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PurposeOptimal heparin titration during cardiopulmonary bypass (CPB) may reduce coagulation system activation and preserve hemostatic function post-CPB. Our objective was to assess if the Heparin Management System (HMS) Plus improves heparin titration, thereby leading to higher thrombin generation post-CPB compared with activated clotting time (ACT)-guided management.MethodsWe conducted a randomized controlled trial of 100 patients undergoing cardiac surgery with CPB at a single center. A total of 50 patients were randomized to conventional ACT-guided management, and 50 to the HMS Plus system. The primary outcome was change in thrombin generation post-CPB compared with baseline, as assessed by calibrated automated thrombography. Secondary outcomes included intraoperative blood loss, chest drain output up to 72 hr, and transfusions. In an exploratory analysis, we compared the quintile of patients with the highest average heparin concentration on CPB (≥ 4.0 mg⋅kg-1) with the rest of the cohort.ResultsA total of 100 patients were included in an intent-to-treat analysis. We observed no difference in post-CPB thrombin generation or secondary outcomes. However, patients in the HMS Plus group had higher average heparin concentrations while on CPB than patients in the conventional management group did (mean difference, -0.21; 95% confidence interval, -0.42 to -0.01). The quintile of patients with the highest average heparin concentration (4.0 mg⋅kg-1) had higher thrombin generation post-CPB than the rest of the cohort did.ConclusionsThe HMS Plus system did not show significant benefits in thrombin generation, bleeding outcomes, or transfusion in patients undergoing cardiac surgery with CPB. Higher average heparin concentrations on CPB were associated with higher post-CPB thrombin generation.Study registrationwww.ClinicalTrials.gov (NCT03347201); first submitted 12 October 2017.

Background. Symptomatic postoperative spinal epidural hematoma (PSEH) is a devastating complication that could develop after lumbar decompression surgery. PSEH can also develop after biportal endoscopic spine surgery (BESS), one of the recently introduced minimally invasive spine surgery techniques. Gelatin-thrombin matrix sealant (GTMS) is commonly used to prevent PSEH. This study aimed at analyzing the clinical and radiological effects of GTMS use during BESS. Methods. A total of 206 patients with spinal stenosis who underwent decompression by BESS through a posterior interlaminar approach from October 2015 to September 2018 were enrolled in this study. Postoperative magnetic resonance imaging (MRI) was performed in all patients for evaluation of PSEH. Patients in whom GTMS was not used during surgery were assigned to Group A, and those in whom GTMS was used were classified as Group B. In the clinical evaluation, the visual analog scale (VAS) of the leg and back, Oswestry Disability Index (ODI), and modified MacNab criteria were used. The incidence rate and degree of dural compression of PSEH on postoperative MRI were measured. Results. The average age of the patients was 68.1±11.2 (42–89) years. The overall incidence rate of PSEH was 20.9% (43/206). The incidence rates in Groups A and B were 26.4% and 13.6%, respectively, showing a significant difference (p=0.023). The VAS-leg and ODI improvement was significantly different depending on the intraoperative use of GTMS. However, there was no statistically significant difference between the two groups in terms of the VAS-back improvement. Groups A and B showed “good” and “excellent” rates according to the modified MacNab criteria in 79.4% and 87.6% of patients, respectively, showing statistically significant difference (p=0.049). In Group A, two patients underwent revision surgery due to PSEH, while none in Group B had such event. Conclusion. Intraoperative use of GTMS during BESS may be related to reduction in the occurrence rate of PSEH. Specifically, patients with GTMS appliance showed marked decrease in the occurrence of PSEH and had better clinical outcomes.

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe . The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 × 10 −21 mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

Mutant isocitrate dehydrogenase 1 ( IDH1 ) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II–IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26–30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85–90 % in wild-type versus 2–6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

The worsening crisis of antibiotic resistance demands innovative therapies to combat multidrug-resistant pathogens. In this study, we report the rational design and optimization of VFR12 (VFRLKKWIQKVI), a thrombin C-terminus-derived 12-mer peptide, via systematic amino acid substitutions and stereochemical modifications. A series of 12-mer analogs were synthesized and assessed for antimicrobial activity, cell selectivity, biofilm disruption, and immunomodulatory effects. Six lead candidates (VFR12-a7, VFR12-a8, VFR12-a7(L), VFR12-a8(L), VFR12-a7(L)-d, VFR12-a8(L)-d) demonstrated potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa with minimal hemolytic activity. Notably, D-amino acid variants VFR12-a7(L)-d and VFR12-a8(L)-d showed significantly improved therapeutic indices, complete resistance to proteolysis, and enhanced serum stability compared to their L-isomers. These peptides effectively inhibited biofilm formation and disrupted preformed biofilms while maintaining excellent biocompatibility. Furthermore, Anti-inflammatory assessment revealed significant suppression of LPS-induced production of TNF-α, IL-6, and nitric oxide, along with strong endotoxin neutralization. Among the analogs, VFR12-a8(L)-d emerged as the most promising candidate, combining potent antimicrobial activity with excellent safety and multifaceted therapeutic properties. These findings provide a valuable framework for the development of next-generation host defense peptides with integrated antimicrobial, antibiofilm, and anti-inflammatory properties, offering a multifaceted approach to tackling antibiotic resistance and sepsis.

