Prekallikrein-deficient Plasma Research Articles

Streptococcal pyogenic exotoxin B (SpeB) boosts the contact system via binding of α-1 antitrypsin

The Streptococcus pyogenes cysteine protease SpeB (streptococcal pyrogenic exotoxin B) is important for the invasive potential of the bacteria, but its production is down-regulated following systemic infection. This prompted us to investigate if SpeB potentiated the host immune response after systemic spreading. Addition of SpeB to human plasma increased plasma-mediated bacterial killing and prolonged coagulation time through the intrinsic pathway of coagulation. This effect was independent of the enzymatic activity of SpeB and was mediated by a non-covalent medium-affinity binding and modification of the serpin A1AT (α-1 antitrypsin). Consequently, addition of A1AT to plasma increased bacterial survival. Sequestration of A1AT by SpeB led to enhanced contact system activation, supported by increased bacterial growth in prekallikrein deficient plasma. In a mouse model of systemic infection, administration of SpeB reduced significantly bacterial dissemination. The findings reveal an additional layer of complexity to host-microbe interactions that may be of benefit in the treatment of severe bacterial infections.

High-Molecular-Weight Hydroxyethyl Starch Accelerates Kallikrein-Dependent Clot Initiation

A decrease in reaction time (R; seconds) has been considered a thrombelastographic hallmark of hypercoagulability. However, the cause of changes in R has been influenced by the method of activation (e.g., celite) and the clinical/laboratory setting (e.g., hemodilution). Although antithrombin deficiency has been implicated as a cause of decreased R in unactivated samples after crystalloid dilution, dilution with hydroxyethyl starch (HES) solutions such as Hextend (6% HES solution; average molecular weight 450 kDa) or Voluven (6% HES solution; average molecular weight 130 kDa) has decreased R values in celite-activated samples in vitro and in vivo, with modulation of these R values observed after aprotinin exposure. Thus, this study proposed to define whether HES affects kallikrein-dependent clot initiation. Citrated human plasma was subjected to 0% or 30% dilution with 0.9% NaCl, Hextend, or Voluven, in the absence or presence of aprotinin (200 KIU/mL final concentration). Prekallikrein-deficient (<1% activity) plasma was similarly diluted. After recalcification and celite activation, thrombelastography was performed for determination of R. R in samples without aprotinin diluted with Hextend (mean +/- SD, 132 +/- 6 seconds) was significantly smaller than that in samples with 0% dilution (155 +/- 5 seconds) and 30% dilution with 0.9% NaCl (162 +/- 9 seconds), but was not less than that in Voluven-diluted samples (149 +/- 14 seconds). R significantly increased (28%-68%) in all conditions with aprotinin compared to samples without aprotinin, and Hextend had significantly smaller R compared with that in the other fluids. Lastly, R was not different in experiments with prekallikrein-deficient plasma. These data indicate that Hextend accelerates kallikrein-dependent clot initiation compared with 0.9% NaCl or Voluven.

Effects of aprotinin on plasma coagulation kinetics determined by thrombelastography: role of Factor XI

Aprotinin is commonly administered in settings involving cardiopulmonary bypass and liver transplantation to decrease peri-operative bleeding. Thrombelastography has been utilized to monitor coagulation in these settings, and aprotinin delays clot initiation, presumably by inhibiting kallikrein; however, aprotinin also inhibits Factor XI (FXI), a contact system protein. Thus, it was hypothesized that celite-activated thrombelastography coagulation kin-etics would be decreased via aprotinin-mediated FXI inhibition. Citrated normal plasma and prekallikrein-deficient (<1% normal activity) plasma were exposed to 0, 200, 400 or 800 kallikrein inhibitory units (KIU)/ml (n = 6 per condition). Samples were recalcified and celite-activated in a thrombelastograph, with clot initiation (R, s) determined. To confirm contact system specificity, additional prekallikrein-deficient samples with 0 or 800 KIU/ml aprotinin were activated with tissue factor (n = 4 per condition). Exposure of celite-activated, normal plasma to aprotinin 0, 200, 400 or 800 KIU/ml resulted in R values of 167 +/- 14, 253 +/- 10, 293 +/- 22 and 349 +/- 21 s, respectively, which were significantly different from one another (P < 0.05). Exposure of celite-activated, prekallikrein-deficient plasma to aprotinin 0, 200, 400 or 800 KIU/ml resulted in R values of 366 +/- 15, 630 +/- 64, 698 +/- 46 and 850 +/- 47 s, respectively, which were significantly different from one another (P < 0.05). There were no significant differences in R values between tissue factor-activated, prekallikrein-deficient plasma samples with 0 or 800 KIU/ml aprotinin. These data support a role for the inhibition of FXI as the mechanism for aprotinin-mediated delayed contact system clot initiation determined by thrombelastography.

