The effect of C1 inhibitor upon Hageman factor autoactivation

Using components purified from human plasma, we have examined the effects of C1 inhibitor (C1 INH), the primary inhibitor of activated Hageman Factor (HFa) and Hageman factor fragment (HFf), on Hageman Factor (HF) autoactivation. When Hageman factor was exposed to a negatively charged surface, provided by either a glass cuvette or dextran sulfate, the addition of C1 INH gave a dose-dependent inhibition of the activity observed. The ability of C1 INH to decrease the maximal enzymatic activity generated was markedly temperature dependent with inhibition increasing as the temperature was raised from 4 degrees C to 37 degrees C. Although the rates of both autoactivation and inhibition were decreased at lower temperatures (4 degrees C), the latter rate was more sensitive to temperature modulation. When HF (final concentration 1 mumol/L) was incubated with C1 INH (0.54, 1.07, and 2.14 mumol/L) in the absence of an initiating surface, no increases in enzymatic activity were observed for up to 48 hours regardless of the C1 INH concentration. However, SDS polyacrylamide gel electrophoresis of the incubation mixture revealed that HF autodigestion had occurred by 48 hours despite the presence of C1 INH. In addition, the appearance of a new band suggested that a complex had been formed between the inhibitor and activated HF. Our findings indicate that C1 INH does not prevent HF autoactivation but rather inactivates the products of HF autodigestion.     

Production and characterization of a murine monoclonal antibody against a heavy chain of Hageman factor (factor XII)

A murine hybridoma cell line that produces a monoclonal antibody to human Hageman factor (HF, factor XII) is described. The antibody (P 5-2- 1) consists of mouse IgG2b heavy chains and lambda light chains, selectively neutralizes HF procoagulant activity, and prevents the proteolytic cleavage of HF during contact activation in plasma. When HF is exposed to P 5-2-1 before the absorption of HF to kaolin, HF procoagulant activity is markedly inhibited. In contrast, P 5-2-1 does not interfere with HF activity after the adsorption of HF to kaolin. P 5-2-1 does not inactivate the prekallikrein-activating activity of 28,000-mol wt HF fragments (HFf). P 5-2-1 binds exclusively to the 40,000-mol wt portion of a heavy chain of HF and inhibits the adsorption of HF to negatively charged surfaces. P 5-2-1 immobilized on Sepharose can be used to deplete HF from normal human plasma. This immunoaffinity-depleted plasma is indistinguishable from congenital HF- deficient plasma and can be used as the substrate for HF procoagulant activity assay.     

The role of HF cofactor in the Hageman factor-dependent fibrinolytic mechanism

In 1969, Ogston et al. reported that the normal activation of fibrinolysis by surface contact requires, in addition to Hageman factor and plasminogen, a HF cofactor which is present in the euglobulin fraction and other factor(s) present in the supernatant. It has also been suggested that the glass-treated plasma is deficient in HF cofactor, In our laboratory the glass-treated plasma was found not to be deficient in HF or in a streptokinase-activated proactivator or in plasminogen. The glass-treated plasma was found deficient in prekallikrein in kininogen and in clotting factors XI, IX, VIII and V. The results presented indicate that HF cofactor activity is not different from that of kallikrein and that HF cofactor does not act as a plasminogen proactivator. Furthermore, the results indicate that the "other factors' present in the supernatant are not involved in contact-activated fibrinolysis.     

The Assay of a Plasma Component Necessary for the Generation of a Plasminogen Activator in the Presence of Hageman Factor (Hageman Factor Co-factor)

S ummary . Kaolin-induced generation of fibrinolytic activity requires, in addition to factor XII (Hageman factor) and plasminogen, at least one further plasma factor which has been termed Hageman factor co-factor (HF co-factor). A technique for the assay of HF co-factor in plasma is described using plasma specifically depleted of HF co-factor by treatment with glass. The kaolin-induced lysis time bears log:log relationship to the concentration of untreated test plasma diluted glass-adsorbed plasma. The influence of the plasma fibrinogen concentration is small, and the assay is not affected by exercise-induced changes in the plasma plasminogen-activator level. Using this technique the range of HF co-factor in the plasma of 20 healthy subjects was found to lie between 76 and 150% of pooled normal plasma.     

Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor.

