Heparin Inhibition Research Articles

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.

Drug Interactions in the Treatment of Chronic Heart Failure: Analysis of Clinical Guidelines

Background. According to Russian epidemiological studies, the prevalence of chronic heart failure (CHF) in the general population is high and amounts to 7–10%. Therapy of any disease, and especially chronic disease, is associated with the prescription of drugs. With the development of evidence-based medicine and implementation of its achievements in real clinical practice all over the world there is an increase in the number of prescribed drugs. This explains the high relevance of the problem of drug-drug interactions. Aims — analysis of interactions of drugs recommended for prescription to patients suffering from CHF. Methods. Based on the Clinical Guidelines (CG), all possible interdrug interactions of recommended medicinal products were analysed. Information on potential drug-drug interactions was obtained from the specialised website Drugs.com. Know more. Be sure (https://www.drugs.com/interactions/list/). Results. ACE inhibitors / ARA II / valsartan+sacubitril, beta-adrenoblockers and aldosterone antagonists are recommended as part of combination therapy for treatment according to CG for all patients with symptomatic heart failure (class II–IV) and reduced LV ejection fraction 40%. Amiodarone, verapamil and diltiazem are among the drugs not recommended for use in patients with the diagnosis of CHF. Also, according to clinical guidelines, HMG-CoA reductase inhibitors (atorvastatin, lovastatin, pitavastatin, rosuvastatin, simvastatin, fluvastatin), direct renin inhibitors (heparin), COX-2 inhibitors (parecoxib, polmacoxib, celecoxib, etoricoxib) are among the unrecommended drugs in chronic heart failure.For ACE inhibitors, no adverse effects from interaction with beta-blockers have been identified. This combination is widely used and recommended by the CG. According to the “Major” type for ACE inhibitors with drugs indicated for use in CHF according to CG, 4 potential interactions were identified: with valsartan-sacubitril; angiotensin II receptor antagonists; with aldosterone antagonist (spironolactone); with loop and thiazide diuretics. Therefore, the appointment of angiotensin II receptor antagonists is carried out in case of ineffectiveness of initial therapy with ACE inhibitors, when changing the treatment tactics. These drugs are not used in combination with each other due to the risk of hyperkalemia. The prescription and use of diuretics while taking ACE inhibitors should be controlled by a physician also due to the risk of hyperglycaemia. In Moderate type, potential interactions with dapagliflozin, eplerenone, cardiac glycosides and heparin have been identified for ACE inhibitors. For beta-blockers, no potential Major-type interactions were identified with drugs from CG. Combinations with dapagliflozin, loop and thiazide diuretics, cardiac glycosides, spironolactone and ivabradine require special attention. It is recommended to avoid the combination of beta-blockers with valsartan. Of the recommended angiotensin II receptor antagonists, no risk of potential interactions with beta-blockers has been identified for candesartan and losartan.

Efficacy and Safety of a DOAC Compared to Unfractionated Heparin and A Low Molecular Weight Heparin in the Prevention of Thromboembolism in Hematopoietic Stem Cell Transplant Recipients.

Hematopoietic stem cell transplantation (HSCT) patients are at risk of thromboembolic events, making thromboprophylaxis crucial. This study aimed to compare apixaban, a direct factor Xa inhibitor (DOAC), with dalteparin and unfractionated heparin for thromboprophylaxis in HSCT recipients. The safety outcome included the assessment of hemorrhagic events. In this open-label randomized clinical trial, 182 HSCT recipients were divided into three groups: Apixaban (n = 61, 2.5 mg two times a day), dalteparin (n = 59, 5000 IU daily), and unfractionated heparin (n = 62, 5000 IU twice daily). These anticoagulant regimens were administered after central vein catheterization and during hospitalization. The primary clinical outcome was the risk of thrombosis, and the secondary outcome was the rate of bleeding. Relevant laboratory results were analyzed using appropriate statistical tests. Among the 61 patients in the apixaban group, six experienced thrombosis (9.83%), with four (6.65%) of them on anticoagulants. In the dalteparin group, three patients (5%) developed thrombosis, two of whom (3.38%) were on anticoagulants. In the heparin group, all four thrombosis cases (6.4%) occurred in patients on anticoagulants (P = 0.543 overall and P = 0.776 in anticoagulant users). Only two cases of bleeding were reported (1.09% overall), one in the dalteparin group (1.69%) and the other in the apixaban group (1.63%). Apixaban, dalteparin, and heparin demonstrated similar effectiveness in preventing thromboembolism in HSCT recipients. Furthermore, the comparison of bleeding rates across the study groups did not reveal significant differences. Larger studies with higher event rates may yield more precise conclusions.

