. MATERIALS AND METHODS: Paired TEG5000 (fresh and citrated whole blood, kaolin and kaolin-heparinase) and TEG6S (citrated whole blood) samples were obtained, together with standard coagulation laboratory tests. Bland-Altman analysis and Lin's concordance correlation coefficient were used to assess agreement. RESULTS: Thirteen consecutive ECMO patients and eight consecutive non-ECMO patients were enrolled and TEG was performed for a total of 84 paired samples. ECMO patients received 19.2 (12.6-25.8) U/kg/h of heparin. Five of the non-ECMO patients did not receive heparin, two of them received a very low prophylactic dose (1.6 and 2.9 IU/kg/h, respectively), and one of them 13.1 U/kg/h of heparin. Using TEG®5000, TEG-R was 21.0 (-23.4; 65.5) min longer on fresh compared to citrated blood in patients receiving heparin while only 1.58 (-5.5; 8.7) min longer in patients not-receiving heparin. These differences were reverted by heparinase. CONCLUSIONS: Using citrated-recalcified blood to perform TEG might lead to underestimation of the effect of heparin.

Unified Methodology for the Primary Preclinical In Vivo Screening of New Anticoagulant Pharmaceutical Agents from Hematophagous Organisms.

The development of novel anticoagulants requires a comprehensive investigational approach that is capable of characterizing different aspects of antithrombotic activity. The necessary experiments include both in vitro assays and studies on animal models. The required in vivo approaches include the assessment of pharmacokinetic and pharmacodynamic profiles and studies of hemorrhagic and antithrombotic effects. Comparison of anticoagulants with different mechanisms of action and administration types requires unification of the experiment scheme and its adaptation to existing laboratory conditions. The rodent thrombosis models in combination with the assessment of hemostasis parameters and hematological analysis are the classic methods for conducting preclinical studies. We report an approach for the comparative study of the activity of different anticoagulants in vivo, including the investigation of pharmacodynamics and the assessment of hemorrhagic effects (tail-cut bleeding model) and pathological thrombus formation (inferior vena cava stenosis model of venous thrombosis). The reproducibility and uniformity of our set of experiments were illustrated on unfractionated heparin and dabigatran etexilate (the most common pharmaceuticals in antithrombic therapy) as comparator drugs and an experimental drug variegin from the tick Amblyomma variegatum. Variegin is notorious since it is a potential analogue of bivalirudin (Angiomax, Novartis AG, Basel, Switzerland), which is now being actively introduced into antithrombotic therapy.

M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

COMPARING THE EFFECTS OF DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID AND LITHIUM HEPARIN ON HEMATOLOGIC VALUES IN BLANDING'S TURTLES (EMYDOIDEA BLANDINGII), PAINTED TURTLES (CHRYSEMYS PICTA), AND COMMON SNAPPING TURTLES (CHELYDRA SERPENTINA).

Hematology is a routine component of clinical management in veterinary patients. Anticoagulant choice can profoundly influence morphologic assessment of erythrocytes, leukocytes, thrombocytes, and their subsequent quantification. Previous chelonian studies suggest that lithium heparin (LH) is a superior anticoagulant due to hemolysis resulting from dipotassium ethylenediaminetetraacetic acid (dEDTA) in some species. The aim of this study was to compare the effects of dEDTA and LH on hematologic values in Blanding's turtles (Emydoidea blandingii, n = 35), painted turtles (Chrysemys picta, n = 34), and common snapping turtles (Chelydra serpentina, n = 36). We collected samples from free-ranging turtles and immediately divided whole blood into LH and dEDTA tubes. Packed cell volume, total solids, erythrocyte sedimentation rate, white blood cell counts, and differential leukocyte counts were determined. Hemolysis was observed macro- and microscopically in dEDTA samples from painted turtles and common snapping turtles. Packed cell volume and heterophil:lymphocyte was lower and erythrocyte sedimentation rate was higher in LH samples from painted turtles (p, 0.05). In snapping turtles, the PCV, number of monocytes, and number of eosinophils was lower in LH samples (p, 0.05). In Blanding's turtles, the number of eosinophils and basophils was higher in LH samples, while heterophil counts were lower (p, 0.05). Anticoagulant choice created constant and proportional bias for multiple analytes in a species-dependent fashion. LH is the recommended anticoagulant for hematology in painted turtles and common snapping turtles. Either LH or dEDTA may be used in Blanding's turtles, though anticoagulant-specific reference intervals may be necessary.

COMPARING THE EFFECTS OF LITHIUM HEPARIN AND DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID ON HEMATOLOGIC VALUES IN PRAIRIE RATTLESNAKES (CROTALUS VIRIDIS) AND LAKE ERIE WATER SNAKES (NERODIA SIPEDON INSULARUM).

Anticoagulants prevent clotting of blood samples and preserve cellular morphology for hematologic evaluations, but studies comparing anticoagulants are limited in snakes. The objective of this study was to evaluate the effects of lithium heparin (LH) and dipotassium ethylenediaminetetraacetic acid (EDTA) on hematologic values in prairie rattlesnakes (PR; Crotalus viridis, n = 16) and Lake Erie water snakes (LEWS; Nerodia sipedon insularum, n = 21). Venipuncture was performed and blood samples were immediately aliquoted into LH and EDTA microtainers. Packed cell volume (PCV), total solids (TS), 100-cell differential counts, and Avian Leukopet white blood cell counts (WBC) were determined for each anticoagulant. Passing-Bablok regression and Bland-Altman plots revealed that anticoagulant choice did not constantly or proportionally bias the values of any WBC parameter. Mixed models demonstrated that blood anticoagulated with EDTA had higher PCV in PR (P = 0.04) and TS in both species (P < 0.05). However, the magnitude of the differences attributable to anticoagulant choice was relatively small and likely not clinically important. Hemolysis was not appreciated in any samples. Our findings demonstrate that LH and EDTA are equally appropriate for use in PR and LEWS, but may require separate reference values.

Heparin as a Potential Therapeutic Substance for Post-Traumatic Stress Disorder.

In sexually mature male Wistar rats with modeled post-traumatic stress disorder, personalized characteristics of neurobiological reactions in the population of predator-induced stress-resilient and stress-susceptible heparinized animals were determined. Characteristics of the systemic response of immune mechanisms, hypothalamic-pituitary-adrenal axis, behavioral manifestations, as well as basic properties of the CNS (excitation/inhibition) are presented. The study demonstrated encouraging positive results of the course administration of unfractionated heparin at a dose below the therapeutic and prophylactic doses. The inclusion of heparin drugs into the clinical practice for the treatment of post-traumatic stress disorder will not require large-scale clinical trials, because many effects of heparin as a nonspecific adaptogen are well studied. Moreover, these properties were confirmed at a higher technological level during the COVID-19 pandemic.