The mechanisms by which human high molecular weight kininogen (HMKrK) contributes to the surface-dependent activation of the Hageman factor systems have been studied. The ability of various mixtures of purified human Hageman factor (coagulation factor XII), HMrK, prekallikrein, and kaolin to activate coagulation factor XI was determined with factor XIa (activated factor XI) clotting assays. Hageman factor, HMrK and prekallikrein were required for maximal rates of activation of factor XI. A certain optimal mixture of purified Hageman factor, HMrK, prekallikrein, and kaolin gave the same rapid initial rate of activation of purified factor XI as an equivalent aliquot of factor XI-deficient plasma. This suggests that potent, surface-mediated activation of factor XI in plasma is explicable in terms of Hageman factor, HMrK, and prekallikrein. By studying separately some of the surface-dependent reactions involving Hageman factor, it was found that HMrK accelerated by at least an order of magnitude the following reactions: (i) the activation of factor XI by activated Hageman factor; (ii) the activation of prekallikrein by activated Hageman factor; and (iii) the activation of Hageman factor by kallikrein. Stoichiometric rather than catalytic amounts of HMrK gave optimal activation of factor XI. These results are consistent with the hypothesis that HMrK and Hageman factor form a complex on kaolin which renders Hageman factor more susceptible to proteolytic activation by kallikrein and which facilitates the action of activated Hageman factor on its substrate proteins, factor XI and prekallikrein.     

Initiation of plasma prorenin activation by Hageman factor-dependent conversion of plasma prekallikrein to kallikrein.

Plasma prorenin is an inactive form of renin (EC 3.4.99.19) that can be converted to active renin in acid-treated plasma by an endogenous serine protease that is active at alkaline pH (alkaline phase activation). To identify this enzyme we first tested the ability of Hageman factor fragments, plasma kallikrein (EC 3.4.21.8), and plasmin (EC 3.4.21.7) to activate prorenin in acid-treated plasma. All three enzymes initiated prorenin activation; 50% activation was achieved with Hageman factor fragments at 1 microgram/ml, plasma kallikrein at 2-4 microgram/ml, or plasmin at 5-10 microgram/ml. We then showed that the alkaline phase of acid activation occurred normally in plasminogen-free plasma but was almost completely absent in plasmas deficient in either Hageman factor or prekallikrein; alkaline phase activation was restored to these latter plasmas when equal parts were mixed together. Therefore, both Hageman factor and prekallikrein were required for alkaline phase activation to occur. We then found that, although plasma kallikrein could activate prorenin in plasma deficient in either Hageman factor or prekallikrein, Hageman factor fragments were unable to activate prorenin in prekallikrein-deficient plasma. These studies demonstrate that alkaline phase prorenin activation is initiated by Hageman factor-dependent conversion of prekallikrein to kallikrein which, in turn, leads to activation of prorenin. In this fashion, we have revealed a possible link between the coagulation-kinin pathway and the renin-angiotensin system.     

A monoclonal antibody that inhibits activation of human Hageman factor (factor XII)

A monoclonal antibody to human Hageman factor (HF, factor XII) was derived from BALB/c mouse spleen cells fused with NS-1 mouse myeloma cells. This antibody, purified from ascites fluid, reacted with HF to inhibit the activation of HF, purified or in normal pooled plasma, as measured by a coagulation assay. The antibody did not inhibit the coagulant activity of activated HF. The antibody also inhibited the generation of amidolytic activity in HF-ellagic acid mixtures, but failed to inhibit the amidolytic properties of the carboxy-terminal fragment of HF (HFf). Amidolytic activity, absent in an HF-monoclonal antibody mixture, was generated upon treatment with insoluble trypsin. Monoclonal antibody, bound to CNBr Sepharose 4B gel (Pharmacia Fine Chemicals, Piscataway, NJ), reversibly bound HF in plasma or in buffer, without activating it. HF was then eluted with 4 mol/L guanidine HCI. The passage of 125I-labeled HF enzymatically cleaved by trypsin through a column of monoclonal antibody-CNBr Sepharose 4B gel resulted in flow- through of HFf with a molecular weight (mol wt) of 30,000 and HF fragments of mol wt 12,000. Elution with 4 mol/L guanidine HCI yielded several HF fragments (mol wt 80,000, 52,000, and 40,000) but not HFf. These data suggest that the single determinant recognized by the murine monoclonal antibody is not on HFf, but rather on the amino-terminal fragment thought to be involved in the binding activity of HF. The monoclonal anti-HF bound to CNBr-activated Sepharose 4B gel could be used to artificially deplete plasma samples of HF.     