Platelets provide robustness of spatial blood coagulation to the variation of initial conditions

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 105/μL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions.

The HABIT: Heparin and Aspirin on Birth in Inherited Thrombophilia, an International Multicenter Phase IIb Randomized Triple Blinded Clinical Trial

Background/Objective: In pregnant women with inherited thrombophilia (IT) and recurrent pregnancy loss, there is no higher-level evidence proving the beneficial effects of anticoagulation or platelet inhibition in preventing miscarriages. We hypothesize that anticoagulation with low molecular weight heparin (LMWH) and/or platelet aggregation inhibition with aspirin will increase the proportion of live birth in this population. Methods: In this phase IIb, factorial, randomized, triple blinded, placebo-controlled (double dummy) clinical trial, pregnant women age 18 to 40 with a history of IT and 2 or more previous miscarriages will be randomized and stratified by age and number of miscarriages in a 2x2 factorial design will be allocated equally to one of the four arms. The primary outcome of live birth will be analyzed through logistic regression analysis, controlling for strata, and results will be reported as odds ratio (OR) and 95% confidence intervals (CIs). Analyzed similarly, the secondary outcomes will include pregnancy loss, maternal mortality, major bleeding events, medication-associated adverse events, placental abruption, preterm birth, and gestational age at delivery. We will perform subgroup analysis for smoking status, weight, age, number of miscarriages, and type of thrombophilia. Discussion: There is lack of evidence for the use of anticoagulants to prevent pregnancy loss in women with inherited thrombophilia, despite the common diverging prescribing practice predominantly extrapolated from observations in acquired thrombophilia. With this study, we aim to provide an evidence base to create a standard of care in cases of recurrent pregnancy loss in women with IT. Keywords: heparin; aspirin; inherited thrombophilia; pregnant; live birth

Experimental and Computational Study of Hyaluronidase Interactions with Glycosaminoglycans and their Ligands

Covalent conjugation of hyaluronidase with copolymeric glycosaminoglycans (GAG, heparin and dermatan sulfate) considerably inactivates the enzyme, while conjugation with polymeric GAG (chondroitin sulfate and hyaluronan) improves its stability. These effects are associated with structural differences of these GAG caused by С-5 epimerization of glucuronic and iduronic acid residues and different effects of (α[1 - 4] and α[1 - 3] relative to β[1 - 4] and β[1 - 3]) glycosidic bonds. Pronounced effects of galactose C-4 epimers (in comparison with glucose) and disaccharide mixture (lactose, cellobiose, maltose) on endoglycosidase activity of hyaluronidase emphasize the importance of its diversified multi-contact microenvironment. For a better understanding of the mechanisms regulating hyaluronidase activity, molecular docking and molecular dynamics were chosen. Stabilization effect of chondroitin ligands on heat inactivation of hyaluronidase was demonstrated. An increase in denaturation temperature by 10-15oC hampers blocking of the active site entrance and prevents the enzyme inactivation. Enzyme-GAG interactions were examined by molecular docking with molecular dynamic elaboration. Gradual chemical modification of hyaluronidase was based on the calculated sequence of preferential binding of GAG. Theoretically, covalent binding of chondroitin sulfate trimers at cs7 or cs7, cs1 and cs5 on the enzyme surface provides complete protection against heparin inhibition. Computational investigation of hyaluronidase microenvironment and interactions which limit the enzyme activity allows identification of the best GAG regulators of hyaluronidase endoglycosidase activity and their experimental verification.

Use of heparin to rescue immunosuppressive monocyte reprogramming by glioblastoma-derived extracellular vesicles.

