The Relation of 'Fletcher Factor' to Factors XI and XII

S ummary . Further evidence is presented for the existence of a new coagulation factor which is closely related to Hageman factor (XII) and plasma thromboplastin antecedent, PTA (XI). This factor has been tentatively designated 'Fletcher factor'. Coagulant activity of Fletcher factor was separated from the clotting activity of factors XI and XII by C-M Sephadex column chromatography of intact normal plasma. Other studies showed that the prolonged partial thromboplastin time or plasma recalcification time of Fletcher-deficient plasma could be 'corrected' by prolonged contact with celite, glass, kaolin, or ellagic acid; all are known activators of factor XII. Cytochrome c, known to inhibit the contact activation of factor XII, completely abolished this contact 'correction' of Fletcher-deficient plasma. Thus, the clotting times of plasmas deficient in Fletcher factor (presently found in seven individuals from four unrelated families) are readily corrected by activated factors XII and XI. None of these individuals has any bleeding tendencies.
Fletcher factor activity is deficient in the plasma of newborn infants; the factor is probably produced in the liver and not dependent on vitamin K for its synthesis.     

Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.     

Changes in high molecular weight kininogen levels during and after cardiopulmonary bypass surgery measured using a chromogenic peptide substrate assay.

High molecular weight kininogen (HK) is a co-factor in the blood-contact activation system. A chromogenic peptide substrate assay for HK (HKcs) has been developed in which test plasmas are mixed with diluted HK-deficient plasma and incubated with a soluble contact system activator that activates prekallikrein and factor XII. Calcium chloride, a synthetic thrombin inhibitor and a chromogenic peptide substrate for activated factor X (FXa) are then added. The FXa generated cleaves the FXa substrate releasing p-nitroanaline, which is measured photometrically. Test plasma HK values were calculated from a standard curve generated using a pooled normal plasma. Acceptable intra-assay and inter-assay precision values were obtained and levels of HK up to 200% were measurable. The assay measured HK in plasmas deficient in factor XII, prekallikrein and factor XI, was not affected by antiphospholipid antibodies and gave an acceptable correlation (r = 0.95) when normal plasmas and mixtures of HK-deficient and normal pooled plasma, calculated to give HK levels of 25 and 50%, were compared using HKcs and a HK one-stage clotting assay. The HKcs was used to measure HK levels in seven patients undergoing cardiopulmonary bypass (CPB). HK levels fell significantly during CPB (P = 0.0014) and were significantly higher (P = 0.016) 6 days after CPB, suggesting that HK may be a positive acute-phase reacting protein.     

Pseudo-factor-XI deficiency: effect of an inhibitor of factor XI adsorption to surface

Recently we have described a normal plasma activity that modulates contact activation by inhibiting adsorption of factor XI to activating surfaces. Here we report the first identified case in which a patient has abnormal clotting tests due to an excess of a similar activity. The patient's plasma had a prolonged partial thromboplastin time and low apparent factor XI assay. His plasma prolonged the partial thromboplastin time of normal plasma and partially neutralized normal factor XI activity in vivo and in vitro. Analysis in dilute plasma revealed normal amounts of factor XI activity and antigen. Factor XI adsorption from plasma to activating surfaces was tested by adding a small amount of 125I-labeled purified factor XI to plasma, exposing the mixture to a glass tube or kaolin, and determining the amount of factor XI adsorbed to the surface. Whereas normal plasma and plasmas deficient in factor XII, factor XI, or Fletcher factor yielded about 4% adsorption to glass, factor XI adsorption from patient's plasma was less than 1%, indicating the presence of an adsorption inhibitor. This inhibitor did not affect factor XI activation or the activity of preformed factor XIa. It was not adsorbed by AI(OH)3 and was present in serum and the macroglobulin peak on gel filtration of the plasma through Sephadex G-200. The patient's history does not allow a definitive conclusion as to whether this inhibitor was associated with abnormal bleeding.     

