Endogenous thrombin potential and time-dependent thrombin generation parameters are independent risk factors for mortality in the general population.

BACKGROUND: Thrombin generation (TG) is used as a global test of coagulation and is an indicator of thrombosis and bleeding risk. Until now, data on the association of TG and mortality are inconclusive. OBJECTIVES: We investigated the association between TG and mortality in the prospective Moli-sani cohort (n = 21 920). METHODS: TG was measured using calibrated automated thrombinography using PPP-Reagent Low. Lag time (LT), endogenous thrombin potential (ETP), peak height, time-to-peak (TTP), and velocity index were quantified. The association of TG and mortality was studied by Cox regression and adjusted for sex, age, body mass index, smoking, contraceptives, and medical history (cardiovascular diseases, hypertension, hypercholesterolemia, diabetes, and cancer). RESULTS: LT and TTP were 4.1 ± 1.0 minutes and 6.6 ± 1.5 minutes, on average. The peak height was 364 ± 88 nM, velocity index was 163 ± 63 nM/min, and ETP was 1721 ± 411 nM·min. ETP was negatively associated with all-cause mortality (hazard ratio [HR], 0.86; 95% CI, 0.81-0.92; P < .001). Subjects in the lowest quintile of the ETP (ETPQ1) had a 1.3-fold higher mortality rate. Additionally, a high TTP/LT ratio was negatively associated with mortality (HR, 0.71; 95% CI, 0.57-0.89; P = .003). Individuals in quintile 1 of the TTP/LT ratio had a 1.4-fold higher mortality rate compared with the remainder of the cohort. Subjects that were both in ETPQ1 and TTP/LTQ1 had a 1.8-fold higher mortality rate, regardless of whether they reported history of cardiovascular disease at baseline (HR, 1.61 [CI: 1.07-2.42]) or not (HR, 1.89 [CI: 1.51-2.36]). CONCLUSION: Low ETP and TTP/LT ratios are independent risk factors for all-cause mortality in the general population.

Altered whole blood thrombin generation and hyperresponsive platelets in patients with pancreatic cancer.

BACKGROUND: Thromboembolic disease is a major complication in patients with pancreatic ductal adenocarcinoma (PDAC). Patients with PDAC often have altered blood cell counts, which are associated with venous thromboembolism (VTE) development. The high thrombotic risk in patients with PDAC may be partially caused by procoagulant blood cells. OBJECTIVES: The aim of this study was to compare blood cell-dependent coagulation between patients with PDAC (n = 18) and healthy controls matched for age and sex (n = 18). METHODS: Thrombin generation (TG) was measured in whole blood (WB) and plasma. The capacity of platelets to release granules (PGRCs) was measured in WB. We explored the occurrence of thromboembolic events in patients with PDAC during a 6-month follow-up. RESULTS: Patients showed an increased endogenous thrombin potential in WB compared with controls. This difference was not observed in plasma, indicating a procoagulant effect of blood cells. Both in WB and plasma, the lag time was prolonged in patients compared with controls. Patients had hyperresponsive platelets, with a shorter time to peak granule release. Of the 18 patients with PDAC, 4 developed a venous thromboembolism (22%) and 1 developed an arterial thrombosis (6%). A shorter lag time in WB, but not in plasma, and an increased PGRC were associated with thromboembolic events. CONCLUSION: Patients with PDAC have an increased and delayed WB TG coagulation profile compared with controls. A shorter lag time in WB TG and increased PGRC are associated with the incidence of thromboembolic events. Platelets appear to be key players in thrombosis development. Measuring hemostasis in WB could improve thrombosis risk estimation in patients with PDAC.

Low factor XIII levels and altered fibrinolysis in patients with multiple myeloma.

