Spectrum of changes in endogenous thrombin potential due to heritable disorders of coagulation

The modern thrombin generation tests describe different phases of generation of thrombin that is initiation, amplification and inhibition of thrombin generation as well as the integral amount of generated thrombin. We investigated 55 patients with congenital deficiencies of different coagulation factors and analysed the relationship between the nature and the concentration of clotting factors, with different parameters of thrombin generation curve that is lag time, peak, time to peak and the area under curve or endogenous thrombin potential. The endogenous thrombin potential was unaffected by severe deficiency of factors XI and XII, and reduced in factor IX, VII and factor V and VIII deficiencies. The lag time was significantly prolonged in cases of severe factor VII, X and V deficiencies, and was almost normal in cases of factors VIII, IX, combined factors V and VIII, factor XI, XII and XIII deficiencies. The peak height was severely affected in cases of severe factor X, V, VIII and IX deficiency and combined deficiency of multiple vitamin K dependant coagulation factors, and significantly reduced in factor VIII, V, X, XIII and combined vitamin K deficiency.In all the patients with less than 40% thrombin generation, the clinical symptoms were severe. Bleeding symptoms were restricted to epistaxis and ecchymosis when thrombin generation was more than 90% of the normal. In the cases of combined deficiency of factors V and VIII all the values were intermediate as they exhibit mild deficiencies of both factors V and VIII and correlated well with the clinical symptoms. Endogenous thrombin potential of inherited isolated deficiencies of coagulation factors may thus provide an interesting insight about involvement of the deficient factor(s) at different phases of thrombin generation.     

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.     

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.     

Effect of DDAVP on plasma level of factor XII

The effect of DDAVP on blood coagulation factors was investigated after its intravenous infusion into normal subjects. A marked increase in factor XII was observed in addition to the expected rise of factor VIII coagulant activity (VIII:C), factor VIII related antigen (VIIIR:Ag) and plasminogen activator, DDAVP also produced a concomitant but less pronounced rise of factor VII, but there was no change in factors V, IX, X and XI.     

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 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.     

Cross-laboratory audit of normal reference ranges and assessment of ABO blood group, gender and age on detected levels of plasma coagulation factors.

We have assessed the influence of the ABO blood group, gender and age on plasma levels of the coagulation factors II, V, VII, VIII, IX, X, XI and XII as part of a quality audit of laboratory activities. There was no statistically significant difference in gender donor age (total normal donors: n = 406, mean/median age = 46.0/47.0 years, range = 16-77 years; females: n = 177, mean/median age = 44.7/46.0 years, range = 16-75 years; males: n = 229, mean/median age = 47.0/48.0 years, range = 17-77 years). With increasing age, we observed small but statistically significant rises (linear correlation; P < 0.01 for all parameters) in factors V, VII, VIII, IX and XI. With gender, we observed higher levels (P < 0.05) in females for factors II, VII, X, IX, XI and XII. With the ABO group, we observed lower levels in the O group (versus non-O group; P < 0.05) for factors VIII, IX and XII. We could therefore define differing normal reference ranges based on the differing study data. Study findings are compared with previously published literature, and this has identified a wide diversity in normal reference ranges both between different factors and between different studies. Finally, we also performed a cross-laboratory audit of peer laboratory practice and similarly show a wide diversity in normal reference ranges used between different laboratories.     

Changes in coagulation factors following acute myocardial infarction in man.

The whole blood clotting time, plasma fibrinogen and individual coagulation factors II, V, VII, VIII, IX, X, XI, and XII were serially measured in 14 patients over a 10-day period following acute myocardial infarction. A further six patients with similar symptoms but without evidence of myocardial infarction were also studied. The whole blood clotting time was significantly shorter within the first 24 h after infarction than on subsequent days. There were significant increases in the levels of fibrinogen and of factors VIII, IX, and XI and a significant decrease in factor XII in the patients who had sustained a myocardial infarction. The patients without myocardial infarction had no significant change in any of the coagulation factors measured.     