The profound immunosuppression found in glioblastoma (GBM) patients is a critical barrier to effective immunotherapy. Multiple mechanisms of tumor-mediated immune suppression exist, and the induction of immunosuppressive monocytes such as myeloid-derived suppressor cells (MDSCs) is increasingly appreciated as a key part of this pathology. GBM-derived extracellular vesicles (EVs) can induce the formation of MDSCs. The authors sought to identify the molecular consequences of these interactions in myeloid cells in order to identify potential targets that could pharmacologically disrupt GBM EV-monocyte interaction as a means to ameliorate tumor-mediated immune suppression. Heparin-sulfate proteoglycans (HSPGs) are a general mechanism by which EVs come into association with their target cells, and soluble heparin has been shown to interfere with EV-HSPG interactions. The authors sought to assess the efficacy of heparin treatment for mitigating the effects of GBM EVs on the formation of MDSCs. GBM EVs were collected from patient-derived cell line cultures via staged ultracentrifugation and cocultured with monocytes collected from apheresis cones from healthy blood donors. RNA was isolated from EV-conditioned and unconditioned monocytes after 72 hours of coculture, and RNA-sequencing analysis performed. For the heparin treatment studies, soluble heparin was added at the time of EV-monocyte coculture and flow cytometry analysis was performed 72 hours later. After the initial EV-monocyte coculture period, donor-matched T-cell coculture studies were performed by adding fluorescently labeled and stimulated T cells for 5 days of coculture. Transcriptomic analysis of GBM EV-treated monocytes demonstrated downregulation of several important immunological and metabolic pathways, with upregulation of the pathways associated with synthesis of cholesterol and HSPG. Heparin treatment inhibited association between GBM EVs and monocytes in a dose-dependent fashion, which resulted in a concomitant reduction in MDSC formation (p < 0.01). The authors further demonstrated that reduced MDSC formation resulted in a partial rescue of immune suppression, as measured by effects on activated donor-matched T cells (p < 0.05). The authors demonstrated that GBM EVs induce broad but reproducible reprogramming in monocytes, with enrichment of pathways that may portend an immunosuppressive phenotype. The authors further demonstrated that GBM EV-monocyte interactions are potentially druggable targets for overcoming tumor-mediated immune suppression, with heparin inhibition of EV-monocyte interactions demonstrating proof of principle.

Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. We determined whether negatively-charged heparin enhanced TMPRSS2 inhibition by alpha-1-antitrypsin (AAT). TMPRSS2 activity was determined in HEK293T cells overexpressing TMPRSS2. We quantified infection of primary human airway epithelial cells (hAEc) with human coronavirus 229E (HCoV-229E) by immunostaining for the nucleocapsid protein and by the plaque assay. Detailed molecular modeling was undertaken with the heparin–TMPRSS2–AAT ternary complex. Enoxaparin enhanced AAT inhibition of both TMPRSS2 activity and infection of hAEc with HCoV-229E. Underlying these findings, detailed molecular modeling revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2–AAT interface, neutralizing otherwise repulsive forces. In conclusion, enoxaparin enhances AAT inhibition of both TMPRSS2 and coronavirus infection. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.

Dynamic Combinatorial Optimization of In Vitro and In Vivo Heparin Antidotes.

Heparin-like macromolecules are widely used in clinics as anticoagulant, antiviral, and anticancer drugs. However, the search of heparin antidotes based on small synthetic molecules to control blood coagulation still remains a challenging task due to the physicochemical properties of this anionic polysaccharide. Here, we use a dynamic combinatorial chemistry approach to optimize heparin binders with submicromolar affinity. The recognition of heparin by the most amplified members of the dynamic library has been studied with different experimental (SPR, fluorescence, NMR) and theoretical approaches, rendering a detailed interaction model. The enzymatic assays with selected library members confirm the correlation between the dynamic covalent screening and the in vitro heparin inhibition. Moreover, both ex vivo and in vivo blood coagulation assays with mice show that the optimized molecules are potent antidotes with potential use as heparin reversal drugs. Overall, these results underscore the power of dynamic combinatorial chemistry targeting complex and elusive biopolymers.

