Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI^-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI^-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI^-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.     

Activation of the contact pathway of blood coagulation on the circulating microparticles may explain blood plasma coagulation induced by dilution

Recent studies have shown that the contact activation of blood coagulation can be initiated on the surface of circulating microparticles–particles formed as a result of the activation or apoptosis of blood cells or endothelial cells. In the present work, by means of a mathematical model, we investigated the mechanism of the activation of contact pathway of blood plasma coagulation. The model describes membrane-dependent reactions of the activation of factors XII and XI with account of the presence of blood plasma inhibitors. All reactions were described by ordinary differential equations integrated by an implicit multistep method. The current mathematical model is based on our previous model of factor XII activation on the platelet surface. The initial model is modified by the addition of factor XI, kallikrein, and blood plasma inhibitors. We show that the amidolytic activity of the contact pathway factors associated with the microparticles is proportional to the concentration of microparticles. In previous studies, an increase in the overall solution amidolytic activity after the dilution of plasma was observed. Computational analysis of the contact pathway activation in the diluted plasma shows that the increase in the activation appears from the dilution of blood plasma inhibitors. Thus, a well-known experimental phenomenon of the hypercoagulability of plasma after dilution can be explained by an increased activation of the blood plasma coagulation through the contact pathway on the circulating microparticles. In addition, the computational analysis reveals that a rapid stop of the contact pathway activation on the microparticles observed in the experiments could be explained by the rapid depletion of the free activation surface.     

Control of human coagulation by recombinant serine proteases. Blood clotting is activated by recombinant factor XII deleted of five regulatory domains.

The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge. This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.     

Contact phase of blood coagulation is not activated in edema of high altitude

To examine whether bradykinin generated by the activation of the contact phase of blood coagulation is involved in the pathogenesis of edema occurring after acute exposure to high altitude, 15 mountaineers were examined at 490 m and 1, 3, and 5 days after arrival at 4,559 m. The clotting activity levels of factor XII, factor XI, plasma prekallikrein, and high-molecular-weight kininogen (HMWK) were measured, and plasma kallikrein-induced proteolytic cleavage of HMWK was assessed by ligand blotting by use of radiolabeled factor XI. After an ascent on foot from 1,170 to 4,559 m in 3 days, three subjects developed high-altitude pulmonary edema, and four subjects presented facial edema. There was no evidence for activation of the contact system in any subject as demonstrated by the lack of proteolytic cleavage of HMWK at high altitude. The absence of contact system activation was further supported by stable plasma levels of the individual factors of contact activation. Therefore, we conclude that bradykinin generated by plasma kallikrein-induced cleavage of HMWK is not involved in the pathogenesis of edema due to acute exposure to high altitude.     

Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.     

Contact phase of coagulation in diabetes mellitus after aspirin administration

The purpose of our study was to investigate the connection between platelets and the contact phase of coagulation. In 17 patients affected by diabetes mellitus we studied the behaviour of prekallikrein, factor XII and factor XI before and after aspirin administration. To evaluate the activity of aspirin we measured platelet production of thromboxane B2 using a radioimmunoassay. In our diabetic patients a hypercoagulable state was confirmed: PK, factor XII and XI levels were significantly higher as compared with normal controls. After aspirin administration a significant decrease of PK levels was found. After 7 days of aspirin administration and 7 days after stopping aspirin a modest but not significant improvement of factor XII and factor XI was observed. In conclusion, we believe that the contact phase of coagulation is another index of the hypercoagulable state in diabetes mellitus. In addition, the decrease of PK obtained by aspirin administration could support the possible connection between the contact phase of coagulation and platelets.     

Acceleration of surface-dependent autocatalytic activation of blood coagulation factor XII by divalent metal ions

