Extracellular Matrix Mimicking Wound Microenvironment Responsive Amyloid-Heparin@TAAgNP Co-Assembled Hydrogel: An Effective Conductive Antibacterial Wound Healing Material.

Bioengineered composite hydrogel platforms made of a supramolecular coassembly have recently garnered significant attention as promising biomaterial-based healthcare therapeutics. The mechanical durability of amyloids, in conjunction with the structured charged framework rendered by biologically abundant key ECM component glycosaminoglycan, enables us to design minimalistic customized biomaterial suited for stimuli responsive therapy. In this study, by harnessing the heparin sulfate-binding aptitude of amyloid fibrils, we have constructed a pH-responsive extracellular matrix (ECM) mimicking hydrogel matrix. This effective biocompatible platform comprising heparin sulfate-amyloid coassembled hydrogel embedded with polyphenol functionalized silver nanoparticles not only provide a native skin ECM-like conductive environment but also provide wound-microenvironment responsive on-demand superior antibacterial efficacy for effective diabetic wound healing. Interestingly, both the cytocompatibility and antibacterial properties of this bioinspired matrix can be fine-tuned by controlling the mutual ratio of heparin sulfate-amyloid and incubated silver nanoparticle components, respectively. The designed biomaterial platform exhibits notable effectiveness in the treatment of chronic hyperglycemic wounds infected with multidrug-resistant bacteria, because of the integration of pH-responsive release characteristics of the incubated functionalized AgNP and the antibacterial amyloid fibrils. In addition to this, the aforementioned assemblage shows exceptional hemocompatibility with significant antibiofilm and antioxidant characteristics. Histological evidence of the incised skin tissue sections indicates that the fabricated composite hydrogel is also effective in controlling pro-inflammatory cytokines such as IL6 and TNFα expressions at the wound vicinity with significant upregulation of angiogenesis markers like CD31 and α-SMA.

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.

An evaluation of a Stenotrophomonas maltophilia outbreak due to commercial arterial blood gas collection kit.

BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacterium that can cause hospital infections and outbreaks within hospitals. This study aimed to evaluate an outbreak of Stenotrophomonas maltophilia, caused by ready-to-use commercial syringes containing liquid lithium and heparin for arterial blood gas collection in a university hospital. METHODS: Upon detecting an increase in Stenotrophomonas maltophilia growth in blood cultures between 15.09.2021 and 19.11.2021, an outbreak analysis and a case-control study (52 patients for the case group, 56 patients for the control group) were performed considering risk factors for bacteremia. Samples from possible foci for bacteremia were also cultured. Growing bacteria were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The genetic linkage and clonal relationship isolates were investigated with pulsed-field gel electrophoresis (PFGE) in the reference laboratory. RESULTS: In the case-control study, the odds ratio for the central venous catheter [3.38 (95% confidence interval [CI]: 1.444, 8.705 ; p = 0.006)], for surgery [3.387 (95% confidence interval [CI]: 1.370, 8.373 ; p = 0.008)] and for arterial blood gas collection history [18.584 (95% confidence interval [CI]:4.086, 84.197; p < 0.001)] were identified as significant risk factors. Stenotrophomonas maltophilia growth was found in ready-to-use commercial syringes used for arterial blood gas collection. Molecular analysis showed that the growths in the samples taken from commercial syringes and the growths from blood cultures were the same. It was decided that the epidemic occurred because the method for sterilization of heparinized liquid preparations were not suitable. After discontinuing the use of the kits with this lot number, the outbreak was brought under control. CONCLUSIONS: According to our results, disposable or sterile medical equipment should be included as a risk factor in outbreak analyses. The method by which injectors containing liquids, such as heparin, are sterilized should be reviewed. Our study also revealed the importance of the cooperation of the infection control team with the microbiology laboratory.

Radiosensitizing effects of heparinized magnetic iron oxide nanoparticles in colon cancer.