Role of surface in surface-dependent activation of Hageman factor (blood coagulation Factor XII)

The mechanism by which negatively charged substances such as celite, kaolin, or ellagic acid contribute to the surface-dependent activation of Hageman factor (Factor XII) was studied. Kinetic studies of the proteolytic activation of (125)I-labeled human Hageman factor by human plasma kallikrein, plasma, activated Factor XI, and trypsin were performed in the presence and absence of high molecular weight kininogen and surface materials such as celite, kaolin, or ellagic acid. The results showed that surface-bound Hageman factor was 500 times more susceptible than soluble Hageman factor to proteolytic activation by kallikrein in the presence of high molecular weight kininogen. Surface binding of Hageman factor enhanced its cleavage by plasmin, activated Factor XI, and trypsin by 100-fold, 30-fold, and 5-fold, respectively. On a molar basis, trypsin was twice as potent as kallikrein in the cleavage of the surface-bound Hageman factor, while plasmin and activated Factor XI were an order of magnitude less potent than kallikrein. Kallikrein even at concentrations as low as 0.5 nM (i.e., 1/1000th of the concentration of prekallikrein in plasma) was very potent in the limited proteolysis of the surface-bound Hageman factor. These results suggest that substances classically known as "activating surfaces" promote the activation of Hageman factor indirectly by altering its structure such that it is much more susceptible to proteolytic activation by other plasma or cellular proteases.     

Molecular assembly in the contact phase of the Hageman factor system.

Data obtained in the past few years have defined the molecular mechanisms of contact activation of the Hageman factor pathways of plasma, i.e., the kinin-forming, intrinsic clotting and fibrinolytic systems. Involved are four molecules: Hageman factor, high molecular weight (MW) kininogen, prekallikrein and factor XI. High MW kininogen serves as a surface cofactor to assemble prekallikrein or factor XI in proximity to surface-bound Hageman factor. Reciprocal proteolytic activation of Hageman factor and prekallikrein represents an essential step in the rapid activation of the contact phase. Although Hageman factor does undergo cleavage and activation in the absence of prekallikrein or high MW kininogen, the rate is approximately 50 and 100 times slower than when these molecules are present. Once Hageman factor is activated on the surface, it cleaves and activates clotting factor XI. Activated Hageman factor (HFa) exhibits two molecular forms. One of these, alpha HFa, activates prekallikrein and factor XI, and the intrinsic clotting system on the surface. alpha HFa and clotting factor XI remain surface bound. The other form of activated Hageman factor, beta HFa, leaves the surface, going into solution where it readily activates additional prekallikrein but not factor XI. Of perhaps even greater importance, kallikrein rapidly dissociates from the surface. Thus the formation of bradykinin and fibrinolysis is disseminated whereas clotting via the intrinsic system remains localized. Reviewed here is the molecular mechanism of contact activation of the Hageman factor pathways and discussed in the interaction of Hageman factor with the negatively charged surface, prekallikrein, factor XI and high MW kininogen. The multiple forms of activated Hageman factor and their potential biologic significance are also discussed.     

In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E

A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein- deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.     

Interactions of C1(-)-inhibitors from normal persons and patients with type II hereditary angioneurotic edema with purified activated Hageman factor (factor XIIa)

Activated high molecular weight Hageman factor (75 Kd) and Hageman factor carboxy-terminal fragments both formed complexes with purified C1(-)-inhibitor, but the Hageman factor fragments appeared to have a higher affinity for the C1(-)-inhibitor than activated Hageman factor. Therefore, the clot-promoting activity of activated Hageman factor might be relatively unimpaired if Hageman factor fragments are also present. Normal C1(-)-inhibitor was cleaved by Hageman factor fragments. Clot-promoting activity was not generated in Hageman factor by exposure to Hageman factor fragments, nor was Hageman factor cleaved by Hageman factor fragments. When Hageman factor was cleaved by streptokinase-activated plasminogen, a 40 Kd fragment was released. In contrast to their interactions with other proteinases, which are blocked by normal C1(-)-inhibitor, Type II C1(-)-inhibitors from plasmas of affected members of eight different kindred with this form of hereditary angioneurotic edema all inhibited the specific coagulant activity of activated Hageman factor to some degree. They did not all form complexes with activated Hageman factor that were stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Role of high-molecular-weight kininogen in surface-binding and activation of coagulation Factor XI and prekallikrein.