The effect of divalent metal ions on the rate of dextran sulfate dependent autocatalytic activation of human blood coagulation factor XII was studied at pH 7.4 and 25 degrees C. Zn2+ and Cu2+, but not Co2+, increased the rate of factor XII activation induced by dextran sulfate with optimum effects at approximately 5 and 1 microM, respectively, while Ca2+ acceleration required much higher concentrations (millimolar). Further investigation of the effect of Zn2+ on factor XII activation demonstrated a complete dependence on the presence of dextran sulfate, lack of inhibition by soybean trypsin inhibitor, the appearance of alpha-XIIa as the primary reaction product, and reaction kinetics characteristic of an autocatalytic process. These results were consistent with Zn2+ affecting only the rate of surface-mediated factor XII autoactivation. The initial turnover velocity of dextran sulfate induced factor XII autoactivation increased linearly with factor XII concentration in the absence of Zn2+ up to 0.9 microM factor XII but showed saturation behavior over this same concentration range in the presence of 5 microM Zn2+, indicating that Zn2+ increased the reaction rate primarily by lowering the apparent Km. Comparison of the kinetics of autoactivation at mu = 0.15 and 0.24 revealed that the enhancement in the apparent kcat/Km brought about by Zn2+ increased from 19-fold to 520-fold, respectively, due to a differential dependence of the Zn2+-stimulated and unstimulated reactions on ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)

We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.     

Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

Procoagulant activity of collagen. Effect of difference in type and structure of collagen

Procoagulant activities of different types and structures of collagen were examined. Collagens used were types I (including its methylated and succinylated forms), II, III, IV and V. Each collagen was coated on an inner surface of a glass tube. The change of fluidity during coagulation of blood in the tube was measured by means of a new rheological technique. For monomeric collagen, the procoagulant activity of the succinylated form (negatively charged) was higher than that of the methylated form (positively charged). The procoagulant activity of type IV (dry) was lower than that of other types of collagen. For fibrillar collagens, the initiation of coagulation for type V (non-banded) was fairly delayed compared to those for types I, II and III (banded). An increase in water content in both monomeric and fibrillar forms promoted procoagulant activity. For most of the collagen forms, the addition of factor XII inhibitor (Polybrene) to blood brought about a remarkable delay of the initiation of coagulation, suggesting that the activation of factor XII on the collagen surface is one of main factors governing procoagulant activity. In addition, our data suggest that large numbers of adherent platelets to the collagen surface promote activation of the intrinsic coagulation system.     

Human annexin V binds to sulfatide: contribution to regulation of blood coagulation

Annexin V is a calcium-dependent phospholipid-binding protein that exhibits anticoagulant activity on binding to phosphatidylserine exposed on the activated surfaces of endothelial cells and platelets, inhibiting activation of factor X and prothrombin in the blood coagulation cascade. Sulfatide (galactosylceramide I(3)-sulfate), one of the glycosphingolipids of the platelet cell membrane, is thought to be involved in blood coagulation systems via activation of factor XII. In this study, we examined whether or not annexin V binds to sulfatide and affects the coagulant activity of sulfatide. Solid phase assaying of annexin V revealed that it binds specifically to sulfatide, i.e. not to galactosylceramide or gangliosides, in the presence of calcium ions. Affinity analysis by means of surface plasmon resonance showed that the K(D) of the interaction between annexin V and sulfatide is 1.2 micro M. Kinetic turbidometric assaying of plasma coagulation initiated by CaCl(2) revealed that the coagulation rate in the presence of sulfatide or phosphatidylserine was decreased by annexin V. These results suggest that annexin V regulates coagulability in the blood stream by binding not only to phosphatidylserine but also to sulfatide.     

Possible basis for the apparent surface selectivity of the contact activation of human blood coagulation factor XII

The activation of factor XII by the proteases factor XIIa and kallikrein is known to be greatly enhanced by certain negatively charged surfaces. Studies that compared factor XII surface binding to factor XII activation found that binding alone was insufficient to account for surface enhancement of the activation rate. The temperature dependence of the reaction showed unusual behavior that may be related to the conformational change of factor XII following binding; the rate of factor XII activation had a relatively low temperature optimum (0-47 degrees C) that was sensitive to choice of surface and salt concentration. In temperature studies, below 47 degrees C, the decrease in the activation rate was not related to the thermal denaturation of enzyme or substrate, nor to the choice of activator enzyme (factor XIIa or kallikrein), nor to the species of factor XII (human or bovine) but to a behavior, designated a thermal transition, associated with the surface or the protein-surface interaction. The previously reported surface selectivity of contact activation is possible due to the temperature characteristics and other properties of the thermal transition; a surface that has a low-temperature thermal transition and that is highly sensitive to salt will be a "poor" contact surface under the usual choice of reaction conditions (approximately 150 mM ionic strength and 37 degrees C). However, solution conditions were identified that allowed the following negatively charged surfaces to function, in nearly equal potency, in the activation of factor XII: phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol 4-phosphate, heparin, and 5-kDa dextran sulfate, as well as the previously characterized sulfatide and 500-kDa dextran sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of activation and autoactivation of human factor XII