A hitherto undescribed plasma factor acting at the contact phase of blood coagulation (Flaujeac factor): case report and coagulation studies.

This paper reports an asymptomatic coagulation defect responsible for an abnormality at the contact phase of blood coagulation in vitro, distinct from Hageman factor and Fletcher factor deficiencies. Coagulation studies in a 50-yr-old French woman without bleeding tendency revealed the following results: whole-blood clotting time in glass tubes and activated partial thromboplastin time with kaolin and ellagic acid were greatly prolonged; one-stage prothrombin was normal; no circulating anticoagulant was detected, and the infusion of normal plasma corrected the coagulation defect with an estimated half-life of 6.5 days; the levels of factor VIII, IX, XI, and XII were normal; mutual correction was obtained with a Fletcher factor-deficient plasma; the level of whole complement was normal. Studies of the contact phase of blood coagulation and contact-induced fibrinolysis showed the same abnormalities as in Hageman factor- and Fletcher-deficient plasmas. These results indicate that the patient's plasma is deficient in a previously undescribed coagulation factor, which participates in the initial stage of the blood coagulation process in vitro. Family studies revealed consanguinity in the propositus' parents. The assay of this newly described factor in the propositus' children revealed a partial defect, compatible with a heterozygous state, in three of the four tested children. This indicates a recessive inheritance of this new blood coagulation defect.     

Hereditary Giant Platelet Syndrome

The platelets from two related patients with the hereditary giant platelet syndrome were examined. They were larger than normal but otherwise ultrastructurally normal; they contained increased storage pools of adenine nucleotides and heparin-neutralizing activity and took up serotonin at an increased rate. They aggregated normally with ADP and collagen but failed to aggregate with bovine factor VIII and Ristocetin. Some change in shape occurred with ADP, and the reduction in adenylate energy change after addition of ADP to platelet-rich plasma was smaller than normal. Platelet coagulant activities including contact product forming activity, intrinsic factor-Xa forming activity and platelet factor 3 activity were normal or increased, but collagen-induced coagulant activity was absent whether tested in washed platelet suspensions or platelet-rich plasma. Platelet washing experiments showed decreased binding of factors V and VIII to hereditary giant platelets and no detectable factor XI in washed platelet suspensions. It is concluded that (1) the hereditary giant platelets studied lacked a binding mechanism for factors, V, VIII and XI; (2) the normal development of collagen-induced coagulant activity apparently depends upon the binding of factor XI to the platelet membrane; and (3) the defective prothrombin consumption observed in these patients may have resulted from the failure of their platelets to bind factor XI.     

Preparation, Characterization, and Activation of a Highly Purified Factor XI: Evidence that a Hitherto Unrecognized Plasma Activity Participates in the Interaction of Factors XI and XII

S ummary . Highly purified native factor XI has been prepared from normal plasma using a five step purification scheme. Purified factor XI is essentially free of factors II, V, VII, VIII, IX, X, XII, and Fletcher factor. No plasminogen-plasmin could be detected. Purified factor XI decays rapidly but can be stabilized with human serum albumin. Factor XI migrates between the β and γ globulins on starch block electrophoresis. It has an apparent molecular weight on gel filtration of 210 000. Purified factor XI is activated by weak trypsin. No detectable BAEe esterase activity accompanies this activation. Neither factor XII adsorbed to kaolin nor activated factor XII in solution could activate purified factor XI; both reagents activate factor XI in dilute factor XII deficient plasma. Hence, plasma appears to supply a third activity which facilitates the interaction of factors XI and XII. This activity is shown to be distinct from Fletcher factor.     

