Determinants of kallikrein proteolysis of apolipoprotein B-100 in human blood plasma

Abstract. The formation of apolipoprotein B-74, a fragment of apolipoprotein B-100, in blood plasma in vitro is shown to occur only at temperatures below 15°C, and is promoted by exposure to glass and other surfaces known to activate factor XII. Removal of C-I esterase inhibitor from plasma permits formation of apolipoprotein B-74 at room temperature. These observations are consistent with conversion of prekallikrein to kallikrein by factor XII, and with evidence that kallikrein proteolysis in vitro is activated in the cold by alteration of its binding to C-I esterase inhibitor. Since the two proteolytic fragments of apolipoprotein B-100 produced by kallikrein digestion are identical to apolipoproteins B-74 and B-26, it is concluded that apolipoproteins B-74 and B-26 are in-vitro products produced in low density lipoproteins of normal plasma in the cold by kallikrein.     

Prorenin-renin conversion by the contact activation system in human plasma: role of plasma protease inhibitors

Acid-pretreated normal human plasma generates renin activity at 0 degree C and neutral pH by the activation of prorenin. The activation is caused by kallikrein generated from prekallikrein by activated factor XII. Nonacidified plasma also generates renin at 0 degree C, but at a lower rate (cold-promoted activation). In normal plasma, 14% +/- 1% of prorenin (mean +/- SEM, n = 30) was activated during incubation at 0 degree C for 7 days (range 6% to 26%). Cold-promoted activation of prorenin was within the normal range in plasma deficient in factor XI, X, IX, VIIIC, VII, V, prothrombin, or high mol wt kininogen. Cold-promoted activation of prorenin was less than or equal to 1% in plasma deficient in factor XII or prekallikrein. Reconstitution of these plasmas with highly purified factor XII or prekallikrein restored normal prorenin activation. Correction of high mol wt kininogen deficiency had no effect. Thus cold-promoted activation of prorenin depends on the presence of factor XII and prekallikrein, whereas the other clotting factors are not essential. The influence of the inhibitors C1 esterase-inhibitor, alpha 2-macroglobulin, antithrombin III, and alpha 1-antitrypsin on the activation of prorenin was studied in factor XII-deficient plasma from which one or more of these inhibitors had been selectively removed by immunoadsorption. Factor XII was subsequently added, and the generation of renin at 37 degrees C was observed after complete factor XII-high mol wt kininogen-mediated activation of prekallikrein induced by dextran sulfate. No activation of prorenin was observed at 37 degrees C after depletion of C1 esterase inhibitor, alpha 2-macroglobulin, antithrombin III, or alpha 1-antitrypsin. When prekallikrein was activated in plasma depleted of both C1 esterase-inhibitor and alpha 2-macroglobulin, 6% of prorenin was activated in 2 hours at 37 degrees C. After additional depletion of antithrombin III, the activation increased to 47%. These results indicate that the contact activation system is capable of activating prorenin in plasma at physiologic pH and temperature when the three most important kallikrein inhibitors, C1 esterase-inhibitor, alpha 2-macroglobulin, and antithrombin III, are absent.     

Contact activation of human plasma prorenin in vitro

Acid activation of plasma prorenin occurs during dialysis to pH 3.3. and also during subsequent dialysis to pH 7.4. The latter, alkaline phase, involves Hageman factor-dependent formation of kallikrein, which in turn activates prorenin. The present study evaluates whether prorenin activation always occurs whenever kallikrein is activated in plasma. TAME esterase activity was used as a measure of plasma kallikrein activity an increase was observed during the alkaline phase of acid activation of prorenin. TAME esterase activity was absent when Hageman factor- or prekallikrein-deficient plasmas were similarly assayed and prorenin was not activated. Kaolin treatment of normal plasma rapidly increased TAME esterase activity at both 25 degrees and -4 degrees C, but no prorenin activation occurred. Similar changes in TAME esterase activity were observed in acid-treated plasma, in which setting prorenin was activated. No change in TAME esterase or renin activity occurred after addition of kaolin to acid-treated plasma deficient in Hageman factor; however, both enzymatic activities increased slightly in acidified prekallikrein-deficient plasma. Mixtures of these deficient plasmas exhibited normal kaolin activation of both TAME esterase and prorenin after acidification. Thus both Hageman factor and prekallikrein are needed for optimal contact activation of prorenin. These results demonstrate that prorenin activation does not always occur when active kallikrein is present in plasma. Prior acidification appears to be a prerequisite. Acidified prorenin may be more susceptible to cleavage; alternatively, competing substrates and/or inhibitors of kallikrein may be destroyed at acid pH, thereby permitting kallikrein to activate prorenin. Under normal conditions, activation of the plasma kallikrein-kinin system appears unlikely to result in activation of prorenin in vivo.     

