High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

Prostaglandin E1 inhibitis platelet aggregation by a pathway independent of adenosine 3',5'-monophosphate.

Aggregation of human blood platelets induced by adenosine diphosphate or 1-epinephrine was inhibited when the platelets were suspended in plasma which had been previously exposed to an insolubilized omega-aminohexylagarose derivative of prostaglandin E1. This decrease of platelet aggregation was not accompanied by a change in the concentration of adenosine 3',5'-monophosphate (cyclic AMP) in platelets. The results demonstrate the existence of an alternative pathway independent of cyclic AMP for the inhibition of platelet aggregation by plasma.

Studies on the prekallikrein (kallikreinogen)--kallikrein enzyme system of human plasma. I. Isolation and purification of plasma kallikreins.

By measurement of its arginine esterase activity, plasma kallikrein was purified from fresh frozen ACD plasma. The steps involved alcohol fractionation, isoelectric precipitation, and carboxymethyl (CM) Sephadex and DEAE cellulose chromatography. Three enzymatically active fractions were finally isolated and termed plasma kallikreins I, II, and III; they represented purifications of 970,320- and 590-fold, respectively. All three kallikreins were active biologically; they increased vascular permeability in the guinea pig and released a kinin from human plasma, as measured in the rat uterus bioassay. Bradykinin and/or closely related kinins were identified in the kallikrein I plasma digest by radioimmunoassay. Kallikreins I, II, and III had similar ratios of hydrolytic activity on a variety of arginine and lysine esters and were immunochemically related. However, differences were present on physicochemical characterization: kallikrein I had S(20,[unk]) of 5.7, a mol wt of 99,800, and migrated as a slow gamma globulin; kallikrein II migrated as a fast gamma globulin with a mol wt of 163,000, but the evidence suggested that it was closely related, if not interconvertible, with kallikrein I. Kallikrein III, on the other hand, migrated as an alpha globulin and reacted quite differently with inhibitors.

Studies on the prekallikrein (kallikreinogen)--kallikrein enzyme system of human plasma. II. Evidence relating the kaolin-activated arginine esterase to plasma kallikrein.

Evidence is presented in this paper that the kaolin-activated arginine esterase of plasma is related to plasma kallikrein activity. Such a relationship is based on studies that (1) establish a constant ratio of esterase activity on various synthetic substrates for the kaolin-activated arginine esterase, purified kallikrein(s), and preparations obtained during the fractionation procedure; (2) exclude other known plasma and tissue arginine esterases; (3) confirm the requirement for factor XII in the activation of the enzyme precursor; and (4) show similarities in behavior between the plasma esterase and purified kallikrein(s) toward a variety of inhibitors. Based on this probable identification, evidence is provided that the concentration of active factor XII determines the rate of activation of plasma kallikreinogen, and that the activation may be blocked by polybrene. Once activated, plasma kallikrein is rapidly inactivated by the naturally occurring plasma inhibitor, but the inhibition is incomplete. Acid or chloroform treatment of plasma rapidly inactivates the plasma inhibitor without affecting the concentration of plasma kallikreinogen. Another plasma arginine esterase with properties suggestive of permeability factor is activated by factor XII in the presence of synthetic substrates, but only at low ionic strength. The data suggest that this enzyme is closely related to plasma kallikrein and that it arises from a common precursor.

Function and immunochemistry of prekallikrein-high molecular weight kininogen complex in plasma.

Plasma from individuals with high molecular weight (HMW) kininogen deficiency has been reported to be deficient in prekallikrein as measured by radial immunodiffusion, prekallikrein coagulant activity, and/or kaolin-activated arginine esterase activity. The discovery that prekallikrein and HMW kininogen circulate as a complex in plasma led us to reevaluate the antigenic and functional properties of prekallikrein in HMW kininogen-deficient plasma as well as in normal plasma. The low prekallikrein antigen level in an individual with HMW kininogen deficiency was corrected to the normal range (80-95%) by the addition of 0.2 U/ml of purified HMW kininogen. A similar increase in apparent prekallikrein antigen was observed when purified prekallikrein and HMW kininogen were combined. The correction of the apparent prekallikrein defect in this HMW kininogen-deficient plasma coincided with the formation of a prekallikrein-HMW kininogen complex as demonstrated by immunoelectrophoresis. Similar findings were demonstrated with purified prekallikrein and HMW kininogen by immunoelectrophoresis as well as crossed immunoelectrophoresis. The coagulant activity in HMW kininogen-deficient plasma was increased in a dose-dependent manner by the addition of HMW kininogen, reaching 85% of normal level at a concentration of 0.2 U/ml. Kaolin-activated arginine esterase activity (kallikrein) in HMW kininogen-deficient plasma was fully corrected when HMW kininogen was added to the deficient plasma after depletion of kallikrein inhibitors. The functional and antigenic concentration of prekallikrein in plasma from four other HMW kininogen-deficient individuals was similarly corrected to normal after adding HMW kininogen. Addition of HMW kininogen increased the apparent prekallikrein activity in native normal plasma (as measured by esterase activity) but not in normal plasma in which inhibitors were inactivated. The apparent prekallikrein antigen concentration (as measured by radial immunodiffusion or electroimmunodiffusion) increased upon addition of HMW kininogen. Immunoelectrophoresis as well as gel filtration of normal plasma revealed the presence of free prekallikrein (17-38% of the total) in addition to the HMW kininogen-prekallikrein complex previously reported. This study emphasizes the influence of HMW kininogen on both functional and immunologic determinations of prekallikrein.

