The contact activation mechanism in human plasma: activation induced by dextran sulfate

Incubation of normal human plasma with dextran sulfate for 7 min at 4 degrees C generates kallikrein amidolytic activity. No kallikrein activity is generated in factor XII or prekallikrein-deficient plasma and only small amounts (8%) in high molecular weight (HMW) kininogen- deficient plasma. Addition of specific antisera directed against prekallikrein or HMW kininogen to normal plasma blocked the generation of kallikrein activity by dextran sulfate. Thus, factor XII, prekallikrein, and HMW kininogen are essential components for optimal activation of prekallikrein. The role of limited proteolysis in the activation of prekallikrein induced by dextran sulfate was studied by adding 125I-prekallikrein to plasma. The generation of kallikrein activity paralleled the proteolytic cleavage of prekallikrein as judged on SDS gels in the presence of reducing agents. The same cleavage fragments were observed as obtained by activation of purified prekallikrein by beta-factor-XIIa. Addition of 131I-HMW kininogen and 125I-factor XII or 131I-HMW kininogen and 125I-prekallikrein to normal plasma followed by activation with dextran sulfate and analysis on SDS gels indicated that the observed cleavage of prekallikrein and HMW kininogen is fast compared to the observed cleavage of factor XII, which is much slower and less extensive. During the first minutes of incubation of normal plasma with dextran sulfate, mainly alpha-factor- XIIa is formed. During prolonged incubation, beta-factor-XIIa is also formed.     

Structural and functional characterization of factor XII

In this article we have reviewed the current knowledge regarding the involvement of Factor XII in contact activation. Clearly in the past decade an overwhelming amount of data and hypotheses have been published regarding the central role of this zymogen in the initiation and further propagation of contact activation reactions. Therefore we feel that it will be helpful to conclude this article with a figure that summarizes those interactions and reactions that are generally believed to reflect the major molecular events occurring during surface-dependent contact activation. The contact factors are capable of very efficient interation with each other, provided a suitable negatively charged surface is present. Such surfaces are thought to stimulate the interactions between the contact factors through binding of the proteins and thus bringing the proteins together. Factor XII readily binds to the negatively charged surface, but for the binding of prekallikrein and Factor XI, the cofactor HMW kininogen is likely to be necessary. Bound at the surface, the zymogens Factor XII and prekallikrein are thought to be involved in a so-called reciprocal activation mechanism in which Factor XIIa activates prekallikrein to kallikrein, which in turn converts Factor XII to Factor XIIa. The formation of Factor XIIa is further promoted by the fact that surface-bound Factor XII is likely more susceptible to proteolytic cleavage and by the fact that the activated Factor XIIa is capable of auto-activating its own zymogen Factor XII. However, the latter effect, although undoubtedly contributing to the formation of Factor XIIa at the surface, seems to be of less importance than the reciprocal activation mechanism. This is underscored by the fact that Factor XII activation is rather slow in prekallikrein-deficient plasma. Surface-bound Factor XIIa is then responsible for the activation of Factor XI to Factor XIa, thereby propagating the initial trigger. Presumably, Factor XIa must leave the surface in order to be able to become involved in the activation of blood coagulation Factor IX.     

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.     

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.     

A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein- HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).     

Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII).

Factor VIII functions in the intrinsic pathway of coagulation as the cofactor for factor IXa proteolytic activation of factor X. Proteolytic cleavage is required for activation and may be responsible for inactivation of cofactor activity. To identify which of the multiple cleavages are required for activation and inactivation of factor VIII, site-directed DNA-mediated mutagenesis of the factor VIII cDNA was performed and the altered forms of factor VIII were expressed in COS-1 monkey cells and characterized. Conversion of arginine residues to isoleucine residues at the aminoterminal side of the cleavage sites at positions 740, 1648, and 1721 resulted in cleavage resistance at the modified site with no alteration in the in vitro procoagulant activity and the susceptibility to thrombin activation. Similar modification of the thrombin cleavage sites at either position 372 or position 1689 resulted in molecules with residual factor VIII activity but resistant to thrombin cleavage at the modified site and not susceptible to thrombin activation. Modification of the arginine to either an isoleucine or a lysine at residue 336, the site postulated for proteolytic inactivation by activated protein C, resulted in a factor VIII molecule with increased procoagulant activity. This increased activity may result from greater resistance to proteolytic inactivation. A model for the activation and inactivation of factor VIII is proposed.     

