Expression and purification of recombinant serine protease domain of human coagulation factor XII in Pichia pastoris.

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human ßFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.

Expression, refolding, and activation of the catalytic domain of human blood coagulation factor XII.

Human blood coagulation factor XII (FXII; 80 kDa) contains a C-terminal serine protease zymogen domain, which becomes activated upon contacting a negative surface. Activated FXII (alphaFXIIa) brings about reciprocal activation of FXII and kallikrein that by further hydrolysis produces the free catalytic domain (betaFXIIa; 28 kDa). Increased levels of alphaFXIIa are associated with coronary heart disease, sepsis, and diabetes. Biophysical investigation of the structural basis of activation, substrate specificity, and regulation of FXII requires an efficient bacterial system for producing the wild-type and mutant recombinant proteins. Here, the cDNA of the zymogen domain of FXII (betaFXII) was cloned into the pET-28a(+) vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) and overexpressed. The multi-disulfide, recombinant protein, His(6)-betaFXII (rbetaFXII), expressed as an inclusion body, was purified by means of a Ni(2+)-charged resin. The matrix-bound rbetaFXII was subjected to refolding with the glutathione redox system and activated by the in vivo activator, kallikrein. The active form, rbetaFXIIa, obtained in milligram quantities, exhibited similar structural and comparable functional properties relative to human betaFXIIa, as indicated by circular dichroism spectroscopy and kinetics of substrate hydrolysis. Thermodynamics of enzyme:inhibitor complex formation, including the expected 1:1 stoichiometry, was determined for rbetaFXIIa by isothermal calorimetric titration with a specific recombinant protein inhibitor, Cucurbita maxima trypsin inhibitor-V (rCMTI-V; 7kDa).

Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests.

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.

[Isolation, purification, and properties of factor XII and its active fragment (beta-XIIa)]. / Poluchenie, ochistka, svoistva faktora XII i ego aktivnogo fragmenta (beta-XIIa).

A procedure of isolation of human blood plasma prekallikrein, coagulation factor XII and active fragment beta-XIIa has been developed. This procedure includes the traditional chromatography steps and FPLC. Disc-electrophoresis revealed that the preparations of factor XII and beta-XIIa were homogeneous. Their specific activity was 70.6 U and 2.5 U, respectively. The procedure described is less time consuming and it allows to isolate these factors in the preparative quantities.

Studies on factor XII in porcine plasma: purification and its conversion to activated form by porcine plasma kallikrein.

When porcine plasma was subjected to four steps of ion-exchange column chromatographies, factor XII (F. XII) was separated into two fractions, F. XII-1 and F.XII-2, and about 8.5 mg of F. XII-1 and 2.3 mg of F. XII-2 were obtained from 675 ml of the plasma. The purified proteins were found to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular masses of F. XII-1 and XII-2 were estimated to be about 84 kDa by SDS-PAGE, and consisted of a single polypeptide chain. However, F. XII-1 converted to active form after removing Polybrene, but F. XII-2 was not activated. Activation of F. XII-2 took place on incubation with porcine plasma kallikrein, and the activation rate was increased only in the presence of negatively charged surfaces, such as sulfatide. When the preparation was incubated with sulfatide, spontaneous activation was not observed on the condition that we used. Activation of porcine F. XII-2 by porcine plasma kallikrein was found to involve the cleavage of the peptide bond on the disulfide-bridged polypeptide chain, and further fragmentation of activated form was observed during prolonged incubation time.

Activation of factor XI in plasma is dependent on factor XII.

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.

The recognition site for hepatic clearance of plasma kallikrein is on its heavy chain and is latent on prokallikrein.

