CXCL16/CXCR6-mediated adhesion of human peripheral blood mononuclear cells to inflamed endothelium.

The endothelial chemokine CXC motif ligand 16 (CXCL16) is involved in the recruitment and firm adhesion of CXCR6+ cells to the atherosclerosis-prone aortic vessel wall. Recently we showed that CXCR6+ platelets from flowing blood attach to CXCL16 expressed by activated endothelium on the luminal side of the blood vessel. With this study we supplement these findings with the observation that platelets bound to the inflamed endothelium are presenting CXCR6 to CXCL16-positive peripheral blood mononuclear cells (PBMCs) and, thus, are mediating an increased adhesion of PBMCs to the arterial wall. Furthermore we identified endothelial CXCL16 as an important adhesion molecule promoting the firm adhesion of CXCR6-positive PBMCs to inflamed endothelium. Our results demonstrate that endothelial CXCL16 as well as platelet CXCR6 are acting as potent PBMC-adhesion ligands, inducing PBMC-adhesion to the atherosclerosis-prone vessel wall and thus promoting the progression of atherosclerosis.

Activated Platelets Induce an Anti-Inflammatory Response of Monocytes/Macrophages through Cross-Regulation of PGE2 and Cytokines.

Platelets are well known for their role in hemostasis and are also increasingly recognized for their roles in the innate immune system during inflammation and their regulation of macrophage activation. Here, we aimed to study the influence of platelets on the production of inflammatory mediators by monocytes and macrophages. Analyzing cocultures of platelets and murine bone marrow-derived macrophages or human monocytes, we found that collagen-activated platelets release high amounts of prostaglandin E2 (PGE2) that leads to an increased interleukin- (IL-) 10 release and a decreased tumor necrosis factor (TNF) α secretion out of the monocytes or macrophages. Platelet PGE2 mediated the upregulation of IL-10 in both cell types via the PGE2 receptor EP2. Notably, PGE2-mediated IL-10 synthesis was also mediated by EP4 in murine macrophages. Inhibition of TNFα synthesis via EP2 and EP4, but not EP1, was mediated by IL-10, since blockade of the IL-10 receptor abolished the inhibitory effect of both receptors on TNFα release. This platelet-mediated cross-regulation between PGE2 and cytokines reveals one mechanism how monocytes and macrophages can attenuate excessive inflammatory responses induced by activated platelets in order to limit inflammatory processes.

Microfluidic coagulation assay for monitoring anticoagulant therapy in acute stroke patients.

Reliable detection of anticoagulation status in patients treated with non-vitamin K antagonist oral anticoagulants (NOACs) is challenging but of importance especially in the emergency setting. This study evaluated the potential of a whole-blood clotting time assay based on Surface Acoustic Waves (SAW-CT) in stroke-patients. The SAW-technology was used for quick and homogenous recalcification of whole blood inducing a surface-activated clotting reaction quantified and visualised by real-time fluorescence microscopy with automatic imaging processing. In 20 stroke or transient ischaemic attack (TIA)-patients taking NOACs kinetics of SAW-CT were assessed and correlated to other coagulation parameters (PT, aPTT) and NOAC-plasma concentration measured by tandem mass spectrometry (LC-MS/MS). In 225 emergency patients with suspicion of acute stroke or TIA, SAW-CT values were assessed. Mean (± SD) SAW-CT in non-anticoagulated stroke patients (n=180) was 124 s (± 21). In patients on dabigatran or rivaroxaban, SAW-CT values were significantly higher 2 and 8 hours (h) after intake rising up to 267 seconds (s) (dabigatran, 2 h after intake) and 250 s (rivaroxaban, 8 h after intake). In patients on apixaban, SAW-CT values were only moderately increased 2 h after intake (SAW-CT 153 s). In emergency patients, SAW-CT values were significantly higher in NOAC and vitamin K antagonist (VKA)-treated as compared to non-anticoagulated patients. In conclusion, the SAW-CT assay is capable to monitor anticoagulant level and effect in patients receiving dabigatran, rivaroxaban and the VKA phenprocoumon. It has a limited sensitivity for apixaban-detection. If specific SAW-CT results were used as cut-offs, SAW-CT yields high diagnostic accuracy to exclude relevant rivaroxaban and dabigatran concentrations in stroke-patients.