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Although platelets contain Factor V, localized primarily in the alpha-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chiu, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098 +/- 0.018 micrograms/10(5) cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234 +/- 0.180 micrograms/10(8) platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0 +/- 3.0 micrograms/ml) than that of Factor V antigen in human plasma (11.1 +/- 0.4 micrograms/ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01 +/- 1.09 micrograms/10(8) platelets) than do guinea pig were 2.85 +/- 0.30 U/ml plasma, 0.022 +/- 0.012 U/10(8) platelets, and 0.032 +/- 0.03 U/10(5) megakaryocytes, compared with human values of 0.98 +/- 0.02 U/ml plasma and 0.124 +/- 0.064 U/10(8) platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with [35S]methionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of 350,000. Both forms of Factor V were substrates for thrombin. Possible explanations for the higher molecular weight and different thrombin sensitivity and stability observed are that a precursor of Factor V was isolated or that the megakaryocyte Factor V had not been fully processed before isolation.

Platelet C1- inhibitor. A secreted alpha-granule protein.

In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways.

Human platelet collagenase.

The presence of proteolytic enzymes such as cathepsin and elastase in platelets and the important role of collagen in platelet aggregation suggested that collagenase might be present in platelets. Epinephrine, ADP, and collagen liberate collagenase from platelets in plasma as measured by the hydrolysis of [(14)C]glycine-labeled collagen fibrils. The collagenase activity appeared in an early phase of platelet aggregation and was not a part of the release reaction. However, only 50% of the total collagenase could be liberated by the aggregating agents used. Sucrose density gradient analysis of platelet homogenates using appropriate sub-cellular markers indicated that collagenase appeared in both the granule and membrane fractions. Gel-filtered platelets failed to show collagenase activity before exposure to aggregating agents but released more collagenolytic activity than was found in platelet-rich plasma. This observation was explained by the finding that collagenolytic activity was inhibited by normal human plasma. One of the inhibitors is alpha(1)-antitrypsin as demonstrated by decreased inhibition in plasma from a patient with homozygous alpha(1)-antitrypsin deficiency. Platelet collagenase activity could also be demonstrated by its ability to decrease the viscosity of collagen solutions and to produce collagen fragments similar to those produced by other mammalian collagenases on disk gel electrophoresis. The observation that partially purified platelet collagenase could destroy the platelet-aggregating activity of collagen suggests that the enzyme might function in a negative feedback mechanism limiting thrombus formation.

Critical role of the carbohydrate side chains of collagen in platelet aggregation.

The reaction between human platelet membrane glucosyl transferase and collagen has recently been proposed as the mechanism for pletelet-collagen adhesion. Collagen contains glucosyl-galactose and galactose side chains linked through the galactose to hydroxylysine. Oxidation of the 6-hydroxymethyl position of the galactosyl residue to aldehydes with galactose oxidase completely abolishes platelet aggregation. This enzymatic modification of collagen can be fully reversed by reduction of the aldehydes formed by NaBH(4) with complete restoration of platelet aggregating ability. Limited digestion with bacterial collagenase abolishes the ability of collagen to aggregate platelets. Removal of the N-terminal telopeptides from collagen with trypsin does not affect platelet aggregation. Tertiary structure of soluble collagen is essential for platelet aggregation. Normal collagen is less effective than lathyritic collagen, which contains only a small number of cross-links. The decreased number of aldehyde groups in the lathyritic collagen are not responsible for the increase in aggregating ability, since reduction with NaBH(4) does not alter platelet aggregation. These results suggest that integrity and accessibility of the galactose receptor site may be crucial for the formation of a ternary collagenenzyme-platelet membrane complex which must precede platelet aggregation.

Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants.

Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG(4)lambda) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M alpha-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (M(r), 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity. The isolated Factor V neutralized the inhibitory activities of the monoclonal antibody that was used to purify it, as well as the rabbit antibodies produced by immunizing the animals with the purified Factor V. Immunoelectrophoresis of purified Factor V against the polyclonal rabbit antiserum resulted in a single precipitin arc of identical mobility to the Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive enzyme-linked immunosorbent assay using the rabbit antibody against Factor V was applied to quantify Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified Factor V gave parallel dose-response curves. In 14 normal plasma the coagulant activity was 0.98+/-0.02 U/ml (mean+/-SEM) and antigen concentration was 11.1+/-0.4 mug/ml. A pool of 14 patients with congenital Factor V deficiency were studied. 10 patients had Factor V antigen ranging from 1.0 to 2.4 mug/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with coagulant activity (0-0.08 U/ml) had Factor V antigen concentrations ranging from 4.4 to 6.1 mug/ml, indicating the presence of a reduced concentration of abnormal Factor V protein. The presence of patients with antigen similar in concentration to coagulant activity and antigen in excess of Factor V activity indicates the heterogeneity of congenital Factor V deficiency.

Contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma.

Although Cl-inhibitor (Cl-INH) and alpha(2)-macroglobulin (alpha(2)M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in Cl-INH, or artificially depleted of alpha(2)M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min(-1). This rate constant was reduced to 0.35, 0.30, and 0.06 min(-1), in plasma deficient respectively in Cl-INH, alpha(2)M, or both inhibitors. Thus Cl-INH (42%) and alpha(2)M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, (125)I-kallikrein (M(r) 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of (125)I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M(r) 370,000) with Cl-INH, while 48% formed a complex (M(r) 850,000) with alpha(2)M. After inactivation by Cl-INH-deficient plasma, >90% of the active (125)I-kallikrein was associated with alpha(2)M. A similar proportion of the label was associated with Cl-INH in plasma deficient in alpha(2)M. After inactivation by plasma deficient in both Cl-INH and alpha(2)M, (125)I-kallikrein was found to form a complex of M(r) 185,000. This latter complex, which may involve antithrombin III, alpha(1)-protease inhibitor, and/or alpha(1)-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either Cl-INH or alpha(2)M, even after the addition of heparin (2 U/ml). These observations demonstrate that Cl-INH and alpha(2)M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either Cl-INH or alpha(2)M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.

Response of the kallikrein-kinin and renin-angiotensin systems to saline infusion and upright posture.

The possibility that bradykinin, a potent vasodilator, might be a physiological antagonist of the renin-angiotensin system was investigated. 11 norman subjects, ranging in age from 21 to 33 yr were studied. Seven of the subjects were given a 10 meq sodium, 100 meq potassium, 2500 ml isocaloric diet. After metabolic balance was achieved, they were infused with either 1 liter of 5 per cent glucose over 2 h or 2 liters of 0.9 per cent saline over 4 h. During the infusions, plasma renin activity (PRA), angiotensin II (A II), prekallikrein, bradykinin, and aldosterone levels were frequently determined. Plasma prekallikrein and kallikrein inhibitor did not change during the infusion of either glucose or saline. In subjects receiving saline, plasma bradykinin fell from 3.9 plus or minus 1.5 (SEM) ng/ml at 0 min to 0.93 plus or minus 0.2 at 30 min and 0.95 plus or minus 0.3 at 120 min. These changes paralleled the decrease in PRA over the same period (7.9 plus or minus 1.3 ng/ml/h to 5.6 plus or minus 0.8 at 30 min and 3.5 plus or minus 0.7 at 120 min). Similarly, A II fell from 113 plus or minus 12 pg/ml to 62 plus or minus 10 and 48 plus or minus 5, respectively, at 30 and 120 min. In contrast, the control group infused with glucose showed no change in bradykinin, A II, or PRA. Another four subjects were given a constant 200 meq sodium/100 meq potassium isocaloric diet. After metabolic balance was achieved, they were kept supine and fasting overnight. At 9 a.m. they assumed an upright position and began walking a fixed distance (200 ft) at a normal rate (3-4 ft/s). Plasma prekallikrein and kallikrein inhibitor did not change during the posture study. The plasma bradykinin rose from a base line of 0.54 plus or minus 0.01 (SEM) ng/ml to 0.96 plus or minus 0.13 at 20 min. 0.77 plus or minus 0.18 at 60 min, and 0.96 plus or minus 0.07 at 120 min. These changes parallel the increase in PRA over the same period (1.65 plus or minus 3.3 ng/ml/h to 3.6 plus or minus 0.85 at 20 min, 5.3 plus or minus 0.9 at 60 min, and 5.35 plus or minus 0.55 at 120 min). Likewise, the A II rose from 32.5 plus or minus 1.82 pg/ml to 50.8 plus or minus 3.6 at 20 min, 54.3 plus or minus 3.2 at 60 min, and 61.3 plus or minus 5.9 at 120 min. Thus, in sodium-depleted individuals, saline infusion produces a rapid fall of plasma bradykinin at a rate similar to that observed for a II and PRA. Conversely, in sodium-loaded individuals, assumption of upright posture leads to a parallel rise in A II, TPRA, and bradykinin. These studies indicate that there is a close correlation of bradykinin levels with renin activity and angiotensin II, in both acute sodium loading and assumption of upright posture, suggesting that these two systems may be physiologically interrelated.

