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 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 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.     

THE FIRST COMPONENT OF THE KININ-FORMING SYSTEM IN HUMAN AND RABBIT PLASMA : ITS RELATIONSHIP TO CLOTTING FACTOR XII (HAGEMAN FACTOR)

The isolation and characterization of the first component of the kinin-forming system in human and rabbit plasma are presented. Functionally, the molecule is the precursor of the activator of prekallikrein (Pre-PKA) and evidence is presented that it is identical with Hageman factor (clotting factor XII). The component from each plasma possessed similar characteristics. This molecule was found to have a mol wt of 110,000 and sedimentation rate of 4.6S. It migrated in electrophoresis as a β-globulin, having an isoelectric point of 6.1. Upon activation with glass, kaolin, diatomaceous earth, ellagic acid, or trypsin, the activated molecule converted purified prekallikrein (prokininogenase) to the active enzyme. Clot-promoting activity was associated with the capacity to activate prekallikrein through each procedure of isolation. The clot-promoting factor was in precursor form, requiring treatment with kaolin or trypsin to gain activity. Evidence indicated that the protein was Hageman factor (factor XII): it promoted clotting of factor XII-deficient, but not Factor XI- or IX-deficient plasma, and did not convert fibrinogen to fibrin it bound to and was activated by kaolin or other negatively charged particles in the presence of chelating agents; the activation by kaolin could be prevented by pretreating the kaolin with hexadimethrine bromide (H Br); prekallikrein-activating and clot-promoting activities were identical in their physical properties; and the prekallikrein activator could not be detected in Hageman factor-deficient plasma. Activation of Hageman factor was accompanied by cleavage of the molecule into several fragments, one of which possessed prekallikrein-activating (PKA) and clot-promoting properties. The PKA fragment sedimented at 2.6S and by gel filtration was found to have a molecular weight of 32,000. The PKA possessed only 1/50 the clot-promoting capacity of the freshly activated native molecule.     

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.     

The Binding and Cleavage Characteristics of Human Hageman Factor during Contact Activation: A COMPARISON OF NORMAL PLASMA WITH PLASMAS DEFICIENT IN FACTOR XI, PREKALLIKREIN, OR HIGH MOLECULAR WEIGHT KININOGEN

The ability of human Hageman factor (coagulation factor XII) to bind to a glass surface and its susceptibility to limited proteolytic cleavage during the contact activation of plasma have been studied using normal human plasma and plasmas genetically deficient in factor XI, prekallikrein, or high molecular weight kininogen (HMWK). When diluted normal plasma containing ¹²⁵I-Hageman factor was exposed to a glass surface for varying times, the Hageman factor was found to bind to the surface, and within 5 min became maximally cleaved from its native 80,000 mol wt to yield fragments of 52,000 and 28,000 mol wt. Hageman factor in factor XI-deficient plasma behaved similarly. In prekallikrein-deficient plasma, the binding of Hageman factor to the glass surface occurred at the same rate as in normal plasma but the cleavage was significantly slower, and did not reach maximum until 60 min of incubation. Cleavage of Hageman factor in HMWK-deficient plasma occurred at an even slower rate, with greater than 110 min of incubation required for maximal cleavage, although the rate of binding to the glass was again the same as in normal plasma. Normal rates of cleavage of Hageman factor were observed for the deficient plasmas after reconstitution with purified human prekallikrein or HMWK, respectively. These observations suggest that normal contact activation in plasma is associated with proteolytic activation of surfacebound Hageman factor.     

PREKALLIKREIN DEFICIENCY IN MAN

Blood plasma obtained from an individual with abnormal thromboplastin formation, due to deficiency of Fletcher factor, was fully corrected by 2% of normal, Hageman factor- or PTA-deficient plasma. It was also reconstituted by addition of highly purified human or rabbit prekallikrein. The plasma failed to generate kinin upon exposure to kaolin, a defect which was also corrected by addition of prekallikrein. Prekallikrein antigen was not detectable in this plasma. Fletcher factor-deficient plasma did not support the normal generation of PF/dil when dilute plasma was incubated in glass vessels and injected intracutaneously. Small quantities of Fletcher factor-deficient or Hageman factor-deficient plasma corrected the ability of the other to generate PF/dil. The formation of plasmin in dilute, acidified plasma incubated with kaolin was also abnormal in Fletcher factor-deficient plasma. Plasmin generation was normalized by addition of prekallikrein or small quantities of Hageman factor-deficient plasma. The data support the identity of Fletcher factor and prekallikrein.     

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.     

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.     

