The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments.

Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surface-mediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen.     

Synthesis and release of Hageman factor (Factor XII) by the isolated perfused rat liver.

The site of synthesis of Hageman factor (HF, Factor XII) has not been previously demonstrated with certainty. We have studied the production and release of HF in the isolated perfused rat liver and have compared rates of synthesis in this system with absolute rates of degradation measured in vivo. Rat livers, perfused for 5 h with a recycling fluid consisting of a perfluorochemical emulsion (Fluosol 43), were used to demonstrate a cumulative increase of HF in the perfusate as measured by a specific and sensitive radioimmunoassay. The rate of increase in the perfusate pool of HF during the final 4 h of perfusion yielded a mean synthetic rate of 3.5 micrograms/h per 100 g body wt, which was approximately 0.2% of the synthetic rate of albumin in the same system. The cumulative appearance of albumin and transferrin was linear after 1 h and calculated rates of synthesis were 2,012 micrograms/h per 100 g and 263 micrograms/h per 100 g body wt, respectively. De novo synthesis of HF was confirmed by demonstrating incorporation of [14C]lysine into specific immunoprecipitates of HF, and by the observations that both specific incorporation of labeled amino acid and net release of immunoassayable HF were inhibited by the administration of cycloheximide. Finally, it was evident that the rates of synthesis observed in the isolated perfused liver agreed closely with absolute rates of degradation of HF measured in vivo with 125I-rat HF (4.0 micrograms/h per 100 g). From these data we conclude that the liver is the principal site of synthesis of HF.     

Inhibition of Hageman factor (factor XII) by popcorn inhibitor

A protein derived from sweet corn or popcorn inhibits the enzymatic activity of the carboxy-terminal fragment of Hageman factor (HFf) and of ellagic acid-activated Hageman factor (HF, factor XII). Not clarified is whether the inhibitor is directed at the active site of HF. Filtration of normal plasma or purified HF through columns of popcorn inhibitor bound to agarose gels demonstrated that HF was bound to these gels and could then be eluted by buffers containing 2.0 mol/L sodium chloride. The eluted HF was in the precursor form. Thus, popcorn inhibitor appeared to attach to a point on the carboxy-terminal HFf that was distinct from the enzymatically active site of this clotting factor.     

Inhibition of the activation of Hageman factor (factor XII) by aprotinin (Trasylol)

Aprotinin (Trasylol; Bayer AG, Leverkusen, Germany), a protease inhibitor resembling or identical with Kunitz' pancreatic trypsin inhibitor, is said to have anticoagulant properties, but these are not clearly defined. The present study provides evidence that one action of aprotinin is inhibition of the activation of Hageman factor (factor XII).     

Inhibition of the activation of Hageman factor (factor XII) by platelet factor 4

Platelet factor 4 is a polypeptide constituent of platelet alpha granules that is released during platelet aggregation and inhibits heparin-mediated reactions. Hageman factor (factor XII) is a plasma proenzyme that, when activated by certain negatively charged agents, initiates clotting via the intrinsic pathway of thrombin formation. In earlier studies using crude systems, platelet factor 4 inhibited activation of Hageman factor by dextran sulfate or cerebrosides, but not activation of Hageman factor by kaolin or ellagic acid. In the present study we examined the mechanisms of inhibition by platelet factor 4, using purified reagents. Platelet factor 4 inhibited activation of Hageman factor by ellagic acid, as measured by amidolysis of a synthetic substrate of activated Hageman factor, an effect inhibited by heparin or by an anti-platelet factor 4 antiserum. Coating glass tubes with platelet factor 4 before addition of normal plasma significantly lengthened the partial thromboplastin time of normal plasma. In addition, the clot-promoting properties of kaolin were inhibited by its prior exposure to platelet factor 4. Thus, the inhibitory properties of platelet factor 4 directed against the activation of Hageman factor were confirmed in a purified system. In this purified system, in contrast to earlier studies using crude systems, platelet factor 4 inhibited activation of Hageman factor by glass, ellagic acid, or kaolin.     

