Role of contact system activation in hemodialyzer-induced thrombogenicity.

BACKGROUND: The contact system is generally believed to be the main trigger of the coagulation cascade during extracorporeal circulation. However, the extent of contact activation, its role for intradialytic thrombin generation as well as the influence of different dialyzer membranes have not been well established. METHODS: In a novel full-scale ex vivo recirculation dialysis model, we investigated the thrombogenicity of three widely used hemodialyzers (Cuprophan Renak RA15-U, Polysulfone F6HPS and AN69XT Nephral 200). The activation of the contact system was evaluated using a newly developed ELISA for factor XIIa-C1-inhibitor complexes. Additionally, we determined free FXIIa (ELISA), thrombin-antithrombin (TAT) complexes, platelet factor 4 (PF4), complement activation (C5a), granulocyte elastase and blood cell counts. The findings in blood from normal volunteers were compared with factor XII-deficient blood. RESULTS: With normal blood AN69 exhibited the highest thrombogenicity in comparison to Cuprophan and Polysulfone, as assessed by TAT generation and platelet consumption. AN69 caused a rapid increase of the FXIIa-C1-inhibitor complexes and of free FXIIa. Despite significant TAT generation with Cuprophan and Polysulfone free FXIIa remained unchanged and the FXIIa-C1-inhibitor complexes stayed below the detection limit. With factor XII-deficient blood Polysulfone exhibited the same TAT generation, whereas the thrombogenicity of AN69 was greatly reduced. CONCLUSIONS: Our data challenge the common assumption that activation of the contact system with generation of FXIIa is the main trigger for coagulation and thrombus formation in hemodialysis. Only the negatively charged AN69 membrane with enhanced thrombogenicity strongly induced contact activation.

Glutardialdehyde induced fluorescence technique (GIFT): a new method for the imaging of platelet adhesion on biomaterials.

One of the major limitations of biomaterials used in medicine is the adhesion and subsequent activation of platelets upon contact with blood. The development of new or modified materials necessitates adequate methods for the detection and quantification of platelet/material interactions. These interactions are commonly investigated by means of scanning electron microscopy (SEM), radioisotope and immunological techniques, or by quantification of released platelet contents. Given the lack of a simple, rapid, and inexpensive assay, we developed a novel method for the accurate assessment of platelet adhesion after contact with foreign surfaces, which enables quantitative measurements as well as imaging of the platelet shape change, and which omits conventional or immunological staining and time-consuming preparative steps. The glutardialdehyde induced fluorescence technique (GIFT) uses the epifluorescence of glutardialdehyde-fixed platelets detected by fluorescence microscopy and is suitable for opaque and transparent materials. Combined with computer-aided image analysis, numbers of adherent platelets, platelet-covered surface, and average platelet spread area can be determined as markers of surface thrombogenicity. To validate the technique, four materials of different thrombogenicity [polypropylene (PP), poly(D,L-lactide) (PDLLA), 2-hydroxyethyl-methacrylate-grafted PDLLA (PDLLA-HEMA), and heparin-coupled PDLLA-HEMA] were investigated by GIFT and SEM. We found concordant results with SEM and GIFT with the following ranking of thrombogenicity: PP > PDLLA > PDLLA-HEMA > or = PDLLA-HEMA-heparin. GIFT significantly discriminated between the investigated materials. The surface modifications led to improved thromboresistance with reduced platelet adhesion and shape change. The main advantages of GIFT as compared with SEM are: no vacuum-drying or dehydration, less time-consuming procedure, fixation and fluorescence "staining" in one step, and suitability for computer-aided image analysis allowing quantitative assessment of platelet adhesion as well as imaging of the platelet shape change with high-contrast images. In conclusion, GIFT is a valid, rapid, and simple method for the quantitative determination of platelet/material interactions intended for the evaluation of thrombogenicity of biomaterials surfaces.