Platelet-derived extracellular vesicles (P-EV) are thought to facilitate the transfer of information from platelets to target cells, playing a role in both physiologic and pathophysiologic processes, particularly in regulating immune responses and healing processes. In addition, P-EV show promise as drug carriers and biomarkers for disease. However, the procedures for isolation, purification and fluorescent labeling of P-EV remain unstandardized. Moreover, the requirement to use freshly obtained platelets for generating EV presents a logistical challenge for their study. In this study, we isolated, characterized, and compared P-EV analogues by sonication of freshly obtained and lyophilized platelets, investigated fluorescent labeling methods, and monitored cellular uptake. We found that P-EV analogues derived from fresh or lyophilized platelets showed similar characteristics regarding size, surface proteins and content. Among the fluorescent labeling methods, CFSE and DiO-C6 were most effective in labeling P-EV analogues from both fresh and lyophilized platelets. All labeling methods led to an increase in P-EV analogue's size, with CFSE and DiO-C6 resulting in the smallest increase. The addition of P-EV analogues to cultured immortal endothelial cells revealed that P-EV analogues were effectively internalized and directed to the lysosomal compartment. The results indicate that P-EV analogues from lyophilized platelets have similar functional properties as those from freshly isolated platelets and these are retained after labeling with CFSE. Thus, lyophilized platelets can serve as a source of P-EV analogues for functional studies.

Chemokines are secreted blood proteins that steer leukocyte migration in the inflammatory response. Neutralization of chemokines is believed to be a beneficial therapeutic strategy for the treatment of inflammation-associated diseases. Proteolytically stable chemokine-binding peptides could be suitable candidates for the development of chemokine-neutralizing agents. Here, we report the mirror-image phage display selection of cyclic all-D-peptides against the C-X-C motif chemokine ligand 8 (CXCL8). Selection yielded structurally diverse all-D-peptides with submicromolar affinity to the target CXCL8 chemokine and different selectivity to related chemokines. Binding of these all-D-peptides caused dissociation of the native CXCL8 dimer and disruption of its binding to GAGs, without an effect on in vitro cell migration. This work demonstrates the example of mirror-image phage display selection of cyclized all-D-peptides and its utility for the development of chemokine-binding agents.

