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.     

Kallikrein modification with affinity to IgG present in higher amounts than normal in plasma from patients with Crohn's disease

This study presents immunological and functional evidence to suggest that the activation of prekallikrein (PK) in human plasma yields two modifications of kallikrein. One of these modifications showed amidolytic properties strongly deviating from those registered for the main part of the enzyme. The substrates were S-2302, Bz-Pro-Phe-Arg-pNA, S-2366 and S-2222. In PAGE immunoblots the PK heavy chain mAb 13G11 was found to detect the kallikrein 85 kD double band and bands with mol. weights of about 152 and 135 kD. Such a 152 kD band could be removed together with an IgG fraction on a Protein G column. In this study the kallikrein identity was confirmed by an estimation of the levels obtained in amidolytic assays of mixtures of normal plasma and plasma deficient in FXI, which in immunological assays showed a PK level of 140-150%, of normal. A comparison in amidolytic assays of normal plasma and plasma from patients with Crohn's disease showed that patients' plasma contained a significantly higher level than normal of modified kallikrein. An IgG removal procedure removed all modified kallikrein, and did not affect ordinary kallikrein levels.     

Kallikrein modification with affinity to IgG present in higher amounts than normal in plasma from patients with Crohn's disease

This study presents immunological and functional evidence to suggest that the activation of prekallikrein (PK) in human plasma yields two modifications of kallikrein. One of these modifications showed amidolytic properties strongly deviating from those registered for the main part of the enzyme. The substrates were S‐2302, Bz‐Pro‐Phe‐Arg‐pNA, S‐2366 and S‐2222. In PAGE immunoblots the PK heavy chain mAb 13G11 was found to detect the kallikrein 85?kD double band and bands with mol. weights of about 152 and 135?kD. Such a 152?kD band could be removed together with an IgG fraction on a Protein G column. In this study the kallikrein identity was confirmed by an estimation of the levels obtained in amidolytic assays of mixtures of normal plasma and plasma deficient in FXI, which in immunological assays showed a PK level of 140–150% of normal. A comparison in amidolytic assays of normal plasma and plasma from patients with Crohn's disease showed that patients' plasma contained a significantly higher level than normal of modified kallikrein. An IgG removal procedure removed all modified kallikrein, and did not affect ordinary kallikrein levels.     

Urinary excretion of diazepam metabolites in healthy volunteers and drug users

Protein G columns were used to remove IgG from human plasma, and the effect on levels of factor XII, factor XI and prekallikrein was studied in functional tests. IgG was detected in PAGE immunoblot experiments with Fc-specific antibodies. Removal of the bulk of IgG in a procedure based on a low plasma dilution (1+2.5) allowed the passage of an IgG fraction along with the contact factors. This fraction was found to be present in higher amounts in plasma from patients with Crohn's disease (n=5) than in control plasma (n=12). In a previous study, PAGE immunoblot experiments showed that part of the prekallikrein was removed along with IgG when a higher plasma dilution (1+10.8) was used (Scand J Clin Lab Invest 1999; 59: 55-64). This observation was supported by results in the present work based on parallel assays with the peptide substrates S-2302 and Bz-Pro-Phe-Arg-pNA. The prekallikrein fraction removed was present in a functional state differing from the main part of prekallikrein by yielding kallikrein with a significantly increased activity against the substrate S-2366. This prekallikrein fraction was present in higher amounts in patient plasma than in control plasma. Part of the corresponding amidase activity was blocked by lima bean trypsin inhibitor, suggesting its presence in association with factor XI. The results also indicated that prekallikrein activator activity was connected with this fraction. With the high dilution procedure an extensive removal of IgG from the patient plasma was obtained compared to the control plasma.     

