Autoactivation of blood factor XII at hydrophilic and hydrophobic surfaces

Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution is shown not to be specific for anionic hydrophilic procoagulants as proposed by the accepted biochemistry of surface activation. Rather, FXII activation in the presence of plasma proteins leads to an apparent specificity for hydrophilic surfaces that is actually due to a relative diminution of the FXII-->FXIIa reaction at hydrophobic surfaces. FXII activation in neat-buffer solution was effectively instantaneous upon contact with either hydrophilic (fully water-wettable clean glass) or hydrophobic (poorly water-wettable silanized glass) procoagulant particles, with greater FXIIa yield obtained by activation with hydrophobic procoagulants. In sharp contrast, both activation rate and yield was found to be significantly attenuated at hydrophobic surfaces in the presence of plasma proteins. Putative FXIIa produced by surface activation with both hydrophilic and hydrophobic procoagulants was shown to hydrolyze blood factor XI (FXI) to the activated form FXIa (FXIFXIIa-->FXIa) that causes FXI-deficient plasma to rapidly coagulate.     

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.     

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.     

Mathematical modeling of material-induced blood plasma coagulation

Contact activation of the intrinsic pathway of the blood coagulation cascade is initiated when a procoagulant material interacts with coagulation factor XII, (FXII) yielding a proteolytic enzyme FXIIa. Procoagulant surface properties are thought to play an important role in activation. To study the mechanism of interaction between procoagulant materials and blood plasma, a mathematical model that is similar in form and in derivation to Michaelis-Menten enzyme kinetics was developed in order to yield tractable relationships between dose (surface area and energy) and response (coagulation time (CT)). The application of this model to experimental data suggests that CT is dependent on the FXIIa concentration and that the amount of FXIIa generated can be analyzed using a model that is linearly dependent on contact time. It is concluded from these experiments and modeling analysis that the primary mechanism for activation of coagulation involves autoactivation of FXII by the procoagulant surface or kallikrein-mediated reciprocal activation of FXII. FXIIa-induced self-amplification of FXII is insignificant.     

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.     

Surface modification with polyethylene glycol-corn trypsin inhibitor conjugate to inhibit the contact factor pathway on blood-contacting surfaces

Blood contacting surfaces bind plasma proteins and trigger coagulation by activating factor XII (FXII). The objective of this work was to develop blood contacting surfaces having the dual properties of protein resistance and inhibition of coagulation. Gold was used as a model substrate because it is amenable to facile modification using gold-thiol chemistry and to detailed surface characterization. The gold was modified with both polyethylene glycol (PEG) and corn trypsin inhibitor (CTI), a potent and specific inhibitor of activated FXII (FXIIa). Two methods of surface modification were developed; sequential and direct. In the sequential method PEG was first chemisorbed on gold; CTI was then attached to the PEG. In the direct method a conjugate of PEG and CTI was first prepared; the conjugate was then immobilized on gold. The surfaces were characterized by water contact angle and XPS. Biointeractions with the modified surfaces were assessed by measuring fibrinogen adsorption from buffer and plasma and by immunoblot analysis of eluted proteins after plasma exposure. Inhibition of FXIIa, autoactivation of FXII, and clotting times of plasma in contact with the surfaces were also measured. Both the sequential and direct surfaces showed reduced protein adsorption, increased FXIIa inhibition and longer clotting times compared with controls. Although the CTI density was lower on surfaces prepared using the sequential method, surfaces so prepared exhibited greater CTI activity than those generated by the direct method. It is concluded that the activity of immobilized PEG-CTI depends on the method of attachment and that immobilized CTI may be useful in rendering biomaterials more blood compatible.     

