Erwin A. Vogler

Contact activation of blood-plasma coagulation.

This opinion identifies inconsistencies in the generally-accepted surface biophysics involved in contact activation of blood-plasma coagulation, reviews recent experimental work aimed at resolving inconsistencies, and concludes that this standard paradigm requires substantial revision to accommodate new experimental observations. Foremost among these new findings is that surface-catalyzed conversion of the blood zymogen factor XII (FXII, Hageman factor) to the enzyme FXIIa (FXII [surface] --> FXIIa, a.k.a. autoactivation) is not specific for anionic surfaces, as proposed by the standard paradigm. Furthermore, it is found that surface activation is moderated by the protein composition of the fluid phase in which FXII autoactivation occurs by what appears to be a protein-adsorption-competition effect. Both of these findings argue against the standard view that contact activation of plasma coagulation is potentiated by the assembly of activation-complex proteins (FXII, FXI, prekallikrein, and high-molecular weight kininogen) directly onto activating surfaces (procoagulants) through specific protein/surface interactions. These new findings supplement the observation that adsorption behavior of FXII and FXIIa is not remarkably different from a wide variety of other blood proteins surveyed. Similarity in adsorption properties further undermines the idea that FXII and/or FXIIa are distinguished from other blood proteins by unusual adsorption properties resulting in chemically-specific interactions with activating anionic surfaces. IMPACT STATEMENT: This review shows that the consensus biochemical mechanism of contact activation of blood-plasma coagulation that has long served as a rationale for poor hemocompatibility is an inadequate basis for surface engineering of advanced cardiovascular biomaterials.

Surface-Energy Dependent Contact Activation of Blood Factor XII

Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution exhibits a parabolic profile when scaled as a function of silanized-glass-particle activator surface energy (measured as advancing water adhesion tension tau(a)(o)=gamma(lv)(o)cos theta in dyne/cm, where gamma(lv)(o) is water interfacial tension in dyne/cm and theta is the advancing contact angle). Nearly equal activation is observed at the extremes of activator water-wetting properties -36<tau(a)(o)<72 dyne/cm (0 degrees <or=theta<120 degrees), falling sharply through a broad minimum within the 20<tau(a)(o)<40 dyne/cm (55 degrees <theta<75 degrees) range over which activation yield (putatively FXIIa) rises just above detection limits. Activation is very rapid upon contact with all activators tested and did not significantly vary over 30 min of continuous FXII-procoagulant contact. Results suggest that materials falling within the 20<tau(a)(o)<40 dyne/cm surface-energy range should exhibit minimal activation of blood-plasma coagulation through the intrinsic pathway. Surface chemistries falling within this range are, however, a perplexingly difficult target for surface engineering because of the critical balance that must be struck between hydrophobicity and hydrophilicity. Results are interpreted within the context of blood plasma coagulation and the role of water and proteins at procoagulant surfaces.

Contributions of Contact Activation Pathways of Coagulation Factor XII in Plasma

Activation of human blood plasma coagulation by contact with hydrophilic or hydrophobic surfaces (procoagulants) is dominated by kallikrein (Kal)-mediated activation of the blood zymogen FXII (Hageman Factor). Mathematical modeling of prekallikrein (PK)-deficient platelet-poor plasma (d(PK)PPP) and PK-reconstituted d(PK)PPP (Rd(PK)PPP) coagulation shows that autoactivation of FXII (FXII-->[surface]FXII) produces no more than about 25% of the total FXIIa produced by the intrinsic pathway. Autoactivation and reciprocal-activation increase in the same proportion with procoagulant surface energy (water-wettability), whereas total amount of FXIIa produced per-unit-area procoagulant remains roughly constant for any particular procoagulant. These results suggest that procoagulant surfaces initiate the intrinsic cascade by producing a bolus of FXIIa in proportion to surface energy or surface area but play no additional role in subsequent molecular events in the cascade. Results further suggest that reciprocal-activation occurs in proportion to the amount of FXIIa produced by the initiating autoactivation step.

A comparison of blood factor XII autoactivation in buffer, protein cocktail, serum, and plasma solutions.

Activation of blood plasma coagulation inxa0vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a mechanochemical reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway.

Moderation of prekallkrein-factor XII interactions in surface activation of coagulation by protein-adsorption competition.

Traditional biochemistry of contact activation of blood coagulation suggesting that anionic hydrophilic surfaces are specific activators of the cascade is inconsistent with known trends in protein adsorption. To investigate contact activation reactions, a chromogenic assay was used to measure prekallkrein (PK) hydrolysis to kallikrein (Kal) by activated factor XII (FXIIa) at test hydrophilic (clean glass) and hydrophobic (silanized glass) surfaces in the presence of bovine serum albumin (BSA). Hydrolysis of PK by FXIIa is detected after contact of the zymogen FXII with a test hydrophobic surface only if putatively-adsorbed FXIIa is competitively displaced by BSA. By contrast, FXIIa activity is detected spontaneously following FXII activation by a hydrophilic surface and requires no adsorption displacement. These results (i) show that an anionic hydrophilic surface is not a necessary cofactor for FXIIa-mediated hydrolysis of PK, (ii) indicate that PK hydrolysis does not need to occur by an activation complex assembled directly on an anionic, activating surface, (iii) confirms that contact activation of FXII (autoactivation) is not specific to anionic hydrophilic surfaces, and (iv) demonstrates that protein-adsorption competition is an essential feature that must be included in any comprehensive mechanism of surface-induced blood coagulation.