BACKGROUND: Acquired factor FXIII (FXIII) deficiency can be immune- or non-immune mediated and may cause severe bleeding symptoms. The incidence of acquired FXIII deficiency and its etiology in patients with multiple myeloma (MM) are poorly understood. OBJECTIVES: To assess FXIII levels and the balance of fibrinolysis in newly diagnosed, untreated MM and monoclonal gammopathy of undetermined significance (MGUS) patients. METHODS: FXIII activity, mixing studies, FXIII-A2B2 antigen, total FXIII-B antigen were measured in platelet-poor plasma from 17 untreated MM patients, 33 untreated MGUS patients, and 30 age and sex-matched healthy controls. Besides routine laboratory measurements, the balance of coagulation and fibrinolysis was evaluated using quantitative fibrin monomer (FM) test, thrombin-antithrombin assay, α2-antiplasmin activity, plasmin-α2-antiplasmin (PAP) complex, D-dimer, plasmin generation assay, clot lysis assay, and ClotPro-TPA test. RESULTS: FXIII-A2B2 levels were significantly lower in MM patients compared to controls [median (IQR):14.6 (11.2-19.4) vs. 21.8 (17.1-26.4) mg/L, p = 0.0015], whereas total FXIII-B did not differ between groups. Decrease in FXIII activity was parallel to the decrease in FXIII-A2B2. An immune-mediated inhibitory mechanism was ruled out. Free/total FXIII-B was significantly higher in MM patients compared to MGUS and healthy controls, suggesting an etiology of FXIII-A consumption. In MM and MGUS patients, FM, D-dimer, and PAP complex were significantly elevated compared to controls, indicating hypercoagulability and ongoing fibrinolysis. CONCLUSIONS: Low FXIII levels due to consumption were observed in MM patients at diagnosis. Hypercoagulability and ongoing fibrinolysis were detected in MM and MGUS, indicating that a disturbed hemostasis balance is already present in the latter benign condition.

Crucial roles of red blood cells and platelets in whole blood thrombin generation.

Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.

Hyperresponsive Platelets and a Reduced Platelet Granule Release Capacity Are Associated with Severity and Mortality in COVID-19 Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is often associated with mild thrombocytopenia and increased platelet reactivity. OBJECTIVE: The aim of the current study was to investigate the adenosine triphosphate (ATP) release kinetics of platelets in hospitalized SARS-CoV-2-infected patients. METHODS: We studied time-dependent platelet activation in whole blood by monitoring the ATP release kinetics upon stimulation with a PAR1 receptor agonist in 41 hospitalized critically ill COVID-19 patients, 47 hospitalized noncritically ill COVID-19 patients, and 30 healthy controls. RESULTS: Our study demonstrated that platelets of critically ill COVID-19 patients were hyper-responsive with a shorter platelet response time (PRT) and a reduced platelet granule release capacity (GRC), probably due to chronic activation. The median PRT of COVID-19 patients admitted to the critical care unit was 10 and 7 seconds shorter than the median PRT in healthy controls and noncritical COVID-19 patients, respectively. Both PRT and GRC were also associated with D-dimer (Spearman r [r s] = -0.51, p < 0.0001 and r s = -0.23, p < 0.05), C-reactive protein (CRP) (r s = -0.59, p < 0.0001 and r s = -0.41, p < 0.01), and neutrophil-to-lymphocyte ratio (NLR) (r s = -0.42, p < 0.0001 and r s = -0.26, p < 0.05). Moreover, an increased PRT and a reduced GRC were associated with an increased mortality (odds ratio [OR]: 18.8, 95% confidence interval [CI]: 6.5-62.8, p < 0.0001 and OR: 4.0; 95% CI: 1.6-10.4, p < 0.01). These relationships remained significant after adjustment for age, sex, D-dimer, CRP, and NLR. CONCLUSION: Using an accessible agonist-induced platelet granule ATP release assay, we show that platelet hyper-responsiveness and reduced platelet GRC in COVID-19 patients were associated with critical illness and mortality.

Patients With Multiple Myeloma Have a Disbalanced Whole Blood Thrombin Generation Profile.

Background: Multiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis. Objectives: To get better insight in the disbalanced hemostasis of MM patients. Methods: We conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls. Results: In WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls. Conclusion: Patients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients.

Osteoprotegerin modulates platelet adhesion to von Willebrand factor during release from endothelial cells.

BACKGROUND: Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. OBJECTIVES: To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain. METHODS: The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF. RESULTS: Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma. CONCLUSIONS: Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.

Aprotinin Inhibits Thrombin Generation by Inhibition of the Intrinsic Pathway, but is not a Direct Thrombin Inhibitor.

