Contact activation of the plasma coagulation cascade. III. Biophysical aspects of thrombin-binding anticoagulants.

A biophysical model linking fibrin polymerization kinetics (following release from a thrombin-fibrinogen complex), coagulation time, and competitive inhibition of thrombin illustrates the utility of thrombin-binding ligands as anticoagulants in blood collection applications. The resulting mathematical relationship connecting fibrinogen, ligand, and thrombin concentrations was tested against experimentally observed anticoagulation of whole, platelet-poor porcine plasma induced by short, single-stranded DNA oligonucleotides originally found to bind thrombin by screening combinatorial libraries. The thrombin-fibrinogen dissociation constant Ks served as the single adjustable parameter in a least-squares fitting of the model to experimental anticoagulation data. Best-fit Ks values corroborated microM values measured in plasma-free systems, and application of the model to a ligand challenge to the intrinsic pathway of plasma coagulation corroborated nM endogenous thrombin concentrations measured in porcine blood activated by endotoxin insult in vivo. The model fit to data suggests that only about 20% conversion of blood fibrinogen to fibrin is required to coagulate (gel) porcine plasma. This prediction is consistent with the common clinical laboratory observation of latent fibrin formation in "serum" separated from blood before fibrinogen is fully converted to fibrin. It was concluded that the thrombin-binding anticoagulation model was a reasonable simulation of the situation in which an initial bolus of either exogenous or endogenous thrombin is rapidly partitioned between fibrinogen-bound and ligand-bound forms with little or no additional free thrombin created over time.

Contact activation of the plasma coagulation cascade. I. Procoagulant surface chemistry and energy.

Contact activation of the intrinsic pathway of porcine blood plasma coagulation is shown to be a steep exponential-like function of procoagulant surface energy, with low activation observed for poorly water-wettable surfaces and very high activation for fully water-wettable surfaces. Test procoagulants studied were a system of oxidized polystyrene films with varying wettability (surface energy) and glass discs bearing close-packed self-assembled silane monolayers (SAMs) with well-defined chemistry consisting of 12 different terminating chemical functionalities. A monotonic trend of increasing coagulation activation with increasing water wettability was observed for the oxidized polystyrene system whereas results with SAM procoagulants suggested a level of chemical specificity over and above the surface energy trend. In particular, it was noted that coagulation activation by SAMs terminated with--CO2H was much higher than anticipated based on surface wettability whereas--NH3(+)-terminated SAMs exhibited very low procoagulant activity. SAMs terminated in--(CH2)2(CF2)7CF3 behaved as anticipated based on surface energy with very low procoagulant activity and did not exhibit special properties sometimes attributed to perfluorinated compounds. Quantitative ranking of the inherent coagulation activation properties of procoagulant surfaces was obtained by application of a straightforward phenomenological model expressed in a closed-form mathematical equation relating coagulation time to procoagulant surface area. Fit of the model with a single adjustable parameter to experimental measurements of porcine platelet-poor plasma coagulation time was very good, implying that assertions and simplifications of the model adequately simulated reality. Two important propositions of the model were that (1) the number of putative "activating sites" scaled linearly with procoagulant surface area, and (2) contact activation of the plasma coagulation cascade was catalytic in the sense that these activating sites were not consumed or "poisoned" by irreversible or slowly reversible protein adsorption during coagulation. An extension of the coagulation model proposed that procoagulant activation properties scale exponentially with the surface density of polar (acid-base) sites, which, in turn, was related to procoagulant wettability.

Contact activation of the plasma coagulation cascade. II. Protein adsorption to procoagulant surfaces.

A study of blood protein adsorption to procoagulant surfaces utilizing a coagulation time assay, contact angles, Wilhelmy balance tensiometry, and electron spectroscopy (ESCA) is presented. Using a new contact angle method of measuring protein adsorption termed "adsorption mapping" it was demonstrated that protein-adsorbent surfaces were inefficient activators of the intrinsic pathway of the plasma coagulation cascade whereas water-wettable, protein-repellent surfaces were efficient procoagulants. Repeated use of fully water-wettable (spreading) glass procoagulants in the coagulation time assay demonstrated that putative "activating sites" were not consumed in the coagulation of platelet-poor porcine plasma. Furthermore, these procoagulant surfaces retained water-wettable surface properties after incubation with blood proteins and saline rinse. The interpretation of these observations was that plasma and serum proteins were not adsorbed to water-wettable surfaces. However, ESCA of these same surfaces revealed the presence of a thin protein layer. Wilhelmy balance tensiometry resolved these seemingly divergent observations by demonstrating that protein was "associated" with a bound hydration layer, but not formally adsorbed through a surface dehydration or ionic interaction mechanism.