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.     

Studies of binding of prekallikrein and Factor XI to high molecular weight kininogen and its light chain.

Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW)-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 x 10(7) M-1 and 4.2 x 10(8) M-1, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMW-kininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity.     

Monoclonal antibody to human high-molecular-weight kininogen recognizes its prekallikrein binding site and inhibits its coagulant activity

We developed a mouse monoclonal antibody (MoAb 115-21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115-21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115-21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115-21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115- 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115-21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115-21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology.     

The kinin-forming enzyme system in pregnancy and obstetrical DIC

Levels of prekallikrein and HMW kininogen that had increased during pregnancy decreased with start of labor. The role of the kinin-forming system with oxytocin in the mechanism of labor was suggested from the results of decreased prekallikrein and HMW kininogen, appearance of a free kallikrein-like enzyme during labor, and from the case of arrested labor in which both prekallikrein and HMW kininogen were markedly decreased. Prekallikrein was markedly decreased in patients with acute obstetrical DIC and severe toxemia of pregnancy. The excessive activation of prekallikrein in DIC seemed to be of help for understanding such clinical signs as shock, abnormal labor, and increased permeability in obstetrical DIC.     

Fibrinogen blocks the autoactivation and thrombin-mediated activation of factor XI on dextran sulfate.

The intrinsic pathway of blood coagulation is activated when factor XIa, one of the three contact-system enzymes, is generated and then activates factor IX. Factor XI has been shown to be efficiently activated in vitro by surface-bound factor XIIa after factor XI is transported to the surface by its cofactor, high molecular weight kininogen (HK). However, individuals lacking any of the three contact-system proteins--namely, factor XII, prekallikrein, and HK--do not suffer from bleeding abnormalities. This mystery has led several investigators to search for an "alternate" activation pathway for factor XI. Recently, factor XI has been reported to be autoactivated on the soluble "surface" dextran sulfate, and thrombin was shown to accelerate the autoactivation. However, it was also reported that HK, the cofactor for factor XIIa-mediated activation of factor XI, actually diminishes the thrombin-catalyzed activation rate of factor XI. Nonetheless, it was suggested that thrombin was a more efficient activator than factor XIIa. In this report we investigated the effect of fibrinogen, the major coagulation protein in plasma, on the activation rate of factor XI. Fibrinogen, the preferred substrate for thrombin in plasma, virtually prevented autoactivation of factor XI as well as the thrombin-mediated activation of factor XI, while having no effect on factor XIIa-catalyzed activation. HK dramatically curtailed the autoactivation of factor XI in addition to the thrombin-mediated activation. These data indicate that factor XI would not be autoactivated in a plasma environment, and thrombin would, therefore, be unlikely to potentiate the activation. We believe that the "missing pathway" for factor XI activation remains an enigma that warrants further investigation.     

Effect of surfaces on fluid-phase prekallikrein activation

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.     

Activation of factor XII by dextran sulfate: the basis for an assay of factor XII

A system was developed for studying the activation of factor XII (Hageman factor) in the presence of dextran sulfate (DS). Salient features of the system included low ionic strength (0.08), low concentration of factor XII (approximately 1/10,000 that in normal plasma), and an excess of exogenous prekallikrein (PK). In this system, factor XII was rapidly converted to the 80,000 molecular weight (mol wt) form of factor XIIa (alpha-factor-XIIa). Once formed, the factor XIIa converted PK to kallikrein at a rate that was proportional to the amount of factor XII originally present in the incubation mixture. This system was used to construct a simple sensitive assay for factor XII in plasma and other biologic samples. The kallikrein produced was measured spectrophotometrically with the chromogenic substrate (H-D-Pro-Phe-Arg- p-nitroanilide (S-2302). This assay was shown to be independent of the high molecular weight kininogen and the PK content of the sample being analyzed. The measurements obtained were consistent with fundamental enzymologic principles and, if desired, could be processed with a simple calculator program to achieve linear standard curves. When applied to the quantitation of factor XII in plasma, the assay yielded values in close agreement with those determined by coagulant assay or by radial immunodiffusion.     

Activation of factor XI in plasma is dependent on factor XII [see comments]

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.     