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.     

HUMAN PLASMA ALPHA 2-MACROGLOBULIN : AN INHIBITOR OF PLASMA KALLIKREIN

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified α₂-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the α₂-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the α₂-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, α₂-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the α₂-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The α₂-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the α₂-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The α₂-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the α₂-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.     

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.     

Separation of Plasma Thromboplastin Antecedent from Kallikrein by the Plasma α2-Macroglobulin, Kallikrein Inhibitor

Plasma thromboplastin antecedent (PTA, factor XI) is an important intermediate in the intrinsic coagulation system, and plasma kallikrein has been implicated as a mediator of the inflammatory process. Whereas their biologic activities are functionally distinct, their identity as separate entities in plasma has not been fully established, and the nature of their plasma inhibitors has not been completely characterized. A partially purified preparation containing the clotting, tosyl arginine methyl ester (TAMe) esterase and kinin-producing activities of these substances has been prepared by DEAE-cellulose chromatography of a Celite eluate obtained from acid-treated human plasma. These activities were not separable by acrylamide gel electrophoresis nor by isoelectric focusing, their pI being approximately 8.7. Human plasma alpha(2)-macroglobulin has been shown to inhibit the proteolytic activity of kallikrein and to inhibit partially its TAMe esterase activity. An alpha(2)-macroglobulin, PTA, kallikrein incubation mixture was separated by gel filtration chromatography. The alpha(2)-macroglobulin formed a high molecular weight complex with kallikrein and appeared in early chromatographic fractions. The PTA-clotting activity was not inhibited by the alpha(2)-macroglobulin; 64% of the initial PTA activity was isolated in later fractions free of kallikrein-induced kinin-like activity. In contrast, clotting, TAMe esterase, and kinin-forming activities were inhibited after gel filtration chromatography of an incubation mixture of these activities and partially purified C1 inactivator (C1 esterase inhibitor). Electrofocusing of an incubation mixture of an activated PTA, kallikrein preparation, and alpha(2)-macroglobulin resulted in the isolation of a PTA fraction free of kallikrein proteolytic activity, and with 4% of the original TAMe esterase activity. In this manner, activated PTA and plasma kallikrein have been shown to be distinct substances, and methods have been introduced for the further purification of active coagulation factor XI.     

Recombinant alpha 1-antitrypsin Pittsburgh (Met 358----Arg) is a potent inhibitor of plasma kallikrein and activated factor XII fragment.

In normal plasma, the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) plays little or no role in the control of plasma kallikrein or activated Factor XII fragment (Factor XIIf), this function being performed by Cl-inhibitor. Recently, an alpha 1-AT variant was described with a Met----Arg mutation at the reactive center P1 residue (position 358) which altered the specificity of inhibition from the Met- or Val-specific protease neutrophil elastase to thrombin, an Arg-specific protease. We have now examined the inhibition of plasma kallikrein and Factor XIIf, both Arg-specific enzymes, with recombinant alpha 1-AT(Met358----Arg) produced by an Escherichia coli strain carrying a mutated human alpha 1-AT gene. The engineered protein was a very efficient inhibitor of both enzymes. It was more effective than Cl-inhibitor by a factor of 4.1 for kallikrein and 11.5 for Factor XIIf. These results suggest that recombinant alpha 1-AT(Met358----Arg) has therapeutic potential for disease states where activation of the plasma kinin-forming system is observed, for example in hereditary angioedema or septic shock.     

