Cheryl F. Scott

Prekallikrein Activation and High-Molecular-Weight Kininogen Consumption in Hereditary Angioedema

Patients with hereditary angioedema lack C-1 inhibitor, a plasma alpha 2-glycoprotein that inhibits both the proteolytic action of C1, the activated first component of the complement system, and the activity of components of the contact phase of coagulation: kallikrein, factor XIa, and factor XIIa. Such patients have been shown to have low levels of C4 and C2, the natural substrates for C-1, but the levels were not correlated with the presence of symptoms. We studied three patients with angioedema for evidence of activation of the contact system and found that during a symptomatic period they had decreased levels of prekallikrein, a substrate for the activated forms of factor XII, and reductions in high-molecular-weight kininogen, a substrate for plasma kallikrein. These observations suggest that zymogens of the contact system are activated during attacks of hereditary angioedema and that some of the clinical manifestations may be mediated through products of this pathway, such as kinins.

Prognostic value of assessing contact system activation and factor V in systemic inflammatory response syndrome

OBJECTIVE To test if serially sampled determinations of the contact system proteins and factor V have prognostic value for death in patients who develop the systemic inflammatory response syndrome. DESIGN Prospective, observational study with sequential measurements in an inception cohort. SETTING Medical intensive care unit (ICU) in a community hospital. PATIENTS Over a 1-yr period, a population base sample of 23 patients was selected from all ICU admissions who met established criteria for the systemic inflammatory response syndrome. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Components of the contact system, factor XII, prekallikrein, high-molecular-weight kininogen, factor XI, alpha 2-macroglobulin-kallikrein complexes and factor V values were measured in plasma samples collected serially (day of admission, and at 2, 12, 24, 48 and/or 72 hrs or at discharge). Data were analyzed to determine if admission values or serially obtained values within 48 hrs were useful in predicting outcome. Fourteen patients survived and nine died. At admission, in all patients, assay values indicated that prekallikrein, high-molecular-weight kininogen, and factor V were significantly lower than normal (as observed in a range of 20 to 23 healthy adults), alpha 2-macroglobulin-kallikrein complexes were higher than normal, while concentrations of factor XII and factor XI were in the normal range. No differences were detected in the admission values between survivors and nonsurvivors, nor between patients with positive or negative blood cultures. However, subsequent values demonstrated a difference in values between survivors and nonsurvivors. Survivors showed improvement in high molecular weight kininogen values and higher than normal factor V values, as compared with nonsurvivors. CONCLUSIONS Low or persistently low serial factor XII, high-molecular-weight kininogen and factor V values are associated with a poor prognosis, whereas high or increasing values of factor XII, high-molecular-weight kininogen, prekallikrein, and factor V all correlate with a favorable outcome.

Effect of Heparin on the Inhibition of the Contact System Enzymes

1. One can accurately predict the contribution of each inhibitor to the total inactivation of an enzyme in plasma once its pseudo-first-order reaction rate constant and concentration are known. 2. Because the mechanism of augmentation of the inactivation rate of an enzyme by ATIII occurs via formation of an ATIII-heparin complex, the degree of potentiation can be predicted by knowing the binding capacity (sites per mole) of the heparin preparation and the concentration of heparin in the reaction (to calculate the concentration of the ATIII-heparin complex). 3. The augmentation by heparin of the inactivation rate of a particular enzyme by ATIII is dependent upon the presence of other enzymes with higher kassoc, since these would strongly compete for the ATIII-heparin complex. 4. In a plasma environment, using therapeutic levels of heparin, there is no augmentation of the inactivation rate of any of the contact enzymes.

