Contact activation of blood-plasma coagulation

Abstract

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.

Figure 1

Simplified line diagram of the plasma-coagulation cascade showing intersection of the intrinsic and extrinsic pathways (many mediators and cofactors involved in hemostasis are not shown and the interaction with platelets has been ignored for simplicity). Contact-activation reactions that are proposed to be surface mediated by the consensus mechanism are expanded in Fig. 2. Blood zymogens are listed in Table 1 and activated forms are denoted by an “a” suffix. Calcium-dependent reactions are suspended in the presence of calcium-chelating anticoagulants such as citrate used to prepare platelet-poor plasma for coagulation studies. Activation of the intrinsic pathway by contact activation of FXII (upper-right, Intrinsic Cascade) is the focus of this Leading Opinion.

Figure 2

Surface-mediated interactions in contact activation of plasma coagulation according to the consensus mechanism involving the participating proteins: prekallikrein (PK), high molecular weight kininogen (HK), factor XII (FXII), factor XI (FXI), kallikrein (Kal). Suffixes “a” and “f” represent activated and fragmented forms, respectively. According to this traditional biochemical theory, FXII “binds” to a surface (represented by the grey box) bearing negatively-charged functional groups through domains rich in positively-charged lysine residues [100, 101]. Binding purportedly induces a conformational change in FXII, ultimately leading to a transformation into an active-enzyme form FXIIa through a process known as autoactivation [102, 103]. Structural changes of FXII upon binding is thought to sharply increase susceptibility to proteolytic cleavage by Kal [104]. FXIIa generated at the surface can, in turn, cleave PK bound to the surface as a complex with HK [105]. This mutual activation of PK and FXII at the surface is referred to as reciprocal-amplification. FXIIa apparently can also hydrolyze FXII by an “autohydrolysis” reaction, sometimes referred to as self-amplification [94, 106]. FXIIa is also implicated in an “autoinhibition” reaction whereby FXIIa itself inhibits production of FXIIa [8, 52]. Ultimately, FXIIa activates FXI bound at the surface as a complex with HK to generate FXIa, leading to propagation of subsequent coagulation cascade reactions diagrammed in Fig. 1 [107, 108]. The exact chemistry of putative autoactivation, autohydrolysis, and autoinhibition reactions is unknown.

Figure 3

Zhuo model of FXII activation (FXII → FXIIa) at a hypothetical procoagulant particle [8] (compare to Fig. 2). All biochemical reactions are proposed to occur within a vicinal “interphase” region that surrounds a procoagulant particle immersed in aqueous FXII solutions (grossly out of scale). Adsorption/desorption is viewed as a partitioning of species between interphase and bulk-solution phases indicated by the double-headed arrows labeled “FXII Partition” and “FXIIa Partition”. Partitioning establishes the interphase concentrations FXII·S and FXIIa·S that ultimately control rate and yield of FXIIa production in solution. FXII activation in buffer solution occurs by autoactivation ( $\mathrm{FXII}\stackrel{\mathrm{surface}}{\to }\mathrm{FXIIa}$) and autohydrolysis ( $\mathrm{FXII}\stackrel{\mathrm{FXIIa}}{\to }2\mathrm{FXIIa}$) whereas FXII activation in plasma occurs by autoactivation and reciprocal activation (kallikrein mediated hydrolysis, not indicated in diagram; see Fig. 2) [48, 109]. FXII activation competes with an autoinhibition reaction indicated by the double-headed curved arrow in which FXIIa itself inhibits FXII→FXIIa [8, 52]. Partitioning concentrates protein within the interphase of hydrophobic procoagulant particles but not within the interphase surrounding hydrophilic procoagulants. As a consequence, surface contact activation of FXII is moderated by the protein composition of the fluid phase in which FXII is dissolved (buffer, protein cocktail, or plasma). The exact chemistry of putative autoactivation, autohydrolysis, and autoinhibition reactions is unknown.

Figure 4

Kinetics of thrombin (FIIa) production by contact activation of recalcified plasma using glass procoagulant particles (following activation protocol outlined in refs. [45, 47]). Panel A collects results obtained with water-wettable particles (water contact angle θ < 10°) at three different surface areas: 353 mm² (solid line), 78 mm² (dotted line), and 15 mm² (dashed line). Panel B collects results obtained with water-wettable glass particles (solid line), aminopropyltriethoxysilane-treated glass particles (θ ~ 70°, dotted line), or octadecyltrichlorosilane-treated glass particles (θ ~ 110°, dashed line) at either 353 mm² (upper series) or 78 mm² (bottom series) procoagulant surface area (see annotations). Ordinate plots fluorescence intensity (FIIa concentration) at various time points following procoagulant-plasma contact. FIIa kinetics exhibit dependence on both procoagulant surface area and surface energy, with higher surface area of any particular procoagulant yielding an earlier onset of FIIa production and increased magnitude of FIIa bolus produced (Panel A). Plasma coagulation was coincident with peak FIIa output, after which FIIa concentration decayed. Decreasing hydrophilicity (decreasing procoagulant efficiency, see Section 1.2) correlated with decreasing FIIa bolus and delayed onset of FIIa production (Panel B). FIIa concentration was measured using a fluorogenic assay that employed Z-Gly-Gly-Arg-(7-amido-4-methylcoumarin) as a substrate (Bachem Biosciences; 20 μl of Hepes Buffer and 10 μl of a 2.5mM stock solution of the fluorogenic substrate were added to a polystyrene culture dish well containing a 40 μl aliquot of plasma. After 15 min incubation at 37°C, 50ml of 20% glacial acetic acid was used to quench the reaction. Fluorescence intensity was measured in sterile Corning black-polystyrene clear-bottom 96-well plates in an excitation wavelength of 390 nm and an emission wavelength of 460 nm).