The link between high-fat meals and postprandial activation of blood coagulation factor VII possibly involves kallikrein

Contrary to low-fat meals, high-fat meals are known to cause postprandial factor VII (FVII) activation, but the mechanism is unknown. To study the postprandial FVII activation in detail, 18 young men consumed in randomized order high-fat or low-fat test meals. Fasting and non-fasting blood samples were collected. The high-fat test was associated with an increase in plasma triglyceride and kallikrein concentrations and postprandial FVII activation (p<0.001). Plasma kallikrein was strongly associated with triglycerides in fasting and non-fasting samples (r2=0.74-0.87, p<0.0001), suggesting that triglyceride-rich lipoproteins may activate prokallikrein. Neither plasma triglycerides nor kallikrein and activated FVII were statistically associated. This may suggest that additional factors are involved in the postprandial FVII activation. No clear evidence for a role of tissue factor expression by monocytes, factor XII or insulin in postprandial FVII activation was observed. Tissue factor pathway inhibitor and prothrombin fragment 1+2, a marker of thrombin generation, were not affected postprandially after either the high-fat or the low-fat meals. Our findings indicate that triglyceride-rich lipoproteins activate prokallikrein postprandially, which might form an important initial event in FVII activation after consumption of high-fat meals.     

Dissemination of contact activation in plasma by plasma kallikrein

The dissemination of contact activation of plasma was examined by measuring the cleavage of Hageman factor (HF) molecules on two separate sets of kaolin particles, one of which contained all of the components of the contact activation system, HF, prekallikrein (PK) and high molecular weight kininogen (HMWK) in whole normal plasma, and the second set of particles containing only HF and HMWK, being prepared with PK-deficient plasma. After mixing of the particles, cleavage of HF on the second set of particles occurred at a rate similar to that occurring on the first set of particles. This indicated that rapid dissemination and burst of activity of the contact reaction takes place in fluid phase. A supernatant factor, responsibel for the dissemination of the contact reaction, was identified as kallikrein. A rapid appearance of cleaved PK (kallikrein) and HMWK on both the kaolin surface and in the supernate was observed. Within 40 s, > 70-80% of the PK and HMWK in the supernate was cleaved. On the surface, approximately 70% of each radiolabeled protein was cleaved at the earliest measurement. Cleavage of PK by activated HF occurred at least 17 times faster on the surface than in the fluid phase, as virtually no cleavage of PK occurred in fluid phase. Each molecule of surface-bound, activated HF was calculated to cleave at a minimum, 20 molecules of PK per minute. It is concluded that the contact activaton of plasma may be divided into three phases: (a) the reciprocal activation of a few molecules of zymogen HF and PK on the surface, with HMWK acting as cofactor to bring these molecules into apposition; (b) the rapid release of kallikrein into the fluid phase and the continued conversion of PK to kallikrein by each surface-bound molecule of activated HF; and (c) the activation by fluid-phase kallikrein of multiple surface-bound HF molecules, and the cleavage of multiple molecules of MHWK both in fluid phase and on the surface by the soluble kallikrein. The evidence suggests that steps b and c account for a great majority of the generation of contact activation of plasma.     

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.     

Inactivation of kallikrein in human plasma

Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.     

Purified human plasma kallikrein aggregates human blood neutrophils.