We partially purified the glycoproteins prokallikrein and kallikrein from rat plasma. The purification of rat plasma kallikrein may result in two forms: an intact form (alpha, M(r) 84-87 kDa) and a partially degraded form (beta, M(r) 46-51 kDa). The alpha-form is composed of a heavy chain (M(r) 50 kDa) and a light chain (M(r) 34-37 kDa) linked by a disulfide bond. The catalytic site is found on the light chain. The beta-form has a partially degraded heavy chain (M(r) 28 kDa). Using a preparation of exsanguinated and perfused rat liver, we verified that rat plasma prokallikrein is not activated by the liver and that neither the proenzyme nor the light chain is removed by the organ. Both forms (alpha and beta) of the active enzyme are similarly removed from the perfusate. We also observed that the clearance of plasma kallikrein is temperature-dependent, and not affected by substances that inhibit binding to galactosyl-, mannosyl-, fucosyl- or phosphomannosyl-specific lectins, but inhibited by beta-galactosides. We suggest that: (a) the binding site to hepatocytes is latent on prokallikrein and is located on its heavy chain, more specifically on the 28-kDa fragment still present in the beta form of the active enzyme and (b) plasma kallikrein is recognized by an S-type lectin.

One-step preparation of Hageman factor fragment concentrate.

The Hageman factor fragment (HFf) concentrate was prepared from acetone and dextran sulphate activated human plasma by chromatography on a CM-Sephadex column. The preparation was free of the main kallikrein inhibitors--Cl-inactivator, alpha 2-macroglobulin, antithrombin III and alpha 1-antitrypsin. The HFf concentrate can serve as an activator of prekallikrein in patient plasma.

Activation of the contact system in ascites from patients with gastrointestinal cancer.

Our observations indicates that the plasma contact system is activated in ascites from patients with gastrointestinal cancer: Factor XII is activated, plasma kallikrein is present in complex with the protease inhibitor alpha 2-macroglobulin, and the plasma kallikrein substrate high molecular weight kininogen, is highly degraded. Contact activation seems to take place in spite of a high level of inhibition. Activation of the contact system generates mediators, which may play a role in the accumulation of ascites.

Effect of heparin on the activation of factor XII and the contact system in plasma.

We examined in purified systems and in human plasma whether heparin serves as a contact system activating compound. Purified human factor XII zymogen was not activated by heparin through an autoactivation mechanism, but was activated in the presence of purified prekallikrein. Zn2+ (12 microM) did not support autoactivation by heparin. The activation of factor XII and the contact system by heparin in plasma anticoagulated with citrate or with hirudin (not chelating ions) was examined by the cleavage of 125I-labeled factor XII and high molecular weight kininogen (HK). Heparin at 1.6 and 16 USP U/ml was not able to produce activation, in contrast to dextran sulfate (20 micrograms/ml) which supported activation of both factor XII and HK. This study indicates that heparinized plasma does not support activation of the contact system mediated through activation of factor XII. It is not expected that heparin anticoagulant therapy will contribute to activation of the contact system.

Purified factor XII has a higher specific activity than the parent molecule in plasma.

The specific clot promoting activity of factor XII (F XII) in plasma samples from 50 healthy adults was between 30 and 48 U/mg, whereas the specific activity of purified F XII ranged from 55 to 66 U/mg. This difference was neither due to partial proteolytic activation during purification of F XII nor to the influence of plasma protease inhibitors. Purified F XII showed normal size and charge, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, respectively. The increase of the specific F XII activity during the purification process mainly occurred after anion exchange chromatography on DEAE-Sephadex and after the final gel filtration step. Upon dextran sulfate activation, proteolytic cleavage of F XII and generation of kallikrein-like amidolytic activity was faster in F XII deficient plasma containing purified F XII than in F XII deficient plasma containing a corresponding amount of pooled normal plasma (NHP). The binding to kaolin was similar for both, purified F XII and plasma F XII. In conclusion, purification alters the properties of F XII in an unknown way, resulting in an increased specific clot promoting activity.

Contact activation factors in plasma from pregnant women--increased level of an association between factor XII and kallikrein.

