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Aims To investigate a correlation of the platelet activation marker CD62 and secretion of the growth factor PDGF from platelets in coronary patients under therapy with the GPIIb/IIIa-inhibitor abciximab. Methods Flow cytometric assessment of fibrinogen binding (GPIIb/IIIa-binding site) and CD62 expression, as well as PDGF release of human platelets (immunoassay) and platelet aggregation with 20 µm ADP and 2 µg ml-1 collagen were evaluated in nine patients with stable coronary artery disease. Patients were undergoing elective balloon angioplasty and were treated with aspirin (100 mg day-1), heparin (ACT < 220 s) and abciximab (bolus and infusion over 12 h). Blood samples were obtained before initiation of abciximab therapy (under aspirin and heparin) (I), 3 h after angioplasty under abciximab (II) and 12 h after termination of abciximab infusion (III). Results Compared with sample I before abciximab therapy, fibrinogen binding was reduced to 37% (± 34 s.d., P < 0.05) (II) and 55% (± 40 s.d., P < 0.05) (III). Reduced fibrinogen binding also led to a significant reduction of the aggregation response to ADP (down to 37% ± 20) and collagen (down to 0%). Mean fluorescence intensity of CD62-expression was 78 units (± 20 s.d.) (I), 72 units (± 14 s.d.) (II) and 64 units (± 12 s.d., P < 0.05) (III). PDGF release from isolated, washed platelets was 99 (± 33 s.d.) ng/109 platelets at (I), 82 (± 31 s.d.) ng/109 platelets and 96 (± 30 s.d.) ng/109 platelets. Conclusions The results indicate that despite a strong reduction of GPIIb/IIIa-binding and platelet aggregation, CD62 as a marker of platelet secretion and the secretion product PDGF were only slightly reduced under abciximab treatment. No direct correlation between CD62 expression and PDGF release could be demonstrated. [ABSTRACT FROM AUTHOR]

Thrombolytic agents activate plasminogen and induce a systemic fibrinolytic and anticoagulant state. Interaction of fibrinolysis with coagulation and platelet aggregation might be important for synergistic interactions with other antiplatelet or anticoagulant drugs. Thrombolytic agents are most often used in patients with coexisting cardiovascular medication, including various antihypertensives, β-blocking agents, nitrates and aspirin (acetylsalicylic acid). In acute coronary syndromes, anticoagulants and antiplatelet compounds such as clopidogrel or glycoprotein IIb/IIIa receptor antagonists might be given. Inducers or inhibitors of the cytochrome P450 system are not reported to affect the pharmacokinetics of any thrombolytic agent. Since the elimination of the recombinant plasminogen activators saruplase and alteplase is dependent on liver blood flow, drugs affecting hepatic blood flow could theoretically affect the hepatic clearance of these agents. In fact, a reduction in thrombolytic activity has only been demonstrated for alteplase with nitroglycerin (glyceryl trinitrate). Pharmacodynamic interactions occur more often. The additive and beneficial effect of aspirin as concomitant therapy to thrombolysis has been demonstrated without excessive bleeding rates. No data are available on the interaction between ticlopidine or clopidogrel and thrombolytic agents in humans. Anticoagulation by heparin concomitantly with thrombolysis improves the patency rate of the occluded coronary vessel, but bleeding complications are seen more frequently. Although there has been no controlled study on the interaction between oral anticoagulants and thrombolytic agents, patients with myocardial infarction who were taking an oral anticoagulant before admission seem to be at higher risk for intracranial haemorrhage during thrombolytic therapy. Currently, no recommendations can be given for possible dose adjustment of thrombolytic therapy in patients receiving antiplatelet comedication. For comedication with heparin, it has been advised to monitor activated partial thromboplastin time frequently and to avoid values >2.5-fold normal. Patients receiving thrombolytic treatment should be monitored frequently for bleeding and the physician should be aware of any comedication exerting antiplatelet (e.g. aspirin, clopidogrel and ticlopidine) or anticoagulant (e.g. warfarin) effects. [ABSTRACT FROM AUTHOR]