Diagnostic Utility of High Dose Heparin Confirmation Step in Heparin Induced Thrombocytopenia ELISA Assay

Introduction:Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening condition that could occur following exposure to heparin. Accurate and timely diagnosis is critical for appropriate clinical management. Laboratory testing for suspected cases is based on screening for the presence of serum anti-PF4/heparin antibodies using solid-phase enzyme-linked immunosorbent assay (ELSIA) which is known to be sensitive but less specific. A positive ELISA test is followed by functional testing to demonstrate the platelet activating properties and heparin dependence of the pathogenic antibodies. Serotonin release assay (SRA) is considered the gold standard functional test for the diagnosis of HIT. In most anti-PF4/heparin ELISA assays, a high-dose heparin buffer (100U/mL) confirmation step is recommended to demonstrate heparin dependence of detected antibodies and increase the specificity of the assay. The necessity of this confirmation step is controversial with some reports suggesting it could lead to misinterpreting positive ELISA results as negative or indeterminate, especially in cases of very strong and high titer HIT activating antibodies. We hence aimed to investigate the utility of applying this confirmation step as part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA).Materials:Three hundred archived serum samples were tested using anti-PF4/heparin IgG antibody ELISA (Immucor Diagnostics, GA, USA). High-dose heparin (100U/mL) confirmation step was performed on all samples with OD units ≥0.4 as recommended by the manufacturer. Samples with OD ≥ 0.4 and ≥50% OD inhibition in the high dose heparin confirmation step are interpreted positive. Mayo-SRA results were compared to a reference 14C SRA method. The 4T clinical score was retrospectively calculated for all patient (range 0-8 points).Results:Of the 300 tested samples, 57 samples were interpreted positive by the anti-PF4/heparin screening ELISA. 33 of the 57 samples were positive using the reference 14C SRA method, whereas 43 samples were positive by Mayo-SRA assay (≥20% serotonin release). Three additional samples were positive by Mayo-SRA, but negative by both screening ELISA and the reference 14C SRA method. All samples with OD units ≥0.4 displayed >50% inhibition in the high-dose heparin regardless of the intensity of the initial OD value or the HIT 4T score, with the exception of one that was negative by both SRA methods and of 1.35 OD value and 6 4T HIT score (Fig-1A). Importantly, thirteen samples were anti-PF4/heparin positive, but SRA negative (by Mayo-SRA and reference method). These samples also displayed positive %heparin inhibition (≥50% OD inhibition) (Fig-1B). Lastly, there were no differences in the degree of %inhibition in samples positive by both reference and Mayo-SRA or Mayo-SRA only (Fig-1B).Conclusion:In our patient cohort, addition of the high dose heparin inhibition confirmation step to the screening anti-PF4/heparin ELISA assay was of no additional diagnostic utility. We hence propose eliminating the heparin inhibition step which would improve laboratory turnaround time, reduce costs, and importantly speed up urgent clinical management decisions.Figure Legend:Fig-1A. No correlation between initial OD values and %OD inhibition using high dose heparin. Scatter plot of all ELISA positive samples (OD ≥0.4) grouped according to OD values. Samples include all SRA-positive and thirteen SRA-negative ELISA-positive samples. Fig-1B. Scatter plot of %OD inhibition comparing samples positive by Mayo-SRA and reference SRA method, positive by Mayo-SRA only, and ELISA-positive SRA-negative by both methods. [Display omitted] DisclosuresPruthi: Bayer Healthcare AG: Honoraria; CSL Behring: Honoraria; Merck: Honoraria; Genentech: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Optimal timing of intervention in NSTE-ACS without pre-treatment

