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.     

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.     

Comparative studies of measuring condition of activated partial thromboplastin time and contact factors in experimental animals

For establishing the optimal incubation time (OIT) for measurement of the activated partial thromboplastin time (APTT) in dogs, rabbits, guinea pigs, rats and mice, we determined the shortest clotting time of the plasma from each animal species and compared them with that of human plasma. The OIT for APTT determination was 15 to 30 sec in guinea pigs, rats and mice and 5 to 10 minutes in dogs and rabbits. The mouse APTT (about 30 sec) with the OIT thus determined was similar to human APTT, and relatively longer than APTT in other animal species (10-20 sec). To elucidate the mechanism of the species differences in OIT, we examined the plasma of each animal species for the activity of the contact factors such as factor XII, factor XI, high molecular weight kininogen (HMWK) and prekallikrein (PK) and their effect on the coagulation of contact factor-deficient plasma. The total activity of contact factors was higher in dogs and guinea pigs and lower in rabbits and mice than that in humans. Species difference with the factor XII, Factor XI and HMWK was noted in clotting time but not in OIT. These results suggest that the species difference in OIT for APTT is probably due to difference in activity of the plasma contact factors and in the mode of coagulation for each contact factor.     

Activated platelets but not endothelial cells participate in the initiation of the consolidation phase of blood coagulation

To address the question of whether initiation of the consolidation phase of coagulation occurs on platelets or on endothelium, we have examined the interaction of coagulation factor XI with human umbilical vein endothelial cells (HUVEC) and with platelets. In microtiter wells factor XI binds to more sites in the absence of HUVEC (1.8 x 10(10) sites/well, K(D) = 2.6 nm) than in their presence (1.3 x 10(10) sites/well, K(D) = 12 nm) when high molecular weight kininogen (HK) and zinc are present. Binding was volume-dependent and abrogated by HUVEC or Chinese hamster ovary cells and was a function of nonspecific binding of HK to the artificial plastic surface. Factor XI did not bind to HUVEC or to HEK293 cell monolayers anchored to microcarrier beads. Activation of HUVEC resulted in von Willebrand's factor secretion, but factor XI binding was not observed. Only activated platelets supported factor XI binding in the presence of HK and zinc (K(D) = 8 nm, B(max) = 1319 sites/cell). Activation of factor XI was observed in plasma in the presence of platelets activated by the thrombin receptor activation peptide but not with activated HUVEC. These results support the concept that activated platelets, but not endothelial cells, expose a procoagulant surface for binding and activating factor XI, thereby initiating the consolidation phase of coagulation.     

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.     

Effect of enzymes involved in the contact phase of blood clotting on the functional activity of neutrophils related to phagocytosis

The influence of the product of blood coagulation contact phase of factor XII and of its activated form on the reduction reaction of nitrobule terazolium by human neutrophils was investigated. In all the cases the product of blood coagulation contact phase induced substantial stimulation of neutrophils. The neutrophil reactions with factor XII were irregular. They were more regular and intensive after the activation of this factor. An assumption was made on the mediated influence of the product of blood coagulation contact phase on neutrophils through the activation of intercommunicated blood plasma enzyme systems.     

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.     

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.     

The influence of cytostatic treatment on the initiation of plasma coagulation in patients with neoplastic diseases

The above described changes in the haemostatic system in acute leukemias are well known and underlined by many authors [1, 5, 6, 9]. It should be stressed that the results of particular nonspecific hemostatic tests in some patients may be within the normal range in spite of significant alterations in the activity of some blood coagulation factors and the presence of hemorrhagic symptoms. In the observations of some authors the factor VIII level is distinctly increased in the majority of acute leukemic cases, whereas the vitamin K-dependent blood coagulation factors show a low activity in some patients [6, 9]. It is not easy to interpret the different behaviour of the factor XI and XII level especially before antileukemic treatment. In 3/4 of all studied cases the factor XI activity was low, whereas the factor XII level was high in 1/4 of patients above the normal range. It may be that there is a specific inhibitor against the factor XI that is produced in acute leukemia. It must be stressed that the level of factor XI shows normal values during hematological remission.     