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.

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.

Activation and function of human Hageman factor. The role of high molecular weight kininogen and prekallikrein.

The activation and function of surface-bound Hageman factor in human plasma are dependent upon both high molecular weight (HMW) kininogen and prekallikrein. HMW kininogen does not affect the binding of Hageman factor to surfaces, but it enhances the function of surface-bound Hageman factor as assessed by its ability to activate prekallikrein and Factor XI. The initial conversion of prekallikrein to kallikrein by the surface-bound Hageman factor in the presence of HMW kininogen is followed by a rapid enzymatic activation of Hageman factor by kallikrein. The latter interaction is also facilitated by HMW kininogen. Kallikrein therefore functions as an activator of Hageman factor by a positive feedback mechanism and generates most of the activated Hageman factor during brief exposure of plasma to activating surfaces. HMW kininogen is a cofactor in the enzymatic activation of Hageman factor by kallikrein and it also augments the function of the activated Hageman factor generated. The stoichiometry of the Hagman factor interaction with HMW kininogen suggests that it enhances the activity of the active site of Hageman factor. Since HMW kininogen and prekallikrein circulate as a complex, HMW kininogen may also place the prekallikrein in an optimal position for its reciprocal interaction with Hageman factor to proceed. The surface appears to play a passive role upon which bound Hageman factor and the prekallikrein-HMW kininogen complex can interact.

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.

Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin.

Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor.

High molecular weight kininogen (HMW)-kininogen, the cofactor of contact-activated blood coagulation, accelerates the activation of Factor XII, prekallikrein, and Factor XI on a negatively charged surface. Although prekallikrein and Factor XI circulate as a complex with HMW-kininogen, no physical association has been demonstrated between Factor XII and HMW-kininogen, nor has the order of adsorption to surfaces of these proteins been fully clarified. In this report we explore the requirements for adsorption of HMW-kininogen to a clot-promoting surface (kaolin), in purified systems, as well as in normal plasma and plasma genetically deficient in each of the proteins of the contact system. The fraction of each coagulant protein associated with the kaolin pellet was determined by measuring the difference in coagulant activity between the initial sample and supernatants after incubation with kaolin, or by directly quantifying the amount of 125I-HMW-kininogen that was associated with the kaolin pellet. In normal plasma, the adsorption of HMW-kininogen to kaolin increased as the quantity of kaolin was increased in the incubation mixture. However, the HMW-kininogen in Factor XII-deficient plasma did not absorb appreciably to kaolin. Furthermore, the quantity of HMW-kininogen from prekallikrein-deficient plasma that adsorbed to kaolin was decreased as compared with normal plasma. These observations suggested that HMW-kininogen in plasma must be altered by a reaction involving both Factor XII and prekallikrein in order for HMW-kininogen to adsorb to kaolin, and to express its coagulant activity. Subsequently, the consequence of the inability of HMW-kininogen to associate with a negatively charged surface results in decreased surface activation. This assessment was derived from the further observation of the lack of prekallikrein adsorption and the diminished Factor XI adsorption in both Factor XII-deficient and HMW-kininogen-deficient plasmas, since these two zymogens (prekallikrein and Factor XI) are transported to a negatively charged surface in complex with HMW-kininogen. The percentage of HMW-kininogen coagulant activity that adsorbed to kaolin closely correlated (r = 0.98, slope = 0.97) with the amount of 125I-HMW-kininogen adsorbed, suggesting that adsorption of HMW-kininogen results in the expression of its coagulant activity. Since kallikrein, which is known to cleave HMW-kininogen, is generated when kaolin is added to plasma, we tested the hypothesis that proteolysis by kallikrein was responsible for the enhanced adsorption of HMW-kininogen to kaolin. When purified HMW-kininogen was incubated with purified kallikrein, its ability to absorb to kaolin increased with time of digestion until a maximum was reached. Moreover, (125)I-HMW-kininogen, after cleavage by kallikrein, had markedly increased affinity for kaolin than the uncleaved starting material. Furthermore, fibrinogen, at plasma concentration (3 mg/ml), markedly curtailed the adsorption of a mixture of cleaved and uncleaved HMW-kininogen to kaolin, but was unable to prevent fully cleaved HMW-kininogen from adsorbing to the kaolin. Addition of purified kallikrein to Factor XII-deficient plasma, which bypasses Factor XII-dependent contact-activation amplified the ability of its HMW-kininogen to adsorb to kaolin. These observations indicate that HMW-kininogen is a procofactor that is activated by kallikrein, a product of a reaction which it accelerates. This cleavage, which enhances its association with a clot-promoting surface in a plasma environment, is an event that is necessary for expression of its cofactor activity. These interactions would allow coordination of HMW-kininogen adsorption with the adsorption of Factor XII, which adsorbs independently of cleavage, to the same negatively charged surface.