Abstract Introduction In patients (P) with non-ST segment elevation acute coronary syndromes (NSTE-ACS), an invasive strategy is recommended to reduce adverse outcomes. The optimal timing to perform coronary angiography (CA) remains undetermined, particularly in our era attending to the new European guidelines restricting pre-treatment (PT). Objective To evaluate the prognostic value of an early strategy (ES; &amp;lt;24h) versus a delayed strategy (DS; &amp;gt;24h) when no loading dose of a P2Y12 antagonist was given as PT in NSTE-ACS. Material and methods Retrospective analysis of P data admitted with NSTE-ACS at multicentric national registry between 2015–19. Compared demographic and clinical characteristics of P with an ES versus DS. A multivariate logistic regression was performed to evaluate predictor factors of in-hospital and 1-year endpoints. Survival was evaluated through Kaplan-Meier curve and Cox multivariate regression. Results 691P were included, mean age 64±11 years, 77.4% male. 59.2% performed CA as an ES. P proposed to a DS presented higher KK class, higher levels of creatinine and lower of hemoglobin. They also needed more frequently invasive (1.1 vs 0.7%, p=0.692) or non-invasive ventilation (1.8 vs 0.7%, p=282). A higher proportion of DS patients used the transfemoral access (5.5 vs 9.3%, p=0.058). On CA, 6.2% had normal coronary arteries, 49.2% 1-vessel disease and 45.1% multivessel disease. Revascularization was performed in 88.2%: PCI in 86.2%, CABG in 1.7% and both in 0.3%, with no significant differences. Pending CA, 98.4% were medicated with aspirin, 64.8% ticagrelor and 44% clopidogrel, with no differences. P proposed to an ES were more medicated with glycoprotein inhibitor (36.3 vs 26.4%, p=0.015) and non-fractioned heparin (6.4 vs 2.1%, p=0.01) and less with fondaparinux (56.2 vs 65.2%, p=0.017). A higher percentage of calcium-channel blockers (25.2 vs 11.7%, p&amp;lt;0.001) and nitrates (74.1 vs 53.3%, p&amp;lt;0.001) was observed in the DS. No difference was observed in beta-blockers (p=0.581). Discharge medication followed these tendencies. There was a trend to worse in-hospital outcomes in the DS regarding heart failure, shock, ventricular arrhythmias, cardiac arrest and death, although not significatively different, except for major bleeding (1.8 vs 0.2%, p=0.044). 1-year composite endpoint of mortality and cardiovascular rehospitalization occurred in 9.9%, with no difference between groups (p=0.181). Predictor factors, evaluated through Cox multivariate regression, were ejection fraction &amp;lt;50% (p=0.001), KK class &amp;gt;I (p=0.002) and nitrate prescription at discharge (p=0.001). A DS was not a predictor factor (p=0.812). Conclusion Our results are in accordance with available data. In P with higher-risk NSTE-ACS in the absence of P2Y12 antagonist PT, an ES was not associated with a reduction in the composite of global mortality and rehospitalization for cardiovascular causes. Funding Acknowledgement Type of funding sources: None.

Global Thrombosis Test: Occlusion Is Attributable to Shear-Induced Platelet Thrombus Formation

Herein, we set out a rebuttal to the publication by Claveria and co-workers published in TH Open this month entitled “Global Thrombosis Test: Occlusion by Coagulation or SIPA?” We strongly believe that the conclusions of their paper, suggesting that occlusion (OT) in the Global Thrombosis Test (GTT) is due to coagulation, rather than shear-induced platelet thrombus formation, is incorrect and the evidence and arguments they present are fundamentally flawed, with major errors both in the experimental approach and in the interpretations of the results. The evidence which they demonstrate, shows that occlusion in the GTT is, in fact, caused by high shear induced platelet thrombus formations. We set out herein the evidence for that, based on histology of the thrombus from the GTT in earlier work using electron microscopy showing large platelet aggregates, the very brief timescale of OT in the GTT compared to coagulation time and the sensitivity of the OT in the GTT to the effects of heparin, t-PA and P2Y12 inhibitors. In addition, we revisit the known pathomechanism of high shear-mediated platelet aggregation to underpin our rationale and show that the modifications to the instrument proposed by Claveria and co-authors would render the technique unphysiological. We highlight several methodological concerns and apparent misinterpreted of the data obtained. We present evidence predominantly from the authors’ own data, together with our earlier published data and evidence from the literature, showing that occlusion in the GTT occurs do to shear-induced platelet aggregation.

Molecular Mechanism of Inhibition of Polysialyltransferase Domain (PSTD) by Heparin.

The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes how the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia take place, and how these interactions are inhibited by LMWH and DP4. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

Role for PARS and Thrombin-Gpibα Interaction for Thrombin Induced Procoagulant Platelet Formation