Practical application of a chromogenic FXIIa assay

Autohydrolysis of blood factor XII (FXII+FXIIa-->2FXIIa) is found to be a facile reaction in neat-buffer buffer solutions of FXII but an insignificant reaction in the presence of plasma proteins. Autohydrolysis causes a chromogenic assay for FXIIa in buffer solution to strongly deviate from the traditional plasma-coagulation assay. Autohydrolysis can be accommodated by performing chromogenic detection of FXIIa as a rate assay in swamping concentrations of FXII. Rate-assay results performed in this way are shown to be in analytical agreement with the plasma-coagulation assay. Autohydrolysis can be used as a means of amplifying FXIIa produced by contacting neat-buffer solutions of FXII with biomaterials, suggesting a route to highly sensitive measurement of biomaterial hemocompatibility.     

Procoagulant activity of surface-immobilized Hageman factor

Procoagulant activity of surface-immobilized coagulation factor XIIa (activated Hageman factor) is reported. Activity of FXIIa immobilized onto the surfaces of three silanized-glass procoagulants spanning a wide range of wettability was assayed in normal and FXII-deficient plasmas. Previously published mathematical models were used to characterize the procoagulant activity of protein-immobilized materials and soluble enzymes. Results show that FXIIa activity is unrelated to underlying procoagulant surface chemistry and is similar to soluble FXIIa activity. The uninfluential role of the surface on FXIIa suggests that the solid surface activates FXII in biomaterial-induced blood coagulation but is not otherwise involved in FXIIa activity as described by the classical mechanism.     

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 ∼80 kDa, confirming Oscar Ratnoff's previous finding of a single-chain activated form of FXII that he called ‘HF_ea’. 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 FXIIa + FXII → 2FXIIa 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).     

Interactions of antithrombin and proteins in the plasma contact activation system with immobilized functional heparin

The interactions of antithrombin (AT) and the contact phase clotting factors with two commercially available heparinized surfaces are reported. The Carmeda (CBAS) and Corline surfaces along with controls (a sulfonated polyethylene surface and a CBAS analog in which the heparin used was devoid of specific AT-binding sequences) were exposed to human plasma. Adsorbed proteins were eluted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The CBAS and Corline surfaces adsorbed large amounts of AT, whereas adsorption on the controls was negligible. Immunoblots for the four contact phase clotting factors indicated less contact activation on the CBAS and Corline surfaces than on the controls. Determination of adsorbed functional AT using a FXa inhibition assay showed that the CBAS surface adsorbed about 4 times as much AT as the Corline surface. Adsorption of AT to the control surfaces was minimal. Assays for adsorbed FXII and FXIIa based on kallikrein generation showed that all four surfaces adsorbed similar amounts of FXII. However, on the controls, most of the FXII was in activated form, whereas on the CBAS and Corline surfaces very little activation occurred.     

Differential Roles for the Coagulation Factors XI and XII in Regulating the Physical Biology of Fibrin

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.     

FⅫ在促凝表面的接触活化动力学研究

目的为了探索病理性凝血血浆间相互作用的微观机制,设计内源性凝血接触激活实验,研究促凝性表面诱发的内源性凝血的条件以及诱发的主要因素。方法本文采用了干净的玻璃珠和硅烷化玻璃珠,从而得到亲水性能不同的促凝颗粒表面,来确定因子Ⅶ的激活与亲水性的关系;利用缓冲液PBS冲洗促凝剂表面,再与乏血小板血浆反应,对比各凝血时间的变化,研究血浆蛋白对凝血时间的影响。结果 (1)在亲水表面FⅫ-FⅫa的激活表现出特异性,在疏水表面的活化反应相对明显减弱(P0.0001),FⅫ与疏水促凝剂接触激活获得更多FⅫa;(2)在疏水表面FⅫ的激活率和产物都是显著衰减,但是活化FⅫ与疏水表面结合紧密(P0.0001),与蛋白质和疏水表面黏附更紧密的结论一致;(3)FⅫ与500mm~2促凝表面在孵育30min时凝血时间和FⅫ的活化产物更为稳定。结论促凝剂表面能量依赖"催化潜力"促使FⅫ-FⅫa的激活;凝血表面积相同的情况下,PPP血浆内FⅫ的活化率及活化量在疏水促凝剂表面明显低于亲水性表面;与蛋白质和疏水表面更易吸附活化相反,在含疏水促凝剂表面的全血浆中FⅫ的活化明显比亲水性的慢,从而产生了一种表面能依赖的催化潜力的外观。     

