Introduction

Since 1991, the opinion on the role of factor XI in coagulation has changed from an intermediary between the contact system and factor IX to an important protease in both the coagulation and the fibrinolytic systems. This change was initiated by two publications that demonstrated the capability of thrombin to activate factor XI.^1,2 This activation pathway provided an explanation for an already longer existing clinical enigma. Patients deficient in factor XII, which should be the main activator of factor XI, do not suffer from a bleeding tendency, in contrast to factor XI-deficient patients who do have a mild hemorrhagic diathesis.^3,4 In the revised model of coagulation, factor XI is activated by thrombin which is formed by the extrinsic pathway. This activation pathway is one of the feed-back loops by which thrombin maintains its own formation after tissue factor pathway inhibitor (TFPI) blocks the tissue factor/factor VIIa-factor Xa complex.⁵ Later studies have shown that this additional formed thrombin is not only important for the generation of fibrin but also for the activation of a fibrinolysis inhibitor called thrombin activatable fibrinolysis inhibitor (TAFI). Activated TAFI downregulates the fibrinolytic system resulting in protection of the fibrin clot.⁶ In this Chapter we will discuss the role and molecular mechanisms of factor XI and TAFI activation with special attention to disseminated intravascular coagulation (DIC).

Biochemistry of Factor XI

Factor XI is a dimer, consisting of two identical subunits of 80 kDa connected by a single disulfide bond (Fig. 1). The plasma concentration is 3 to 7 μg/ml (20 to 40 nM), almost all of which circulates as a complex with high molecular weight kininogen (HMWK).⁷ Like most clotting factors, factor XI is a zymogen that requires proteolytic activation to become an active serine protease. The proteolytic activation is achieved by cleavage at the Arg 369-Ile 370 peptidyl bond, to yield two heavy chains of 50 kDa and two light chains of 35 kDa linked by disulfide bonds.^1,8

Figure 1

Structure of factor XI. Factor XI consists of two identical disulfide bridge linked polypeptide chains. Each chain consists of 4 apple domains and a catalytic site. The arrow identifies the site of cleavage during activation. (Reprinted with permission (more...)

The amino acid sequence and domain structure of factor XI have been elucidated in 1986.⁹ Each light chain contains an active site consisting of a catalytic triad of His-413, Asp-462 and Ser-557.¹⁰ Each heavy chain consists of four repeat sequences or apple domains called A1, A2, A3, A4 and is 58% identical to the corresponding region of prekallikrein.⁹ The A1 domain harbors the binding site for HMWK as well as the site for interaction with (pro)thrombin.^11–13 The A2 domain contains a site necessary for the Ca²⁺-dependent activation of factor IX by factor XIa,^14,15 although recent studies have shown that the binding site for factor IX is on the A3 domain.¹⁶ The A3 domain is capable of binding platelets in the presence of HMWK,¹⁷ and also contains a binding site for unfractionated heparin.¹⁸ The binding sites on factor XI identified for the interactions with factor IX and platelets are in close proximity to each other and that raises questions about factor XI binding simultaneously to platelets and factor IX via the same A3 domain. Recently, Sun et al suggested that because factor XI is a dimer it might bind to platelets via one A3 domain and uses the other A3 domain to interact with factor IX.¹⁹ Finally, the A4 domain binds factor XII and mediates the dimer formation between the two polypeptide chains.^20-22

Activation of Factor XI

Factor XI can be activated in vitro by factor XIIa, factor XIa and thrombin. During contact activation, factor XII becomes auto-activated upon binding to negatively charged surfaces where it can activate factor XI. HMWK is an important co-factor in this reaction assembling and stabilizing factor XI in a conformation facilitating its activation.²³ HMWK also forms a complex with prekallikrein, and the interaction of this complex with factor XIIa results in the formation of kallikrein, which in turn releases the nonapeptide bradykinin from HMWK.⁴ As was demonstrated in a baboon model of lethal E. coli septic shock, inhibition of factor XIIa activity en thereby the formation of bradykinin, prevented hypotension but not DIC,²⁴ strongly suggesting that factor XIIa is not involved in the activation of factor XI in DIC.