Activation of Hageman factor (factor XII) by bismuth subgallate, a hemostatic agent

Bismuth subgallate is an effective agent in preventing hemorrhage after adenotonsillectomy. The experiments described demonstrate that this may occur through the activation of Hageman factor by this agent. Bismuth subgallate shortened the clotting time of whole blood, an action localized to an effect on the early steps of the intrinsic pathway; bismuth subgallate did not accelerate the thrombin time or prothrombin time of normal plasma, but could be substituted for kaolin as an activator of coagulation in assays of the partial thromboplastin time. The action of bismuth subgallate was localized to an effect on Hageman factor. It did not induce coagulation of plasma samples deficient in any of the recognized factors participating in the intrinsic pathway of thrombin formation, but it shortened the clotting time of plasma deficient in factor VII, a component of the extrinsic pathway. Evidence was obtained that Hageman factor exposed to bismuth subgallate corrected the defect of Hageman factor-deficient plasma and acquired amidolytic properties in the absence of other clotting factors. These studies provide a rationale for the hemostatic properties of bismuth subgallate.     

Structural Changes Accompanying Enzymatic Activation of Human Hageman Factor

The structure of Hageman factor, isolated from human plasma, was analyzed before and after enzymatic activation. The purified molecule is a single polypeptide chain of 80,000 molecular weight (mol wt) sedimenting at 4.5S. An amino acid analysis has been performed. The concentration of Hageman factor in normal human plasma was found to be 29 mug/ml with variation between individuals ranging from 15 to 47 mug/ml. Treatment of the molecule with kallikrein, plasmin, or trypsin resulted in cleavage at two primary sites, yielding fragments of 52,000, 40,000, and 28,000 mol wt. No further changes occurred in the fragments with subsequent reduction. Prekallikrein-activating ability was associated exclusively with the 28,000 moiety.     

Inhibition of the activation of Hageman factor (factor XII) by beta 2-glycoprotein I

beta 2-Glycoprotein I (apolipoprotein H), a constituent of normal human plasma, has been shown to inhibit the generation of amidolytic activity in plasma that has been exposed to negatively charged agents. Studies with purified Hageman factor (factor XII) demonstrate that this inhibitory property is directed against the activation of Hageman factor. In this study beta 2-glycoprotein I inhibited the kaolin-induced generation of clot-promoting properties in solutions of Hageman factor. This effect was localized to an interaction between beta 2-glycoprotein I and kaolin. In contrast, once Hageman factor was activated by kaolin, its clot-promoting properties were not inhibited by beta 2-glycoprotein I. Further, beta 2-glycoprotein inhibited the generation of amidolytic activity against H-D-prolyl-L-phenylalanyl-L-arginine p-nitroanilide dihydrochloride in mixtures of Hageman factor and ellagic acid. The specificity of the action of beta 2-glycoprotein I was confirmed by its neutralization by immunoglobulin fractions of antiserums directed against this protein.     

Isolation and properties of an abnormal Hageman factor (Factor XII) molecule in a cross-reacting material-positive hageman trait plasma.

We have previously described two unrelated individuals with homozygous Hageman trait (Factor XII deficiency) whose plasmas contained nonfunctional material immunologically indistinguishable from normal Hageman factor (HF). Abnormal HF from the plasma of one these subjects has now been purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, alkaline disc gel electrophoresis, and immunoelectrophoresis. Purified abnormal HF had no clot-promoting activity, but showed the same specific antigenicity as purified normal HF by an immunoassay. The abnormal HF was of a single chain polypeptide with the same molecular weight (80,000) as normal HF and was positively stained by periodic acid-Schiff reagent. Both normal and abnormal HF had similar amino acid compositions and isoelectric points (pI 6.5 approximately 7.1). When 125I-labeled abnormal HF and 131I-labeled normal HF were mixed with normal plasma and exposed to glass, both HF underwent an identical pattern of cleavage, yielding 52,000- and 30,000-mol wt fragments. Similarly, abnormal HF was fragmented by trypsin in the same way as normal HF, but no prekallikrein-activating activity was generated after cleavage. [3H]Diisopropyl phosphorofluoridate was incorporated into a 29,000-mol wt fragment of the trypsin-cleaved normal HF, but not into that of the trypsin-cleaved abnormal HF. These data suggest that the molecular defect in this abnormal HF resides at or near the active site serine residue in the 30,000-mol wt part of the molecule.     

Inhibition of the activation of Hageman factor (factor XII) by peripheral blood cells.

Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in PBMC supernates were not identified with known agents, but had properties of a nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin, prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments suggest that peripheral blood cells may impede intravascular coagulation. Whether this property helps maintain the fluidity of blood is unclear.     

Inhibition of the activation of Hageman factor (factor XII) by extracts of Schistosoma mansoni.