Prime-2-CoV&#95;Beta is a novel Orf virus (ORFV)-based COVID-19 vaccine candidate expressing both the nucleocapsid and spike proteins of SARS-CoV-2 with the receptor-binding domain (RBD) of the Beta strain. This candidate was shown to be safe and immunogenic in a first-in-human Phase I clinical trial. With the shift in the immune landscape toward the Omicron variant and the widespread vaccine- and/or infection-derived immunity, further pre-clinical research was needed to characterize Prime-2-CoV. Here, we quantified the humoral and cellular response to Prime-2-CoV&#95;Beta in pre-immunized mice and compared the protective efficacy of mono- and bivalent variant-based Prime-2-CoV vaccine candidates in hamsters. Prime-2-CoV&#95;Beta induced robust humoral and cellular immune responses in naïve animals but did not further boost antibody titers in the tested setting when given as repeat booster at short interval. We furthermore showed that Prime-2-CoV&#95;Beta-based mono- and bivalent immunization strategies produced comparable immunogenicity and protection from infection. Our results highlight the potential of the Orf virus as a vaccine platform against SARS-CoV-2 and potentially other infectious viruses.

Background: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses., Methods: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61)., Results: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions., Conclusion: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors., Trial Registration: ClinicalTrials.gov identifier: NCT01915524 .

Mathematical models of tumor dynamics generally omit information on individual target lesions (iTLs), and consider the most important variable to be the sum of tumor sizes (TS). However, differences in lesion dynamics might be predictive of tumor progression. To exploit this information, we have developed a novel and flexible approach for the non-parametric analysis of iTLs, which integrates knowledge from signal processing and machine learning. We called this new methodology ClassIfication Clustering of Individual Lesions (CICIL). We used CICIL to assess similarities among the TS dynamics of 3,223 iTLs measured in 1,056 patients with metastatic colorectal cancer treated with cetuximab combined with irinotecan, in two phase II studies. We mainly observed similar dynamics among lesions within the same tumor site classification. In contrast, lesions in anatomic locations with different features showed different dynamics in about 35% of patients. The CICIL methodology has also been implemented in a user-friendly and efficient Java-based framework., (© 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of αvβ3 and αvβ5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet αIIbβ3 and platelets express αv integrins, the effect of cilengitide on platelet pro-coagulative response and adhesion is of interest. Flow-based adhesion assays were performed to evaluate platelet adhesion and rolling on von Willebrand factor (vWf), on fibrinogen and on human umbilical vein endothelial cells (HUVECs). Flow cytometry was used to detect platelet activation (PAC1) and secretion (CD62P) by cilengitide and light transmission aggregometry was used to detect cilengitide-dependent platelet aggregation. Cilengitide inhibited platelet adhesion to fibrinogen at concentrations above 250 µM [which is the Cmax in human studies] and adhesion to vWf and HUVECs at higher concentrations under physiologic flow conditions. Platelet aggregation was already impaired at cilengitide concentrations >10 µM. Activation of αIIbβ3 integrin was inhibited by 250 µM cilengitide, whereas platelet secretion was unaffected by cilengitide. No evidence of cilengitide-induced platelet activation was found at all tested concentrations (0.01-1500 µM). At higher concentrations, platelet activation was inhibited, predominantly due to αIIbβ3 inhibition.

A universal coagulation test that reliably detects prolonged coagulation time in patients, irrespective of the anticoagulant administered, has not been available to date. An easily miniaturised, novel μ-fluidic universal coagulation test employing surface acoustic waves (SAW) is presented here. SAW was employed to instantly mix and recalcify 6 μl citrated whole blood and image correlation analysis was used to quantify clot formation kinetics. The detection of clinically relevant anticoagulant dosing with old anticoagulants (unfractionated heparin, argatroban) and new anticoagulants (dabigatran, rivaroxaban) has been tested and compared to standard plasma coagulation assays. The applicability of this novel method has been confirmed in a small patient population. Coagulation was dose-proportionally prolonged with heparin, argatroban, dabigatran, and rivaroxaban, comparable to standard tests. Aspirin and clopidogrel did not interfere with the SAW-induced clotting time (SAW-CT), whereas the strong GPIIb/IIIa-inhibitor abciximab did interfere. Preliminary clinical data prove the suitability of the SAW-CT in patients being treated with warfarin, rivaroxaban, or dabigatran. The system principally allows assessment of whole blood coagulation in humans in a point-of-care setting. This method could be used in stroke units, emergency vehicles, general and intensive care wards, as well as for laboratory and home testing of coagulation.