In the contact phase of activation of the kinin-forming, intrinsic clotting, and fibrinolytic systems, high-molecular-weight kininogen acts as a cofactor for the activation of Factor XI, prekallikrein, and Hageman factor. One mechanism by which high-molecular-weight kininogen acts as a cofactor has been studied by using 125I-labeled Factor XI and prekallikrein in kaolin-activated normal human plasma and plasmas deficient in high-molecular-weight kininogen and Hageman factor. High-molecular-weight kininogen was found to be essential for normal binding and cleavage of both Factor XI and prekallikrein on the kaolin surface. Hageman factor was essential for cleavage but not for binding of Factor XI and prekallikrein to kaolin. In normal plasma 80% of the activated Factor XI remained surface-bound, whereas 80% of the kallikrein was not surface-bound. These findings are consistent with the hypothesis that, in the initial phase of contact activation, high-molecular-weight kininogen links both Factor XI and prekallikrein to the exposed surface where they are activated by surface-bound activated Hageman factor. Once activated, the Factor XI molecules remain localized at the site of activation, in contrast to the kallikrein molecules which are found largely in the surrounding plasma.     

Soluble mediators of injury of the microvasculature; Hageman factor and the kinin forming, intrinsic clotting and the fibrinolytic systems

Studies on Hageman factor have revealed that this protein of approximately 80,000 MW is activated in both solid and fluid phase. In solid phase, the molecule interacts with negatively charged particles without undergoing cleavage. Enzymatic activity is acquired, presumably following a conformational change in the structure of Hageman factor. In fluid phase, the enzymes kallikrein, plasmin, and plasma thromboplastin antecedent (clotting Factor XI) all activated Hageman factor, and in human plasma, the Hageman factor is readily cleaved during this activation. Evidence is presented indicating that kallikrein is the most important fluid phase activator and that the activation with kallikrein is essential for the normal function of the intrinsic clotting, fibrinolytic and kinin forming systems. Information on the role of these systems in immunopathology awaits careful analyses of the function of individual components and means of their accurate detection and quantitation.     

Hageman factor (HF) deficiency

A patient with Hageman factor (HF) deficiency is described. This syndromeis characterized by complete absence of any hemorrhagic tendency in the presence of laboratory findings which, as a rule, are associated with severe disturbances in the hemostatic mechanism. The clotting time was markedly prolonged,the plasma prothrombin time was normal, but prothrombin consumption wasdecreased. The thromboplastin generation test revealed that HF is essential forblood thromboplastin formation at least in vitro.

Procedures for the differentiation of HF deficiency from AHF, PTC and PTAdeficiency syndromes are outlined.

Transfusions of as little as 50 cc. of 20 day old blood normalized the abnormalclotting tests immediately for a period of about 36 hours.

The basis for the apparent lack of need for preoperative preparation withblood transfusions in HF deficiency is discussed.

Submitted on November 21, 1955 Accepted on February 6, 1956     

Induction of expression of monocyte interleukin 1 by Hageman factor (factor XII).

The results reported here indicate that activated species of Hageman factor (HF, factor XII), a protein that mediates blood clotting, fibrinolysis, and activation of the complement cascade, induce elaboration of interleukin 1 (IL-1) by human monocytes. Augmentation of IL-1 production in mononuclear cell cultures was observed when HF was present along with lipopolysaccharide (LPS) but was not observed with HF alone. Furthermore, antiserum to HF abrogated the enhancement of IL-1 in cultures containing HF and LPS. Total IL-1 activity, which represents secreted and cell-associated IL-1, was enhanced in LPS-stimulated mononuclear cultures by HF. In the absence of LPS, the initial activation product of HF, HFa, which contains the serine protease enzyme activity and the surface-binding domains of the protein, induced IL-1 beta protein and mRNA. In the presence of LPS, the enzymatic moiety (HFf), which is also contained in HF and HFa, amplified IL-1 production. Induction and amplification of monocyte IL-1 by HF provides further evidence for establishing a role for HF in the acute-phase reaction and the cellular immune response.     

Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent.