Thrombin-mediated factor XI activation and auto-activation of factor XI were first described on non-physiological surfaces like dextran sulfate. Thrombin can activate factor XI in the absence of co-factors or a surface (Fig. 2), but kinetic studies indicate that this reaction is too slow to be physiologically relevant.² Moreover, fluid-phase thrombin is inhibited by antithrombin within seconds while fibrin-bound thrombin is still active but protected from the inhibitory actions of antithrombin.^25,26 Therefore, the activation of factor XI by thrombin in vivo is probably a localized reaction which can take place on one of the various surfaces in the human vasculature, for example on the surface of the fibrin clot.

Figure 2

Activation of factor XI by thrombin. Factor XI was incubated with different concentrations of thrombin for 10 minutes. Thrombin was inactivated with hirudin and the amplification reaction was initiated by adding factors VIII, IX, and X. Aliquots were (more...)

Besides dextran sulfate also more physiological polyanions like glycosaminoglycans, which are present on the vascular subendothelium, have been demonstrated to act as co-factors in thrombin mediated activation of factor XI. Heparin at a concentration of 5 μg/ml induced a 60-fold increase in activation rate, while dermatan sulphate increased a 12-fold increase in factor XI activation.²⁷

Platelets can also serve as a physiological surface for thrombin-mediated factor XI activation. Factor XIa can bind to platelets in the presence of HMWK and retains full capacity to activate factor IX.²⁸ Furthermore, activated platelets promote thrombin mediated factor XI activation in the absence of factor XII at rates greater than obtained with dextran sulfate.^29,30

Questions were raised about factor XI activation by thrombin in a plasma-milieu since it was demonstrated that HMWK and fibrinogen can inhibit this reaction.^2,31 However, these experiments were performed using high concentrations of dextran sulfate as co-factor, which is a poor choice because dextran sulfate has an antithrombin effect in plasma. Furthermore, HMWK inhibits strongly in the presence, yet only slightly on the absence of dextran sulfate.³² In the presence of thrombin, fibrin rather than fibrinogen will be present in plasma and it was demonstrated that factor XI activation by thrombin is not affected by binding of thrombin to fibrin.³³

Factor XI activation in a plasma milieu has been demonstrated using gel electrophoresis, autoradiography and a detection system measuring change in turbidity as a measure of fibrin formation.^34,35 Since already less than 1% activation of factor XI can result, via the amplification system of the intrinsic pathway, in significant amounts of thrombin it is easier to measure factor XI activation indirectly. Using this indirect system of measuring factor XI activation, von dem Borne et al clearly demonstrated a role of factor XI not only in fibrin formation but also in fibrinolysis.³⁵ In a series of experiments it was shown that factor XI activation could contribute to formation of fibrin when coagulation was initiated with low concentrations of tissue factor. At higher tissue factor concentrations factor XI had no effect on the amount of fibrin formed. A key observation was that the presence of factor XI inhibited clot lysis, induced by adding tissue type plasminogen activator (t-PA), even at tissue factor concentrations at which factor XI had no effect on fibrin formation. It was demonstrated that the inhibitory effect of factor XI on fibrinolysis was caused by the generation of additional thrombin, formed after clot formation has taken place.³⁵ So, on the one hand only small amounts of thrombin provided by the extrinsic pathway were sufficient for clot formation, whereas on the other hand large amounts of thrombin formed by the intrinsic pathway were necessary for protecting the clot against lysis.

The intermediary between factor XI-dependent thrombin formation and fibrinolysis was later elucidated as a carboxypeptidase called thrombin activatable fibrinolysis inhibitor (TAFI).