Exposure of human blood polymorphonuclear leukocytes (PMN) to purified active plasma kallikrein resulted in PMN aggregation when kallikrein was present at concentrations ranging from 0.4 to 0.6 U/ml (0.18-0.27 microM). Kallikrein-induced PMN aggregation was not mediated through C5-derived peptides, because identical responses were observed whether or not kallikrein had been preincubated with an antibody to C5. Moreover, kallikrein was specific for aggregating PMN, because no aggregation was observed with Factor XII active fragments (23 nM), Factor XIa (0.6 U/ml or 15nM), thrombin (1.6 microM), plasmin (2 microM), porcine pancreatic elastase (2 microM), bovine pancreatic chymotrypsin (2 microM), or bradykinin (1 microM). Bovine pancreatic trypsin (2 microM) aggregated PMN, but to a lesser extent than kallikrein (0.18 microM). Kallikrein was a potent aggregant agent for PMN because similar responses were observed with kallikrein (0.5 U/ml or 0.23 microM) and an optimal dose (0.2 microM) of N-formyl-methionyl-leucyl-phenylalanine. In addition, PMN incubation with kallikrein resulted in stimulation of their oxidative metabolism as assessed by an increased oxygen uptake. Neutropenia and leukostasis observed in diseases associated with activation of the contact phase system may be the result of PMN aggregation by plasma kallikrein.     

Specificity of the amidase and kininogenase methods for the determination of rat urinary kallikrein

The specificity of the amidase and kininogenase methods for determining rat urinary kallikrein was studied. Male and female rat urine was employed. Esterase A1, A2 and kallikrein were separated by DEAE-Sephadex A-50 chromatography. Esterase A1 showed no amidase activity towards the substrate H-D-Val-Leu-Arg-p-nitroanilide. In contrast, esterase A2 and kallikrein attacked the substrate, and the activity of kallikrein was especially inhibited by aprotinin, while esterase A2 was more sensitive to soybean trypsin inhibitor. Esterase A1 did not show kininogenase activity, whereas esterase A2 showed this activity, but only towards the dog plasma substrate. Kallikrein possessed kininogenase activity towards both dog and rat plasma kininogen. We believe that the most specific method for measuring rat urinary kallikrein activity is the kininogenase method using partially purified rat plasma kininogen.     

Activation of inactive plasma renin by plasma and tissue kallikreins.

1. Normal human plasma contains a proactivator of inactive renin. The pro-activator is activated at physiological pH in plasma that has been pretreated with acid. This activation in vitro leads to the conversion of inactive renin into the active form with simultaneous generation of kallikrein activity. 2. The endogenous activator of inactive renin has the same pH profile and inhibitor spectrum as plasma kallikrein. 3. Inactive renin can also be activated by exposure of plasma to exogenous trypsin, and in normal plasma the quantities of inactive renin that are activated after acidification and with trypsin are identical. Prekallikrein (Fletcher factor)-deficient plasma, however, has much lower renin activity after acidification than with trypsin. Thus acid activation of inactive renin depends on plasma prekallikrein, whereas the action of trypsin is independent of prekallikrein. 4. Highly purified tissue (pancreatic) kallikrein, in a concentration of less than 2 X 10(-8) mol/l, activates inactive renin that has been isolated from plasma by ion-exchange chromatography. In this respect it is at least 100 times more potent than trypsin. 5. It is therefore possible that plasma and/or tissue (renal) kallikreins are also involved in the activation of inactive renin in vivo.     

Activation of Human Factor VII in Plasma and in Purified Systems: ROLES OF ACTIVATED FACTOR IX, KALLIKREIN, AND ACTIVATED FACTOR XII

Factor VII can be activated, to a molecule giving shorter clotting times with tissue factor, by incubating plasma with kaolin or by clotting plasma. The mechanisms of activation differ. With kaolin, activated Factor XII (XII(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-deficient plasma, was partially activated in prekallikrein and high-molecular weight kininogen (HMW kininogen)-deficient plasmas, but was activated in other deficient plasmas. After clotting, activated Factor IX (IX(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-,HMW kininogen-, XI-, and IX-deficient plasmas, but was activated in Factor VIII-, X-, and V-deficient plasmas. In further studies, purified small-fragment Factor XII(a) (beta-XII(a)), kallikrein, and Factor IX(a) were added to partially purified Factor VII and to plasma. High concentrations of beta-XII(a) activated Factor VII in a purified system; much lower concentrations of beta-XII(a) activated Factor VII in normal plasma but not in prekallikrein or HWM kininogen-deficient plasmas. Kallikrein alone failed to activate partially purified Factor VII but did so when purified Factor IX was added. Kallikrein also activated Factor VII in normal, Factor XII-, and Factor IX-deficient plasmas. Purified Factor IX(a) activated partially purified Factor VII and had no additional indirect activating effect in the presence of plasma. These results demonstrate that both Factor XII(a) and Factor IX(a) directly activate human Factor VII, whereas kallikrein, through generation of Factor XII(a) and Factor IX(a), functions as an indirect activator of Factor VII.     