The synthetic direct thrombin inhibitor argatroban has a rapid onset and offset of anticoagulation. However, there are no data about the pharmacokinetic-pharmacodynamic (PK-PD) relationship of argatroban in patients undergoing contemporary percutaneous coronary intervention (PCI) and no data about other coagulation parameters than activated clotting time (ACT) in this setting. In the ARG-E04-trial, 140 patients were randomly assigned to argatroban (250, 300, or 350 µg/kg as bolus before PCI, followed by 15, 20, or 25 µg/kg/min infusion) or unfractionated heparin (70-100 IU/kg bolus). A 2-compartment model with first-order elimination adequately described the pharmacokinetic profile of argatroban over all 3 dosing groups. Clearance (CL) and distribution volumes (V1 and V2) were 21 L/h, 9.2 L, and 6.6 L, respectively. A significant sigmoidal E(max) relationship was established between the argatroban plasma concentration and the response in ACT and the endogenous thrombin potential (ETP), whereas the response in activated partial thromoplastin time (aPTT), ecarin time (ECA-T), and prothrombinase-induced clotting time (PiCT) could be described by a nonsigmoidal E(max) model. This study proves a relatively small interindividual variability of both PK and PK-PD properties of argatroban even at high doses and supports the profile of argatroban as a drug with a predictive dose-effect relationship and therefore good controllability.

The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with P-selectin expressed on endothelial cells and platelets. PSGL-1 extracellular mucin-like domain displays a variable number of tandem repeats (VNTRs) polymorphism. The wildtype consists of 16 decameric repeats (designated A isoforms) and variants with 15 (B allele) and 14 (C allele) repeats that are assumed to be associated with reduced risk of vascular disease. We investigated the adhesion of these natural variants to P-selectin in native human neutrophils. Healthy volunteers were genotyped and the adhesion of neutrophils expressing the PSGL-1 isoforms A/A, A/B and B/B were studied under static and physiologic flow conditions. Homozygous B/B neutrophils attached significantly weaker to P-selectin at elevated shear rates from 24 up to 64 dyn/cm(2) than A/A and A/B neutrophils. No difference in adhesion rate was found under static conditions and shear stress below 24 dyn/cm(2), but B/B neutrophils rolled significantly faster than A/A neutrophils at shear stress ≥ 12 dyn/cm(2). There was no difference in the adhesive capacity between A/A an A/B neutrophils. These data support the view that the role of the decamers is to extend the ligand binding domain far above the cell surface to support stable leukocyte adhesion and rolling., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This article intends to close the gap between the abundance of regular articles focusing on adhesive mechanisms of cells in a flow field and purely technical reports confined to the description of newly developed algorithms, not yet ready to be used by users without programming skills. A simple and robust method is presented for analysing raw videomicroscopic data of flow-based adhesion assays using the freely available public domain software ImageJ. We describe in detail the image processing routines used to rapidly and reliably evaluate the number of adherent and translocating platelets in videomicroscopic recordings. The depicted procedures were exemplified by analysing platelet interaction with immobilized von Willebrand factor and fibrinogen in flowing blood under physiological wall shear rates. Neutralizing GPIbalpha function reduced shear-dependent platelet translocation on von Willebrand factor and abolished firm platelet adhesion. Abciximab, Tirofiban and Eptifibatide completely inhibited GPIIb/IIIa-dependent stable platelet deposition on fibrinogen. The presented method to analyse videomicroscopic recordings from flow-based adhesion assays offers the advantage of providing a simple and reliable way to quantify flow-based adhesion assays, which is completely based on ImageJ and can easily be applied to study adhesion mechanisms of cells in non-fluorescent modes without the need to deviate from the presented protocol.