Evaluation of Prothrombin Time with Use of Highly Diluted Tissue Factor Reagent

Prothrombin time (PT) is an established screening method for hemorrhagic disorders. Recent progress of the biochemistry of tissue factor (TF)-dependent coagulation pathway revealed that TF/factor VIIa complex activated factor IX rather than factor X. However, PT does not reflect the activity of factors IX and VIII, because of excess amount of TF reagent in the assay system. In this study, we evaluated PT using highly diluted TF reagent (Dil-PT) and its clinical application. Coexistence of magnesium with calcium ion resulted in the shortening of Dil-PT. Dil-PT prolonged in accordance with the decrease of TF reagent, and prolongation was observed in factor VIII-and factor IX-deficient plasmas similarly to the factor X-, factor V-, factor VII-and factor II-deficient plasmas. On the contrary, prolonged clotting time of factor XI-, factor XII-, high molecular weight kininogen-and plasma prekallikrein-deficient plasmas were the same as that of normal pooled plasma in the Dil-PT system. In the clinical samples, significant shortenings of Dil-PT and PT were observed in the patients with rhabdomyolysis. On the other hand, Dil-PT showed significant shortning in gestational toxicosis, but PT did not. These results suggest that Dil-PT reflect the activity of factors IX and VIII besides factors VII, X, V and II, and Dil-PT is an useful screening method that does not require specific reagents and apparatus for the detection of hypercoagulable state.

High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: role for antithrombin in the regulation of kallikrein.

The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with heparin on the regulation of kallikrein by the heparin-activated inhibitor, antithrombin, were investigated. H-kininogen, at levels sufficient to fully complex kallikrein, greatly potentiated the acceleration of antithrombin inhibition of kallikrein produced by heparin with high affinity for antithrombin. At I = 0.15, pH 7.4, 25 degrees C, kininogen thus maximally increased the heparin enhancement of the second-order rate constant for the antithrombin-kallikrein reaction from 13-fold (1.6 x 10(2) M-1 s-1 to 2.1 x 10(3) M-1 s-1) to 1200-fold (1.9 x 10(5) M-1 s-1). In contrast, H-kininogen had no effect on the antithrombin-kallikrein reaction in the absence of heparin, nor did the protein enhance the rate constants of 1.7 x 10(4) and 3.4 x 10(4) M-1 s-1 for kallikrein reactions with its primary plasma inhibitors C1-inhibitor and alpha 2-macroglobulin, respectively, in the absence or presence of heparin. Consistent with these results, SDS gel electrophoresis of the 125I-labeled kallikrein-inhibitor complexes formed in a mixture of these kallikrein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha 2-macroglobulin for kallikrein, accounting for 54% of the total kallikrein complexes, only when both heparin and H-kininogen were present. Similarly, the presence of therapeutic levels of heparin (approximately 1 unit/mL) in normal, factor XII-deficient, and prekallikrein-deficient plasmas enhanced the rate of inactivation of added kallikrein by 2.3-fold and significantly altered the partitioning of radiolabeled kallikrein from predominantly C1-inhibitor and alpha 2-macroglobulin complexes (86-92%) to mostly antithrombin complexes (50-53%). Experiments in antithrombin-deficient and H-kininogen-deficient plasmas confirmed that the enhanced kallikrein inactivation rate and predominant formation of antithrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kallikrein inhibition in plasma remained significant (approximately 40-70%) at optimal concentrations of unfractionated or size- and antithrombin affinity-fractionated heparin, in the presence of plasma levels of calcium and zinc ions, at 37 degrees C, and with minimal plasma dilution. These results suggest that antithrombin and H-kininogen may play important roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.

Activation of the Kallikrein-Kinin System in Human Plasma by a Serine Protease from Mites.