Fibronectin degradation products containing the cytoadhesive tetrapeptide stimulate human neutrophil degranulation.

We investigated whether adhesive glycoproteins, such as fibronectin or fibrinogen, could function to provide a nidus for neutrophil degranulation. Elastase release in recalcified plasma was normal in afibrinogenemic plasma, but 73% less in plasma depleted of fibronectin. Proteolytic digests of fibronectin, but not intact fibronectin (50-1,000 micrograms/ml), induced a concentration-dependent release of neutrophil elastase and lactoferrin. MAbs N293, which recognized the mid-molecule of fibronectin, N294, which was directed toward the 11-kD cell adhesive fragment, and N295, generated against the amino terminal of the 11-kD fragment, inhibited the release of elastase by 7, 24, and 60%, respectively. The cytoadhesive tetrapeptide portion of fibronectin, Arg-Gly-Asp-Ser (250-1,000 micrograms/ml), released 1.94 +/- 0.10 micrograms/ml of elastase from 10(7) neutrophils, in contrast to the lack of release by the control hexapeptide, Arg-Gly-Tyr-Ser-Leu-Gly. Plasmin appeared to be the enzyme responsible for fibronectin cleavage, since neutrophil elastase release in plasma that had been depleted of plasminogen was decreased and reconstitution of plasminogen-deficient plasma with purified plasminogen corrected the abnormal release. Plasmin cleaved fibronectin to multiple degradation products, each less than 200 kD. This fibronectin digest released 1.05 microgram/ml of elastase from 10(7) neutrophils. We suggest that the activation of plasminogen leads to the formation of fibronectin degradation products capable of functioning as agonists for neutrophils.

Modulation of the human monocyte binding site for monomeric immunoglobulin G by activated Hageman factor.

Macrophage Fc gamma receptors play a significant role in inflammation and host defense. One monocyte/macrophage Fc gamma receptor, Fc gamma RI, the binding site for monomeric IgG, appears to be especially responsive to modulatory signals by hormones and mediators. Since Factor XIIa is generated during inflammation, we studied the effect of XIIa on Fc gamma RI. Factor XIIa, in a concentration-dependent manner (0.01-0.19 microM), reduced the number of monocyte binding sites for monomeric IgG up to 80% without altering the affinity of binding. Its precursor, Factor XII, and the low molecular weight fragment of XIIa, lacking most of the heavy chain region, did not reduce the expression of Fc gamma RI. Neither corn trypsin inhibitor (36 microM) nor diisopropylfluorophosphate (3.6 mM) diminished the effect of Factor XIIa on Fc gamma RI, although each completely inhibited the coagulant and amidolytic activity contained on the light chain of Factor XIIa. Protein synthesis was not a requirement for this effect of Factor XIIa, nor was internalization of Fc gamma RI necessary. In contrast to similar concentrations of IgG, Factor XIIa failed to displace significantly monomeric IgG from the monocyte surface, suggesting that Factor XIIa does not directly compete for Fc gamma RI. The data suggest that the heavy chain of XIIa, which contains domains that may have cell hormone activity, also contains a domain that regulates Fc gamma RI on monocytes. In addition to other hormones and mediators, Factor XIIa may serve a regulatory function in modulating Fc gamma receptor expression during inflammation.