Procoagulant stimulus processing by the intrinsic pathway of blood plasma coagulation

Potentiation of the intrinsic pathway of human blood plasma coagulation in vitro by contact with a solid procoagulant surface leads to bolus release of thrombin (FIIa) in concentration proportion to the intensity of activation as measured by procoagulant surface area or energy (water wettability). This rather remarkable finding is confirmed using two different assays: one triggering coagulation substantially through the intrinsic pathway alone and the second triggering coagulation through the intrinsic pathway in the presence of exogenous FIIa spikes. Similarity of experimental outcomes of these assays strongly suggests that endogenous FIIa production through the intrinsic pathway is independent of the absolute amount of FIIa present in plasma. Furthermore, we corroborate previous work indicating that procoagulant surfaces remain activating after repeated use and are not poisoned or denatured in the process of activating plasma coagulation. It is concluded that the sharp control mechanism that gives rise to bolus-production of FIIa from the intrinsic pathway must occur between surface activation of FXII and the FII --> FIIa step, is not related to inhibition by FIIa, and does not involve deactivation of procoagulant surfaces.     

A direct cbromogenic peptide substrate assay for hageman factor (FXII)

A direct chromogenic peptide substrate assay for Hageman factor (FXII) has been developed using a novel soluble activator of FXII. In this assay, plasma samples are treated with acetone to destroy plasma inhibitors of α and β FXIIa and diluted with methylamine-containing buffer to block enzyme binding sites on α₂-macroglobulin. Following incubation for 10 minutes at 37°C with the FXII activator and the addition of buffer containing kallikrein inhibitor, the FXIla activities generated are detected by recording the release of pNA from the chromogenic peptide substrate S-2222. Analysis of plasma fractions in the assay (obtained by Sephadex G-150 gel filtration) revealed a major peak which yielded activity on activation. This peak corresponded with peak levels of FXII antigen and eluted at a molecular weight of approximately 80 000. The activity generated could be inhibited by corn inhibitor. Comparative studies with normal and FXII-deficient plasmas in this assay and a clotting assay resulted in a good correlation between the two assays.     

Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces

Activated human plate lets trigger FXII-mediated contact activation, which leads to the generation of FXIIa–antithrombin (AT) and FXIa–AT complexes. This suggests that contact activation takes place at different sites, on activated platelets and material surfaces, during therapeutic procedures involving biomaterials in contact with blood and is differentially regulated. Here we show that activation in platelet-poor plasma, platelet-rich plasma (PRP), and whole blood induced by glass, kaolin, and polyphosphate elicited high levels of FXIIa-C1-inhibitor (C1INH), low levels of FXIa–C1INH and KK–C1INH, and almost no AT complexes. Platelet activation, in both PRP and blood, led to the formation of FXIIa–AT, FXIa–AT, and kallikrein (KK)–AT but almost no C1INH complexes. In severe trauma patients, FXIIa–AT and FXIa–AT were correlated with the release of thrombospondin-1 (TSP-1) from activated platelets. In contrast, FXIIa–C1INH complexes were detected when the FXIIa–AT levels were low. No correlations were found between FXIIa–C1INH and FXIIa–AT or TSP-1. Inhibition of FXIIa on material surfaces was also shown to affect the function of aggregating platelets. In conclusion, formation of FXIIa–AT and FXIIa–C1INH complexes can help to distinguish between contact activation triggered by biomaterial surfaces and by activated platelets. Platelet aggregation studies also demonstrated that platelet function is influenced by material surface-mediated contact activation and that generation of FXIIa–AT complexes may serve as a new biomarker for thrombotic reactions during therapeutic procedures employing biomaterial devices.     