Functional domains in the heavy-chain region of factor XI: a high molecular weight kininogen-binding site and a substrate-binding site for factor IX

To probe the molecular interactions of factor XI we have prepared two monoclonal antibodies (MoAbs; 5F7 and 3C1), each of which binds the heavy chain of reduced and alkylated factor XIa. Competitive solid phase radioimmunoassay (RIA) binding studies revealed that 5F7 and 3C1 are directed against different epitopes within factor XI. One antibody (5F7) blocked the surface-mediated proteolytic activation of factor XI and its binding to HMW kininogen, but had no effect on factor-XIa- catalyzed factor IX activation. The other antibody (3C1) is a competitive inhibitor of factor-IX activation by factor XIa, but blocked factor-XI binding to HMW kininogen only at 1,000-fold higher concentration than 5F7. Moreover, HMW kininogen had no effect on the kinetics of factor-XIa-catalyzed factor-IX activation. Furthermore, factor XI CNBr peptide fragments that bind to the 5F7 and 3C1 antibodies were isolated. The peptides that bound to the 5F7 antibody blocked the binding of HMW kininogen to factor XI but did not inhibit factor-XIa-catalyzed factor-IX activation. However, the peptides isolated by the 3C1 antibody inhibited factor-XIa-catalyzed factor-IX activation and had no effect on factor-XI binding to HMW kininogen. Our results indicate that distinct functional domains within the heavy chain region of factor XI are important for the binding of factor XI to HMW kininogen and for activation of factor IX by factor XIa.     

Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma.

Prekallikrein and high-molecular-weight kininogen were found associated in normal human plasma at a molecular weight of 285,000 as assessed by gel filtration on Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was 115,000. This prekallikrein could be isolated at a molecular weight of 285,000 after plasma deficient in high-molecular-weight kininogen was combined with plasma that is congenitally deficient in prekallikrein. Addition of purified 125I-labeled prekallikrein and high-molecular-weight kininogen to the respective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated by incubating prekallikrein with high-molecular weight kininogen. This study demonstrates that prekallikrein and high-molecular-weight kininogen are physically associated in plasma as a noncovalently linked complex and may therefore be adsorbed together during surface activation of Hageman factor. The complex is disrupted when these proteins are isolated by ion exchange chromatography.     

Plasma kallikrein/kinin system: a revised hypothesis for its activation and its physiologic contributions

Recent studies indicate that assembly of high molecular weight kininogen on its multiprotein receptor allows for prekallikrein activation. On endothelial cells, factor XII activation is secondary to prekallikrein activation and amplifies it. The immediate consequence of plasma prekallikrein activation is the cleavage of high molecular weight kininogen (HK) with liberation of bradykinin. Cleaved high molecular weight kininogen is antiangiogenic. Bradykinin stimulates tPA liberation and nitric oxide formation. In addition, formed plasma kallikrein promotes single-chain urokinase activation and subsequent plasminogen activation. Kininogens and their breakdown products also are antithrombins. The angiotensin converting enzyme breakdown product of bradykinin prevents canine coronary thrombosis. The author presents a new hypothesis for physiologic assembly and activation of the plasma kallikrein/kinin system and discusses its influence on vascular biology.     

Recombinant prolylcarboxypeptidase activates plasma prekallikrein

The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment.     

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.     

A different cleavage site for high molecular weight kininogen in vivo following intravenous injection of dextran sulfate in the rabbit

Purified radiolabeled rabbit Hageman factor, prekallikrein, and high molecular weight kininogen were used to examine Hageman factor system molecular dynamics after the intravenous injection of heparin-like dextran sulfate polymer in the rabbit. Hageman factor system proteins rapidly disappeared from the circulation following dextran sulfate injection, as measured by radial immunodiffusion, by kaolin-releasable kinin formation, and by measuring circulating levels of radiolabeled Hageman factor, prekallikrein, and high molecular weight kininogen. 125I-Hageman factor was distributed mainly to lung, liver, and spleen following dextran sulfate injection. Proteolysis of circulating 125I-Hageman factor occurred at a site within a disulfide loop into fragments of 50,000 and 30,000 molecular weight. Proteolysis of 125I-prekallikrein also occurred with visualization of a 50,000 molecular weight fragment. Although extensive proteolysis of 131I-high molecular weight kininogen was observed, the cleavage fragments were not the same as those generated during contact activation in vitro. The major fragment of high molecular weight kininogen observed in vivo was at 80,000 molecular weight, in contrast to the 65,000 molecular weight fragment generated by kallikrein in vitro. These results indicate that high molecular weight kininogen can undergo proteolysis in vivo into fragments not known to be associated with kinin release.