The combination of radiation treatment and chemotherapy is currently the standard for management of cancer patients. However, safe doses do not often provide effective therapy, then pre-treated patients are forced to repeat treatment with often already increased tumor resistance to drugs and irradiation. One of the solutions we suggest is to improve primary course of radiation treatment via enhancing radiosensitivity of tumors by magnetic-guided iron oxide nanoparticles (magnetite). We obtained spherical heparinized iron oxide nanoparticles (hIONPs, ∼20 nm), characterized it by TEM, Infrared spectroscopy and DLS. Then hIONPs cytotoxicity was assessed for colon cancer cells (XTT assay) and cellular uptake of nanoparticles was analyzed with X-ray fluorescence. Combination of ionizing radiation (IR) and hIONPs in vitro caused an increase of G2/M arrest of cell cycle, mitotic errors and decrease in survival (compared with samples exposed to IR and hIONPs separately). The promising results were shown for magnetic-guided hIONPs in CT26-grafted BALB/C mice: the combination of intravenously administrated hIONPs and IR showed 20,8% T/C ratio (related to non-treated mice), while single radiation had no shown significant decrease in tumor growth (72,4%). Non-guided by magnets hIONPs with IR showed 57,9% of T/C. This indicates that ultra-small size and biocompatible molecule are not the key to successful nano-drug design, in each case, delivery technologies need to be improved when transferred to in vivo model.

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.

Heparin-Modified Superparamagnetic Iron Oxide Nanoparticles Suppress Lithium Chloride/Pilocarpine-Induced Temporal Lobe Epilepsy in Rats through Attenuation of Inflammation and Oxidative Stress.

The development of antiepileptic drugs is still a long process. In this study, heparin-modified superparamagnetic iron oxide nanoparticles (UFH-SPIONs) were prepared, and their antiepileptic effect and underlying mechanism were investigated. UFH-SPIONs are stable, homogeneous nanosystems with antioxidant enzyme activity that are able to cross the blood-brain barrier (BBB) and enriched in hippocampal epileptogenic foci. The pretreatment with UFH-SPIONs effectively prolonged the onset of seizures and reduced seizure severity after lithium/pilocarpine (LP)-induced seizures in rats. The pretreatment with UFH-SPIONs significantly decreased the expression of inflammatory factors in hippocampal tissues, including IL-6, IL-1ß, and TNF-α. LP-induced oxidative stress in hippocampal tissues was in turn reduced upon pretreatment with UFH-SPIONs, as evidenced by an increase in the levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and a decrease in the level of lipid peroxidation (MDA). Moreover, the LP-induced upregulation of apoptotic cells was decreased upon pretreatment with UFH-SPIONs. Together, these observations suggest that the pretreatment with UFH-SPIONs ameliorates LP-induced seizures and downregulates the inflammatory response and oxidative stress, which exerts neuronal protection during epilepsy.

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.

Generalized Multifunctional Coating Strategies Based on Polyphenol-Amine-Inspired Chemistry and Layer-by-Layer Deposition for Blood Contact Catheters.

Blood-contacting catheters play a pivotal role in contemporary medical treatments, particularly in the management of cardiovascular diseases. However, these catheters exhibit inappropriate wettability and lack antimicrobial characteristics, which often lead to catheter-related infections and thrombosis. Therefore, there is an urgent need for blood contact catheters with antimicrobial and anticoagulant properties. In this study, we employed tannic acid (TA) and 3-aminopropyltriethoxysilane (APTES) to create a stable hydrophilic coating under mild conditions. Heparin (Hep) and poly(lysine) (PL) were then modified on the TA-APTES coating surface using the layer-by-layer (LBL) technique to create a superhydrophilic TA/APTES/(LBL)4 coating on silicone rubber (SR) catheters. Leveraging the superhydrophilic nature of this coating, it can be effectively applied to blood-contacting catheters to impart antibacterial, antiprotein adsorption, and anticoagulant properties. Due to Hep's anticoagulant attributes, the activated partial thromboplastin time and thrombin time tests conducted on SR/TA-APTES/(LBL)4 catheters revealed remarkable extensions of 276 and 103%, respectively, when compared to uncoated commercial SR catheters. Furthermore, the synergistic interaction between PL and TA serves to enhance the resistance of SR/TA-APTES/(LBL)4 catheters against bacterial adherence, reducing it by up to 99.9% compared to uncoated commercial SR catheters. Remarkably, the SR/TA-APTES/(LBL)4 catheter exhibits good biocompatibility with human umbilical vein endothelial cells in culture, positioning it as a promising solution to address the current challenges associated with blood-contact catheters.