Fletcher factor deficiency. A diminished rate of Hageman factor activation caused by absence of prekallikrein with abnormalities of coagulation, fibrinolysis, chemotactic activity, and kinin generation

Fletcher factor-deficient plasma is deficient in prekallikrein and therefore generates no bradykinin upon activation with kaolin. It also possesses a diminished rate of kaolin-activable coagulation and fibrinolysis and possesses a defect in kaolin-activable chemotactic activity. These abnormalities are also corrected by reconstitution with purified prekallikrein. Addition of intact activated Hageman factor corrected the coagulation, fibrinolytic, and chemotactic defects and addition of Hageman factor fragments corrected the fibrinolytic defect and partially corrected the chemotactic defect; neither of these corrected the kinin-generating defect. Although the Hageman factor-dependent pathways appear to be initiated by contact activation of Hageman factor, the kallikrein generated activates more Hageman factor; this feedback is necessary for the Hageman factor-dependent pathways to proceed at a normal rate. It is the absence of this feedback in Fletcher factor-deficient plasma that accounts for the diminished rate of activation of Hageman factor and therefore a diminished rate of activation of the coagulation and fibrinolytic pathways. The ability of prekallikrein to correct the coagulation, fibrinolytic, kinin-generating, and chemotactic defects of Fletcher factor-deficient plasma is consistent with the identity of the Fletcher factor and prekallikrein.     

Inhibition of the activation of Hageman factor (factor XII) by soluble human placental collagens types III, IV, and V.

The initial step in the formation of thrombin via the intrinsic pathway is the activation of Hageman factor (factor XII). Some, but not all, studies have shown that this activation may be brought about by collagen. We examined the effect of three types of soluble human placental collagen on Hageman factor. Collagen types III, IV, and V did not appear to activate Hageman factor under the conditions tested. To the contrary, these collagen species inhibited activation of Hageman factor by glass or ellagic acid. These studies suggest that some types of collagen may play an inhibitory role in blood coagulation.     

The autoactivation of rabbit hageman factor

Proteolytic cleavage and activation of isolated, single chain, zymogen Hageman factor was observed in the presence of kaolin alone. The rate of cleavage of kaolin-bound Hageman factor was enhanced 50-fold by the presence of prekallikrein and high molecular weight kininogen. The two-chain 82,000 dalton form of activated Hageman factor (α-HF(a)) also cleaved kaolin- bound single-chain Hageman factor in a dose-dependent manner, yielding fragments of 28,000 and, 50,000 dahons under reducing conditions. Cleavage of kaolin-bound single-chain Hageman factor was not inhibited by preincubation with diisopropylfluorophosphate (12 mM) for 10 min, but long-term incubation of Hageman factor with diisopropylfluorophosphate (up to 48 h) resulted in inhibition of cleavage of kaolin-bound Hageman factor to an extent proportional to the inhibition of procoagulant Hageman factor activity. Hageman factor cleavage was maximal when the kaolin concentration was {approximately} 10-fold greater than the Hageman factor concentration (wt:wt), and was partially inhibited by high molecular weight kininogen. Kaolin-bound Hageman factor cleaved clotting factor XI in an amount which correlated with the extent of cleavage of the Hageman factor. These findings are compatible with the concept that single-chain Hageman factor and α- HF(a), are both capable of cleaving and activating kaolin-bound Hageman factor and that a close molecular association of kaolin-bound Hageman factor molecules is required for this reaction.     

Inhibition of Hageman factor activation

A method for studying inhibitors of the contact stages of blood coagulation is described. A number of positively charged substances were shown to inhibit the contact stages. The inhibitory substances include spermine, cytochrome c, ribonuclease, and lysozyme. The inhibitory effect of these substances was neutralized by the addition of an activated plasma thromboplastin antecedent, factor XI, (PTA) fraction. Other positively charged substances including protamine, hexadimethrine, polylysine, polyornithine, methylene blue, and ortho-toluidine blue also inhibited the contact stages of coagulation, but the inhibitory effect on coagulation was not neutralized by the activated PTA fraction. Negatively charged substances such as heparin and insulin did not inhibit the contact stages of coagulation.Cytochrome c inhibited Celite adsorption of a partially purified Hageman factor fraction, and cytochrome, ribonuclease, spermine, and lysozome inhibited the adsorption of Hageman factor from PTA-deficient plasma. Very much smaller quantities of Celite completely adsorbed Hageman factor from the fraction rather than from whole plasma, which suggested the possibility that plasma contains an inhibitor or inhibitors of Hageman factor adsorption.Furthermore cytochrome c, spermine, ribonuclease, and lysozyme inhibited the coagulant activity of the following activators of the Hageman and PTA factors: Celite, kaolin, sodium stearate, ellagic acid, and skin. It is suggested that negatively charged sites on these activators are critical for adsorption and activation and that inhibition results from neutralization of the negatively charged sites by the adsorbed inhibtor. Tests with polylysine polymers indicate that inhibitory activity is directly related to molecular size over the molecular weight range of 4000 to 100,000.     