Procoagulant platelets, a subpopulation of platelets which support thrombin generation, play a role in regulating pathological thrombosis. Using a novel flow cytometry assay (Hua et at, Blood 2015) to identify procoagulant platelets based upon the cell death marker GSAO and activation marker P-selectin, we found that patients with angiogram proven coronary artery disease had heightened thrombin procoagulant platelet formation compared to healthy controls, (25.2% vs 12.2%, p<0.001). Inhibition of this is an attractive target for atherothrombosis prevention. However, the mechanism of thrombin induced procoagulant platelet formation remains controversial with discrepant results regarding requirement for the thrombin binding domain of N terminal GPIbα (Dorman et al, Blood 2000 and Ravanat et al, Blood 2010) and recent reports implicating PAR4 (French et al JTH, 2016). Here we use a recently described novel assay to investigate the roles of PARs and GPIbα measuring procoagulant platelets by FACS (GSAO+/P-selectin+) in whole blood, plasma and washed platelets after treatment with heparin, PAR and GPIbα inhibitors prior to thrombin or PAR agonist stimulation.First, using active site inhibited PPACK-thrombin we demonstrated that catalytically active thrombin is required for procoagulant platelet formation in our in vitro system. PAR1 and PAR4 are implicated in procoagulant platelet formation, however, preincubation with combination of PAR1 and PAR 4 inhibitors (vorapaxar and a PAR4 polyclonal inhibitory antibody), resulted in only partial inhibition of thrombin induced procoagulant platelets (reduction 40%±5.7%, p<0.01) suggesting a role for an additional non-PAR pathway. Thrombin-GPIbα interaction was implied when competition studies with recombinant soluble GPIbα, glycocalicin showed a 49%±7.7% reduction. Involvement of the N-terminus of GPIbα was confirmed by a 39.5%±2.9% reduction after cleavage of GPIbα proximal to the thrombin binding site by NK protease. Pretreatment with PAR1 inhibitor after NK protease cleavage of GPIbα reduced thrombin stimulated procoagulant platelet formation to unstimulated levels implying both PAR cleavage and GPIbα-thrombin interaction is required for thrombin induced procoagulant platelet formation.Consistent with a role of thrombin-GPIbα interaction, we noted a reduction in thrombin induced procoagulant platelets after heparin bolus in patients undergoing coronary angiogram (16.3±10.5% vs 6.1%±2.4%, p<0.001). Heparin is known to known to displace thrombin from GPIbα in addition to anti-thrombin effects via enhancement of antithrombin (AT). Using platelets resuspended in AT-deficient plasma, we demonstrated high dose heparin suppressed procoagulant platelet formation via an AT-independent mechanism, this was confirmed by heparin suppression of thrombin induced procoagulant platelets in washed platelets. In similar experiments, the heparinoid danaparoid, which has no anti-thrombin effect, also inhibited thrombin induced procoagulant platelet formation. Heparin inhibition was not due to PAR inhibition as heparin had no effect on direct PAR activation by PAR1 agonist TRAP or PAR4 agonist AYPGKF. These data suggest heparin has a direct effect on platelets in suppression of thrombin induced procoagulant platelet formation, most likely relating to displacement of thrombin from GPIbα.Together this data indicates that a thrombin-GPIbα interaction via the N terminal fragment is required in addition to thrombin mediated PAR activation. This mechanism is a potential target for modifying atherothrombotic risk.Figure 1: Whole blood was treated with PAR1 and PAR4 inhibitors and washed platelets were treated with glycocalicin, NK protease or NK and PAR1 inhibitor prior to thrombin stimulation (2 U/mL). Data are expressed as fold change from vehicle control ((*p<0.05; **p<0.01, statistics are calculated for n≥3). Figure 2: Plasma, washed platelets or whole blood were treated or not with heparin (12.8 U/ml) prior to thrombin stimulation (2 U/mL), PAR1 (20 µM) or PAR4 (300 µM) agonists. Data are expressed as the ratio of heparin-treated sample to untreated (***p<0.001, statistics are calculated for n≥3). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Host-Guest Complex for Heparin Binding and Sensing