Amustaline (S-303, 200 µM) in combination with glutathione (GSH) quencher inactivates pathogens in red blood cell concentrates with retention of RBC therapeutic function. S-303 efficiently inactivates a broad spectrum of pathogens in whole blood which may facilitate further preparation of components with a single pathogen inactivation process. However, S-303 impacts platelet function (Pongerard et al) in a dose-dependent manner. We have characterized, in detail, the effects of S-303 on plasma coagulation factor function and the mechanism by which S-303 inactivates specific coagulation factor activities. Whole blood was treated with S-303 (100, 200 and 400 µM) and platelet-free plasma was isolated from blood by centrifugation. Global coagulation assays, prothrombin time (PT) and activated thromboplastin time (aPTT) were prolonged with 200 and 400 µM S-303 in dose-dependent fashion. This suggested that certain factors in the extrinsic, intrinsic and common pathways were impacted by higher concentrations of S-303. To understand which factors may be impacted, clotting assays with factor-deficient plasma and model systems using purified proteins were performed. Factor (F)X activation by the extrinsic Xase complex (FVIIa-TF-FX) at both low and high tissue factor concentrations was inhibited by S-303 (200 µM) in purified protein assays. Similarly, FX activation by the intrinsic Xase complex (FIXa-FVIIIa-FX) and prothrombin activation by the prothrombinase complex (PTase: FXa-FVa-FII) was also attenuated by higher concentrations of S-303. These results were in accordance with the S-303-dependent inhibition of activities of factors X, IX, VIII, VII and II, assessed by factor-deficient plasma clotting assays. Notably, FV activity from (1:1000 diluted) plasma, but not purified FV, was preserved in PTase complex and FV-deficient plasma clotting assays. Further, FV activation by thrombin was also preserved in the presence of S303. These data suggest that the inhibition of FII activation by S-303 in PTase is decreased by the presence of plasma. Plasma FVa inactivation by activated protein C (APC) anticoagulant pathway (APC-protein S-PL) was preserved in the presence of S-303. However, pre-treatment of APC and protein S with S-303 prior to inactivation of FVa in purified system assays inhibited the action of APC and protein S. These differences between the plasma assays and purified assays are likely due to other off-target effects of S-303 that are absent in purified assays. S-303 did not inhibit either the antithrombin (AT)-dependent inactivation of thrombin in plasma or the tissue factor pathway inhibitor-dependent inactivation of thrombin generation by the FVIIa-FXa-FVa-Protein S complex at both low and high TF suggesting that the anticoagulant pathways are left intact whereas the procoagulant pathways are attenuated by S-303 treatment. Compared with untreated controls, clot formation time was prolonged and mean clot formation was attenuated in thromboelastometry (EXTEM®) on WB treated with S-303, supporting observations above. Lysis onset time was also prolonged in the presence of added tissue plasminogen activator. To understand the molecular bases of S-303-dependent alterations in function, S-303 was screened for off-target adduct formation in four 5-mer peptide libraries using mass spectrometry. S-303 selectively formed adducts with thiol groups on cysteine residues in these assays. Further S-303 did not break preformed disulfides in these model systems. To better understand the selective inhibition of factors X, IX, VII, prothrombin, APC and protein S that lack free thiols, proteomics was performed on a FX that was profoundly affected by S-303. S-303 formed an adduct with residue Cys375, spatially proximal to the catalytic triad and active site Ser380 in the protease domain of FX. Cys375 is part of a disulfide bond with Cys403. As a negative control, AT that was unaffected by S-303 was analyzed in the same proteomic assays in which no Cys adducts were detected, in accordance with the functional data. Our results suggest that S-303 forms adducts with disulfide bonds in select proteins that are labile or otherwise accessible. This might suggest that functionally important disulfides exist in several coagulation factors and that S-303 and derivatives can be used to probe for these disulfides.

Background: The small family of vitamin K-dependent proteins are characterized by posttranslational modification of specific glutamic acid residues to yield γ-carboxyglutamic acid (Gla). Gla residues give these proteins calcium ion-binding properties, which are essential for a number of coagulation factors and mineralization processes. Biophysical characteristics of Gla are, however, incomplete, hindering molecular dynamics simulations and protein structure predictions. Objectives: This study aimed to elucidate the general biophysical characteristics (pKa and KD) of calcium binding to γ-carboxyglutamic acid in a protein environment and determine how positioning of γ-carboxyglutamic acid influences cooperative calcium binding and protein structure. Methods: Residue-based pKa of Gla carboxyl groups in model peptides was individually determined by measuring 1H and 13C nuclear magnetic resonance chemical shift changes as a function of pH. In addition, residue-based KD values of Ca2+ binding were determined using Ca2+ nuclear magnetic resonance titrations. Secondary structure of peptides and proteins was assessed using circular dichroism and nuclear magnetic resonance. Results: Carboxylic acid groups present on Gla residues have 2 different pKa values of 2.62 ± 0.07 and 5.02 ± 0.05. In presence of calcium ions, pKa values drop to 2.54 ± 0.02 and 4.55 ± 0.04. Affinity of a single Gla residue for calcium is low (∼15 mM); 2 Gla residues show cooperativity, resulting in a 25-fold increased affinity for calcium ions (0.6 mM). Finally, cooperative calcium ion binding led to increased α-helical content in model proteins. Conclusion: Vitamin K-dependent proteins present Gla residues in a different manner but benefit from cooperative calcium ion binding. Experimentally determined pKa and KD values can be used for interpretation of binding interactions or for molecular dynamics simulations of Gla domains with unknown structure.