Introduction: Thromboembolic complications under antiretroviral therapy (ART) have been described in the past. In particular, the influence of protease inhibitors (PIs) on platelet activation and coagulation is currently under discussion., Methods: HIV-1-infected, PI-naive adults (n = 18) were investigated before and 4 weeks after the start of the ART, consisting either of boosted PI regimens (n = 13) plus reverse transcriptase inhibitors (RTIs) or a double PI regimen (n = 5) without RTI co-medication. Administered PIs were saquinavir (n = 15), lopinavir (n = 4), fosamprenavir (n = 2) and atazanavir (n = 2). Platelet CD62P, CD40L (%+ cells) and PAC-1 binding [mean fluorescence intensity (MFI)] as well as monocyte CD11b (MFI) and monocyte-associated CD41 (%+ cells and MFI) expression were assessed by flow cytometry with or without platelet stimulation. To investigate the influence of platelets on coagulation, the endogenous thrombin potential (ETP) [render fluorescence units (RFI)] was determined., Results: CD62P, PAC-1 binding and CD11b expression remained unchanged. In contrast, the mean+/-SD MFI of CD40L (from 18.2+/-9.0 to 25.5+/-10.4, P = 0.038) and CD41 (from 446.1+/-213.8 to 605.0+/-183.8, P = 0.010) as markers for increased platelet-leucocyte interaction increased significantly. The collagen-induced ETP time-to-peak was altered significantly from 23.8+/-11.4 to 17.0+/-4.2 min (P = 0.028), although the ETP RFI peak showed no evidence for increased procoagulatory capacity (47.1+/-18.6 to 57.3+/-19.9, P = 0.085)., Conclusions: Effects of the evaluated PI HIV therapy on platelet function assessed under field conditions seem to be minor, not affecting all investigated parameters. We found no evidence for increased platelet activation under PI-containing ART. However, CD41 as a marker for increased platelet-leucocyte interaction and CD40L, which can contribute to atherosclerosis, increased significantly.

We have recently shown that in polymorphonuclear leukocytes, 11-keto boswellic acids (KBAs) induce Ca2+ mobilisation and activation of mitogen-activated protein kinases (MAPK). Here we addressed the effects of BAs on central signalling pathways in human platelets and on various platelet functions. We found that beta-BA (10 microM), the 11-methylene analogue of KBA, caused a pronounced mobilisation of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal-regulated kinase (ERK)2, and Akt. These effects of beta-BA were concentration dependent, and the magnitude of the responses was comparable to those obtained after platelet stimulation with thrombin or collagen. Based on inhibitor studies, beta-BA triggers Ca2+ mobilisation via the phospholipase (PL)C/inositol-1,4,5-trisphosphate pathway, and involves Src family kinase signalling. Investigation of platelet functions revealed that beta-BA (> or =10 microM) strongly stimulates the platelet-induced generation of thrombin in an ex-vivo in-vitro model, the liberation of arachidonic acid (AA), and induces platelet aggregation in a Ca2+-dependent manner. In contrast to beta-BA, the 11-keto-BAs (KBA or AKBA) evoke only moderate Ca2+ mobilisation and activate p38 MAPK, but fail to induce phosphorylation of ERK2 or Akt, and do not cause aggregation or significant generation of thrombin. In summary, beta-BA potently induces Ca2+ mobilisation as well as the activation of pivotal protein kinases, and elicits functional platelet responses such as thrombin generation, liberation of AA, and aggregation.

Increased platelet-leukocyte-aggregate (PLA) formation has been reported in acute coronary syndromes (ACS) and during cardiopulmonary bypass, and PLA formation has been acknowledged as a possible target for antiplatelet therapy in ACS and coronary interventions. It has also been suggested as a monitoring tool for risk stratification parameters. In a controlled study design as well as under clinical conditions we investigated the effect of antiplatelet agents by flow cytometric measurement of PLA formation. We were able to demonstrate considerable reduction in PLA formation under experimental and clinical clopidogrel therapy alone or in combination with aspirin. In healthy volunteers the percentage of monocyte-PLAs decreased significantly to 55 to 75% of the baseline under clopidogrel, depending on the type and concentration of the activating agent. In patients with severe peripheral artery disease, formation of monocyte-PLAs at baseline and after stimulation with thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP) was significantly lower under combined therapy when compared with patients under aspirin alone or without antiplatelet treatment. Flow cytometric measurement of PLA formation appears to be well suited for dose response of antiplatelet agents in healthy volunteers and a valuable tool in establishing the clinical significance of circulating PLAs. It may also be a qualified method to monitor platelet function in long-term treatment with antiplatelet agents that interfere with the degranulation process. It is not suited for acute situations.