Removal of IgG from normal plasma and plasma from untreated patients with active Crohn's disease--effect on levels of contact factors

Protein G columns were used to remove IgG from human plasma, and the effect on levels of factor XII, factor XI and prekallikrein was studied in functional tests. IgG was detected in PAGE immunoblot experiments with Fc-specific antibodies. Removal of the bulk of IgG in a procedure based on a low plasma dilution (1+2.5) allowed the passage of an IgG fraction along with the contact factors. This fraction was found to be present in higher amounts in plasma from patients with Crohn's disease (n=5) than in control plasma (n=12). In a previous study, PAGE immunoblot experiments showed that part of the prekallikrein was removed along with IgG when a higher plasma dilution (1+10.8) was used (Scand J Clin Lab Invest 1999; 59: 55-64). This observation was supported by results in the present work based on parallel assays with the peptide substrates S-2302 and Bz-Pro-Phe-Arg-pNA. The prekallikrein fraction removed was present in a functional state differing from the main part of prekallikrein by yielding kallikrein with a significantly increased activity against the substrate S-2366. This prekallikrein fraction was present in higher amounts in patient plasma than in control plasma. Part of the corresponding amidase activity was blocked by lima bean trypsin inhibitor, suggesting its presence in association with factor XI. The results also indicated that prekallikrein activator activity was connected with this fraction. With the high dilution procedure an extensive removal of IgG from the patient plasma was obtained compared to the control plasma.     

Part of prekallikrein removed from human plasma together with IgG--immunoblot experiments and functional tests

With the present study, evidence is provided that prekallikrein (PK) in human plasma might be present in two different states, one of them removed along with IgG on Protein G columns. At a plasma dilution of 1 + 2.5, small amounts of an IgG fraction were left in plasma along with all of the PK. At a dilution of 1 + 11, nearly all IgG was removed. The removal in parallel of part of the PK was shown in immunoblot experiments and confirmed in amidase assays. One monoclonal antibody against PK (13G11) and two preparations of polyclonal antibodies were used for the immunoblot experiments. Different peptide substrates (S-2302, S-2222, Bz-Pro-Phe-Arg-pNA), along with protease inhibitors (soybean trypsin inhibitor, corn trypsin inhibitor, lima bean trypsin inhibitor) were used for the amidase assays. The amidase assays indicated that factors XII and XI were reduced by Protein G columns. In all experiments with extensive removal of IgG, protein recognized by the factor XII light chain mAb C6B7 was removed at the same time. This antibody preparation did not detect purified contact factors, but it did recognize a preparation of purified beta-FXIIa, and also significant amounts of protein present in plasma deficient in factor XII and not detectable in plasma deficient in PK. This protein accordingly seems to be connected with the PK fraction removed with IgG.     

Enzymes of the contact phase of blood coagulation: kinetics with various chromogenic substrates and a two-substrate assay for the joint estimation of plasma prekallikrein and factor XI

Kinetic constants (Km and kcat) of kallikrein and factor XIa for the chromogenic substrates H-D-L-prolyl-L-phenylanyl-L-arginine-p-nitroanilide (S-2302) and L-pyroglutamyl-L-propyl-L-arginine-p-nitroanilide (S-2366) were determined. The determined constants allow the use of S-2302 and S-2366 in an assay that leads to the joint estimation of factor XI and prekallikrein in activated plasma. The assay reports approximately 3.1 micrograms/ml factor XIa and 34.5 micrograms/ml kallikrein in kaolin-activated plasma (kaolin content 2 mg/ml). The dual-substrate amidolytic assay shows good correlation with the coagulant assay of both factors (0.92 with the prekallikrein assay and 0.98 with the factor XI assay). It is capable, through the joint estimation of factor XI and prekallikrein levels, of differentiating among plasma samples deficient in components of the contact phase of blood coagulation. Kinetic constants of factor beta-XIIa (factor XII fragment) for these substrates and for N-benzol-L-isoleusyl-L-glutamyl-glycyl-L-arginine-P-nitro ani lide (S-2222) were determined, and they allowed the assessment of the contribution of this factor to this assay and its estimation in the activated phase.     

Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302

The amidolytic activity of activated kallikrein in plasma can be measured by use of the chromogenic substrate, S-2302 (H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated to kallikrein by exposure to 50 mg/L dextran sulfate in acetone/water (35/65 by vol) at 0 degrees C for 15 min. The acetone slows anti-kallikrein activity and increases the kallikrein activity by 30%. The 37 degrees C reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris buffer (pH 7.5 at 37 degrees C). We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma deficient in prekallikrein (Fletcher trait) diluted with pooled normal human plasma. We recommend collecting blood specimens with sodium citrate as the anticoagulant and with use of a double-syringe technique and all-plastic containers. Plasma kallikrein activity with Chromozym-PK (Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with S-2302 as substrate (x-axis) gave the relation: y = 0.28x + 0.82 (r = 0.94). Day-to-day analytical variation was 2.4% for a pooled plasma with a mean value of 85.9 mukat/L. The mean (and 2 SD) for 50 healthy adults was 86.4 (32.4) mukat/L.     

Plasma prekallikrein: quantitative determination by direct activation with Hageman factor fragment (beta-XIIa)

This report describes a plasma prekallikrein assay which, unlike methods that employ contact activation, is not affected by the factor XII or HMW kininogen content of the plasma analyzed. In this assay beta-XIIa, a potent fluid-phase activator of prekallikrein, is added to diluted plasma in the presence of 20% acetone (to inactivate kallikrein inhibitors) at 30 degrees C and the kallikrein generated is measured with the chromogenic substrate S-2302. Prekallikrein is fully activated under these conditions and the activity remains stable for at least 30 hr. The mean prekallikrein concentration in plasma samples from 24 healthy individuals was 1.50 +/- 0.35 (S.D.) S-2302 U/ml, corresponding to 20.3 +/- 4.7 micrograms/ml prekallikrein (the specific activity of highly purified human prekallikrein was determined to be 74 S-2302 U/mg). In contrast, the mean concentration in five plasma samples from patients deficient in HMW kininogen was 0.38 +/- 0.02 S-2302 U/ml. No activity was generated in prekallikrein-deficient plasma, and essentially normal levels (1.35 +/- 0.18 S-2302 U/ml) were measured in plasmas from three patients with factor XII deficiency. Plasma prekallikrein was also quantitated by radial immunodiffusion, which gave results similar to those obtained by functional assay with beta-XIIa. The determination of plasma prekallikrein by direct activation with beta-XIIa in the presence of acetone offers several advantages over the use of contact activators such as dextran sulfate. These advantages include complete inactivation of kallikrein inhibitors and total activation of prekallikrein (even in plasmas deficient in other contact factors) without simultaneous generation of plasmin.     

Evaluation of a microassay for human plasma prekallikrein

Current methods for determining plasma prekallikrein, one of three zymogens of the contact phase of plasma proteolysis, are laborious and impractical for general use in a clinical laboratory. Therefore, we have developed a simple, reliable assay using commercially available reagents. By use of the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCI (S-2302), a functional assay, performed in a 96-well microplate, was designed to measure prekallikrein in plasma. Measures were taken to destroy the naturally occurring plasma protease inhibitors of kallikrein without affecting the integrity of the plasma prekallikrein, which allowed complete activation of the zymogen to virtually 100% of predicted activity when compared with that of purified kallikrein. Besides permitting full activation, the use of low pH to destroy critical plasma protease inhibitors allowed the conversion of prekallikrein to kallikrein in as many as 44 plasma samples at one time without the tedious individual timing step usually required to activate each sample. An excellent correlation was found (r = 0.92) when this functional microassay was compared with a functional spectrophotometric assay performed in three subject populations: normal individuals, women receiving oral contraceptives (who frequently exhibit high plasma prekallikrein concentrations), and patients with liver disease (who manifest low plasma prekallikrein levels). This plasma prekallikrein microassay should facilitate the increased determination of plasma prekallikrein in pathophysiologic conditions as well as the monitoring of the progression of various diseases in which contact activation occurs.     