Despite the growing need for small-caliber tissue-engineered vascular grafts (sTEVGs), no clinically approved substitutes exist, largely due to thrombotic failure. We recently purified a fucosylated chondroitin sulfate from two marine invertebrates, Holothuria tubulosa and Sarcotragus spinosulus, which showed strong anticoagulant activity and in vitro cytocompatibility. This study aimed to develop nanofibrous poly(Ɛ-caprolactone) (PCL) electrospun scaffolds functionalized with these marine polysaccharides to improve hemocompatibility and endothelialization. The functionalized scaffolds exhibited anticoagulant and antiplatelet properties and supported endothelial cell colonization. Human microvascular endothelial cells cultured on the scaffolds formed a confluent monolayer within 4 days, confirmed by vascular endothelial cadherin and von Willebrand factor expression. These results demonstrate that crosslinking PCL scaffolds with sulfated marine polysaccharides is a promising strategy for overcoming current sTEVG limitations.

BACKGROUND:Neutrophils are known to externalize their DNA and intracellular contents to neutralize invading pathogens. This process may enhance blood coagulation during inflammation. Tissue factor (TF) pathway inhibitor (TFPI) binds to extracellular DNA and may be citrullinated by peptidylarginine deiminase 4. Citrullination of TFPI reduces its anticoagulant activity toward factor (F)Xa but appears to retain its inhibition of TF-triggered thrombin generation, indicating differential regulation of TFPI functions by peptidylarginine deiminase 4. OBJECTIVES:This work aimed to study the effects of citrullination of TFPI-alpha on the inhibition of FXa, FVIIa/TF, and the cofactor activity of protein S. METHODS:The effect of TFPI citrullination on the inhibition of FXa and FVIIa/TF was measured by chromogenic assays using purified components and by calibrated automated thrombography. Interaction with protein S was assessed by surface plasmon resonance and solid-phase binding assays using immobilized protein S, recombinant TFPI, and synthetic TFPI domains. RESULTS:Citrullination of TFPI abolished its ability to inhibit FXa- and FXIa-triggered thrombin generation. However, its impaired inhibition of TF-triggered thrombin generation was still enhanced by protein S. Chromogenic assays revealed that citrullinated TFPI was essentially inactive as an inhibitor of the FVIIa-TF complex in the absence of protein S but partially restored by protein S. Interaction studies revealed that binding of citrullinated TFPI to protein S was reduced approximately 4-fold. CONCLUSION:Citrullinated TFPI shows impaired natural anticoagulant activity. While anti-FXa activity is essentially absent, its anti-TF/FVIIa activity can still be enhanced by protein S. This enhancement is incomplete; however, protein S binding to citrullinated TFPI is impaired.

Background: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. Objectives: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. Methods: Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. Results: F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. Conclusion: These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.

Vascular calcification is a common phenomenon in various vascular diseases, where its presence heralds increased occurrence of adverse disease events, which invariably lead to increased morbidity and mortality in patients. Although the impact of calcification has become apparent, adequate and early detection of the most damaging form of early microcalcification is still in its infancy, preventing reliable identification of locations that would benefit from intervention. In this review, we will provide an overview of the current state-of-the-art noninvasive calcification imaging and its persisting limitations. We discuss promising approaches that may address these limitations in the future. In this context particular attention will be paid to imaging modalities such as CT, PET, and ultrasonography and molecular and cellular mechanisms and agents involved in physiological bone formation.