How intravascular helminth parasites evade host hemostatic defense mechanisms and survive within the circulating blood has not been adequately explained. Previous reports have described an inhibitor of the intrinsic clotting pathway in extracts of adult Schistosoma mansoni. Using a purified preparation of Hageman factor, we examined the ability of schistosome extracts and secretory products to inhibit the activation of human Hageman factor (factor XII) in an amidolytic assay. Both schistosome extracts and secretory products inhibited the activation of purified Hageman factor by more than 95%. Schistosome extracts inhibited activation of Hageman factor both by ellagic acid and by bovine sulfatides. In contrast, activated Hageman factor retained full activity in the presence of schistosome extracts as tested both on an amidolytic synthetic substrate and a natural substrate, plasma thromboplastin antecedent (factor XI). Our findings indicate that extracts and secretory products of adult Schistosoma mansoni contain a potent inhibitor of the activation of Hageman factor. Knowledge of a site at which schistosomes inhibit the intrinsic clotting pathway provides added insight into the mechanisms by which the parasites avoid the host hemostatic defense mechanisms.     

Activation of Hageman factor (factor XII) by sulfatides and other agents in the absence of plasma proteases

Incubation of purified human HF (factor XII) with sulfatides, EA, kaolin, or glass resulted in the generation of amidolytic activity in the apparent absence of other enzymes. Sulfatides or EA rapidly and efficiently initiated intrinsic coagulation in normal plasma but, under the conditions tested, only trivially corrected the prolonged partial thromboplastin clotting times of plasma deficient in prekallikrein or HMWK. Preliminary incubation of HF with crude IgG directed against plasma kallikrein or SBTI did not influence the results. The presence of albumin greatly enhanced activation of the amidolytic properties of purified HF by EA, even when albumin had been lipid-extracted or treated with DFP or SBTI; albumin also increased activation of HF by sulfatides. Internal cleavage and minimal scission of the HF molecule accompanied the generation of amidolytic properties in mixtures of HF and sulfatides; cleavage was not blocked by SBTI. These experiments demonstrate that negatively charged substances can activate HF in absence of other enzymes and that this activation is accompanied by formation of a two-chain species of HF.     

Inhibition of the activation of Hageman factor (factor XII) by complement subcomponent C1q.

Hageman factor (HF, Factor XII) is activated by glass, collagen, and ellagic acid, and initiates blood coagulation via the intrinsic pathway. C1q inhibits collagen-induced platelet aggregation and adherence of platelets to glass, effects attributable to the collagen-like region of C1q. We examined the actions of C1q on HF activation. Incubation of C1q with HF before addition of HF-deficient plasma extended the activated partial thromboplastin time. Similarly, when glass tubes were coated with C1q before testing, the partial thromboplastin time of normal plasma was increased. C1q reduced the activation of HF by ellagic acid, as measured by the release of p-nitroaniline from the synthetic substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride, an effect inhibited by monoclonal anti-human C1q murine IgG and by digestion of C1q by collagenase. Thus, C1q inhibits activation of HF in vitro in clot-promoting and amidolytic assays and suggests a regulatory mechanism for the inhibition of coagulation.     

The Interaction of Hageman Factor and Immune Complexes

The possible interaction of Hageman factor from human or rabbit plasma with a variety of immunologic reactants was studied. Evidence of an interaction was not obtained and neither binding of radiolabeled Hageman factor to immune aggregates nor depletion of the Hageman factor from the supernate was observed. Cleavage of the labeled Hageman factor molecule into its 30,000 molecular weight-active fragments was not detectable after incubation with immune complexes.Isolated Hageman factor was far more sensitive to activation than Hageman factor in plasma or serum. There was no consistent activation of isolated Hageman factor by immunologic reactants as determined by conversion of prekallikrein to its enzymatic form or by shortening of the clotting time of factor XII-deficient plasma. A variety of immunologic stimuli were tested: (a) antigen-antibody complexes in soluble or precipitated form; (b) particulate antigen-antibody complexes, i.e., zymosan-anti-zymosan in which a surface was presented for activation; (c) human IgM-IgG and IgG-IgG (rheumatoid factor) complexes; (d) immune aggregates consisting of heat or bis-diazotized benzidine-aggregated myeloma proteins of all human immunoglobulin classes and subclasses: IgG(1,2,3,4), IgA, IgD, IgM, and IgE. Absorption with immune aggregates did not reduce the quantity of Hageman factor in solution, nor was the Hageman factor bound to the precipitates. The presence of plasma or serum with immune aggregates did not generate activity of the Hageman factor.The only preparations of immunoglobulins capable of activating Hageman factor were found to be contaminated with bacteria. These bacteria, upon isolation, activated Hageman factor.     