Assessment of Actin FS and Actin FSL sensitivity to specific clotting factor deficiencies

We present a two centre study designed to assess the sensitivity of Actin FS and Actin FSL to deficiencies of factor VIII, IX, XI or XII. The study was undertaken at two centres to avoid bias due to the investigations being undertaken on one analyser. Samples from patients with a factor VIII (n = 36, F VIII = < 1.0–50 iu/dl), factor IX (n = 22, F IX = 2–48 iu/dl), factor XI (n = 23, F XI = 5–50 u/dl) or a factor XII (n = 18, F XII = 1–50 u/dl) deficient state were studied. Activated partial thromboplastin times (APTT) were determined using two batches of Actin FS and of Actin FSL; comparison of APTT results between centres was facilitated by the conversion of clotting times to ratios (test ÷geometric mean normal clotting time). APTT ratios were considered to be elevated if greater than two standard deviations above the mean normal. The factor deficient status of each sample was verified by assaying all samples for factors VIII, IX, XI and XII. Clotting factor assays were performed on a Sysmex CA-1000^? fitted with research software, which permitted the auto-dilution and testing of three serial dilution of both a reference preparation and each patient's sample. Assay results were calculated using parallel-line Bioassay principles. This procedure allowed for variation in clotting times due to the effect of temporal drift of any of the reagents within the assay system. Actin FS and Actin FSL demonstrate acceptable sensitivity to factor VIII deficiency, however, both reagents failed to detect a large proportion of factor XI (17.4% and 30.4% of samples, respectively) and factor XII (66.7% and 72.2%, respectively) deficiencies. The detection rate with Actin FSL for factor IX deficiency was also poor (36.4% not detected). As factor IX and XI deficiencies are both associated with haemorrhagic disorders, the inability of these reagents to detect such abnormalities gave cause for concern.     

Effects of recombinant activated factor VII on thrombin-mediated feedback activation of coagulation

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (< 20 min), and rFVIIa addition had minimal effect, except for improved antifibrinolytic effect in factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase.     

Congenital Haemorrhagic Condition Similar but Not Identical to Factor X Deficiency

A 23-year-old white male with a bleeding tendency since early childhood presented a congenital coagulation defect similar but not identical to factor X deficiency. A first and second stage defect were demonstrated, characterized by a prolonged prothrombin time, prolonged partial thromboplastin time, abnormal thromboplastin generation, abnormal prothrombin consumption. The Stypven clotting time was slightly prolonged on fresh plasma but was normal on frozen plasma. Factors I, II, V, VIII, IX, XI, and XII were all within normal limits; factor VII was at the lower limits of normally or slightly decreased. Mr. Stuart's plasma failed to correct the defect of the patients plasma; however, a known factor VII deficient plasma was able to correct the abnormality. Factor X levels showed low (3–13%) only when assayed using tissue whole thromboplastin or tissue partial thromboplastin; the factor X assay using a Stypven-cephalin mixture yielded normal or near normal values. The factor II + factor X level using a Stypven-cephalin mixture appeared normal also. The significance of the findings is discussed. The results are tentatively interpreted as being due to an abnormal factor X rather than to a real deficiency.     

Liver transplantation: intraoperative changes in coagulation factors in 100 first transplants

Six intraoperative blood samples were obtained at intervals from each of 100 individuals undergoing their first liver transplants. The patients fell into the following diagnostic categories: postnecrotic cirrhosis 28, primary biliary cirrhosis 20, sclerosing cholangitis 19, miscellaneous diseases 14, carcinoma/neoplasia 12 and fulminant hepatitis 7. Coagulation factor values in the initial (baseline) blood samples varied by patient diagnosis. In general, all factor levels were reduced except factor VIII:C, which was increased to almost twice normal. The slight intraoperative changes in factors II, VII, IX, X, XI and XII suggested that a steady-state relationship existed between depletion (consumption/bleeding) and repletion (transfusion, transit from extra- to intravascular space), even in the anhepatic state. In contrast, there were rapid and very significant falls in factor VIII and fibrinogen and a less pronounced decrease in factor V, all reaching their nadirs in early to mid-Stage III. The cause of these coagulation changes appears to be activation of the fibrinolytic system.     

Screening coagulation tests and clotting factors in homozygous beta-thalassemia.