Heparin is a naturally occurring highly charged polyanion and a well-known anticoagulant widely used in surgical practice.[1] More specifically, it is a negatively charged sulfated polysaccharide belonging to the group of glycosaminoglycans. The anticoagulant effect of heparin is based on its ability to activate antithrombin-III, which, subsequently, inactivates vital coagulation cascade substance such as thrombin and factor Xa consequently preventing fibrin and clot formation.[2] To balance and neutralize heparin dosing, a heparin inhibitor is needed, and commonly, arginine-rich protein protamine sulfate (PS) is used for this purpose.[3] However, in recent years there has been an interest to investigate alternatives for protamine due to its adverse effects like hypotension and serious allergic reactions.[4]In this study, a host-guest complex capable of selective heparin binding and sensing is presented.[5] Heparin binding affinity was studied in solution with a variety of polycationic macrocyclic hosts, a pillar[5]arene and multiple resorcin[4]arenes, by dynamic light scattering, dye displacement assay, isothermal titration calorimetry, and anti-Xa assay. The measurements reveal the significant importance of multivalency in the electrostatic host-heparin binding in competitive, application-relevant media. Additionally, to monitor heparin concentration, a host-guest indicator displacement assay was performed by following the free and bound state of methyl orange dye in UV-Vis spectroscopic experiments. Furthermore, this colorimetric sensing based on the tertiary host-guest-heparin supramolecular assembly was utilized in the construction of a calibration curve in a range of blood plasma concentrations. Figure 1

Heparanase modulation by Wingless/INT (Wnt).

Heparanase is an endo-beta-glucuronidase, the only enzyme in mammals capable of cleaving heparan sulfate/heparin chains from proteoglycans. The oligosaccharides generated by heparanase present extensive biological functions since such oligosaccharides interact with adhesion molecules, growth factors, angiogenic factors and cytokines, modulating cell proliferation, migration, inflammation, and carcinogenesis. However, the regulation of heparanase activity is not fully understood. It is known that heparanase is synthesized as an inactive 65kDa isoform and that post-translation processing forms an active 50kDa enzyme. In the present study, we are interested in investigating whether heparanase is regulated by its own substrate as observed with many other enzymes. Wild-type Chinese hamster (Cricetulus griséus) ovary cells (CHO-K1) were treated with different doses of heparin. Heparanase expression was analyzed by Real-time PCR and flow cytometry. Also, heparanase activity was measured. The heparanase activity assay was performed using a coated plate with biotinylated heparan sulfate. In the present assay, a competitive heparin inhibition scenario was set aside. Exogenous heparin trigged a cell signaling pathway that increased heparanase mRNA and protein levels. The Wnt/beta-catenin pathway, judged by TCF-driven luciferase activity, seems to be involved to enhance heparanase profile during treatment with exogenous heparin. Lithium chloride treatment, an activator of the Wnt/beta-catenin pathway, confirmed such mechanism of transduction in vivo using zebrafish embryos and in vitro using CHO-K1 cells. Taken together the results suggest that heparin modulates heparanase expression by Wnt/beta-catenin.

Vasotocin stimulates maturation-inducing hormone, oocyte maturation and ovulation in the catfish Heteropneustes fossilis: Evidence for a preferential calcium involvement

Arginine vasotocin (VT) is the basic neurohypophysial nonapeptide hormone in teleosts. VT is also distributed in the ovary of the catfish Heteropneustes fossilis and induces final oocyte maturation (FOM) and ovulation by stimulating the maturation-inducing hormone (MIH). The present study reports the effects of cAMP (0.5 mM), phosphodiesterase inhibitors (IBMX -0.5 mM and theophylline- 0.5 mM), the inositol triphosphate (IP3) receptor inhibitor heparin (10 μg/mL) and the Ca2+ chelator BAPTA-AM (25 μM) on VT (100 nM) - induced progestin stimulation, FOM and ovulation. Incubation of post-vitellogenic follicles with cAMP, IBMX and theophylline for 0, 8, 16 and 24 h stimulated basal secretion of progesterone (P4), 17-hydroxyprogesterone (17-P) and 17, 20β-dihydroxy-4-pregnen-3-one (MIH) in a duration-dependent manner. The incubation of the follicles with heparin stimulated P4 modestly, and 17-P and MIH levels in a duration-dependent manner. The incubation of the follicles with BAPTA-AM stimulated P4 and MIH levels marginally and 17-P robustly. The stimulation was in the order cAMP > IBMX > theophylline > heparin > BAPTA-AM. The incubation of the follicles with VT stimulated P4, 17-P, MIH, GVBD and ovulation in a duration-dependent manner. The co-incubations with VT and the test compounds inhibited the VT-induced stimulation of P4, 17-P and MIH levels in a time-dependent manner in the order heparin > BAPTA-AM > cAMP > IBMX > theophylline. Concurrently, the VT-induced stimulation of GVBD and ovulation were also inhibited by the test compounds in the same order. The results show that VT induces FOM and ovulation preferentially acting through Ca2+ pathway and a crosstalk between Ca2+ and cAMP signaling pathways seems to integrate the processes.