Amidolytic, procoagulant, and activation-suppressing proteins produced by contact activation of blood factor XII in buffer solution

The relative proportions of enzymes with amidolytic or procoagulant activity produced by contact activation of the blood zymogen factor XII (FXII, Hageman factor) in buffer solution depends on activator surface chemistry/energy. As a consequence, chromogenic assay of amidolytic activity (cleavage of amino acid bonds in s-2302 chromogen) does not correlate with the traditional plasma coagulation time assay for procoagulant activity (protease activity inducing clotting of blood plasma). Amidolytic activity did not vary significantly with activator particle surface energy, herein measured as water adhesion tension τ(o)=γ(lv)(o)cosθ(a) ; where γ(lv)(o) is pure buffer interfacial tension and θ(a) is the advancing contact angle. By contrast, procoagulant activity varied as a parabolic-like function of τ(o), high at both hydrophobic and hydrophilic extremes of activator surface energy and falling through a broad minimum within a 20<τ(o)<40 mJ/m(2) (55°<θ(a) < 75°) range, corroborating and expanding previously-published work. It is inferred from these functional assays that an unknown number of protein fragments are produced by contact activation of FXII (a.k.a. autoactivation) rather than just αFXIIa and βFXIIa as popularly believed. Autoactivation products produced by activator particles within the 20<τ(o)<40 mJ/m(2) (55°<θ(a) < 75°) surface-energy range suppresses production of procoagulant enzymes by activators selected from the hydrophobic or hydrophilic surface-energy extremes through as-yet unknown biophysical chemistry. Suppression proteins may be responsible for the experimentally-observed autoinhibition of the autoactivation reaction.

Enzymes produced by autoactivation of blood factor XII in buffer: A contribution from the Hematology at Biomaterial Interfaces Research Group

High-resolution electrophoresis of FXII-derived proteins produced by contact activation of FXII in buffer solutions (i.e. in absence of plasma proteins) with hydrophilic and silanized-glass activators spanning the observable range of water wettability (hydrophilic to hydrophobic), shows no evidence of proteolytic cleavage of FXII into αFXIIa or βFXIIa. The autoactivation mixture contains only a single-chain protein with a molecular weight of ∼80xa0kDa, confirming Oscar Ratnoffs previous finding of a single-chain activated form of FXII that he called HFea. Functional assays have shown that these autoactivation products exhibit procoagulant potential (protease activity inducing clotting of blood) or amidolytic potential (cleaves amino bonds in s-2302 chromogen but do not cause coagulation of plasma) or both amidolytic potential and procoagulant potential. Some of these proteins also have the remarkable potential to suppress autoactivation (i.e. suppress creation of enzymes with procoagulant potential). It is thus hypothesized that autoactivation of FXII in the absence of plasma proteins generates not just a single type of activated conformer, as suggested by previous researchers, but rather an ensemble of conformer products with collective activity that varies with activator surface energy used in contact activation of FXII. Furthermore, reaction of αFXIIa with FXII in buffer solution does not produce additional αFXIIa by the putative autoamplification reaction FXIIaxa0+xa0FXIIxa0→xa02FXIIa as has been proposed in past literature to account for the discrepancy between chromogenic and plasma-coagulation assays for αFXIIa in buffer solution. Instead, net procoagulant activity measured directly by plasma-coagulation assays, decreases systematically with increasing FXII solution concentration. Under the same reaction conditions, chromogenic assay reveals that net amidolytic activity increases with increasing FXII solution concentration. Thus, although autoamplification does not occur it appears that there is some form of FXII self reaction that influences products of αFXIIa reaction with FXII. Electrophoretic measurements indicate that no proteolytic cleavage takes in this reaction leading us to conclude that change in activity is most likely due to change(s) in FXII conformation (with related change in enzyme activity).

Surface dependent contact activation of factor XII and blood plasma coagulation induced by mixed thiol surfaces

Studies of the activation of FXII in both platelet poor plasma and in neat buffer solutions were undertaken for a series of mixed thiol self-assembled monolayers spanning a broad range of water wettability. A wide spectrum of carboxyl/methyl-, hydroxyl/methyl-, and amine/methyl-thiol modified surfaces were prepared, characterized, and then utilized as the procoagulant materials in a series of FXII activation studies. X-ray photoelectron spectroscopy was utilized to verify the sample surfaces thiol composition and contact angles measured to determine the sample surfaces wettability. These samples were then used in in vitro coagulation assays using a 50% mixture of recalcified plasma in phosphate buffered saline. Alternatively, the samples were placed into purified FXII solutions for 30u2009min to assess FXII activation in neat buffer solution. Plasma coagulation studies supported a strong role for anionic surfaces in contact activation, in line with the traditional models of coagulation, while the activation results in neat buffer solution demonstrated that FXIIa production is related to surface wettability with minimum levels of enzyme activation observed at midrange wettabilities, and no statistically distinguishable differences in FXII activation seen between highly wettable and highly nonwettable surfaces. Results demonstrated that the composition of the solution and the surface properties of the material all contribute to the observation of contact activation, and the activation of FXII is not specific to anionic surfaces as has been long believed.

Contact activation by the intrinsic pathway of blood plasma coagulation

Abstract It is well known that the biomaterial surfaces that comprise biomedical devices will initiate blood coagulation. This can occur through the adhesion and activation of platelets, but also through the activation of the proteins of the intrinsic coagulation cascade. Nominally, this involves activation of the zymogen FXII (Hageman Factor) which in turn activates FXI, prekallikrein and high-molecular-weight kininogen to form an activation complex. The end result of this sequence of zymogen/enzyme conversion steps is the production of thrombin, the conversion of fibrinogen to fibrin and the development of fibrin strands. In this chapter, we discuss the proteins of the intrinsic pathway, the traditional view of contact activation, and finally alternative ideas about the activation of this important pathway. Notably, we attempt to reconcile the inconsistencies between protein adsorption and function at biomaterial surfaces and the traditional views of contact activation.