An investigation has been made of the protein layers formed on hemodialysis membranes during clinical use. Dialyzers having membranes of polymethylmethacrylate (PMMA), Cuprophane, cellulose acetate (CA), and saponified cellulose ester (SCE) were examined. Immediately following dialysis the dialyzers were washed free of blood and the membranes eluted with 2% SDS. The eluates were examined by SDS-PAGE followed by protein immunoblotting. Antisera to 16 common plasma proteins were used to probe for the presence of these proteins in the eluates. Most of the proteins tested for were found in the different eluates, suggesting that the protein layers are extremely complex. The protein compositions were qualitatively different on the different membranes. Except for HMWK the contact phase clotting factors were present in very small amounts and were largely activated. The clear presence of HMWK and the relatively small amounts of fibrinogen provide support for the occurrence of the Vroman effect. Fibrinogen was found to be degraded and this may be related to the observation that plasminogen was activated to plasmin. Complement C3 was an abundant component of all eluates. It was degraded to small fragments in a way which could not be related to complement activation. Many of the other proteins, particularly those of high molecular weight, were extensively degraded. It is speculated that this heretofore unremarked phenomenon may be due to the action of enzymes released by cell damage.

A polyetherurethane (PU) was modified using fluorinated surface-modifying macromolecules (SMMs). A double radiolabel method was used simultaneously to measure the number of adhered platelets ((51)Cr) and the quantity of adsorbed Fg ((125)I), in a cone-and-plate instrument. The objectives were to determine if adsorbed Fg levels correlated to platelet adhesion on the surfaces, and to assess if any reductions in platelet adhesion for the SMM-treated surfaces resulted from surface-induced platelet lysis, rather than changes directly related to lower platelet activation and attachment on the novel surfaces. Platelet lysis was determined from lactate dehydrogenase (LDH) and unbound (51)Cr released into plasma isolated from whole blood exposed to test materials. The corresponding Fg adsorption, evaluated under the same platelet adhesion conditions, did not account for the reduced platelet adhesion on the treated surfaces. LDH and (51)Cr platelet release were very low and indicated no statistically significant differences between the materials. It was therefore concluded that platelet lysis did not contribute to the reduction in platelet adhesion characteristic observed on the SMM-treated surfaces. More importantly, the work emphasizes that the platelet activation cannot be inferred to by assessing the quantity of fibrinogen as is commonly done in the literature. The finding suggests a much more complex mechanism of action for the SMM surface modifiers. On-going work is investigating other Fg parameters such as protein binding affinity and protein conformational state in order to establish the mechanism by which the fluorinated surface modifiers may be reducing platelet adhesion via intermediary changes in initial protein adsorption.

The transient detection of fibrinogen on surfaces has been described (Vroman effect) and high-mol-wt kininogen (HK) has been shown to play a role in this reaction. In this study, we attempted to identify the form of HK responsible for preventing detection of the fibrinogen initially adsorbed from plasma to various artificial surfaces and to determine if other plasma components were involved. We compared 125I-fibrinogen adsorption in the presence of normal plasma to plasma deficient in specific proteins. On all surfaces tested, we found that fibrinogen was displaced from the surface. The extent of displacement was greatly reduced, however, but not eliminated in HK-deficient plasma. Factor XII- deficient plasma also showed reduced fibrinogen displacement. These data indicate that HK can actually displace fibrinogen; however, factor XII, or a factor XII-mediated reaction also appears to be necessary for this displacement to occur. Furthermore, when normal plasma was first subjected to extensive contact activation by dextran sulfate, during which the HK was extensively degraded to components smaller than the light chain (as assessed by Western blotting), we observed greatly reduced displacement of fibrinogen. Extensive contact activation of Factor XI-deficient plasma failed to show low-mol-wt derivatives, however, and displacement of fibrinogen was similar to normal plasma that had not undergone extensive activation. These data indicate that HKa (active cofactor produced during contact activation by factor XIIa or kallikrein) is primarily responsible for displacing fibrinogen, and that HKi (inactive cofactor generated by factor XIa) cannot displace fibrinogen. The fibrinogen from all plasma samples looked similar by Western blot analysis, suggesting that fibrinogenolysis was not a component of the Vroman effect. In addition, experiments performed with plasma prechromatographed on lysine agarose showed that a lysine- agarose adsorbable protein may be minimally involved in fibrinogen desorption and a synergism may exist between HK and that protein.

Adsorption of fibrinogen from buffer as a single protein and from plasma to four materials has been studied. The two NIH-NHLBI primary reference standards, filler free polydimethylsiloxane and low density polyethylene, were used along with polyvinylcholoride and cellulose materials supplied by the IUPAC Working Party. The materials were examined in both film and tubing form, except for polydimethylsiloxane which was studied only in tubing form. Adsorption was measured at room temperature using 125I-labelled fibrinogen. The order of adsorbed amounts in the single protein experiments was found to be: cellulose < PVC < PE = PDMS. Apparent adsorption affinities are in the same order. In plasma, all surfaces except cellulose showed maxima in adsorption as a function of plasma concentration after 5 min contact. This is indicative of initial adsorption followed by displacement of fibrinogen (the Vroman effect). Cellulose showed very low adsorption of fibrinogen from plasma. The Vroman maxima were more pronounced on the tubing samples than on the films, and, as for the single protein experiments, adsorption was found to be less on tubing than on film samples. A tentative interpretation of the Vroman effect data suggests that the order of procoagulant activity of the materials may be: PDMS = PE < PVC < cellulose.

SummaryProsthetic vascular grafts become coated with a layer of fibrin that contributes to graft thrombosis and occlusion. We compared the effect of antithrombin III-independent inhibitors of thrombin with heparin for their ability to prevent fibrin accretion onto a model of a vascular graft formed in vitro by coating polyethylene tubing with thrombin bound to a layer of polymerized fibrin. Equivalent antithrombin concentrations of heparin, D-Phe-Pro-Arg CH2Cl (PPACK), recombinant hirudin (r-hirudin), and Hirulog-1 were added to barium chloride-adsorbed plasma containing radiolabelled fibrinogen. Whereas, PPACK and r-hirudin persistently inhibited fibrin accretion, the inhibition by heparin was transient. Hirulog-1 had no effect on early fibrin accretion and was actually associated with enhanced accretion at 30 min (control 11.7 ± 2.0 μg fibrin/cm2; Hirulog-1, 18.4 ± 3.5 μg fibrin/cm2, p <0.001). Both Hirulog-1 and r-hirudin displaced radiolabelled thrombin from the fibrin surface. Whereas hirudin-thrombin complexes are stable, Hirulog-1 produces only transient inhibition of the displaced thrombin thereby accounting for the enhanced fibrin accretion with this anticoagulant. These studies show that the antithrombin III-independent inhibitors, r-hirudin and PPACK, are more effective inhibitors of fibrin accretion onto fibrin-coated polyethylene than heparin or Hirulog-1. In addition, they emphasize the importance of determining the ability of anticoagulants to displace thrombin from fibrin and to form stable thrombin-inhibitor complexes; lack of stability of thrombin-inhibitor complexes must be countered by levels of anticoagulant that are adequate to maintain its effectiveness.