Unfractionated heparin reverses aspirin inhibition of platelets during coronary artery bypass graft surgery.

Unfractionated heparin (UFH) is an effective antithrombotic during surgery but has known adverse effects, in particular on platelets. A marked increase in platelet responsiveness has previously been observed in patients within minutes of receiving UFH, despite adequate inhibition by aspirin prior to heparin. We studied this phenomenon in patients undergoing cardiac artery bypass grafting (n = 17) to determine whether the effects of heparin were systemic or platelet-specific. All patients' platelets were fully inhibited by aspirin prior to surgery, but within 3 min of receiving heparin spontaneous aggregation and responses to arachidonic acid (AA) and ADP increased significantly (p ≥ 0.0002), and activated platelets were found in the circulation. While there was no rise in thromboxane in the plasma following heparin, levels of the major platelet 12-lipoxygenase product, 12-HETE, rose significantly. Mixing experiments demonstrated that the changes caused by heparin resided primarily in the platelets, while addition of AA pathway inhibitors, and analysis of oxylipins provided evidence that, following heparin, aggregating platelets regained their ability to synthesise thromboxane. These findings highlight potentially unrecognised pro-thrombotic and pro-inflammatory changes during CABG surgery, and provide further evidence of adverse effects associated with UFH.

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.

The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

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.

A New Synthesized Dicarboxylated Oxy-Heparin Efficiently Attenuates Tumor Growth and Metastasis.

Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors that support tumor growth. Heparanase expression is upregulated in human carcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, the only heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI) values ranging from 21 to 70% and extending the survival time of the mice. Of particular significance was the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity, compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation. Heparanase-dependent and -independent effects of XII are being investigated. Collectively, our pre-clinical studies with compound XII strongly justify its examination in cancer patients.

Heparinized Acellular Hydrogels for Magnetically Induced Wound Healing Applications.

The control of angiogenesis has the potential to be used for regulation of several pathological and physiological processes, which can be instrumental on the development of anticancer and wound healing therapeutical approaches. In this study, mesenchymal stem/stromal cells (MSCs) were seeded on magnetic-responsive gelatin, with or without heparin functionalization, and exposed to a static 0.08 T magnetic field (MF), for controlling their anti-inflammatory and angiogenic activity, with the aim of accelerating tissue healing. For the first time, it was examined how the amount of heparin and magnetic nanoparticles (MNPs) distributed on gelatin scaffolds affected the mechanical properties of the hydrogels and the morphology, proliferation, and secretome profiling of MSCs. The findings demonstrated that the addition of MNPs and heparin affects the hydrogel swelling capacity and renders distinct MSC proliferation rates. Additionally, MF acts as a topographical cue to guide MSCs alignment and increases the level of expression of specific genes and proteins that promote angiogenesis. The results also suggested that the presence of higher amounts of heparin (10 µg/cm3) interferes with the secretion and limits the capacity of angiogenic factors to diffuse through the hydrogel and into the culture medium. Ultimately, this study shows that acellular heparinized hydrogels efficiently retain the angiogenic growth factors released by magnetically stimulated MSCs thus rendering superior wound contraction (55.8% ± 0.4%) and cell migration rate (49.4% ± 0.4%), in comparison to nonheparinized hydrogels (35.2% ± 0.7% and 37.8% ± 0.7%, respectively). Therefore, these heparinized magnetic hydrogels can be used to facilitate angiogenesis in various forms of tissue damage including bone defects, skin wounds, and cardiovascular diseases, leading to enhanced tissue regeneration.