Biochemistry of TAFI

The “discovery” of TAFI followed from efforts to find an explanation for the pro-fibrinolytic capacity of the anticoagulant protein activated protein C. Bazjar et al found that activated protein C is not intrinsically profibrinolytic but that by attenuating thrombin formation a fibrin clot becomes more susceptible to fibrinolysis.³⁶ When massive prothrombin activation occurs an antifibrinolytic activity was generated and the search for a thrombin activatable inhibitor of fibrinolysis started. The protein that was found was a zymogen, which could be activated by thrombin to an enzyme with carboxypeptidase B-like activity.³⁷ This enzyme had been described before by other investigators and had been named plasma carboxypeptidase B or carboxypeptidase U (unstable).^38,39 TAFI is a single-chain glycoprotein of about 60 kDa, synthesized by the liver, and circulates in human plasma at a concentration of 4–15 μg/ml (75 to 275 nM).^40,41 TAFI antigen levels are the same in men and women.⁴² TAFI is activated by cleavage at Arginine 92 by thrombin and activated TAFI (TAFIa) can inhibit t-PA induced fibrinolysis.⁴⁰ In normal individuals, the TAFI antigen levels correlated with TAFI activity.⁴¹ The activation of TAFI is strongly stimulated by the thrombin-thrombomodulin complex at an efficiency 1250-fold greater than by thrombin alone.⁴³ Thrombomodulin is a component of the blood vessel wall, but also soluble fragments of thrombomodulin are present in the circulation and both forms of thrombomodulin can potentiate the activation of TAFI.^44,45 Thrombomodulin binds thrombin and changes the specificity of thrombin from fibrinogen to protein C, resulting in an anticoagulant rather than a procoagulant activity. The existence of two substrates for the thrombin-thrombomodulin complex implies a dual function: anticoagulant by activating protein C and antifibrinolytic by activating TAFI. It seems that the local thrombomodulin concentration is an important factor in the regulation of these opposite effects.⁴⁶ Protein C and TAFI both require epidermal growth factor (EGF) domains 4,5, and 6 of thrombomodulin for efficient activation, but TAFI activation requires approximately 13 additional residues amino acids extra in domain 3.^47,48

TAFIa catalyzes removal of C-terminal lysine and arginine residues from fibrin. These sites on the fibrin monomers are generated as a result of partial cleavage by plasmin and are necessary to enhance binding of plasminogen to fibrin, which facilitates fibrinolysis (Fig. 3).⁴⁹ At higher concentrations TAFIa also directly inhibits plasmin.⁵⁰

Figure 3

Inhibition of fibrinolysis by TAFIa. Plasmin generates C-terminal lysine and arginine residues on fibrin monomers. These residues serve as plasminogen binding sites. Binding of plasminogen on these sites upregulates fibrinolysis. TAFIa downregulates fibrinolysis (more...)

No natural inhibitor of TAFIa has been described, but probably its intrinsic instability especially at body temperature is important for its down regulation in vivo.⁵¹

The Intrinsic Pathway and TAFI Activation

It is important to realize that the largest production of thrombin is after initial clot formation has taken place. The fibrin clot itself provides an environment in which the enzyme thrombin is partly protected against inhibition by antithrombin.²⁶

In whole blood systems and in systems working with purified components it has been demonstrated that the intrinsic pathway consisting of clotting factors XI, IX and VIII, is responsible for the major part of the total amount of thrombin formed.^52,53 Deleting factor VIII or IX from a reaction system with purified components had no effect on the initial rate of thrombin observed, but the propagation rate was only one third of that observed from a system including these intrinsic factors at plasma concentrations. The addition of factor XI to this experimental system increased the rate of thrombin formation by 15%.⁵²Using a clot lysis assay von dem Borne et al demonstrated that the mechanism of factor XI dependent inhibition of fibrinolysis is dependent upon TAFI.⁶ Inhibiting the activation of TAFI with a monoclonal antibody or the activity of factor XIa with a monoclonal antibody resulted in the same shortening of the lysis time. In plasma deficient of TAFI the factor XIa-inhibiting antibody had no extra effect on clot lysis (Fig. 4). This antifibrinolytic effect, which is TAFI dependent, has also been demonstrated for clotting factors IX and VIII, the other factors of the intrinsic pathway.⁵⁴ Using a very sensitive functional assay for TAFIa quantification in plasma of 13 individuals it was shown that on average 65% (range 35–89%) of the total amount of TAFIa formed is factor XI dependent.⁵⁵