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.     

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.     

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.     

Purification of High Molecular Weight Kininogen and the Role of This Agent in Blood Coagulation

Recent studies of individuals with high molecular weight (HMW) kininogen deficiency established the importance of this plasma protein for in vitro initiation of blood coagulation. In the present study, HMW-kininogen was highly purified from human plasma by monitoring its clot-promoting activity, using Fitzgerald trait plasma as a substrate. This preparation of HMW-kininogen revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mol wt: 120,000) and released 1% of its weight as bradykinin upon incubation with plasma kallikrein. HMW-kininogen specifically repaired impaired surface-mediated plasma reactions of Fitzgerald trait plasma, but did not affect those of Hageman trait and Fletcher trait plasma. Kinin release from HMW-kininogen by trypsin, but not by plasma kallikrein, resulted in total loss of clot-promoting activity. No inhibitors of coagulation were found when all kinin activity was removed from HMW-kininogen by trypsin. The roles of HMW-kininogen, Hageman factor (HF, Factor XII), plasma prekallikrein (Fletcher factor), and plasma thromboplastin antecedent (PTA, Factor XI) in blood coagulation were studied in a purified system. HMW-kininogen was absolutely required for activation of PTA by HF and ellagic acid. The yield of activated PTA was proportional to the amount of HF, HMW-kininogen, and PTA in the mixtures, suggesting that, to activate PTA, these three proteins might form a complex in the presence of ellagic acid. No fragmentation of HF was found under these conditions. In contrast to HF, HF-fragments (mol wt: 30,000) activated PTA in the absence of HMW-kininogen and ellagic acid. Thus, it appears that in the present study PTA was activated in two distinct ways. Which pathway is the major one in whole plasma remains to be determined.     

The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

Hageman factor (factor XII) is activated by exposure to surfaces such as glass or by solutions of certain compounds, notably ellagic acid. Changes in the structure of Hageman factor accompanying activation have been examined in this study by circular dichroism spectroscopy. The spectrum of unactivated Hageman factor in aqueous solutions suggests that its conformation is mainly aperiodic. Various perturbants altered the conformation of Hageman factor in differing ways, demonstrating the sensitivity of Hageman factor to its environment.After activation of Hageman factor with solutions of ellagic acid, a negative trough appeared in the region of the circular dichroism spectrum commonly assigned to tyrosine residues, along with other minor changes in the peptide spectral region. Some of these changes are similar to changes that occurred upon partial neutralization of the basic residues at alkali pH. Activation of Hageman factor by adsorption to quartz surfaces (in an aqueous environment) also produced changes similar to those in the ellagic acid-activated Hageman factor, including the negative ellipticity in the tyrosine region.These observations suggest that the activation process may be related to a change in status of some of the basic amino acid residues, coupled with a specific change in the environment of some tyrosine residues. The importance of these changes during the activation process remains to be determined. The sensitivity of Hageman factor to its environment is consistent with the view that the initiation of clotting by exposure of plasma to appropriate agents is brought about by alterations in the conformation of Hageman factor that occur in the apparent absence of Fletcher factor or other recognized clotting factors.     

Studies on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation of Hageman factor and additional cofactors

As demonstrated by others, fibrinolytic activity was generated in diluted, acidified normal plasma exposed to kaolin, a process requiring Hageman factor (Factor XII). Generation was impaired by adsorbing plasma with glass or similar agents under conditions which did not deplete its content of Hageman factor or plasminogen. The defect could be repaired by addition of a noneuglobulin fraction of plasma or an agent or agents eluted from diatomaceous earth which had been exposed to normal plasma. The restorative agent, tentatively called Hageman factor-cofactor, was partially purified by chromatography and had an apparent molecular weight of approximately 165,000. It could be distinguished from plasma thromboplastin antecedent (Factor XI) and plasma kallikrein, other substrates of Hageman factor, and from the streptokinase-activated pro-activator of plasminogen. Evidence is presented that an additional component may be needed for the generation of fibrinolytic activity in mixtures containing Hageman factor, HF-cofactor, and plasminogen.The long-recognized generation of plasmin activity in chloroform-treated euglobulin fractions of plasma was found to be dependent upon the presence of Hageman factor. Whether chloroform activation of plasminogen requires Hageman factor-cofactor was not determined, but glass-adsorbed plasma, containing Hageman factor and plasminogen, did not generate appreciable fibrinolytic or caseinolytic activity.These studies emphasize the complex nature of the mechanisms which lead to the generation of plasmin in human plasma.     

Activation of Rabbit Hageman Factor by Homogenates of Cultured Rabbit Endothelial Cells

Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 M(r) form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.