Influence of a preadsorbed terpolymer on human platelet accumulation, fibrinogen adsorption, and ex vivo blood activation in hemodialysis hollow fibers.

Results are presented on kinetics of platelet accumulation in charged polyacrylonitrile (AN69) hollow fibers by continuous data recording under flow conditions (wall shear rate 108-1050 s-1), using suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer, containing washed red blood cells (0-40%). Preadsorption of a terpolymer of acrylonitrile, poly(ethyleneoxide) methacrylate and trimethylaminoethyl chloride methacrylate leads to very efficient passivation with respect to platelet accumulation and fibrinogen adsorption. In human ex vivo tests, evaluation of complement peptide C3a, platelet beta-thromboglobulin, leucocyte-polymorphonuclear neutrophile elastase and fibrinopeptide A shows no detectable activation. Furthermore, preadsorption appears to result in simultaneous improvement in hemocompatibility of the blood lines leading to and from the dialysis module. This single pretreatment of dialysis membranes should allow injection of lower doses of anticoagulant to patients submitted to hemodialysis.

Surface passivation by human albumin of plasmapheresis circuits reduces platelet accumulation and thrombus formation. Experimental and clinical studies.

The contact of flowing blood with an artificial surface leads to adsorption of plasma proteins, followed by platelet adhesion and aggregation and thrombus formation. This phenomenon is enhanced by turbulent flow at joints, bifurcations, and constrictions. In therapeutic plasmapheresis using an IBM blood cell separator, blockage of the extracorporeal circulation system by platelet-fibrin thrombi imposed a halt in treatment for manual clearance of the circuit for 66 in 149 cases (44%). Thus it was decided to passivate the surface of the extracorporeal circuit by filling the tubing with 4% human serum albumin 15-20 min before the treatment session and then displacing the albumin solution with the patient's blood without creating an air-liquid interface. After introduction of this technique, a blockage was observed for only 11 in 239 cases (5%). In vitro measurements of platelet accumulation on the internal surface of the circulation system were carried out using washed human platelets labeled with 111In-oxine in the presence of a 40% hematocrit. Preadsorption of the surface with albumin reduced platelet deposition to 4-5% that observed for an equivalent pretreatment with physiological saline.

Synthesis and adsorption of a poly(N-acetylethyleneimine)-polyethyleneoxide-poly (N-acetylethyleneimine) triblock-copolymer at a silica/solution interface. Influence of its preadsorption on platelet adhesion and fibrinogen adsorption.

The synthesis of a triblock copolymer poly-(N-acetylethyleneimine)-polyethylenoxide-poly(N-acet ylethyleneimine) includes two successive steps: the first is the functionalization of a poly(ethyleneglycol) precursor by creating sulfonic esters at its chain ends, the second uses these esters to initiate the cationic polymerization of 2-methyl-2-oxazoline. Homopolymers appear in the raw product; hence successive selective extractions of the copolymer with benzene and dioxane are necessary. The final yield in pure copolymer was 11%. The copolymer was characterized by UV and 1H-NMR spectrometry and light scattering. Adsorption isotherms were determined on silica, for varying pH and salt concentration. Optimum conditions for coating silica with the polymer were determined. The efficiency of this precoating to reduce the adsorption of fibrinogen was very high (99.2% reduction with respect to bare silica). Steric exclusion chromatography of a variety of proteins gave a satisfactory calibration curve. Platelet accumulation on copolymer precoated glass was reduced to 10-20% of its value on bare glass, a result superior to that obtained by albumin passivation of the same glass surface.

Thrombin stimulated platelet accumulation on protein coated glass capillaries: role of adhesive platelet alpha-granule proteins.

The generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22 degrees C from solutions of thrombin 100 NIH units (33 micrograms)/ml gave surface concentrations in the range 0.019-0.101 micrograms/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37 degrees C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s-1 or 2,000 s-1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet alpha-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s-1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by anti-thrombospondin antibodies.

The use of monoclonal antibodies to human platelet membrane glycoprotein IIb-IIIa to quantitate platelet deposition on artificial surfaces.

A new technique is described to quantitate platelet deposition in vitro on artificial surfaces, based on a surface phase radioimmunoassay using the monoclonal antibody 6C9, directed specifically against the membrane glycoprotein complex IIb-IIIa of human platelets. Results correlate in linear fashion with those obtained using 111Indium labeled platelets. The method offers particular advantages for the measurement of platelet deposition in whole blood, since platelet separation, washing and labeling procedures are eliminated, together with the ensuing possible selection of platelet populations. In vitro perfusion is performed in glass capillaries of precisely defined diameter (0.80 or 0.56 mm i.d.). Blood flow is laminar and accurately controlled over wall shear rates ranging from venous to capillary (50-4,000 s-1). Using glass capillaries precoated with purified human albumin or fibrinogen or bovine collagen, platelet deposition from suspensions of washed human platelets in Tyrode's-albumin buffer in the presence of a 40% hematocrit is (platelets/mm2): 11,000 (albumin), 78,000 (fibrinogen) and 306,000 (collagen) after 5 min perfusion at 2,000 s-1. In heparin, citrate or hirudin anticoagulated whole blood, surfaces are passivated, probably by albumin adsorption from plasma (platelets/mm2): 400 (albumin), 3,600 (fibrinogen) and 48,000 (collagen) after 5 min perfusion in the presence of 13 mM citrate.