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.

Application of a new dynamic flow model for investigating the biocompatibility of modified surfaces.

An in vitro model was developed to compare the biocompatibility of four different coating methods (three heparin and one nonheparin) under hemodynamic conditions. Fresh human donor blood (heparin 5 IU/ml) was recirculated in a standardized experimental circuit. All circuit components were either coated or remained uncoated for control purposes. The aim of the study was to investigate a wide spectrum of effects on blood; coagulation parameters (e.g., fibrinogen, ATIII, thrombin-antithrombin-complex), complement parameters (C1rsC1 Inh, C3b(Bb)P, SC5b-9, C5a), differential blood analyses, platelet activation (flow cytometric investigations), PF 4, and PMN-elastase release were examined by showing possible trends. All heparin coated systems reduced platelet stimulation in comparison to untreated biomaterials. Leukocyte activation was reduced to different degrees depending upon the coating method used. Complement activation was markedly reduced by all coated systems. The results obtained indicate that the pump driven, dynamic blood flow model is suitable to characterize the biocompatibility of surface modified biomaterials. Advantages lie in the integration of the different polymers as parts of the circuit, the low priming volume, and the generation of blood flow conditions similar to those that occur in clinical applications.

Improvement of haemocompatibility of metallic stents by polymer coating.

An alternative to open heart surgery in treating arterial diseases causing restricted blood flow is the implantation of intracoronary metallic stents. In spite of the advances in implantation and in spite of the excellent mechanical properties of metallic stents, there are still limitations because of the thrombogenicity of the metal. We have, hence, directed our attention to the coating of metallic stents with an ultrathin polymer layer by chemical vapor deposition (CVD) polymerization of 2-chloroparacyclophan. In a second step of surface modification the poly(2-chloroparaxylylene) layer is modified by treatment with a sulfur dioxide plasma in order to obtain a more hydrophilic surface with new functional groups. The results demonstrate the stable polymer coating of the stents and the improvement of haemocompatibility after treatment with sulfur dioxide plasma. Platelet adhesion is decreased from 85% for the metal surface to 20% for the CVD-coated and sulfur-dioxide-plasma treated surface.

A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila.

In vertebrates, transcriptional regulators of the GATA family appear to have a conserved function in differentiation and organ development. GATA-1, -2 and -3 are required for different aspects of hematopoiesis, while GATA-4, -5 and -6 are expressed in various organs of endodermal origin, such as intestine and liver, and are implicated in endodermal differentiation. Here we report that the Drosophila gene serpent (srp) encodes the previously described GATA factor ABF. The multiple functions of srp in Drosophila suggest that it is an ortholog of the entire vertebrate Gata family. srp is required for the differentiation and morphogenesis of the endodermal gut. Here we show that it is also essential for Drosophila hematopoiesis and for the formation of the fat body, the insect organ analogous to the liver. These findings imply that some aspects of the molecular mechanisms underlying blood cell development as well as endodermal differentiation are early acquisitions of metazoan evolution and may be common to most higher animals.

Development and characterization of a wettable surface modified aromatic polyethersulphone using glow discharge induced HEMA-graft polymerisation.

The aromatic polyethersulphone (PES) is a well known polymer for the preparation of membranes with excellent thermal stability and chemical resistance. The disadvantage of PES-membranes is their hydrophobic character, which in contact with protein containing solutions leads to high protein adsorption and as a consequence to deterioration of membrane properties. In this report the surface modification of PES by means of glow discharge induced grafting of 2-hydroxyethyl methacrylate (HEMA) is described. Graft polymerisation creates a largely wettable layer of poly(2-hydroxyethyl methacrylate) (PHEMA) on the surface of PES. This has been shown by contact angle measurements using the Wilhelmy plate method. Chemical characterization is carried out by means of X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy/attentuated total reflection (FTIR-ATR). The influence of storage conditions on the surface properties of modified PES samples has been investigated after storage in vacuum, water, and air.