Characterization of proteases in AHF concentrates: effect on factor VIII:von Willebrand protein as assessed by high-pressure gel permeation chromatography

Antihemophilic factor concentrates were surveyed for amidolytic activity on the chromogenic substrates S2238, S2302, S2222, and S2251, which are sensitive to thrombin, kallikrein, factor Xa, and plasmin, respectively. For antihemophilic factor concentrates from two manufacturers, the rates of amidolysis of S2238 and S2302 were approximately an order of magnitude greater than the rates of amidolysis of S2222 and S2251. The S2238 and S2302 activities were characterized by quantitating their interactions with specific substrates or inhibitors. The Km for amidolysis of S2238 was 558 mumol/L, which is 80 times higher than for thrombin but in close agreement to the reported value for activated protein C. The S2238 activity was not inhibited by the thrombin-specific inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, nor by soybean trypsin inhibitor or micromolar concentrations of antithrombin III in the presence of heparin. The S2238 activity was inhibited by D-Phe-Pro-Arg-CH2Cl, but with an estimated second-order rate constant of 3 X 10(5) mol/L-1 minute-1, approximately 1000 times less than for thrombin. These data are consistent with the identity of the S2238 activity as activated protein C. On the other hand, the S2302 activity in antihemophilic factor concentrates was most likely attributable to kallikrein. This was based on the agreement with authentic kallikrein of the Km for S2302 of 154 mumol/L as well as by the rapid inactivation by nanomolar concentrations of the kallikrein-specific inhibitor D-Phe-Phe-Arg-CH2Cl. However, the relative resistance of the S2302 activity to inhibition by soybean trypsin inhibitor or antithrombin III and the partial inhibition by aprotinin suggested that a large proportion of the kallikrein was bound to alpha 2-macroglobulin. This was confirmed by immunoprecipitation using specific anti alpha 2-macroglobulin IgG. The potential for proteolysis of factor VIII:von Willebrand protein during its purification from antihemophilic factor concentrates was demonstrated, and the proteolyzed factor VIII coagulant species was characterized. High-pressure gel permeation chromatography of purified factor VIII:von Willebrand protein at high ionic strength resulted in two sharp peaks of factor VIII procoagulant activity. The earlier eluting peak corresponded with the void volume, and the later peak eluted with an apparent molecular weight of 53,000 daltons. Immediately after separation, the 53,000-dalton factor VIII coagulant had at least a 100-fold higher specific activity than the factor VIII coagulant present in the void volume. However, the 53,000-dalton factor VIII coagulant was labile, with a half-life of 80 minutes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunochemical Studies of Plasma Kallikrein

A monospecific antibody against human plasma kallikrein has been prepared in rabbits with kallikrein further purified to remove gamma globulins. The antisera produced contained antikallikrein and also anti-IgG, in spite of only 8% contamination of kallikrein preparation with IgG. The latter antibody was removed by adsorption of antisera with either Fletcher factor-deficient plasma or with purified IgG. Both kallikrein and prekallikrein (in plasma) cross-react with the antibody with no apparent difference between the precipitation arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified by radial immunodiffusion.Kallikrein antibody partially inhibits the esterolytic and fully inhibits the proteolytic activity of kallikrein. In addition, the antibody inhibits the activation of prekallikrein, as measured by esterase or kinin release. The magnitude of the inhibition is related to the molecular weight of the activator used. Thus, for the four activators tested, the greatest inhibition is observed with kaolin and factor XII(A), while large activator and the low molecular weight prekallikrein activators are less inhibited.With the kallikrein antibody, the incubation of kallikrein with either plasma or partially purified C1 esterase inactivator results in a new precipitin arc, as detected by immunoelectrophoresis. This finding provides physical evidence for the interaction of the enzyme and inhibitor. No new arc could be demonstrated between kallikrein and alpha(2)-macroglobulin, or alpha(1)-antitrypsin, although the concentration of free kallikrein antigen decreases after interaction with the former inhibitor.By radial immunodiffusion, plasma from healthy individuals contained 103+/-13 mug/ml prekallikrein antigen. Although in mild liver disease, functional and immunologic kallikrein are proportionally depressed, the levels of prekallikrein antigen in plasma samples from patients with severe liver disease remains 40% of normal, while the functional kallikrein activity was about 8%. These observations suggest that the livers of these patients have synthesized a proenzyme that cannot be converted to active kallikrein.     