REGULATION OF THE FORMATION AND INHIBITION OF HUMAN PLASMA KALLIKREIN

During the past two decades, remarkable strides have been made in defining the mechanisms by which the intrinsic coagulation, fibrinolytic, and kininforming pathways are activated. Three proteins, factor XI1 (Hageman factor), prekallikrein (Fletcher factor), and high molecular weight (HMW) kininogen (Williams, Fitzgerald, Flaujeac factor), have been shown to be the major factors required for the activation of factor XI and prekallikrein. Hereditary deficiencies have been described for each of these contact factors. Methods have been developed for the purification of each of these proteins, and functional and immunochemical assays have been developed for each component. The molecular events occurring during contact activation have been described but are not yet fully understood. Activation has, in each instance, been shown to involve enzymatic digestion; however, the detailed structural features that permit the binding of these reactants to the surface require further study. How the interaction of the enzymes, factor XIIa, factor XIa, and kallikrein with the cofactor-substrate HMW kininogen results in accelerated reaction rates is still unclear. Each of these three proteins (factor XII, prekallikrein, and HMW kininogen) bears a close functional, and in some cases structural, relationship to the other two. First, factor XI1 and prekallikrein are each involved in a reciprocal scheme for the activation of the other. Factor XI1 may be regarded as an essential component of the plasma kinin-forming system, as well as of the intrinsic pathway of coagulation. Thus, addition of kaolin to factor XIIdeficient plasma is not followed by the release of either kallikrein or bradykinin,l. even though this plasma contains adequate quantities of both prec u r s o r ~ . ~ The formation of kallikrein is initiated by activation of factor XI1 ( M , 80,000) (FIG. 1). Two mechanisms have been distinguished. Factor XI1 may be activated on a surface due to conformational changes and/or cleavage by kallikrein in the presence of HMW kininogen.6 Initially, in the presence of a surface, a cleavage of factor XI1 occurs without fragmentation, since the two chains (52,000 and 28,000) are held together by disulfide bridges. Factor XIIa bound to a surface can then activate either prekallikrein or factor XI bound to the same surface through HMW kininogen. In contrast, cleavage can occur outside the disulfide bridges either after exposure of factor XI1 to surfaces or during purification. Apparently, the 52,000 chain remains

A new assay for high molecular weight kininogen in human plasma using a chromogenic substrate

High molecular weight kininogen (HK), the cofactor of contact-activated plasma proteolysis, is currently assayed by coagulant or immunological methods. The former is limited by the need for rare, congenitally-deficient plasma and a high coefficient of variation (CV), and the latter, by failure to distinguish nonfunctional protein. The surface activation of factor XI requires HK as its cofactor to transport its zymogen form to a negatively-charged surface where it is converted to its enzymatic form by factor XIIa. Based on this principle, we developed an assay for HK using the chromogenic substrate pyroGlu-Pro-Arg-p-nitroanilide (S-2366, KabiVitrum), which is hydrolyzed by factor XIa. Plasma is first acidified to inactivate protease inhibitors. After neutralization and dilution, the plasma is incubated with an excess of factor XI, factor XIIa, and soybean trypsin inhibitor (to inactivate generated kallikrein), in the presence of a negatively-charged surface (kaolin) in order to form factor XIa. EDTA is included in the buffer to prevent calcium-dependent reactions. This activation process is stopped by adding corn trypsin inhibitor to inactivate the enzyme in this reaction, factor XIIa. Then, S-2366 is added and is hydrolyzed by the factor XIa that was formed. Since factor XI and factor XIIa are in excess of the concentration of HK in the diluted plasma, HK is the rate-limiting protein in this assay for the formation of factor XIa (after subtracting the small amount of factor XIa generated in the absence of HK). The assay is specific for HK, since no activity is detected in kininogen-deficient plasma, and when compared with the HK coagulant assay, r = 0.95 and slope = 0.95. The mean of 21 normal donors was 0.98 U/ml (range 0.68 - 1.28 U/ml) as compared with pooled, normal plasma. The CV for 1 U/ml HK for the chromogenic assay was 2% as compared with 9.5% for the coagulant assay. When purified reagents become commercially available, this assay could prove useful in clinical laboratories or intensive care units for monitoring the progression of various disease states in which contact activation occurs.

Human factor XIa cleaves fibrinogen: Effects on structure and function☆

Factor XIa, the enzymatic form of the factor XI zymogen, is generated as a result of factor XII-dependent surface activation in plasma. Factor XIa degrades high molecular weight kininogen, its cofactor for activation (which binds factor XIa to the surface), as well as cleaves and activates coagulation factor IX. In this report, we present evidence that factor XIa can also cleave fibrinogen and decrease the thrombin-catalyzed formation of the fibrin clot. Furthermore, the products of factor XIa-digested fibrinogen markedly inhibited the rate of polymerization of fibrin monomers. Factor XIa initially cleaved the A alpha-chain of fibrinogen and subsequently degraded the B beta-chain. However, the cleavage sites on both chains were distinct from those susceptible to thrombin. The gamma-chain was degraded only after prolonged incubation with factor XIa. Furthermore, the profile of fibrinogen proteolysis by factor XIa was distinctly different from that of plasmin-catalyzed fibrinogenolysis. Unlike plasmin, factor XIa was not able to cleave the NH2-terminus of the B beta-chain of fibrinogen. Moreover, factor XIa, unlike plasmin, failed to hydrolyze fibrin. Further study of the proteolytic digests of fibrinogen produced by factor XIa may give additional insight into the mechanism of polymerization of this protein.