To investigate the earliest steps of the intrinsic clotting pathway, Hageman factor (Factor XII) was exposed to Sephadex gels to which ellagic acid had been adsorbed; Hageman factor was then separated from the gels and studied in the fluid phase. Sephadex-ellagic acid-exposed Hageman factor, whether purified or in plasma, activated plasma thromboplastin antecedent, but only when high molecular weight kininogen was presnet. In the absence of plasma prekallikrein, maximal activation of plasma thromboplastin antecedent was slightly delayed in plasma, a delay not observed with similarly treated purified Hageman factor. Thus, high molecular weight kininogen was needed for expression of Hageman factor's clot-promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor and plasma thromboplastin antecedent.     

Endothelial cells produce a substance that inhibits contact activation of coagulation by blocking the activation of Hageman factor.

Human umbilical vein endothelial cells (HUVECs) produce a property that impairs the generation of coagulant and amidolytic activity initiated when normal human plasma is exposed to glass. This inhibitory property blocks the adsorption of Hageman factor (factor XII) to glass, thereby preventing the activation of Hageman factor, but does not impair the coagulant or amidolytic activity of already activated Hageman factor (factor XIIa). This property in HUVEC lysates could be neutralized by a purified preparation of Hageman factor but not by purified prekallikrein or high molecular mass kininogen. A partially purified inhibitory fraction from cell lysates exhibited a single homogeneous band in SDS/PAGE of approximately 22.5 kDa. Inhibitory activity was also found in concentrates of conditioned media from HUVECs, which also impaired the binding of Hageman factor to a surface; it may not be identical with that found in cell lysates.     

Purification of Hageman factor (factor XII) on columns of popcorn- agarose

Purification of Hageman factor (HF, factor XII) from human plasma is a tedious procedure and the product is not always in the precursor form. Hojima has described a protein derived from corn kernels that inhibits the enzymatic properties of HF. This inhibitor binds to the precursor form of HF. Rapid purification of HF was achieved by using as the major purification step adsorption of this clotting factor to popcorn inhibitor bound to agarose. The product had a specific activity of 50.0 to 67.1 coagulant units of HF per milligram protein, and the yield was 33% to 40% of the HF content of the starting plasma. The purified protein displayed a single band upon unreduced or reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis and less than 0.1% was in an activated form, as measured in coagulant assays. The technique described is more rapid and reliable than methods described earlier.     

Enzymatic activities of activated and zymogen forms of human Hageman factor (factor XII)

Pro-Phe-Arg chloromethylketone (PPACMK) at 5.26 microM inactivated the amidolytic activity of native human Hageman factor with an apparent first-order rate constant of 0.75 min-1. The activated forms of Hageman factor, Hfa and HFf, were also inactivated by PPACMK with rate constants 0.82 and 0.72 min-1. These numbers indicate that the activity detectable in native Hageman factor is due to contamination with activated species. Uncleaved Hageman factor reacts slowly with 40 mM diisopropyl fluorophosphate with concomitant loss of its procoagulant activity. Incubation of native Hageman factor with PPACMK does not destroy its procoagulant activity, even in the presence of the activator dextran sulphate, but PPACMK inhibits autoactivation of Hageman factor, suggesting that no active site is formed in uncleaved, surface-bound Hageman factor. The activation of prekallikrein by Hageman factor under initial-rate conditions occurs after a lag and is prevented by an inhibitor of Hageman factor from corn. The kinetics of prekallikrein activation and the effects of inhibitors provide evidence that the amidolytic and proteolytic activities of human Hageman factor reside in the activated forms derived by limited proteolysis of the native molecule.     

Evidence for a New Plasma Thromboplastin Factor I. Case Report, Coagulation Studies and Physicochemical Properties

Studies of defective plasma thromboplastin formation in four siblings indicated a defect which was different from any of the known coagulation factordeficiency states. Although none of the children had any history of hemorrhagictendencies, a prolonged whole blood clotting time in an 11-year-old girl ledto the findings of a markedly prolonged partial thromboplastin time (PTT),abnormal thromboplastin generation test (TGT), and a normal prothrombintime in the patient and in three of her ten siblings. The abnormal PTT andTGT were corrected by aluminum hydroxide adsorbed fresh plasma and byserum. Using the kaolin-PTT system, equal mixtures of plasma from the patients and normal plasma produced a normal time. In addition, plasmas deficient in plasma thromboplastin antecedent (PTA), Hageman factor (HF),antihemophilic factor (AHF), or plasma thromboplastin component (PTC)corrected the abnormality.

Physical and chemical properties of plasma correcting the defect in vitroindicated that the defect is closely related to that found in PTA and HF deficient plasma.

Submitted on December 18, 1965 Accepted on March 3, 1965