The kinetics of the enzymic reactions that participate in the contact activation system of human plasma were examined. These reactions are potentiated by dextran sulfate, a negatively charged solute that mimics many of the effects of glass or kaolin on this system. The reactions of reciprocal activation, consisting of activation of factor XII by kallikrein and of prekallikrein by activated factor XII, follow Michaelis-Menten kinetics; values of kcat and Km for each of these reactions were determined in the presence of dextran sulfate and in its absence. In the presence of dextran sulfate, the catalytic efficiency for factor XII activation was increased 11 000-fold, and that for prekallikrein was increased 70-fold. Autoactivation of factor XII in the presence of dextran sulfate also follows Michaelis-Menten kinetics with kcat = 0.033 s-1 and Km = 7.5 microM. This finding supports the concept that autoactivation is an enzymic process, initiated by traces of activated factor XII which are invariably present in factor XII preparations. At prekallikrein and factor XII levels equal to those in plasma, reciprocal activation is approximately 2000-fold more rapid than autoactivation. Thus, reciprocal activation is the predominant mode of factor XII activation in normal plasma.     

The state of hemolysis under the effect of a noninfectious natural stressor with a deficit of insulin

An influence of animals immobilization with lack of insulin on the condition of systems depended on factor XII was investigated on the experiments under forty-five rabbits. It was determined that the immobilization causes deep violations in the "polysystem" of factor XII that was expressed in discordance of processes of coagulation, fibrinolysis, kininogenesis.     

Platelet-associated tissue factor contributes to the collagen-triggered activation of blood coagulation

The extravascular localization of tissue factor (TF), the central initiator of coagulation, is thought to ensure that thrombus formation is prevented in the intact vessel. We observed that during a 5-min stimulation of human blood with collagen (type I), TF antigen appeared on the surface of platelets adhering to leukocytes. The rapidly presented intravascular TF was competent to start the coagulation cascade. The isolated platelets from healthy donors contained appreciable amounts of the TF protein, while no TF antigen was detected in the neutrophils and rapidly isolated monocytes. Direct interactions with the neutrophils and monocytes were apparently necessary to activate the platelet-associated TF. This was most likely mediated by inactivation of tissue factor pathway inhibitor through leukocyte elastase. In summary, the leukocyte-elicited activation of the platelet TF participates in the rapid initiation of coagulation by collagen.     

Accelerating effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein

The effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine and human plasmas. The sulfatide-mediated activation of factor XII and prekallikrein in the presence of high-molecular-weight (HMW) kininogen was remarkably accelerated by 10(-5) M zinc ions. This accelerating effect was observed only in the presence of HMW kininogen. The kinetic analysis of the accelerating effect of zinc ions demonstrated that zinc ions reduce the Km values and increase the Vmax values on the activation of factor XII by kallikrein and on the activation of prekallikrein by factor XIIa. The value of Vmax/Km increased 26.4-fold in the former reaction and 2.8-fold in the latter reaction, indicating that zinc ions accelerate mainly the activation of factor XII by kallikrein. In the presence of 5 x 10(-4) M zinc ions, typical difference spectra due to a red shift of tryptophan and/or tyrosine residues were observed for HMW kininogen and its derivatives but not low-molecular-weight (LMW) kininogen. Since the concentration of zinc ions required to induce the difference spectra is comparable with that to enhance the activation of factor XII and prekallikrein, it appears that there is some correlation between the conformational change of HMW kininogen and the enhancement of the activation.     

Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade

Triggered self-activation of factor XII, a blood coagulation protease, was utilized for the amplified visual detection of ss-DNA targets in a non-sequence specific way. Factor XII holds potential as a low-affinity and therefore non-interfering probe for DNA secondary structure and for the screening of protein binding to ss-DNA. The observation that ss-DNA also accelerates coagulation of human blood plasma is relevant to the emerging field of aptamer therapeutics.     

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.     

A mosquito salivary protein inhibits activation of the plasma contact system by binding to factor XII and high molecular weight kininogen

The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their blood-feeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn(2+) ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn(2+) binding. We propose that hamadarin may attenuate the host's acute inflammatory responses to the mosquito's bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely.     

Factor XII in coagulation,inflammation and beyond

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe^−/− mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.