A serine protease was earlier purified to homogeneity from a culture extract of mites and its possible involvement in the allergic reaction was proposed [K. Takahashi et al. (1990): Int. Arch. Allergy Appl. Immun., 91, 80-85]. The serine protease thus obtained was found in the present study to be trypsin-like protease and had high substrate specificity towards the synthetic substrate for blood coagulation factor XIIa. The enzyme gave rise to rhythmic contraction of rat uterine horns in factor XII-deficient human plasma, but not in prekallikrein-deficient plasma. These findings lead us to conclude that this enzyme activates the kallikrein-kinin system in plasma through hydrolysis of prekallikrein in the plasma.

Interaction of Plasma Kallikrein with Protein C Inhibitor in Purified Mixtures and in Plasma

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies.

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.

Interaction of plasma kallikrein with protein C inhibitor in purified mixtures and in plasma

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Purified Human Plasma Kallikrein Does Not Stimulate but Primes Neutrophils for Superoxide Production

In patients with septicemia and septic shock the contact phase of blood coagulation is activated. It has been suggested that polymorphonuclear leukocytes (PMN) are directly activated by purified plasma kallikrein. This has been recently questioned because granulocytic elastase release induced by recalcification of normal and prekallikrein-deficient plasma was similar. We studied the interaction of different preparations of purified human plasma kallikrein with PMN. Cytosolic calcium shifts were measured with the quin2 method, PMN aggregation was assayed in an aggregometer, and superoxide production was quantitated as superoxide dismutase inhibitable cytochrome c reduction in a continuous assay. No increase of cytosolic free calcium was found during at least 5 min after adding 10 micrograms/ml plasma kallikrein to PMN. Similarly, highly purified plasma kallikrein from two different sources did not induce PMN aggregation at all, nor did it stimulate superoxide production. However, sequential exposure of PMN to plasma kallikrein and formylpeptide increased the superoxide production compared to stimulation with formylpeptide alone. This phenomenon which is called priming was observed at plasma kallikrein concentrations greater than or equal to 7 micrograms/ml. The active site of the molecule was required for the priming, because plasma prekallikrein, active site-inactivated plasma kallikrein, and soybean trypsin inhibitor treated kallikrein did not prime PMN. This indicates that the contact activation system may play a role in host defence against bacterial infection.

Rat paw edema induced by trimucase II, a kinin-releasing enzyme from Trimeresurus mucrosquamatus venom

Trimucase II, a proteolytic enzyme isolated from Trimeresurus mucrosquamatus venom, caused rat hind-paw edema dose dependently. Captopril potentiated significantly, while pretreatment with cellulose sulfate suppressed the trimucase II-induced edematous response. Pretreatment with diphenhydramine and methysergide reduced by 42 and 46% the edema induced by trimucase II and kallikrein, respectively. The residual response was significantly further depressed by [Thi 5,8, D-Phe 7]bradykinin and trasylol. Kinin generation by trimucase II was also found in vitro from plasma and was concentration- and time-dependent. Kinin formation was inhibited by soybean trypsin inhibitor, trasylol and endogenous kininase. The kinins released were destroyed by chymotrypsin. Unlike cellulose sulfate, trimucase II caused kinin formation from Factor XII-deficient and prekallikrein-deficient plasma but not from high molecular weight kininogen-deficient plasma. These data indicate that, in addition to the mediators released from mast cells, kinins have an important role in the edema caused by trimucase II, and that kinins are released from plasma, probably due to direct activation of kininogen.

Physicochemical and biochemical aspects of fibrinogen adsorption from plasma and binary protein solutions onto polyethylene and glass

The adsorption of fibrinogen to polyethylene and glass from plasma and binary protein solutions was measured using baboon fibrinogen radiolabled with 125I. The effects of plasma dilution, shear rate, contact time, and plasma fibrinogen concentration were examined. Fibrinogen adsorption to polyethyelen passed through a maximum at times dependent upon the plasma dilution, shifting from 1 h to 10 s as the plasma concentration varied from 0.01 to 100%. The initial fibrinogen adsorption rate from diluted plasma (0.01–1%) to polyethylene was found to be mass transfer limited and a fibrinogen diffusivity of 4.4 ± 0.9 × 10 −7 cm 2s −2 was calculated. Fibrinogen adsorption was also measured under static conditions after 2 h at 37°C as a function of plasma dilution. Adsorption to glass and polyethylene was maximal at intermediate plasma concentrations. Fibrinogen adsorption to glass and polyethylene from both normal and kininogen-deficient plasma was similar but that from prekallikrein-deficient plasma was somewhat enhanced. Fibrinogen adsorption to glass from solutions containing either albumin or hemoglobin was maximal at intermediate mixture concentrations. The maximum shifted to smaller concentrations as the ratio of competing protein to fibrinogen increased. This effect is similar to that caused by plasma dilution and suggests that the observed fibrinogen displacement phenomenon occurring with plasma results from numerous proteins expressing their stronger affinity for a finite number of adsorption sites.