Decreased retention in the retention test Homburg (RTH) indicates a loss of platelet function; increase is associated with an increased activation of platelets, for example, in patients with vascular diseases. Compared with other materials (e.g., collagen, glass pearls, etc.) the filter surface in the retention tubes is nonthrombogenic. Therefore, the RTH seems to be well suited for measuring an in vivo over-reactivity of platelets. In a pilot study using the RTH we evaluated the postoperative over-reactivity of platelets in 14 patients and observed a significant heterogeneity of the platelet population concerning size and stickiness. Reliable platelet function tests are also necessary for "drug monitoring," since they deliver important clinical laboratory parameters for efficient control of a therapy with antiplatelet drugs. Therefore, we evaluated in vitro how, after administration of platelet aggregation-inhibiting medications (such as acetylsalicylic acid, Prostaglandin and ReoPro in various concentrations, the adenosine diphosphate (ADP), collagen, ristocetin, or suprarenin increased retention can be reduced. The reaction of the platelets in platelet-rich plasma of different patients or donors to the addition of ADP is variable. The platelet function inhibitor effect is dose dependent. In a clinical pilot study, a significant platelet-inhibiting effect of clopidogrel using the RTH has been shown.

Adhesion and aggregation are important parameters characterizing the function of intact platelets in flowing blood and in contact with a more or less thrombogenic surface. In the retention test Homburg (RTH), platelets are exposed to a standardized textured surface (Sysmex retention tubes) under defined conditions of flow. Platelet counts are performed before and after the Sysmex retention tube passage. The difference between these values indicates the percentage of retained platelets (retention index). Decreased retention in the RTH indicates a loss of function or defective platelet function; increase is associated with an increased activation of platelets, for example, in patients with vascular diseases. For further evaluation of the retention phenomenon the filters were fixed after cell passage and examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM and TEM micrographs show activated platelets adhering and spreading on the filter surface, comparable with platelets on a disturbed endothelium. Also, we examined the influence of different G forces and centrifugation times on the retention behavior of the platelets in citrated platelet-rich plasma (PRP) and whole blood (WB). G forces influenced the retention index in PRP and WB significantly and in a different way. Finally, we used a platelet standard, as customary in the quality check, to determine the serial as well as the day-to-day precision.

Treatment with the direct thrombin inhibitor argatroban (ARG) is often followed by vitamin K-antagonist treatment (VKA). Phenprocoumon (PC) and acenocoumarol (AC) are frequently used in Europe. The standard monitoring test for VKA, pro-thrombin time (PT), is prolonged by direct thrombin inhibitors. Therefore the International Normalized Ratio (INR) obtained during combined treatment does not reflect the true effect of the VKA. A similar interference of the VKA on the activated partial thromboplastin time (aPTT), a monitoring assay for direct thrombin inhibitors, can occur. In 39 healthy volunteers the effect of ARG alone or combined with PC or AC on PT, INR, aPTT, and Ecarin Clotting Time (ECT) was investigated. 6 groups each of 6-8 volunteers received a 5-hour infusion of either 1.0, 2.0 or 3.0 microg/kg/min ARG (days 1, 3, 4 and 5) before initiation of either PC or AC (day 1) and during continued VKA dosing (target INR 2-3). A linear relationship (INR(ARG+VKA) = intercept + slope * INR (VKA alone)) was observed between the INR measured "on" and "off" ARG. The slope depended on the argatroban dose and on the International Sensitivity Index (ISI) of the PT reagent, the steepest slope (i.e., the largest difference between INR (ARG+VKA) and INR (VKA alone)) was seen with the highest ARG dose and the PT reagent with an ISI of 2.13. There was a close correlation between plasma levels of ARG and aPTT or ECT. Under VKA the ARG-aPTT relationship indicated an increased sensitivity of the aPTT to ARG, VKA treatment had no effect on the prolongation of the ECT induced by argatroban. In conclusion, ARG at doses up to 2 microg/kg/min can be discontinued at an INR of 4.0 on combined therapy with VKA, as this would correspond to an INR between 2.2 and 3.7 for the VKA. If it is necessary to monitor ARG in the critical transition period, the ECT which is not influenced by VKA can be used as an alternative to the aPTT.