Involvement of the contact phase and intrinsic pathway in herpes simplex virus‐initiated plasma coagulation

Summary. Background: A hemostatic response to vascular injury is initiated by the extrinsic pathway of coagulation and amplified by the intrinsic pathway. We previously reported that purified herpes simplex virus type‐1 (HSV1) has constitutive extrinsic pathway tissue factor (TF) and anionic phospholipid on its surface derived from the host cell, and can consequently bypass strict cellular control of coagulation. Objective: The current work addresses the hypothesis that HSV1‐induced plasma coagulation also involves intrinsic pathway, factor VIII (FVIII), and upstream contact activation pathway, factor XII (FXII). Results: HSV1‐initiated clotting was accelerated when purified FVIII was added to FVIII‐deficient plasma and in normal plasma attenuated by an inhibitory anti‐FVIII antibody (Ab). High HSV1 concentrations predictably reduced the effect of FVIII due to the availability of excess viral TF. To further define TF‐independent clotting mechanisms initiated by HSV1, the extrinsic pathway was disabled using factor VII‐deficient plasma. The intrinsic pathway is triggered by activation of FXII associated with surface‐bound kallikrein, which subsequently activates factor XI. Here we found that an inhibitor of activated FXII, corn trypsin inhibitor, and anti‐FXII, anti‐kallikrein and anti‐FXI Abs inhibited HSV1‐initiated clotting. HSV1‐enhanced activation of purified FXII was confirmed by Western blot, but required prekallikrein. Conclusion: The current work shows that HSV1 can trigger and amplify coagulation through the contact phase and intrinsic pathway, and suggests an additional mechanism that may contribute to vascular pathology.     

Chemotactic activity generated from the fifth component of complement by plasma kallikrein of the rabbit

Rabbit plasma kallikrein incubated with rabbit C5 resulted in the generation of chemotactic and secretagogue activity for rabbit neutrophils. This effect on C5 appeared to be due to kallikrein itself and not to a contaminating enzyme, because it could be inhibited by anti-kallikrein IgG or by soybean trypsin inhibitor to the same extent the kinin generation by the same kallikrein preparation was inhibited by these agents. The chemotactic response was consistent with the generation of a C5a-like peptide from C5 because the effect could be partially inhibited by carboxypeptidase N and was related to the generation of a small (approximately 14,000 mol wt) fragment of C5. No direct chemotactic response was detectable for kallikrein, activated Hageman factor, high-molecular weight kininogen, or intact C5. Incubation of Kallikrein, high-molecular weight kininogen, and Hageman factor together, so that activation of all three proteins occurred, did not results in the generation of detectable chemotactic activity.     

Plasma kallikrein and prorenin in patients with hereditary angioedema

Recent evidence indicates that plasma kallikrein is activated during acute attacks of hereditary angioedema. Plasma kallikrein is known to convert inactive renin, or prorenin, into an active proteolytic enzyme in plasma exposed to acid or low temperatures as well as in purified systems. To establish whether plasma kallikrein could activate prorenin under physiologic or pathologic conditions, prorenin to renin conversion was assessed at neutral pH in plasma deficient in C1 inhibitor (hereditary angioedema). In these plasma samples lacking the two major inhibitors of kallikrein and possessing less than 10% of the inhibitory activity of normal plasma, prorenin was not converted to an active enzyme despite conditions under which prekallikrein was completely activated to plasma kallikrein and despite normal prorenin concentrations and activability.     

Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation

When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.