Background Aprotinin is a broad-acting serine protease inhibitor that has been clinically used to prevent blood loss during major surgical procedures including cardiac surgery and liver transplantation. The prohemostatic properties of aprotinin likely are related to its antifibrinolytic effects, but other mechanisms including preservation of platelet function have been proposed. Aim Here we assessed effects of aprotinin on various hemostatic pathways in vitro, and compared effects to tranexamic acid(TXA), which is an antifibrinolytic but not a serine protease inhibitor. Methods We used plasma-based clot lysis assays, clotting assays in whole blood, plasma, and using purified proteins, and platelet activation assays to which aprotinin or TXA were added in pharmacological concentrations. Results Aprotinin and TXA dose-dependently inhibited fibrinolysis in plasma. Aprotinin inhibited clot formation and thrombin generation initiated via the intrinsic pathway, but had no effect on reactions initiated by tissue factor. However, in the presence of thrombomodulin, aprotinin enhanced thrombin generation in reactions started by tissue factor. TXA had no effect on coagulation. Aprotinin did not inhibit thrombin, only weakly inhibited the TF-VIIa complex and had no effect on platelet activation and aggregation by various agonists including thrombin. Aprotinin and TXA inhibited plasmin-induced platelet activation. Conclusion Pharmacologically relevant concentrations of aprotinin inhibit coagulation initiated via the intrinsic pathway. The antifibrinolytic activity of aprotinin likely explains the prohemostatic effects of aprotinin during surgical procedures. The anticoagulant properties may be beneficial during surgical procedures in which pathological activation of the intrinsic pathway, for example by extracorporeal circuits, occurs.

A Synthetic Triple Helical Collagen Peptide as a New Agonist for Flow Cytometric Measurement of GPVI-Specific Platelet Activation.

Synthetic cross-linked collagen-related peptide (CRP-XL) is a glycoprotein VI (GPVI) receptor activator for platelet activation. This triple helical peptide, widely used in platelet function tests, is synthesized and cross-linked through cysteine residues at its N-terminus and C-terminus. Currently, there is only one laboratory, which is capable to produce this valuable peptide for clinical applications. In an attempt to provide a standardized alternative for CRP-XL, we developed a synthetic triple helical collagen peptide (STH-CP) with the same primary sequence as CRP-XL (GPC-(GPO)10-GPCG-amide)3, which was both on the C-terminus and on the N-terminus fixed on a scaffold with a binding side for each of the three peptides. The performance of STH-CP on platelet function was studied using flow cytometry and compared with CRP-XL. We found that platelet activation pattern in response to STH-CP and CRP-XL is similar, although the STH-CP requires sixfold higher concentrations to activate platelets to the same state. The intra-assay percent coefficient of variation of STH-CP and CRP-XL were both < 5% and the interindividual variation measured in 118 individuals for both peptides was around 23 and 21% for αIIbß3 activation and P-selectin expression, respectively. The STH-CP in ready-to-use reaction mix has lower variation than CRP-XL over 1-year storage. In reference values and seasonal variation study, the platelet activation response showed a strong correlation between STH-CP and CRP-XL.Our findings show that this new STH-CP is a stable and potent platelet GPVI agonist which can induce the same reproducible platelet activation as CRP-XL and that STH-CP can be considered as a good alternative for CRP-XL.

Thrombin generation and platelet activation in cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy - A prospective cohort study.

BACKGROUND AND OBJECTIVES: Cytoreductive surgery (CRS) with hyperthermic intraperitoneal peroperative chemotherapy (HIPEC), indicated for patients with peritoneal metastases from digestive or gynecological malignancies alike, demonstrates a considerable impact on hemostatic metabolism, both on platelet and on coagulation level. The potential hemostatic interference in CRS and HIPEC is phase dependent. The hypothesis of this prospective cohort study is that the procedure exposed an increased thrombotic risk, resulting in a faster and increased thrombin generation and hyper platelet function. METHODS: This study explores the combined use of ROTEM (rotational thromboelastometry), PACT (platelet activation test) and CAT (thrombin generation test) assays during CRS and HIPEC with a follow-up of 7 days postoperative in 27 patients with confirmed histological diagnosis of peritoneal disease. RESULTS: Platelet reactivity (relative to before incision values) to CRP (collagen-related peptide) (p value 0.02) and TRAP (thrombin receptor activator peptide) (p value 0.048) seems to be slightly reduced during CRS and HIPEC with regard to αIIbß3 activation, while P-selectin expression is not affected. During surgery, CAT demonstrates that, the LT (lagtime) (p value 0.0003) and TTP (time-to-thrombin peak) values (p value 0.002) decrease while and the TP (thrombin peak) (p value 0.004) and ETP (endogenous thrombin potential) (p value 0.02) increase. Subsequently, after surgery, the LT and TTP increase and ETP and TP decrease in time. ROTEM EXTEM (extrinsic) MCF (maximum clot firmness) (p value 0.005), INTEM (intrinsic) MCF (p value 0.003) and FIBTEM (fibrinogen) MCF (p value <0.001) decreased during CRS. At day 7 INTEM and FIBTEM MCF values (p values of 0.004 and <0.001) were significantly higher than before surgery. No considerable changes in platelet count and hemoglobin concentration and absence of leukopenia are noticed. CONCLUSION: This approach detects changes in coagulation much earlier than noticed by standard coagulation tests.

Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates.

BACKGROUND: Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage. STUDY DESIGN AND METHODS: The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference. RESULTS: Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis. CONCLUSION: PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.

Effect of high or low protamine dosing on postoperative bleeding following heparin anticoagulation in cardiac surgery. A randomised clinical trial.

While experimental data state that protamine exerts intrinsic anticoagulation effects, protamine is still frequently overdosed for heparin neutralisation during cardiac surgery with cardiopulmonary bypass (CPB). Since comparative studies are lacking, we assessed the influence of two protamine-to-heparin dosing ratios on perioperative haemostasis and bleeding, and hypothesised that protamine overdosing impairs the coagulation status following cardiac surgery. In this open-label, multicentre, single-blinded, randomised controlled trial, patients undergoing on-pump coronary artery bypass graft surgery were assigned to a low (0.8; n=49) or high (1.3; n=47) protamine-to-heparin dosing group. The primary outcome was 24-hour blood loss. Patient haemostasis was monitored using rotational thromboelastometry and a thrombin generation assay. The low protamine-to-heparin dosing ratio group received less protamine (329 ± 95 vs 539 ± 117 mg; p<0.001), while post-protamine activated clotting times were similar among groups. The high dosing group revealed increased intrinsic clotting times (236 ± 74 vs 196 ± 64 s; p=0.006) and the maximum post-protamine thrombin generation was less suppressed in the low dosing group (38 ± 40 % vs 6 ± 9 %; p=0.001). Postoperative blood loss was increased in the high dosing ratio group (615 ml; 95 % CI 500-830 ml vs 470 ml; 95 % CI 420-530 ml; p=0.021) when compared to the low dosing group, respectively. More patients in the high dosing group received fresh frozen plasma (11 % vs 0 %; p=0.02) and platelet concentrate (21 % vs 6 %; p=0.04) compared to the low dosing group. Our study confirms in vitro data that abundant protamine dosing is associated with increased postoperative blood loss and higher transfusion rates in cardiac surgery.

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Vascular endothelial growth factor pathway in endometriosis: genetic variants and plasma biomarkers.

OBJECTIVE: To study single nucleotide polymorphisms (SNPs) involved in angiogenesis (VEGF, PLGF, VEGFR1, VEGFR2, HIF-1α) and plasma levels of the corresponding proteins (VEGF, PLGF, sVEGFR1, sVEGFR2) in women with and without endometriosis. DESIGN: Allele frequencies of vascular endothelial growth factor (VEGF) pathway SNPs and plasma levels of the corresponding proteins were investigated in patients with endometriosis and in controls. SETTING: University hospital. PATIENT(S): Samples of DNA from 1,931 Caucasian patients were included (1,109 patients with endometriosis and 822 controls). An additional study group included 973 DNA samples from volunteers, self-reported to be healthy without laparoscopic evaluation. INTERVENTION(S): Women who underwent a laparoscopy for subfertility and/or pain and healthy volunteers without laparoscopic evaluation. MAIN OUTCOME MEASURE(S): Functional SNPs of the VEGF, VEGFR1, VEGFR2, HIF-1α genes and Hap Map tagging SNPs of the PLGF gene were genotyped by using iPLEX technology on a Sequenom MassArray and TaqMan SNP Genotyping Assay. The VEGF levels were determined in ethylenediaminetetraacetic acid plasma samples by using Bio-Plex Protein Array System. PLGF, sVEGFR1, and sVEGFR2 levels were measured in ethylenediaminetetraacetic acid plasma samples by using ELISA Quantikine kits. RESULT(S): A significant association was found between the rs2268613 polymorphism in the PLGF gene and PLGF plasma levels. In all study subjects, women with the AA variant of the rs2268613 PLGF gene had significantly lower PLGF plasma levels (median [interquartile range] 9.36 [8.19-10.43] pg/mL) than those with the AG variant (12.1 [11.81-20.84] pg/mL; P(a)=.0085, P(b)=.04), both before and after multiple testing. Plasma levels of VEGF were elevated in endometriosis patients (especially in minimal-mild endometriosis during the menstrual cycle phase) compared with laparoscopic controls but had a moderate diagnostic performance (area under the curve, 0.73) in this discovery dataset. At a cut-off plasma level of VEGF >3.88 pg/mL, minimal-mild stages of endometriosis were diagnosed with a sensitivity of 74% and a specificity of 80% during the menstrual phase of cycle. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing. CONCLUSION(S): Genetic variants in the PLGF rs2268613 gene may influence plasma levels of the corresponding protein. Plasma levels of VEGF were elevated in endometriosis patients compared with controls. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing.