Neutrophil Secretion During Blood Coagulation: Evidence for a Prekallikrein Independent Pathway

In association with blood coagulation, neutrophils undergo a secretory response (Plow, J Clin Invest 69: 564, 1982) and it has been suggested that plasma kallikrein is responsible for inducing this reaction (Wachtfogel et al., J Clin Invest 72: 1672, 1983). To assess the contribution of kallikrein to this response, the capacity of normal and prekallikrein-deficient blood and plasma to support secretion has been compared utilizing elastase as a marker of secretion. Serial dilutions of prekallikrein-deficient plasma were as effective as normal plasma in supporting neutrophil release of elastase. The extent of elastase release in spontaneously clotting normal and prekallikrein-deficient blood was similar. At 37 degrees C in whole blood or at 22 degrees C in plasma, prekallikrein activators had the same effect in neutrophil secretion in normal and prekallikrein-deficient blood and plasma samples. Taken together, these results provide evidence for the existence of a prekallikrein independent pathway that can function as a predominant mechanism for induction of neutrophil secretion during blood coagulation.

A Monoclonal Anti-Human Plasma Prekallikrein Antibody That Inhibits Activation of Prekallikrein by Factor XIIa on a Surface

AbstractOf five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was ~80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein–HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited ~70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein- HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

Structural and Functional Characterization of Factor XII

In this article we have reviewed the current knowledge regarding the involvement of Factor XII in contact activation. Clearly in the past decade an overwhelming amount of data and hypotheses have been published regarding the central role of this zymogen in the initiation and further propagation of contact activation reactions. Therefore we feel that it will be helpful to conclude this article with a figure that summarizes those interactions and reactions that are generally believed to reflect the major molecular events occurring during surface-dependent contact activation. The contact factors are capable of very efficient interation with each other, provided a suitable negatively charged surface is present. Such surfaces are thought to stimulate the interactions between the contact factors through binding of the proteins and thus bringing the proteins together. Factor XII readily binds to the negatively charged surface, but for the binding of prekallikrein and Factor XI, the cofactor HMW kininogen is likely to be necessary. Bound at the surface, the zymogens Factor XII and prekallikrein are thought to be involved in a so-called reciprocal activation mechanism in which Factor XIIa activates prekallikrein to kallikrein, which in turn converts Factor XII to Factor XIIa. The formation of Factor XIIa is further promoted by the fact that surface-bound Factor XII is likely more susceptible to proteolytic cleavage and by the fact that the activated Factor XIIa is capable of auto-activating its own zymogen Factor XII. However, the latter effect, although undoubtedly contributing to the formation of Factor XIIa at the surface, seems to be of less importance than the reciprocal activation mechanism. This is underscored by the fact that Factor XII activation is rather slow in prekallikrein-deficient plasma. Surface-bound Factor XIIa is then responsible for the activation of Factor XI to Factor XIa, thereby propagating the initial trigger. Presumably, Factor XIa must leave the surface in order to be able to become involved in the activation of blood coagulation Factor IX.

Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme- linked differential antibody immunosorbent assay

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF- deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF- deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time- dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.

Assessment of Hageman Factor Activation in Human Plasma: Quantification of Activated Hageman Factor-C1̄ Inactivator Complexes by an Enzyme-Linked Differential Antibody Immunosorbent Assay

AbstractAn enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1̄ inactivator (HF-C1̄ INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1̄ INH complexes. As little as 0.5 μg/mL of activated HF added to plasma can be detected, corresponding to activation of ~2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF-deficient plasma, indicating competition between unactivated HF and activated HF-C1̄ INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields <1% of the activated HF-C1̄ INH complex formation obtained with normal plasma. Kaolin activation of HF-deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time-dependent increase in formation of activated HF-C1̄ INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1̄ INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1̄ INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.

Effect of Heparin on the Inactivation Rate of Human Activated Factor XII by Antithrombin III

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.

Effect of heparin on the inactivation rate of human activated factor XII by antithrombin III

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.