Adsorption of Hageman factor (factor XII) and other human plasma proteins to biomedical polymers

Protein adsorption to surfaces occurs whenever blood comes into contact with biomaterials, prosthetic devices, and artificial organs. The plasma protein Hageman factor (factor XII) present at the interface between blood and foreign surfaces can be qualitatively and quantitatively detected after in vitro perfusion of anticoagulated human blood through important biomedical polymers. We have determined protein adsorption by a modified radioimmunoassay and by scanning electron microscopy using immunogold labeling techniques. The materials used included vascular graft materials (Dacron and expanded polytetrafluoroethylene) and the National Heart, Lung, and Blood Institute-Devices and Technology Branch reference materials polydimethylsiloxane, polyethylene, and silicone rubber. Factor VIII-von Willebrand factor, another trace plasma protein, and other plasma proteins such as fibrinogen, immunoglobulin G, albumin, fibronectin, and hemoglobin were also detected at the blood-contacting surface. At physiologic flow rates, the adsorption of these proteins from the circulating blood to the surface of these materials appears to be a function of time, with certain materials, as well as of the physical and chemical characteristics of the material surface. Hageman factor adsorption to surfaces, quantified under static conditions, occurs at nanogram concentrations. These data suggest that trace proteins, such as those important in the activation of the coagulation cascade, can significantly affect the blood compatibility or thrombogenicity of an implanted device.     

The role of prekallikrein and high-molecular-weight kininogen in the contact activation of Hageman factor (factor XII) by sulfatides and other agents

Hageman factor (HF, factor XII), adsorbed to negatively charged agents, is transformed to an activated state in which it initiates reactions of the intrinsic pathway of thrombin formation by activating plasma thromboplastin antecedent (PTA, factor XI). High-molecular-weight kininogen (HMWK, Fitzgerald factor) and plasma prekallikrein accelerate these early steps in the clotting process. Questions have been raised about the role of HMWK in the activation of Hageman factor by surfaces. In the present studies, we report that the activation of purified human HF by sulfatides, ellagic acid, kaolin, or glass occurred in the absence of HMWK. Indeed, small amounts of HMWK inhibited activation of HF by ellagic acid. Physiological concentration of HMWK had little or no influence on the activation of HF by sulfatides, kaolin, or glass, but higher concentrations (3 to 6 times more) showed the same inhibitory effect as after activation by ellagic acid. This inhibitory property of HMWK may be attributed to competitive binding of HF and HMWK on the surface of the activating agents. In fact, when HF was added to kaolin or glass that had been incubated with HMWK and then washed, the inhibitory effect persisted, indicating HMWK that was bound to the surface blocked activation of HF. Studies with 125I-HF and 125I-HMWK supported this interpretation. Plasma prekallikrein accelerated activation of the amidolytic properties of HF by sulfatides, kaolin, or glass but did not influence activation of HF by ellagic acid. In the presence of plasma kallikrein, HMWK at moderate concentrations slightly accelerated the rate of activation of HF by activating agents other than ellagic acid. Higher concentrations of HMWK counteracted both the accelerating effect of prekallikrein and the inhibitory effect observed when HF was incubated with an excess of kaolin. These experiments, then, support the view that the procoagulant function of HMWK is localized to a point subsequent to the activation of HF.     

Influence of augmented Hageman factor (Factor XII) titers on the cryoactivation of plasma prorenin in women using oral contraceptive agents.

Prolonged cold storage of plasma may induce the conversion of plasma prorenin (inactive renin) to renin. This phenomenon is exaggerated in oral contraceptive (OC) users; the titer of Hageman factor (HF, Factor XII) in OC users is higher than in nonusers. The present study relates these observations. The increment in plasma renin activity (PRA) during cold storage, as measured by generation of angiotensin I, correlated strongly with the initial plasma titer of HF. Increasing the HF titer of nonusers to that observed in OC users by addition of purified HF increased cold-induced PRA at least twofold, while reducing the plasma HF titer of OC users correspondingly decreased cold-induced PRA. Thus, in OC users, the enhanced conversion of plasma prorenin to renin during cold storage reflects the elevated plasma titer of HF.