Dual surface modification with PEG and corn trypsin inhibitor: effect of PEG:CTI ratio on protein resistance and anticoagulant properties

The objective of this study was to investigate the bioactivity and protein-resistant properties of dual functioning surfaces modified with PEG for protein resistance and corn trypsin inhibitor (CTI) for anticoagulant effect. Surfaces on gold substrate were prepared with varying ratios of free PEG to CTI-conjugated PEG. Two methods designated, respectively, "sequential" and "direct" were used. For sequential surfaces, PEG was first immobilized on gold and the surfaces were incubated with CTI at varying concentration. For direct surfaces, a PEG-CTI conjugate was synthesized and gold surfaces were modified using solutions of the conjugate of varying concentration. The CTI density on these surfaces was measured using radiolabeled CTI. Water contact angles were measured and the thickness of PEG-CTI layers was determined by ellipsometry. Fibrinogen adsorption from buffer and human plasma, and adsorption from binary solutions of fibrinogen and α-lactalbumin were investigated using radiolabeling methods. Bioactivity of the surfaces was evaluated via their effects on FXIIa inhibition and plasma clotting time. It was found that as the ratio of CTI-conjugated PEG to free PEG increased, bioactivity increased but protein resistance was relatively constant. It is concluded that on these surfaces conjugation of PEG to CTI does not greatly compromise the protein resistance of the PEG but results in improved interactions between the CTI and the "target" protein FXIIa. At the same CTI density, sequential surfaces were more effective in terms of inhibiting FXIIa and prolonging clotting time.     

A new enzyme-linked immunosorbent assay recognizing both rat and human activated coagulation Factor XII (FXIIa)

We describe the first sandwich enzyme-linked immunosorbent assay (ELISA) capable of recognizing both rat and human activated coagulation Factor XII (FXIIa). Increased plasma concentrations of FXIIa have been associated with adverse outcomes in several cardiovascular disease states. In humans, the FXIIa antigen in plasma is quantified by a direct sandwich ELISA employing an antibody (mAb 2/215) raised against its β-fragment (β-FXIIa), but this assay is unable to detect rat β-FXIIa antigen. Thus, experimental models are at present limited in their capacity to reveal mechanisms by which FXIIa might contribute to cardiovascular pathology. Consistent with overlap between human and rat FXIIa protein epitope sequences, Western blot analysis and ELISA demonstrate that another previously developed antibody against human FXIIa (mAb 201/9) detects β-FXIIa in both human and rat plasma, with no evidence for cross-reactivity with the FXII zymogen or FXIIa complexed with its endogenous inhibitor. The mAb 201/9 based ELISA identified similar elevations in FXIIa in plasma from rats and humans with chronic renal failure. The capacity of this ELISA to identify rat plasma FXIIa has potential application to a wide range of experimental models of human cardiovascular disease.     

Blood protein adsorption onto chitosan

Chitosan was recently indicated to enhance osteogenesis, improve wound healing but to activate the coagulation and the complement systems. In the present study approximately 10 nm thick chitosan film were prepared on aminopropyltriethoxysilane (APTES) coated silicon. The surfaces were incubated in serum or plasma and subsequently in antibodies towards key complement and contact activation of coagulation proteins. The deposited amounts were compared with those on hydrophilic and hydrophobic silicon, APTES and IgG coated reference samples. Although large amounts of serum deposited to chitosan only a weak transient activation of the complement system and no activation of the intrinsic pathway was observed. Upon acetylation the chitosan layer became a strong activator of the alternative pathway of the complement. After incubation in human plasma anti-fibrinogen deposited onto chitosan but not onto the acetylated chitosan, a finding that may explain previous observations of procoagulant activity by chitosan.