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell-matrix and cell-cell contact. While increased mechanical loading at cell-matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell-cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell-cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell-cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.

Previous studies demonstrated that thrombin is an important factor in brain injury after intracerebral hemorrhage. This study investigated the effect of thrombin on hydrocephalus development in a rat intraventricular hemorrhage (IVH) model. There were three parts in this study. First, male Sprague–Dawley rats had an injection of 200 μL saline, autologous blood or heparinized blood, into the right lateral ventricle. Second, rats had an injection of 50 μL saline or 3U thrombin into the right lateral ventricle. Third, rats had an injection of thrombin (3U) with a protease-activated receptor-1 (PAR-1) antagonist, SCH79797 (0.15 nmol), or vehicle into the right lateral ventricle. Lateral ventricle volumes were measured by magnetic resonance imaging and the brains were used for immunohistochemistry and western blot analyses. Intraventricular injection of autologous blood induced hydrocephalus from day 1 to 28. Heparinized blood injection resulted in less hydrocephalus at all time points compared with blood injection alone (P<0.05). Intraventricular injection of thrombin caused significant hydrocephalus, ventricular wall damage, and periventricular blood–brain barrier disruption. Thrombin-induced hydrocephalus was reduced by co-injection of the PAR-1 antagonist SCH79797 (P<0.05). In conclusion, thrombin contributes to hydrocephalus development after IVH and thrombin-induced hydrocephalus is through PAR-1.

Necroptosis is a form of programmed cell death executed by receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL). Platelets are circulating cells that play central roles in haemostasis and pathological thrombosis. In this study we demonstrate seminal contribution of MLKL in transformation of agonist-stimulated platelets to active haemostatic units progressing eventually to necrotic death on a temporal scale, thus attributing a yet unrecognized fundamental role to MLKL in platelet biology. Physiological agonists like thrombin instigated phosphorylation and subsequent oligomerization of MLKL in platelets in a RIPK3-independent but phosphoinositide 3-kinase (PI3K)/AKT-dependent manner. Inhibition of MLKL significantly curbed agonist-induced haemostatic responses in platelets that included platelet aggregation, integrin activation, granule secretion, procoagulant surface generation, rise in intracellular calcium, shedding of extracellular vesicles, platelet-leukocyte interactions and thrombus formation under arterial shear. MLKL inhibition, too, prompted impairment in mitochondrial oxidative phosphorylation and aerobic glycolysis in stimulated platelets, accompanied with disruption in mitochondrial transmembrane potential, augmented proton leak and drop in both mitochondrial calcium as well as ROS. These findings underscore the key role of MLKL in sustaining OXPHOS and aerobic glycolysis that underlie energy-intensive platelet activation responses. Prolonged exposure to thrombin provoked oligomerization and translocation of MLKL to plasma membranes forming focal clusters that led to progressive membrane permeabilization and decline in platelet viability, which was prevented by inhibitors of PI3K/MLKL. In summary, MLKL plays vital role in transitioning of stimulated platelets from relatively quiescent cells to functionally/metabolically active prothrombotic units and their ensuing progression to necroptotic death.

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.

We evaluated modifications in the hemostatic balance of different concentrations of apixaban (APIX) in 25 healthy donors and 53 patients treated with aspirin (ASA, n = 21), ASA and clopidogrel (ASA + CLOPI, n = 11), or ASA and ticagrelor (ASA + TICA, n = 21). Blood samples from participants were spiked ex vivo with apixaban 0 (APIX0), 40 (APIX40), and 160 ng/mL (APIX160). We assessed the effects of APIX on (1) clot formation, by ROTEM thromboelastometry; (2) thrombin generation primed by platelets; and (3) platelet and fibrin interactions with a thrombogenic surface, in a microfluidic model with circulating blood. APIX caused dose-related prolongations of clotting time with minimal impact on other ROTEM parameters. Thrombin generation was significantly inhibited by APIX160, with ASA + TICA actions showing the strongest inhibition (p < 0.01 vs APIX0). Microfluidic studies showed that APIX160 was more potent at suppressing platelet and fibrin interactions (p < 0.001 vs. APIX0). APIX40 demonstrated a consistent antithrombotic action but with a favorable protective effect on the structural quality of fibrin. APIX potentiated the antithrombotic effects of current antiplatelet regimens. APIX at 40 ng/mL, enhanced the antithrombotic action of single or dual antiplatelet regimens but was more conservative for hemostasis than the 160 ng/mL concentration.