NICTABA and UDA, two GlcNAc-binding lectins with unique antiviral activity profiles.

OBJECTIVES: This study aimed to assess the antiviral properties of a unique lectin (NICTABA) produced by the tobacco plant, Nicotiana tabacum. METHODS: Cellular assays were used to investigate the antiviral activity of NICTABA and Urtica dioica agglutinin (UDA). Surface plasmon resonance (SPR) studies were performed to study the sugar specificity and the interactions of both lectins with the envelope glycoproteins of HIV-1. RESULTS: The N-acetyl-d-glucosamine (GlcNAc)-binding lectins exhibited broad-spectrum activity against several families of enveloped viruses including influenza A/B, Dengue virus type 2, herpes simplex virus types 1 and 2 and HIV-1/2. The IC50 of NICTABA for various HIV-1 strains, clinical isolates and HIV-2 assessed in PBMCs ranged from 5 to 30 nM. Furthermore, NICTABA inhibited syncytium formation between persistently HIV-1-infected T cells and uninfected CD4+ T lymphocytes and prevented DC-SIGN-mediated HIV-1 transmission to CD4+ target T lymphocytes. However, unlike many other antiviral carbohydrate-binding agents (CBAs) described so far, NICTABA did not block HIV-1 capture to DC-SIGN+ cells and it did not interfere with the binding of the human monoclonal antibody 2G12 to gp120. SPR studies with HIV-1 envelope glycoproteins showed that the affinity of NICTABA for gp120 and gp41 was in the low nanomolar range. The specific binding of NICTABA to gp120 could be prevented in the presence of a GlcNAc trimer, but not in the presence of mannose trimers. NICTABA displayed no antiviral activity against non-enveloped viruses. CONCLUSIONS: Since CBAs possess a high genetic barrier for the development of viral resistance and NICTABA shows a broad antiviral activity profile, this CBA may qualify as a potential antiviral candidate with a pleiotropic mode of action aimed at targeting the entry of enveloped viruses.

Broad anti-HIV activity of the Oscillatoria agardhii agglutinin homologue lectin family.

OBJECTIVES: Oscillatoria agardhii agglutinin homologue (OAAH) proteins belong to a recently discovered lectin family. The founding member OAA and a designed hybrid OAAH (OPA) recognize similar but unique carbohydrate structures of Man-9, compared with other antiviral carbohydrate-binding agents (CBAs). These two newly described CBAs were evaluated for their inactivating properties on HIV replication and transmission and for their potential as microbicides. METHODS: Various cellular assays were used to determine antiviral activity against wild-type and certain CBA-resistant HIV-1 strains: (i) free HIV virion infection in human T lymphoma cell lines and PBMCs; (ii) syncytium formation assay using persistently HIV-infected T cells and non-infected CD4+ T cells; (iii) DC-SIGN-mediated viral capture; and (iv) transmission to uninfected CD4+ T cells. OAA and OPA were also evaluated for their mitogenic properties and potential synergistic effects using other CBAs. RESULTS: OAA and OPA inhibit HIV replication, syncytium formation between HIV-1-infected and uninfected T cells, DC-SIGN-mediated HIV-1 capture and transmission to CD4+ target T cells, thereby rendering a variety of HIV-1 and HIV-2 clinical isolates non-infectious, independent of their coreceptor use. Both CBAs competitively inhibit the binding of the Manα(1-2)Man-specific 2G12 monoclonal antibody (mAb) as shown by flow cytometry and surface plasmon resonance analysis. The HIV-1 NL4.3(2G12res), NL4.3(MVNres) and IIIB(GRFTres) strains were equally inhibited as the wild-type HIV-1 strains by these CBAs. Combination studies indicate that OAA and OPA act synergistically with Hippeastrum hybrid agglutinin, 2G12 mAb and griffithsin (GRFT), with the exception of OPA/GRFT. CONCLUSIONS: OAA and OPA are unique CBAs with broad-spectrum anti-HIV activity; however, further optimization will be necessary for microbicidal application.