Figure 4

Effect of TAFI on fibrinolysis in the presence or absence of a factor XIa inhibiting antibody. Plasma deficient in TAFI was preincubated with TAFI (), a monoclonal anti-factor XI antibody, called XI-1 (s), TAFI and XI-1 (D), or buffer (t). Coagulation (more...)

In Vivo Fibrinolysis Models

The antifibrinolytic role of activation of factor XI and TAFI has been studied in several animal models. Minnema et al have demonstrated in a rabbit thrombosis model that inhibiting factor XIa activity within the fibrin clot resulted in an almost two-fold increase in endogenous thrombolysis of jugular vein thrombi compared with controls.⁵⁶ Inhibiting TAFIa activity with a potato carboxypeptidase inhibitor also resulted in a two-fold increase of fibrinolysis and combined inhibition of TAFIa activity and factor XI activation did not further increase fibrinolysis (Fig. 5). In addition, inhibiting systemic factor XIa activity enhanced endogenous thrombolysis in this model suggesting that thrombin formation continues in a factor XI dependent way after initial clot formation has taken place and that this thrombin is capable of activating TAFI.

Figure 5

Effect of potato carboxypeptidase inhibitor on endogenous fibrinolysis. Endogenous fibrinolysis after incubation of the thrombi with an anti-factor XI antibody and/or potato carboxypeptidase inhibitor, an inhibitor of TAFIa activity. White bar represents (more...)

In a dog arterial thrombosis model, Redlitz et al demonstrated that during thrombolysis induced with t-PA, TAFI is activated and that there was a positive correlation with TAFIa activity and the time to restoration of blood flow in this model.⁵⁷ This was further explored by Klement et al in a rabbit arterial thrombolysis model.⁵⁸ The authors demonstrated in their thorough experimental set up that co-administration of potato carboxypeptidase inhibitor, as inhibitor of TAFIa, and t-PA significantly improved t-PA induced thrombolysis measured as a significant increase in original clot lysis, a decrease in the time to aortic patency and an increase in the duration of aortic patency. These results were achieved without side effects like enhanced bleeding. Inhibiting TAFIa activity may be the first clinical application in patients treated with t-PA because of acute arterial thrombosis.

In Vivo DIC Models

Only a few data reports have been published in abstract form about the role of TAFI in experimental models of DIC.^59,60 In these rat models, DIC was induced after administration of endotoxin or tissue factor. TAFIa activity was inhibited with a potato carboxypeptidase inhibitor or with DL-mercaptmethyl-3-guanidinoeththylthio-propanoic acid and the lungs or kidneys were excised and examined for fibrin deposition and compared with controls. Both inhibitors of TAFIa activity reduced the amount of fibrin deposition in the lungs or kidneys. However preliminary, these data imply that also in DIC TAFI is activated and that inhibiting TAFIa can reduce the amount of fibrin deposition.

In a human experimental model of DIC factor XIa activity could be detected already one hour after a single bolus injection of endotoxin to human volunteers (Fig. 6).⁶¹ The primary set up of this study was to examine the activation pathways of factor XI in vivo. Besides measuring factor XIa activity in an enzyme capture assay, activated factor XI complexed to its inhibitors was also measured. Thrombin activity was measured as thrombin-antithrombin complexes and with the prothrombin fragment F1+2. Activation of factor XII and prekallikrein was also measured using ELISAs. Using these sensitive assays no activation of the contact system could be demonstrated, while thrombin generation was detected after one hour to become maximal after 3 to 4 hours. Activation of factor XI persisted for more than 4 hours. This activation was accompanied by a small but significant decline in factor XI antigen levels after 2 hours, which returned to baseline at 24 hours. Using a computer simulation model it was calculated that the amount of activated factor XI generated was approximately 670 pM, which corresponds with 2 to 3% of the plasma concentration of factor XI.⁶¹

Figure 6

Activation of factor XI after endotoxin infusion in human volunteers. Mean ± SEM of factor XIa levels measured in the enzyme capture assay (m) and the total amount of factor XIa-factor XIa inhibitor complexes (l) after endotoxin infusion. The (more...)