The Coagulant Activity of Platelets

When platelet-rich plasma is incubated for 16–20 hours at 37° C. and the platelets are then separated and washed, tissue-factor activity develops in these platelets. The active platelets accellerate the clotting of plasma samples deficient in Factor XII, XI, IX or VIII, and of normal plasma; the coagulant activity for samples deficient in Factors V, VII and X is much less marked. The activity developed will cause activation of Factor X in a serum eluate, whereas no such activity is evident in platelets separated from plasma immediately after collection from the donor.
The development of tissue-factor activity of platelets depends on the presence of Factor XII but not on the presence of any of the other factors tested. The relationship of tissue-factor activity to platelet factor 3 is discussed.     

Effects of treadmill exercise on platelet functions and blood coagulating activities in healthy men

The effects of treadmill exercise (up to 85% of the predicted maximum heart rate) on platelet functions and coagulating activities were studied in 26 normal men. Blood sampling for the measurements were performed from the antecubital vein at rest, at 3, 6, 9, and 12 min during exercise, immediately postexercise, and at 6 and 30 min after exercise. Measurements for blood analysis included the following: platelet sensitivity and percent aggregation to ADP, platelet counts, plasma thromboxane B2 and 6-keto-prostaglandin F1 alpha levels, plasma epinephrine and norepinephrine levels, plasma fibrinogen level, activity of plasma antithrombin III, and of plasma factors VIII, IX, XI, and XII. No significant changes were induced by dynamic leg exercise in platelet sensitivities and the maximum and 3-min percent aggregation. The platelet counts increased during exercise in platelet-rich plasma without a significant change in that in whole blood. During exercise, plasma thromboxane B2 levels showed a tendency to increase, while plasma 6-keto-prostaglandin F1 alpha levels to decrease. Plasma epinephrine levels showed a tendency to increase and norepinephrine levels increased during exercise. Among coagulating factors, factor VIII activities and fibrinogen levels increased without altering activities of factors IX, XI, and XII. Antithrombin III activities also increased during exercise. In spite of significant changes in several coagulating factors, prothrombin time and partial thromboplastin time were not influenced by exercise. In conclusion, dynamic leg exercise of a moderate to high intensity produced a significantly elevated plasma level of factor VIII, fibrinogen, antithrombin III, and catecholamines without affecting the hemostatic balance in normal subjects.     

Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation

Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2μM and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentration-dependent fashion but has no effect on tissue factor-induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn2(+), but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa-mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn2(+) will promote this interaction.     

A study of antihaemophilic globulin (factor VIII) consumption.

A study has been made of the rate of disappearance of factor VIII during the clotting of normal plasma samples and of samples deficient in factors V, VII, IX, X, XI and XII. The rate of disappearance (or consumption) of factor VIII was the same in the normal and factor VII deficient samples. Delayed disappearance of factor VIII was observed in samples deficient in factors V, IX, X, XI and XII. The significance of the findings to the theory of blood coagulation is discussed.     

The Effects of Collagen and Kaolin on the Intrinsic Coagulant Activity of Platelets. EVIDENCE FOR AN ALTERNATIVE PATHWAY IN INTRINSIC COAGULATION NOT REQUIRING FACTOR XII

Collagen and kaolin have been shown by other workers to initiate intrinsic coagulation by activating factor XII in plasma and to have complex effects on platelets. Because of the presence of collagen at sites of vascular injury there is good reason to believe that collagen has physiological importance in haemostasis. The present experiments were done to determine the effects of collagen and kaolin on platelets and to distinguish the platelet effects from the activity which these surface-active agents produce in plasma.
Using an albumin-density-gradient separation (ADGS) method for washing platelets free of loosely adsorbed coagulation factors, it is shown here that collagen can induce a coagulant activity in platelets which initiates intrinsic coagulation. This activity is independent of factor XII, provided factor XI is present. It is postulated that this collagen-induced coagulant activity of platelets provides an alternative pathway, by-passing factor-XII activation, for initiating intrinsic coagulation. The existence of this alternative pathway may provide an explanation for the absence of a haemostatic defect in Hageman trait. The effects of kaolin were similar to those of collagen, but kaolin had greater capacity to activate plasma factor XII and platelet factor 3 and relatively less capacity to activate platelet-associated factor XI.     