A multi-targeted drug candidate with dual anti-HIV and anti-HSV activity.

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4⁺ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing ß-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1ß, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific ß-chemokine upregulation required new mRNA synthesis. The upregulation of ß-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation).

The lantibiotic peptide labyrinthopeptin A1 demonstrates broad anti-HIV and anti-HSV activity with potential for microbicidal applications.

Lantibiotics are peptides, produced by bacteria, that contain the noncanonical amino acid lanthionine and many of them exhibit antibacterial activities. The labyrinthopeptin A1 (LabyA1) is a prototype peptide of a novel class of carbacyclic lantibiotics. Here, we extensively evaluated its broad-spectrum activity against HIV and HSV in vitro, studied its mechanism of action and evaluated potential microbicidal applications. LabyA1 exhibited a consistent and broad anti-HIV activity (EC50s: 0.70-3.3 µM) and anti-HSV activity (EC50s: 0.29-2.8 µM) in cell cultures. LabyA1 also inhibited viral cell-cell transmission between persistently HIV-infected T cells and uninfected CD4(+) T cells (EC50∶2.5 µM) and inhibited the transmission of HIV captured by DC-SIGN(+)-cells to uninfected CD4(+) T cells (EC50∶4.1 µM). Time-of-drug addition studies revealed that LabyA1 acts as an entry inhibitor against HIV and HSV. Cellular and virus binding studies combined with SPR/FLIPR technology showed that LabyA1 interacted with the HIV envelope protein gp120, but not with the HIV cellular receptors. LabyA1 also demonstrated additive to synergistic effects in its anti-HIV-1 and anti-HSV-2 activity with anti(retro)viral drugs in dual combinations such as tenofovir, acyclovir, saquinavir, raltegravir and enfuvirtide. LabyA1 can be considered as a novel lead peptide as it had profound antiviral activity against HIV and HSV. Pre-treatment of PBMCs with LabyA1 neither increased the expression of the activation markers CD69 and CD25, nor enhanced HIV replication, nor significantly induced various inflammatory cytokines/chemokines. LabyA1 also did not affect the growth of vaginal Lactobacilli populations. Based on the lack of toxicity on the vaginal Lactobacillus strains and its synergistic/additive profile in combination with clinically approved anti(retro)virals, it deserves further attention as a potential microbicide candidate in the prevention of sexual transmitted diseases.

Feglymycin, a unique natural bacterial antibiotic peptide, inhibits HIV entry by targeting the viral envelope protein gp120.

Feglymycin (FGM), a natural Streptomyces-derived 13mer peptide, consistently inhibits HIV replication in the lower µM range. FGM also inhibits HIV cell-to-cell transfer between HIV-infected T cells and uninfected CD4(+) T cells and the DC-SIGN-mediated viral transfer to CD4(+) T cells. FGM potently interacts with gp120 (X4 and R5) as determined by SPR analysis and shown to act as a gp120/CD4 binding inhibitor. Alanine-scan analysis showed an important role for l-aspartic acid at position 13 for its anti-HIV activity. In vitro generated FGM-resistant HIV-1 IIIB virus (HIV-1 IIIB(FGMres)) showed two unique mutations in gp120 at positions I153L and K457I. HIV-1 IIIB(FGMres) virus was equally susceptible to other viral binding/adsorption inhibitors with the exception of dextran sulfate (9-fold resistance) and cyclotriazadisulfonamide (>15-fold), two well-described compounds that interfere with HIV entry. In conclusion, FGM is a unique prototype lead peptide with potential for further development of more potent anti-HIV derivatives.

CXCR4 chemokine receptor antagonists: nickel(II) complexes of configurationally restricted macrocycles.

Tetraazamacrocyclic complexes of transition metals provide useful units for incorporating multiple coordination interactions into a single protein binding molecule. They can be designed with available sites for protein interactions via donor atom-containing amino acid side chains or labile ligands, such as H(2)O, allowing facile exchange. Three configurationally restricted nickel(II) cyclam complexes with either one or two macrocyclic rings were synthesised and their ability to abrogate the CXCR4 chemokine receptor signalling process was assessed (IC(50) = 8320, 194 and 14 nM). Analogues were characterised crystallographically to determine the geometric parameters of the acetate binding as a model for aspartate. The most active nickel(II) compound was tested in several anti-HIV assays against representative viral strains showing highly potent EC(50) values down to 13 nM against CXCR4 using viruses, with no observed cytotoxicity (CC(50) > 125 µM).