The integrin antagonist, cilengitide, is a weak inhibitor of αIIbß3 mediated platelet activation and inhibits platelet adhesion under flow.

The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of αvß3 and αvß5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet αIIbß3 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 αIIbß3 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 αIIbß3 inhibition.

A novel µ-fluidic whole blood coagulation assay based on Rayleigh surface-acoustic waves as a point-of-care method to detect anticoagulants.

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.

Biomarkers and coagulation tests for assessing the biosimilarity of a generic low-molecular-weight heparin: results of a study in healthy subjects with enoxaparin.

Low-molecular-weight heparins (LMWHs) differ considerably in their influence on clotting tests and release of tissue factor pathway inhibitor (TFPI). Biosimilarity therefore becomes an issue when generic forms of LMWHs are developed. So far, no bioequivalence study with a generic LMWH has been reported. A generic enoxaparin (test) was compared with the originator (reference) in 20 volunteers after single-dose subcutaneous administration (40 mg enoxaparin sodium, 4000 IU/mL anti-factor Xa (anti-FXa; activity). Target variables were anti-FXa and anti-FIIa activity, activated partial thromboplastin time (aPTT), prothrombinase-induced clotting time (PiCT), and TFPI over 24 hours. The statistical evaluation of the anti-FXa activity profile demonstrated bioequivalence of test and reference with confidence intervals of area under the plasma concentration-time curve (AUC0-tlast) (93%-99%) and Amax (88%-95%). Confidence intervals of AUC(0-tlast) (89%-102%) and Amax (90%-103%) of anti-FIIa activity also fulfill bioequivalence criteria. The 90% confidence interval for the maximum concentration of TFPI ranged from 90% to 113%. The claim of similarity was also supported by aPTT and PiCT profiles. Bioequivalence with the originator enoxaparin could be demonstrated by ex vivo inhibition of FXa and FIIa activity, by coagulation tests (aPTT and PiCT), and by in vivo release of TFPI. Whether such data also prove biosimilarity of the generic enoxaparin needs to be determined.

Monitoring direct FXa-inhibitors and fondaparinux by Prothrombinase-induced Clotting Time (PiCT): relation to FXa-activity and influence of assay modifications.

INTRODUCTION: FXa-activity can be measured by the Prothrombinase induced Clotting time (PiCT). The manufactured assay uses bovine FXa as component and employs a incubation period before re-calcification. Its use with new direct FXa-inhibitors is challenged by reports on decreased sensitivity. METHODS: Blood was incubated with 3 investigational, structurally related (oxazolidinones) direct FXa-inhibitors including the recently approved agent rivaroxaban (0 - 2.0 microM), with the structurally distinct direct FXa-inhibitor DX 9065a (0 - 18 microM) and with the indirect inhibitor fondaparinux (0 - 0.6 microM). We tested modifications of PiCT regarding the source of FXa (bovine or human) and the incubation step (incubation before re-calcification=2-step, no incubation =1-step), and compared results with inhibition of human or bovine FXa-activity. RESULTS: The bovine 2-step PiCT showed a paradoxical decrease with all direct FXa-inhibitors, this effect is surmounted only at high concentrations and is not seen with the bovine 1-step PiCT. The decrease in PiCT is not observed in antithrombin-depleted plasma. The humanized PiCT (1 or 2 step) showed a consistent prolongation under all direct inhibitors. Fondaparinux prolonged PiCT with either assay. The correlation between PiCT and corresponding FXa-activity was significant both for humanized 2-step PiCT or bovine 1 step PiCT (r2=0.80), but the 95% prediction interval was large and covered a span of 40% FXa-activity between one agent and another. CONCLUSIONS: The customary bovine PiCT should only be used to monitor direct FXa-inhibitors when modified as 1-step procedure. PiCT is not suitable to assess similarity of FXa-inhibition when different agents are interchanged.