Heparin Oligosaccharides as Vasoactive Intestinal Peptide Inhibitors via their Binding Process Characterization.

BACKGROUND: It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al. found that by an alanine scan, the heparin- binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP. OBJECTIVE: A variety of experiments were designed to study the binding process and structure-activity relationship between heparin oligosaccharides and VIP. METHODS: Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro. RESULTS: The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP. CONCLUSION: The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity.

A low-anticoagulant heparin suppresses metastatic dissemination through the inhibition of tumor cell-platelets association.

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.

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.

Evaluation of the effect of phenylpropanoids on the binding of heparin to human serum albumin and glycosylated human serum albumin concerning anticoagulant activity: A comparison study.

The nonenzymatic advanced glycation end products (AGEs) and the accumulation of AGEs are the two main factors associated with the long-term pathogenesis of diabetes. Human serum albumin (HSA) as the most abundant serum protein has a higher fortuity to be modified by nonenzymatic glycation. In this study, the interaction of three phenylpropanoids (caffeic acid (Caf), p-coumaric acid (Cou), and cinnamic acid (Cin)) toward HSA and glycosylated HSA (gHSA) was analyzed by multiple spectroscopic techniques combined with molecular docking. The formation of fibrils in HSA and gHSA was confirmed by the Thioflavin T (ThT) assay. The phenylpropanoids have shown anti-fibrillation properties in vitro. The obtained thermodynamic parameters indicated that hydrogen bonding and van der Waals forces are the main forces in the binding interaction, and the quenching mechanism of the protein fluorescence is static. Molecular docking results, as well as the in vitro results, showed that Caf, Cou, and Cin exhibit more stable interactions with HSA, respectively. In addition, molecular docking analysis showed that Caf and Cou interact well with K199. Given the critical role of K199 in HSA glycosylation in diabetic patients, this process inhibits the interaction of stabilizer compounds and thus accelerates gHSA aggregation.

Effect of bivalirudin on coagulation in neonatal (cord) and adult human blood in vitro.

INTRODUCTION: Bivalirudin is recommended as an alternative to heparin in cardiac surgery with cardiopulmonary bypass. Although it has been used in infants and children for this indication, there is a paucity of data on the pharmacologic effects of bivalirudin in neonates. Given the immaturity of the hemostatic system in neonates, we hypothesized that coagulation responses to bivalirudin in this population would be different than in adults. METHODS: Blood samples were drawn from placenta-cord units and from healthy adult donors. The study was carried out in two steps. First, bivalirudin was added to cord and adult blood samples at concentrations of 0, 5, 10, 15, and 20 µg/mL. Activated clotting time and thromboelastographic variables were recorded. Next, we used a Chandler loop system to assess the efficacy of bivalirudin in a simple model of cardiopulmonary bypass. The loops were primed with cord or adult blood and were run until thrombus was detected. Plasma bivalirudin concentrations were measured at 1, 15, 30, 45, 60, and 75 min after initiating rotation of the loops using liquid chromatography/mass spectrometry. RESULTS: Bivalirudin elicited a dose-dependent prolongation inhibition of coagulation in both cord and adult blood samples with greater potency in cord blood in comparison to adult blood (activated clotting time: 627 ± 50 vs. 452 ± 22 s at 15 µg/mL bivalirudin, p < .0001). This relative potency was also demonstrated in the Chandler loop system, but interestingly, cord blood appeared to inactivate bivalirudin more rapidly than adult blood with earlier clotting in loops containing cord blood. CONCLUSIONS: This study demonstrates that bivalirudin has greater potency in cord blood in vitro than in adult blood. Plasma degradation appears to proceed more rapidly in cord blood than in adults. Both of these findings should be considered when planning dosing regimens in neonatal patients.