Platelet and Fibrin Deposition at the Damaged Vessel Wall: Cooperative Substrates for Neutrophil Adhesion Under Flow Conditions

At sites of vessel wall damage, the primary hemostatic reac- tion involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Ad- hered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)- bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion. Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling adhesion and allowed more PMNs to adhere at higher shear (320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. However, at higher shear rates (Û200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interac- tion of PMNs with a fibrin network

Characterization of Eosinophil Adhesion to TNF-a-Activated Endothelium Under Flow Conditions: a4 Integrins Mediate Initial Attachment, and E-Selectin Mediates Rolling

The multistep model of leukocyte adhesion reveals that selectins mediate rolling interactions and that integrins mediate firm adhesion processes. In this study, the interaction between eosinophils and TNF-a-activated HUVEC (second or third passage) was studied under flow conditions (0.8 and 3.2 dynes/cm 2 ). Especially the role of a4 integrins on eosinophils and E-selectin on HUVEC was studied. Inhibition of the integrin a4 chain on eosinophils reduced the number of firmly adhered resting eosinophils to TNF-a-stimulated endothelium by 43% whereas the percentage rolling cells increased 2.2-fold compared with untreated control eosinophils. Blocking of E-selectin on the endothelium reduced the number of adherent eosinophils by only 23% and 16%. In this situation, however, hardly any rolling adhesion was observed, and the few rolling cells showed a low rolling velocity. Blocking both a4 integrin on eosinophils and E-selectin on HUVEC reduced the number of adhered eosinophils by 95%. P-selectin did not significantly participate in eosinophil adhesion to TNF-a-activated HUVEC. Inhibition of both a4 integrins and ß2 integrins on eosinophils resulted in a reduction of adhered cells by 65% and a 3-fold increase in percentage rolling cells. Taken together, these results clearly show that resting eosinophils preferentially use constitutively active a4 integrins ( a4 ß1 , a4 ß7 ) for the first attach-ment to TNF-a-activated HUVEC. In addition, a4 integrins and E-selectin work synergistically in eosinophil adherence to TNF-a-activated HUVEC. Although E-selectin is important for eosinophil rolling under these conditions, P-selectin plays only a minor role

Neutrophil Adhesion to Fibrinogen and Fibrin Under Flow Conditions Is Diminished by Activation and L-Selectin Shedding

The adhesion of neutrophils (polymorphonuclear leukocytes [PMNs]) to immobilized fibrinogen/fibrin is mediated by b2-integrins. However, the influence of physiologic flow con- ditions on neutrophil adhesion to these surfaces is poorly defined. In this report, the effect of flow and neutrophil acti- vation on adhesion to immobilized fibrinogen and fibrin was examined. For the evaluation of (the distribution of) neutro- phil adhesion, real-time video-assisted microscopy and custom- made software were used. Under flow conditions, ad- herent neutrophils appeared to support the subsequent margination of other neutrophils, thereby enhancing the ad- herence of these cells to fibrin. Consequently, neutrophils adhered in clusters, especially at higher shear stresses (eg, cluster index 1.4 at shear 80 mPa). Preactivation of PMNs with fMLP (10Ï7 mol/L) or 4b-phorbol, 12-myristate, 13-ace- tate (PMA; 100 ng/mL) resulted in approximately 50% inhibi- tion of adhesion to fibrin and a more random distribution (cluster index Ú0.5). L-selectin antibodies or neuraminidase treatment of PMNs also inhibited adhesion and clustering, indicating a role for L-selectin. Under static conditions, no clustering appeared and PMN activation with fMLP or PMA caused threefold and sevenfold increased adhesion, respec- tively. Under these conditions, anti-L-selectin antibodies or neuraminidase did not affect adhesion. These results indi- cate that, under flow conditions, adherent neutrophils support adhesion of flowing neutrophils by L-selectin-mediated cell-cell interactions. Preactivated neutrophils, with lowered L-selectin expression, are less susceptible for this interac- tion. By this mechanism, adhered leukocytes can modulate the recruitment of leukocytes to the vessel wall at sites of inflammation

Two novel mutations in the prothrombin gene identified in a patient with compound heterozygous type 1/2 prothrombin deficiency

Thrombosis and Hemostasi

Two novel mutations in the prothrombin gene identified in a patient with compound heterozygous type 1/2 prothrombin deficiency

Thrombosis and Hemostasi

Therapeutic drug monitoring of infliximab : performance evaluation of three commercial ELISA kits

BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab (IFX, Remicade®) can aid to optimize therapy efficacy. Many assays are available for this purpose. However, a reference standard is lacking. Therefore, we evaluated the analytical performance, agreement and clinically relevant differences of three commercially available IFX ELISA kits on an automated processing system. METHODS: The kits of Theradiag (Lisa Tracker Infliximab), Progenika (Promonitor IFX) and apDia (Infliximab ELISA) were implemented on an automated processing system. Imprecision was determined by triplicate measurements of patient samples on five days. Agreement was evaluated by analysis of 30 patient samples and four spiked samples by the selected ELISA kits and the in-house IFX ELISA of Sanquin Diagnostics (Amsterdam, The Netherlands). Therapeutic consequences were evaluated by dividing patients into four treatment groups using cut-off levels of 1, 3 and 7 μg/mL and determining assay concordance. RESULTS: Within-run and between-run imprecision were acceptable (≤12% and ≤17%, respectively) within the quantification range of the selected ELISA kits. The apDia assay had the best precision and agreement to target values. Statistically significant differences were found between all assays except between Sanquin Diagnostics and the Lisa Tracker assay. The Promonitor assay measured the lowest IFX concentrations, the apDia assay the highest. When patients were classified in four treatment categories, 70% concordance was achieved. CONCLUSIONS: Although all assays are suitable for TDM, significant differences were observed in both imprecision and agreement. Therapeutic consequences were acceptable when patients were divided in treatment categories, but this could be improved by assay standardizatio