The activity state of factor VII in plasma: two pathways for the cold promoted activation of factor VII

The apparent amount of factor VII as determined in a one-stage test depends on the type of thromboplastin used: bovine thromboplastin only reacts with human factor VIIa whereas human thromboplastin interacts with unactivated human factor VII as well. Therefore the ratio factor VII activity as measured with bovine thromboplastin divided by the factor VII activity as assessed with human thromboplastin reflects the state of activation of factor VII in plasma. This approach was used to study the process of cold promoted factor VII activation and the involvement of different clotting factors therein. It could be shown that cold promoted activation does not occur in the absence of factors II and XII and is reduced for about 50% in factor IX deficient plasma. The other coagulation factors have a minor influence on the process. The results indicate that the cold promoted factor VII activation is the result of activation by both activated contact products and thrombin.     

Identifying hypocoagulable states with a modified global assay of overall haemostasis potential in plasma.

To test the sensitivity of the global assay of overall haemostasis potential (OHP) in detecting hypocoagulation, the OHP was assayed in plasma containing exogenous thrombin (0.04 IU/ml), tissue-plasminogen activator (330 ng/ml), Ca and a platelet reagent. Commercial plasmas with factor II, V, VIII, IX, X, XI, XII or VII deficiency were mixed with normal plasma in different proportions to imitate different severities. Samples from patients with haemophilia and factor XII deficiency were also examined. No clot was found in the absence of factor II/factor X, indicating that the tiny dose of thrombin worked solely as a trigger for the intrinsic pathway activation. Changed levels of the investigated coagulants, apart from factor XII, influenced the outcome. OHPs were decreased in patients with haemophilia but were unchanged or even increased in those with factor XII deficiency. This modified OHP method may therefore be useful for estimating the bleeding tendency in haemophilic patients and to find suitable doses and intervals for prophylactic treatment. It may also be of use in investigations of the effect of antifibrinolytic drugs as well as for identifying a thrombotic tendency in patients with factor XII deficiency. For detection of other coagulation factor deficiencies, our investigations with the commercial plasmas suggest that the OHP assay is also valuable, especially when the intrinsic pathway of the coagulation cascade is impaired.     

On the mechanism of inhibition of tissue factor pathway by the synthetic pentasaccharide during coagulation of human plasma.

The tissue factor (TF) pathway is preponderant in the initiation of blood coagulation in normal haemostasis and in thrombotic states. In the present study we investigated the mechanisms by which the synthetic pentasaccharide may influence the regulation of the TF pathway during clotting of human platelet poor plasma (PPP). Clotting of normal PPP or plasmas immuno-depleted of a single clotting factor (factor VII, factor XII, factor XI, factor IX, factor VIII, factor X, factor V, factor II) was initiated by triggering the TF pathway in the presence of fondaparinux (0.5 microg/ml). Activated factor VII (FVIIa) levels were measured in serum obtained at several time intervals after re-calcification of PPP. A clotting assay highly specific for FVIIa was used. The synthetic pentasaccharide inhibited the generation of FVIIa by 66%. The inhibitory effect of fondaparinux on FVIIa was completely abolished when antithrombin activity of plasma was inhibited by a specific antibody. Following the activation of TF pathway in plasmas depleted of factor X or factor IX, the inhibitory effect of fondaparinux on FVIIa generation was completely abolished, whereas it was not significantly modified when the other clotting factor-depleted plasmas were clotted. When fondaparinux was added in the serum, after the maximal generation of FVIIa, it inhibited by 20-30% the activity of the FVIIa-TF complex. These data suggest that fondaparinux enhances the antithrombin-dependent downregulation of the TF pathway by decreasing the generation of FVIIa via the inhibition of the generation and the activity of activated factor IX and activated factor X, and by inhibiting the activity of the FVIIa-TF complex.     

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.     