The plasma levels of FXII, prekallikrein (PK), and high- and low molecular weight kininogens (HK and LK) were studied in pregnant women in the last trimester and in non-pregnant controls. FXIIa and plasma kallikrein were assayed in acetone-treated citrated plasma (CPLa) with the tetrapeptide S-2222 as substrate, using soybean trypsin inhibitor and corn inhibitor to exclude kallikrein and FXIIa respectively. No difference in PK-level could be registered for the two kinds of plasma, but the level of FXII had increased to about 150% in the pregnancy plasma. No difference in HK-level was observed, whereas the LK-level was significantly higher in pregnancy plasma, about 250% and 160% in rocket immunoassay and bioassay respectively. In fractions from gel filtration of plasma acetone-activated in the presence of benzamidine (BPLa), kallikrein was assayed as S-2302 amidase, HK and LK were measured in rocket immunoassay, and HK and FXII were studied in PAGE immunoblot experiments. In contrast to previous results obtained upon gel filtration of CPLa, not only kallikrein and HK, but in addition also FXII now appeared together in the same fractions and as two separate peaks. One peak eluting in early fractions (gel mol. wt. 300-400 KD), and one late eluting peak of proteins adsorbed to the gel material. The first peak was notably marked in pregnancy plasma. The results provide support for the assumption of an association in plasma between the three contact activation factors studied.

Separation of the Hageman factor fragment from human serum albumin by chromatography on blue sepharose CL-6B.

The separation of the Hageman factor fragment (HFf) activity from human serum albumin by chromatography on Blue Sepharose CL-6B is described. The complete separation cannot be achieved in a single chromatography step due to complex formation between HFf and albumin.

Interactions of C1(-)-inhibitors from normal persons and patients with type II hereditary angioneurotic edema with purified activated Hageman factor (factor XIIa).

Activated high molecular weight Hageman factor (75 Kd) and Hageman factor carboxy-terminal fragments both formed complexes with purified C1(-)-inhibitor, but the Hageman factor fragments appeared to have a higher affinity for the C1(-)-inhibitor than activated Hageman factor. Therefore, the clot-promoting activity of activated Hageman factor might be relatively unimpaired if Hageman factor fragments are also present. Normal C1(-)-inhibitor was cleaved by Hageman factor fragments. Clot-promoting activity was not generated in Hageman factor by exposure to Hageman factor fragments, nor was Hageman factor cleaved by Hageman factor fragments. When Hageman factor was cleaved by streptokinase-activated plasminogen, a 40 Kd fragment was released. In contrast to their interactions with other proteinases, which are blocked by normal C1(-)-inhibitor, Type II C1(-)-inhibitors from plasmas of affected members of eight different kindred with this form of hereditary angioneurotic edema all inhibited the specific coagulant activity of activated Hageman factor to some degree. They did not all form complexes with activated Hageman factor that were stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A rapid purification with high recovery of factor XII (Hageman factor) on immunoaffinity column: application to an abnormal clotting factor XII (factor XIITORONTO).

A rapid purification procedure with high recovery of blood coagulation factor XII (Hageman factor, HF) was established. Homogeneous HF was isolated in 6 days on a monoclonal antibody-immunoaffinity column chromatography followed by gel filtration. Approximately 4,300-fold purification of HF was attained with 31% yield on average (n = 4). Using this method, an abnormal HF was purified from plasma of a patient with cross-reacting material (CRM)-positive Hageman trait (factor XIITORONTO). The abnormal HF was found to be a single chain polypeptide with the same molecular weight (80,000) as the normal HF. Both abnormal and normal HF had similar amino acid compositions.

Interaction of bovine factor XIIa with an inhibitor from bovine plasma.

An inhibitor of factor XIIa has been purified from bovine plasma and characterized (Thornton, R.D. and Kirby, E.P. (1987) J. Biol. Chem. 262, 12714-12721). This inhibitor interacts with XIIa to form a very stable complex with a 1:1 stoichiometry. The active site of XIIa, located on the light chain, is directly involved in the interaction, and complex formation between factor XIIa inhibitor and XIIa can be blocked by diisopropyl fluorophosphate, corn trypsin inhibitor, or the chromogenic substrate S2302. Incubation of the complex with excess XIIa does not result in cleavage of the complex. The complex does not spontaneously dissociate and is stable to boiling, SDS, thiocyanate, acid, and hydroxylamine or Tris at pH 7-10. In addition to complex formation, a cleaved form of factor XIIa inhibitor can be observed. We suggest that the inhibitor is acting as a mechanism-based inactivator, using the criteria of time-dependent inactivation under pseudo-first-order conditions, 1:1 stoichiometry, active site involvement, kinetic protection by substrate or by an active site inhibitor, and partitioning between cleavage of factor XIIa inhibitor and inactivation by complex formation.