Factor XIa dimer in the activation of factor IX

Factor XI, unlike other coagulation proteins, is a homodimer of two identical subunits linked by a single disulfide bond formed by Cys321. The present study was undertaken to understand the physiological significance of the dimeric nature of factor XI. We have expressed a mutant FXI/G326C in which the Gly326 residue of factor XI has been mutated to Cys326, reasoning that Cys321 would form an intrachain disulfide bond with Cys326 as in prekallikrein, a plasma protein that exists as a monomer even with 58% amino acid sequence identity and a domain structure very similar to factor XI. No free thiol could be detected in the expressed protein, and it migrated as a monomer on nonreduced SDS-PAGE. In physiological buffer, however, the protein was found to exist in a state of monomer-dimer equilibrium as assessed by gel-filtration chromatography and ultracentrifugation studies (K(d) approximately 36 nM). Functional studies revealed that FXI/G326C was indistinguishable from plasma factor XI in a plasma-clotting assay and in a factor IX activation assay both in the presence and absence of activated platelets even at concentrations at which less than 5% of the mutant exists as dimers. We conclude that, for optimal function in the presence of activated platelets, a preformed dimer of factor XI is not required.     

Kallikrein and prekallikrein levels in a large number of congenital clotting deficiencies and abnormalities

Kallicrein (K) and prekallicrein (PK) were assayed in a large number of cases with congenitial clotting factor defects. Patients with factor XII deficiency were separated from other clotting abnormalities. The results were compared with a control group of normal subjects. We found significantly reduced PK activity levels in the factor XII deficient group. Although less evident, the reduction of PK activity in the group of other clotting defects was modest, however, not due to a factor VII defect. In our study we found that in the absence of factor XII, PK is not activated. Further studies will be necessary to show if PK activation is altered or reduced in other congenital clotting abnormalities.     

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.     

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.     

Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa

To study the pathways for initiation of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as surfaces for factor XI activation by factor XIIa, factor XIa, or thrombin. Activated gel-filtered platelets promoted the activation of factor XI (60 nm) by thrombin (0.02-10 nm, EC(50) approximately 100 pm, threshold concentration approximately 10 pm) at initial rates 2- to 3-fold greater than those obtained with dextran sulfate in the presence of either high molecular weight kininogen (45 nm) and ZnCl(2) (25 micrometer) or prothrombin (1.2 micrometer) and CaCl(2) (2 mm). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nm.min(-1) with thrombin, 6 nm.min(-1) with factor XIIa and 2 nm.min(-1) with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nm) were 24-167 (mean = 86) min(-1) for thrombin, 4.6-50 (mean = 21) min(-1) for factor XIIa, and 1.3-14 (mean = 8) min(-1) for factor XIa. A physiological concentration of fibrinogen (9.0 micrometer) inhibited factor XI activation by thrombin (but not by factor XIIa) in the presence of dextran sulfate but not in the presence of gel-filtered platelets. Compared with factors XIIa and XIa, thrombin is the preferred factor XI activator, and activated platelets are a relevant physiological surface for thrombin-mediated initiation of intrinsic coagulation in vivo.     

Binding of coagulation factor XI to washed human platelets

The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces.     

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.     

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.     

Effect of heparin and ASA on changes of haemostasis induced by venous occlusion (author's transl)

The influence of venous occlusion on plasmatic coagulation, on platelets, and on fibrinolysis was examined. After occlusion, activated factors XI and X could be demonstrated. Simultaneously, platelet aggregation induced by both collagen and epinephrine was increased. Fibrinolysis was found to be moderately enhanced. In patients taking acetylsalicylic acid (ASA), platelet functions were not altered by occlusion but the activation of plasmatic clotting factors was not influenced. Low dose heparin inhibited plasmatic activation but had no influence on the increase of platelet activities. By simultaneous administration of both substances, an additive effect was observed resulting in inhibition of plasmatic and platelet activation due to venous occlusion.     

Effect of vitamin E on the function of blood platelets in patients with diabetes mellitus

An effect of vitamin E on blood platelets functioning was studied in 39 patients with diabetes mellitus type 1. Control group included 20 healthy blood donors. Vitamin E in a daily dose of 1000 mg produced statistically significant decrease in platelets aggregation, number of circulating platelet aggregates and release of the platelet factory 4 in diabetics after 7 days of treatment. No adverse reactions were seen in any patient treated with vitamin E. The obtained results indicate that vitamin E inhibits increased platelets activity in the patients with diabetes mellitus type 1 and does not exert toxic reactions during the treatment.     

Normal platelet adhesiveness and aggregation in congenital PTA or Hageman factor deficiency

Platelet adhesiveness and aggregation were studied in two patients with congenital factor XI deficiency and in a patient with congenital factor XII deficiency. A normal aggregation pattern was observed in every instance, regardless of the aggregating agent. The same was true for platelet adhesiveness. It is concluded that factor XI and factor XII play no role in platelet aggregation and adhesiveness.