Alteration of factor VII activity by activated Fletcher factor (a plasma kallikrein): a potential link between the intrinsic and extrinsic blood-clotting systems.

Although surface contact is known to accelerate the one-stage prothrombin time of human plasma through the participation of Hageman factor (factor XII) and factor VII, it has not been clear whether Hageman factor interacts with factor VII directly or indirectly. Recently, Gj?nnaess reported experiments suggesting that plasma kallikrein was an intermediate between Hageman factor and factor VII. The present study was undertaken to elucidate the interaction of plasma kallikrein and factor VII. Incubation of Fletcher-trait plasma (deficient in a plasma prekallikrein) with kaolin at 0 degrees C. did not induce shortening of the Thrombotest time or enhancement of factor VII activity, in contrast to studies of normal plasma. Monospecific rabbit antiserum against plasma kallikrein blocked the shortening of the Thrombotest time of normal plasma brought about by kaolin. Purified Hageman factor fragments (prekallikrein activator) induced an increase in factor VII activity in normal or Hageman-trait plasma, but not in Fletcher-trait plasma. A purified plasma kallikrein preparation enhanced factor VII activity in all plasmas, including that of Fletcher-trait plasma. The effect of the kallikrein preparation was blocked by soybean trypsin inhibitor, Trasylol, or rabbit antiserum against kallikrein, but not by lima bean trypsin inhibitor or antiserum against Hageman factor. The activity of partially purified factor VII was enhanced by purified kallikrein in the presence, but not in the absence of factor VII-deficient plasma. These results further support the idea that the enhancement of factor VII activity by surface contact is via Hageman factor and plasma kallikrein, suggesting a possible link between the intrinsic and extrinsic pathway of blood clotting. The significance of this phenomenon in hemostasis in vivo remains to be elucidated.     

Factor XII-induced fibrinolysis. Diminished proactivator activity and drastic reduction of activator activity in Fletcher factor-deficient plasma gamma globulin

Fibrinolytic studies in gamma G fractions of three Fletcher factor-deficient plasmas (functionally deficient in prekallikrein) revealed weak or no factor XII-independent activator activity. Two of the three Fletcher trait patients showed no plasminogen activator activity in clot lysis, fibrin plate, and amidolytic assays. The third patient showed no activator activity as determined by clot lysis and amidolytic assays but gave 10% of the activator activity detected in normal undiluted gamma G fraction in absence of HFf when determined by fibrin plate assay. Normal plasma gamma G fractions showed detectable and significant plasminogen activator activity. These fractions did not contain kallikrein or activated factor XI activities, thus indicating that the activator activity could not be attributed to the presence in these fractions or trace of these activated factors. Furthermore, factor XI-deficient plasma gamma G fraction, which was shown to contain no activated prekallikrein, showed normal plasminogen activator activity. Finally, specific antibodies to prekallikrein were shown not to quench the activity of plasminogen activator present in normal plasma gamma fraction. A double genetic deficiency to explain the absence in Fletcher factor-deficient plasma gamma G fractions of both prekallikrein proactivator and activator activities is not likely. Thus plasma prekallikrein, besides being a known plasminogen proactivator, appears to be required for the expression of a plasminogen activator activity.     