Background: The C-terminal region of TFPI hosts a FV homology motif, enabling binding to the acidic region within the B-domain of FV and preventing the activation of FV and its FV-short isoform. This region also assists TFPI in binding with its co-factor, protein S. Truncation of TFPI's C-terminal tail by plasmin, thrombin, or the neutrophil enzyme matrix metalloproteinase-8 leads to less efficient coagulation inhibition. This study investigates whether citrullination of arginine residues within the C-terminal region of TFPI affects coagulation parameters. Aim: To examine the effects of C-terminal citrullination of TFPI, C-terminal peptides with specific citrullination patterns were investigated on FV activation and in their interactions with protein S. Methods: We synthesized several variants of the TFPI C-terminal region with arginine replaced by citrulline at different locations (R5, R16, R18, and R22). These variants were assayed for their ability to inhibit prothrombinase activity by preventing FV activation. Their performance was further evaluated using calibrated automated thrombography (CAT) experiments. Additionally, the variants were tested for their interference in the binding between full-length TFPI and protein S and their capacity to inhibit TFPI's anti-FXa activity. Results: Citrullination at R16 or R18 resulted in a substantial reduction (<50%) in prothrombinase inhibition due to the inability of these peptides to prevent FV activation. Variants with multiple citrullinated residues (R16, R18, and R22) showed severe loss of inhibition (<80%). Complete citrullination (R5, R16, R18, and R22) resulted in complete loss of inhibitory activity towards the prothrombinase complex. In thrombin generation assays, variants with citrulline residues near the FV homology motif impaired thrombin generation inhibition, and fully citrullinated variants failed to inhibit thrombin generation completely. The fully citrullinated variant showed a substantial reduction in competition with full-length TFPI for protein S interactions. Conclusion: This study demonstrates that citrullination of arginine residues within the C-terminal region of TFPI significantly impairs its ability to inhibit coagulation. Specifically, citrullination at critical sites (R16 and R18) within the FV homology motif results in a substantial reduction in prothrombinase inhibition and thrombin generation suppression. Further compounding citrullination, especially in constructs where R16, R18, and R22 were citrullinated, leads to severe loss of inhibitory function, with complete citrullination rendering TFPI almost entirely ineffective in these roles. Additionally, fully citrullinated TFPI variants show reduced competition with full-length TFPI for protein S interactions, suggesting compromised co-factor binding. These findings indicate that extracellular post-translational modifications such as citrullination can drastically alter the functional integrity of TFPI, highlighting the importance of the C-terminal arginine residues in maintaining its anticoagulant properties. Further research may explore the therapeutic implications of modulating TFPI citrullination in coagulation disorders.

Background. Tissue factor pathway inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway by inhibiting the TF/FVIIa/FXa complex. Beyond its established role, recent studies have suggested non-canonical functions of TFPI, including protection against atherogenesis in animal models. However, the underlying molecular mechanisms remain unclear. Objective. This study aims to elucidate the impact of TFPI on proliferation and phenotypic switching of vascular smooth muscle cells (VSMC) and endothelial cells (EC) using a novel, genetically modified variant of TFPI. Methods. Pluripotent stem cells were genetically modified to express a partially functional TFPI variant (NP_006278.1:(Tyr137_Arg140del)) and differentiated into endothelial and vascular smooth muscle cells. In silico models were employed to elucidate the binding of this modified TFPI to FXa. TFPI expression and secretion were quantified via Western blot and ELISA. Functionality of the mutant TFPI was assessed using chromogenic assays for FVIIa, FXa, and prothrombinase inhibition, as well as calibrated automated thrombography. Cellular proliferation in VSMC was quantified with an EdU- and an impedance-based assay. Extracellular vesicle secretion by contractile and synthetic VSMC was measured using a particle tracer device. VSMC were analyzed for the expression of contractility markers and vascular calcification in response to high calcium stimuli. Results. EC-derived TFPI exhibited partial functionality, with Kunitz domain 1 and the C terminus remaining effective in inhibiting FVIIa and prothrombinase, respectively, while the functionality of Kunitz domain 2 was impaired. Inhibition of thrombin formation was also reduced with the mutant TFPI variant. Mutant EC displayed aberrant expression of EC markers CD31 and CD144, along with reduced TFPI secretion. VSMC with the TFPI mutation showed decreased TFPI expression and secretion, significantly reduced expression of contractility markers, and increased proliferation, which was reversible upon repair of the mutation or treatment with the FXa inhibitor rivaroxaban. Mutant, synthetic VSMC did not increase TFPI secretion, unlike wild-type cells, but had a 4.4-fold increase in vesicle secretion compared to wild-type cells. Calcification was significantly higher and faster in mutant VSMC, a process that could be partially reversed with extracellular recombinant TFPI. Conclusion. Our findings demonstrate that a Kunitz domain 2-deficient TFPI variant affects the proliferation and phenotypes of endothelial and vascular smooth muscle cells in a FXa-dependent manner. Aberrant marker expression in EC could possibly lead to cell activation or endothelial-to-mesenchymal transition. The altered TFPI and vesicle secretion patterns in mutant VSMC indicate a stronger synthetic phenotype, corroborated by lower expression of contractile markers. These molecular changes could explain the enhanced calcification rate and increased vascular remodeling observed in previous animal studies. This study highlights the critical role of TFPI in maintaining vascular integrity and underscores the need for further research into its non-canonical functions.