Effect of hemodialysis on contact group of coagulation factors,platelets, and leukocytes

Earlier reports have suggested possible activation and consumption of factor XII during hemodialysis. To investigate this possibility, a series of in vivo and in vitro experiments were conducted using different dialysis membranes and two different dialysates (acetate and bicarbonate). Factors XII and XI activities, factor XII concentration, and high-molecular-weight kininogen were measured. In addition, platelet count, white blood cell count, and hematocrit were monitored. Contrary to the previous reports, no discernible consumption of factor XII, factor XI, or high-molecular-weight kininogen was found irrespective of the type of membrane or the composition of the dialysate used. Transient leukopenia was noted with cellulosic membranes, whereas none occurred with polyachrylonitrile dialyzers. The composition of dialysate did not affect the white blood cell count during dialysis.     

Activation of intrinsic coagulation pathway in pre-eclampsia

Disseminated intravascular coagulation, thrombocytopenia, consumption of factors VIII and II, and antithrombin deficiency have been previously demonstrated in pre-eclampsia. However, the precise mechanism responsible for initiation of disseminated intravascular coagulation has not been elucidated. The present study documents activation of the intrinsic coagulation pathway in a patient with severe pre-eclampsia. The studies revealed marked reductions of plasma coagulant activities of all intrinsic pathway factors, i.e., XII, XI, IX, and VIII. In addition, the ratio of plasma factor XII activity to antigen concentration was markedly abnormal, and plasma high-molecular-weight kininogen concentration was diminished. It is suggested that activation of the intrinsic coagulation pathway may be operative in the genesis of disseminated intravascular coagulation in pre-eclampsia.     

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Effects on coagulation factor production following primary hepatomitogen-induced direct hyperplasia

AIM: To investigate the molecular mechanisms involved in coagulation factor expression and/or function during direct hyperplasia (DH)-mediated liver regeneration.METHODS: Direct hyperplasia-mediated liver regeneration was induced in female C57BL/6 mice by administering 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a representative hepatomitogen. Mice were weighed and sacrificed at various time points [Day 0 (D0: prior to injection), 3 h, D1, D2, D3, and D10] after TCPOBOP administration to obtain liver and blood samples. Using the RNA samples extracted from the liver, a comprehensive analysis was performed on the hepatic gene expression profiling of coagulation-related factors by real-time RT-PCR (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIIIβ, plasminogen, antithrombin, protein C, protein S, ADAMTS13, and VWF). The corresponding plasma levels of coagulation factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIII, and VWF) were also analyzed and compared with their mRNA levels.RESULTS: Gavage administration of TCPOBOP (3 mg/kg body weight) resulted in a marked and gradual increase in the weight of the mouse livers relative to the total body weight to 220% by D10 relative to the D0 (control) ratios. At the peak of liver regeneration (D1 and D2), the gene expression levels for most of the coagulation-related factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, XI, XII, XIIIβ, plasminogen, antithrombin, protein C, ADAMTS13, VWF) were found to be down-regulated in a time-dependent manner, and gradually recovered by D10 to the basal levels. Only mRNA levels of factor X and protein S failed to show any decrease during the regenerative phase. As for the plasma levels, 5 clotting factors (prothrombin, factors VIII, IX, XI, and XII) demonstrated a significant decrease (P < 0.05) during the regeneration phase compared with D0. Among these 5 factors, factor IX and factor XI showed the most dramatic decline in their activities by about 50% at D2 compared to the basal levels, and these reductions in plasma activity for both factors were consistent with our RT-PCR findings. In contrast, the plasma activities of the other coagulation factors (fibrinogen, factors V, VII, XIII, and VWF) were not significantly reduced, despite the reduction in the liver mRNA levels. Unlike the other factors, FX showed a temporal increase in its plasma activity, with significant increases (P < 0.05) detected at D1.CONCLUSION: Investigating the coagulation cascade protein profiles during liver regeneration by DH may help to better understand the basic biology of the liver under normal and pathological conditions.