Destabilase complexes--natural liposome produced by medicinal leeches Hirudo medicinalis

Electrophoretic analysis of destabilase preparation demonstrates the presence of protein combinations with MW 12.3, 25 and 50 kD. Fraction (MW 12.3 D) is a monomer of destabilase aggregation having properties of micellar proteins and represents a stable lipid-protein complex, where the role of lipid component is played by the stable analogue of prostacyclin (MW 391 D). The synthesis of a low molecular fraction of destabilase is fulfilled with bacteria--symbiont of leeches Aeromonas hydrophila. When the destabilase (MW 12.3 kD) contacts with blood a process of complexe formation is triggered with hirudin and blood plasma kallikrein inhibitor, forming a stable 'destabilase complex' (DC; MW 25 kD), possessing also a high aggregation capacity. Polymer forms of the destabilase complex form a liposome changing its spatial orientation depending on the nature of the solvent. Such structural organization provides a high stability of DC components and a rapid penetration through cellular membranes (transmembrane transfer) and it also provides prophylactic antithrombotic action in the case of peroral application to animals, due to the blockade of vascular platelets (inhibition of platelet aggregation by prostacyclin analogue) and plasmic (inhibition of thrombin activity and blood plasma kallikrein) links of the hemostasis process. Destabilase fraction with MW 50 kD is a dimer of the destabilase complex. As a result of DC destruction (liposome), hirudin, prostacycline analogue and blood plasma kallikrein inhibitor are released.     

Plasma prekallikrein assay: reversible inhibition of C-1 inhibitor by chloroform and its use in measuring prekallikrein in different mammalian species

The assay of plasma prekallikrein requires activation of prekallikrein to kallikrein and sufficient inactivation of the plasma protease inhibitors of kallikrein to accurately measure the generated kallikrein activity. One method of elimination of the plasma protease inhibitors to kallikrein is to chemically pretreat the plasma. Methylamine has previously been employed to selectively inactivate alpha 2-macroglobulin. Our study examines the effect of sequential preincubation of plasma with chloroform and methylamine on the plasma prekallikrein assay. Chloroform was demonstrated to be a chemical inhibitor of purified C-1 inhibitor, but alpha 2-macroglobulin was not. Chloroform inhibition of C-1 inhibitor was not caused by precipitation of the protein into the interface between the water and organic solvent phase. Greater than 95% of C-1 inhibitor antigen was recovered in the supernatant of chloroform-treated purified C-1 inhibitor, and chloroform-saturated buffer inhibited purified C-1 inhibitor. Chloroform did not dissociate a preformed complex of kallikrein and C-1 inhibitor, but its inhibition of C-1 inhibitor was reversible. The addition of methylamine to plasma pretreated with chloroform in the plasma prekallikrein assay allowed for only a slight increase in the amount of kallikrein measured at 1 minute kaolin activation times, but provided for sustained measurement of activated prekallikrein when kaolin activation times were 5 to 7 minutes. Without chemical pretreatment, prekallikrein was not measurable in rabbit plasma. Both rabbit and pig plasma prekallikrein was measurable after exposure of the plasma to chloroform and methylamine, although the peak activation times and the contribution of each animals' protease inhibitors varied with the species. Our results show that chloroform is a reversible inhibitor of C-1 inhibitor, and that the plasma prekallikrein assay in which it is used is useful for the measurement of prekallikrein in nonhuman mammalian plasma samples.     

Prekallikrein activator (Hageman factor fragment) in human plasma fractions

The recent finding that hypotensive reactions to blood products can be attributed to the presence of a prekallikrein activator in the products, prompted us to measure the distribution of this substance among plasma fractions produced by the cold ethanol method. While the levels observed varied among different production lots, prekallikrein activator was found to be present in four lots of fibrinogen, four lots of immune serum globulin, one lot of immune serum globulin for intravenous use, and nine lots of antihemophilic factor. Nineteen lots of 25% albumin were tested and found to be free of detectable amounts of prekallikrein activator. In addition to 25% albumin and the fraction V rework fractions leading to it, only supernatant III from the fraction II + III reworks was found to be free of the prekallikrein activator. All other fractions contained significant amounts of activity. Chloroform treatment, used to move kallikrein inhibitors was found to destroy the prekallikrein activator in fractions with low protein levels. The prekallikrein activator in antihemophilic factor preparations was formed during contact with glass, and its behavior led to the conclusion that these concentrates contained Hageman factor (Factor XII).     