Patients with Disseminated Intravascular Coagulation

Activation of factor XI has been measured in small patient groups with DIC. In one study factor XI antigen levels in 9 patients with suspected DIC were normal, while in another publication a patient with severe eclampsia and DIC had a marked reduction of factor XI clotting activity.^62,63 Because only small amounts of factor XI are activated in patients it is difficult to measure its activation. After activation factor XI becomes irreversibly complexed to a serine protease inhibitor. The most important inhibitors of factor XIa are C1inhibitor and α₁-antitrypsin. Most of the factor XIa becomes complexed to C1 inhibitor but because of the longer half-life time the factor XIa-α₁-antitrypsin complexes are also a good parameter to assess factor XI activation in clinical samples.^64,65

Using an improved ELISA which measures factor XIa-a₁-antitrypsin complexes Komiyama et al have shown activation of factor XI in 4 patients with DIC as high as 295 ng/ml (Å1,8 nM) which corresponds with activation of 5% of the total factor XI.⁶⁶ This was also observed by Wuillemin et al who found elevated factor XIa-α₁-antitrypsin complexes in 6 patients with DIC.⁶⁵ Moreover, in a group of 13 children with meningococcal septic shock and DIC, factor XIa-α₁-antitrypsin complexes were elevated in all patients and factor XIa-C1inhibitor complexes in 9 patients.⁶⁷

An increase in the TAFI plasma concentration is associated with an increased formation of activated TAFI by thrombin, since the Michaelis-Menten constant for TAFI activation by thrombin is far above the TAFI plasma concentration. In mice and rats, TAFI was identified as an acute phase reactant, which suggests that increased TAFI levels during inflammation contribute to the antifibrinolytic state that is characteristic for DIC.^68,69 Furthermore, plasma levels of soluble thrombomodulin fragments are elevated in patients with DIC and in vitro studies have shown that also soluble thrombomodulin fragments can stimulate TAFI activation.⁴⁵ However, since information of TAFI activation in patients with DIC is lacking, the role of TAFI in the pathofysiology of DIC remains to be investigated.

Conclusion

Although clinical data are not abundant, it is reasonable to state that factor XI is activated in patients with DIC. This activation is mediated by thrombin, which is generated through the extrinsic pathway. By activation of factor XI more thrombin will be formed and this thrombin is important in the activation of TAFI (Fig. 7). The significance of TAFI in the pathogenesis DIC is not clear and awaits further research.

Figure 7

Model of blood coagulation. The initiation of coagulation occurs with the formation of the tissue factor-factor VIIa complex (TF-VIIa). The intrinsic pathway is triggered when thrombin (IIa) is generated leading to the activation of factor XI. More thrombin (more...)

The role of the intrinsic pathway (factor XI, factor IX and factor VIII) as the pathway important for the maintenance of coagulation has started new research for interventions in thrombotic complications. Current antithrombotic strategies are aimed at factor Xa and/or thrombin and carry the risk of hemorrhage. Inhibiting activation of factor XI could probably result in clots that are less thrombogenic and more susceptible to fibrinolysis, without inhibiting tissue factor-dependent hemostasis. In a murine model of intracerebral stroke it was demonstrated that inhibiting factor IXa activity reduced microvascular fibrin deposition, increased cerebral perfusion and reduced the infarct volumes. This could also be achieved by giving t-PA or heparin to the mice but these interventions significantly enhanced intracerebral hemorrhage.⁷⁰ Especially in patients with DIC and high risk on bleeding these kind of highly tailored interventions may turn out to be very promising.

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