BACKGROUND:Cardiac troponin T (cTnT) is key in diagnosing myocardial infarction (MI) but is also elevated in end-stage renal disease (ESRD) patients. Specific larger cTnT proteoforms were identified for the acute phase of MI, while in serum of ESRD patients solely small cTnT fragments were found. However, others allocated this to a pre-analytic effect due to abundant thrombin generation in serum. Therefore, we investigated the effect of various anticoagulation methods on cTnT composition and concentration and compared the cTnT composition of MI and ESRD patients. METHODS:The agreement of cTnT concentrations between simultaneously collected serum, lithium-heparin (LH) plasma, and ethylenediaminetetraacetic acid (EDTA) plasma was studied using the high-sensitivity (hs-)cTnT immunoassay. cTnT proteoform composition was investigated in a standardized time-dependent manner through spike experiments and in simultaneously collected blood matrixes of MI and ESRD patients. RESULTS:Excellent hs-cTnT concentration agreements were observed across all blood matrixes (slopes > 0.98; 95% CI, 0.96-1.04). Time-dependent degradation (40 kDa intact:29 kDa fragment:15 to 18 kDa fragments) was found in LH plasma and EDTA plasma, and serum in ratios (%) of 90:10:0, 0:5:95, and 0:0:100, respectively (48 h after blood collection). Moreover, gel filtration chromatography (GFC) profiles illustrated mainly larger cTnT proteoforms in MI patients, while in ESRD patients mainly 15 to 18 kDa fragments were found for all matrices. CONCLUSIONS:The extent of cTnT degradation in vitro is dependent on the (anti)coagulation method, without impacting hs-cTnT concentrations. Furthermore, mainly larger cTnT proteoforms were present in MI patients, while in ESRD patients mainly small 15 to 18 kDa cTnT fragments were found. These insights are essential when developing a novel hs-cTnT assay targeting larger cTnT proteoforms.

Supplementary Figures 1-5 from Tumor Cells Secrete Galectin-1 to Enhance Endothelial Cell Activity

Figure 1: Comparison of pregnancy, delivery and post-partum data between women included in the Fibrinogest Study and the French general population Conclusion(s):We did not observe a greater risk of miscarriage compared to the general population but found a significant risk of retroplacental hematoma as well as of post-partum haemorrhage and thrombosis.Most women did not received analgesia at the delivery due to the underlying fibrinogen disorder.Our findings highlight the urgent need for international guidance on management of pregnancy in HFD.

Results: HBC with dose escalation most significantly reduced blood pressure and increased perfusion of paw vessels, which was accompanied by complement system activation and secretion of NO and angiotensin 1-7.We observed the respiratory disturbances and increased angiotensin II and thromboxane B2 production after protamine and HBC infusion.Protamineinduced hypotension and vasodilatation were accompanied by the release of prostacyclin metabolite and angiotensin 1-7 (Figure 1A-G).Both compounds elevated vascular permeability in major mouse organs (Figure 1H).PER977 slightly changed cardiorespiratory parameters, what may have been related to prostacyclin release.The data were analyzed using GraphPadPrism8.Conclusion(s): All antidotes at the high dose were found to change cardiovascular and respiratory parameters through distinct mechanisms of action.PER977 seems to be the safest heparin neutralizer.