Structural basis for the design of bisubstrate inhibitors of protein kinase CK2 provided by complex structures with the substrate-competitive inhibitor heparin.

The Ser/Thr kinase CK2, a member of the superfamily of eukaryotic protein kinases, has an acidophilic substrate profile with the substrate recognition sequence S/T-D/E-X-D/E, and it is inhibited by polyanionic substances like heparin. The latter, a highly sulphated glucosamino glycan composed mainly of repeating 2-O-sulpho-α-l-idopyranuronic acid/N,O6-disulpho-α-d-glucosamine disaccharide units, is the longest known substrate-competitive CK2 inhibitor. The structural basis of CK2's preference for anionic substrates and substrate-competitive inhibitors is only vaguely known which limits the value of the substrate-binding region for the structure-based development of CK2 bisubstrate inhibitors. Here, a tetragonal and a monoclinic co-crystal structure of CK2α, the catalytic subunit of CK2, with a decameric heparin fragment are described. In the tetragonal structure, the heparin molecule binds to the polybasic stretch at the beginning of CK2α's helix αC, whereas in the monoclinic structure it occupies the central substrate-recognition region around the P+1 loop. Together, the structures rationalize the inhibitory efficacy of heparin fragments as a function of chain length. The monoclinic CK2α/heparin structure, in which the heparin fragment is particularly well defined, is the first CK2 structure with an anionic inhibitor of considerable size at the central part of the substrate-recognition site. The bound heparin fragment is so close to the binding site of ATP-competitive inhibitors that it can guide the design of linkers and pave the way to efficient CK2 bisubstrate inhibitors in the future.

Protamine Sulfate Neutralization Profile of Various Dosages of Bovine, Ovine and Porcine UFHs and Their Depolymerized Derivatives in Non-Human Primates.

Introduction:Currently used unfractionated heparins (UFHs) and low molecular weight heparins (LMWHs) are derived from porcine intestinal mucosa. However, heparins have also been manufactured from tissues of other mammalian species such as cow (Bovine) and sheep (Ovine). Protamine sulphate (PS) is an effective inhibitor of heparin and is used clinically to neutralize both LMWH and UFH. In this study, we determined the PS neutralization profile of these agents in non-human primate model using anti-Xa and anti-IIa methods.Material and Methods:UFHs obtained from bovine, ovine and porcine mucosal tissues and their respective depolymerized LMWHs were administered at both, gravimetric (0.5 mg/kg) and potency adjusted (100 U/kg) dosages regimen intravenously to individual groups of primates in cross over studies. PS was administered at a fixed dosage and the relative neutralization of these anticoagulants was measured utilizing amidolytic anti-Xa and anti-IIa methods.Results:These studies have demonstrated that, the equi-gravimetric dosages of BMH, PMH and OMH have comparable PS neutralization profiles. At potency adjusted dosages, all UFHs were completely neutralized by PS. Although comparable, the LMWHs were not fully neutralized by PS in both the anti-Xa and anti-IIa assays. PS was more efficient in neutralizing the anti-IIa effects of LMWHs.Conclusion:Heparins of diverse origins showed comparable neutralization profiles by PS in the amidolytic anti-Xa and anti-IIa assays.

Acute Coronary Syndrome in Well Trained Young Adult: A Rare Cases

Acute Coronary Syndrome (ACS) has been observed in the older population (&gt;40 years old), sedentary lifestyle and unhealthy behaviour. Recently, there is an increase of ACS reported in the younger population even without a sedentary lifestyle.We report a case of awell-trained 25-years old man thatcame with late presentation of ST elevation myocardial infarction (STEMI) occurred 10 minutes after vigorous exercise. His traditional risk factor was a smoker.Electrocardiogram (ECG) showed a pathological Q wave in V1-V3 and T wave inversion in V1-V6 with elevated cardiac biomarker. Primary percutaneous coronary intervention (PPCI) was performed due to prolonged chest pain and it showed subtotal occlusion in mid left anterior descending artery (LAD) with high thrombus burden. One drug eluting stent (DES) was implanted at mid LAD and GPIIb/IIIa inhibitor and low molecule weight heparin was given with good clinical result.