Preparation and some properties of human serum kallikrein immobilized on ARM-Sepharose.

A highly purified human serum kallikrein immobilized on CH-Sepharose 4-B was obtained. KM values for N-benzoyl-L-arginine ethyl ester and N-tosyl-L-arginine methyl ester hydrolysis of this preparation were 1.10 x 10(-3) M and 3.6 x 10(-4) M, respectively; pH optimum of hydrolysis of these esters were found to be 8.2 and 8.5, respectively. The immobilized kallikrein possessed kininogenase activity and was capable of activating prekallikrein.     

Surface and fluid phase activities of two forms of activated Hageman factor produced during contact activation of plasma

The ability of the two forms of activated Hageman factor (HFa) produced during contact activation of plasma to activate prekallikrein and factor XI was studied. alpha-HFa, defined as an 80,000 mol wt two-chain enzyme which remains bound to the surface was capable of cleaving surface-bound prekallikrein and factor XI. beta-HFa, a 28,000 mol wt single chain molecule, released from the surface during contact activation was able to cleave prekallikrein but showed no activity on factor XI. Cleavage of prekallikrein by beta-HFa occurred irrespective of whether the substrate was surface-bound or in solution. Cleavage of factor XI occurred only when it was surface bound and only the alpha- form of HFa was capable of this proteolytic action. Factor XI was found to remain bound to the surface while prekallikrein and kallikrein rapidly dissociated from the surface into the supernate. These findings suggest that the initiation of intrinsic coagulation through the activation factor XI is a localized event occurring at the site of contact activation and is the result of the action of alpha-HFa. By contrast, kinin generation and fibrinolysis resulting from the formation of kallikrein can be initiated either at the site of contact activation, by alpha-HFa action, or throughout the plasma, by beta-HFa; further dissemination of these activities is assured by the rapid dissociation of kallikrein itself from the surface.     

Plasma Kallikrein Activation and Inhibition during Typhoid Fever

As an ancillary part of a typhoid fever vaccine study, 10 healthy adult male volunteers (nonimmunized controls) were serially bled 6 days before to 30 days after ingesting 10(5)Salmonella typhi organisms. Five persons developed typhoid fever 6-10 days after challenge, while five remained well. During the febrile illness, significant changes (P < 0.05) in the following hematological parameters were measured: a rise in alpha(1)-antitrypsin antigen concentration and high molecular weight kininogen clotting activity; a progressive decrease of platelet count (to 60% of the predisease state), functional prekallikrein (55%) and kallikrein inhibitor (47%) with a nadir reached on day 5 of the fever and a subsequent overshoot during convalescence. Despite the drop in functional prekallikrein and kallikrein inhibitor, there was no change in factor XII clotting activity or antigenic concentrations of prekallikrein and the kallikrein inhibitors, C1 esterase inhibitor (C1-INH) and alpha(2)-macroglobulin. Plasma from febrile patients subjected to immunoelectrophoresis and crossed immunoelectrophoresis contained a new complex displaying antigenic characteristics of both prekallikrein and C1-INH; the alpha(2)-macroglobulin, antithrombin III, and alpha(1)-antitrypsin immunoprecipitates were unchanged. Plasma drawn from infected-well subjects showed no significant change in these components of the kinin generating system. The finding of a reduction in functional prekallikrein and kallikrein inhibitor (C1-INH) and the formation of a kallikrein C1-INH complex is consistent with prekallikrein activation in typhoid fever. The correlation of these changes with the drop in platelet count suggests that a common mechanism may be responsible.