In 30 children with homozygous beta-thalassemia the hemostasis screening tests (bleeding time, PT, PTT), platelet count and specific assays of clotting factors were carried out 25 days after their last transfusion. PT, PTT, and bleeding time showed minor variations; considerable thrombocytosis was found in splenectomized patients. Factors IX and XII were decreased in a high proportion of patients, the vitamin K-dependent factors (II, VII, IX, X) were slightly reduced and factors I, V and VIII remained within the normal range in a majority of patients. Hepatic failure resulting in defective protein synthesis does not explain the more marked impairment of factors XI and XII, which might be secondary to activation of the intrinsic coagulation and/or kallikrein systems following intravascular haemolysis and multiple blood transfusions.     

Platelets and Initiation of Intrinsic Clotting

S ummary . Comparison of activities in platelet rich and platelet poor plasmas from normal donors and patients deficient in either factor VIII, IX, XI or XII indicates that platelets contain activities which can partially substitute for plasma factors XI and XII. The factor-XI-like activity is expressed in a one-stage activated partial thromboplastin assay and in an intact prothrombin consumption system. The factor-XII-like activity is scarcely detectable in a one-stage assay but markedly enhances the defective prothrombin consumption of factor XII deficient plasma. Intact prothrombin consumption tests with platelet poor plasmas fortified with cephalin show that in the presence of high concentrations of platelet factor 3 activity only trace contact activation is required to promote good prothrombin consumption. The platelet, by supplying both platelet factor 3 and activities bypassing plasma contact activation factors XI and XII, may provide an important route for activating intrinsic clotting.     

Inhibition of platelet prothrombinase activity by a lupus anticoagulant

Lupus anticoagulants are spontaneously occurring antibodies with specificity for negatively charged phospholipids. The plasma of a patient with such a polyclonal antibody of IgM type demonstrated low levels of factor VIII coagulant activity (VIII:C) and factors IX, XI and XII when analyzed by biologic clotting assays, whereas in immunochemical assays, normal levels of VIII coagulant antigen and factor IX were obtained. After immunoadsorption of patient plasma with anti-IgM Sepharose, normal biologic activities were demonstrated in clotting assays for VIII:C, factors IX, XI, and XII. The addition of the patient's isolated IgM to normal plasma resulted in grossly abnormal results in these coagulation assays, and a pattern similar to that of the patient's plasma was obtained. The inhibitory effect of the patient's lupus anticoagulant on blood coagulation was demonstrated also in platelet-rich plasma. The results of the clotting assays indicated that the anticoagulant inhibited several of the reactions in the blood coagulation cascade. The availability of purified components made it possible to demonstrate an inhibiting effect on the activation of prothrombin by factor Xa in the presence of isolated platelets, as well as in a system where purified factor V and well defined phospholipid vesicles were substituted for the platelets.     

Evolution of blood coagulation activators and inhibitors in the healthy human fetus

Blood coagulation proteins were determined in 285 healthy fetuses from 19 to 38 weeks' gestation and compared with those of 60 normal full- term newborns and 40 adult controls. Prolongation of the coagulation screening tests, prothrombin time, activated partial prothrombin time, and thrombin clotting time, in fetuses throughout intrauterine life was explained by low levels of vitamin K-dependent factors (II, VII, IX, and X), contact factors (XI, XII, prekallikrein, and high-molecular- weight kininogen), factor V, factor VIII, and fibrinogen. Low levels of antithrombin III, heparin cofactor II, protein C and protein S, and tissue factor pathway inhibitor were also found, and these probably contributed to a satisfactory hemostatic balance. Some of these parameters were evaluated by both immunologic and functional assays to detect possible "fetal" proteins. An increase in factor levels was observed after the thirty-fourth week of intrauterine life for most of the coagulation activators and inhibitors, but only factors V and VIII reached adult values at birth. This study therefore showed that fetal hemostasis is a dynamic system that evolves gradually toward the neonatal state and then toward the adult state.     

A distinct coagulopathy associated with Interleukin-2 therapy

Summary We studied the coagulation profiles of 14 patients with advanced malignancies treated with Interleukin-2 (IL-2). A 43% prolongation of the PTT (P<0.001) and a significant decrease in functional levels of factors II, IX, X, XI, and XII were observed 6 h post IL-2 treatment in comparison to pretreatment values. These parameters normalized within 2–3 d following IL-2 administration. The PT, factors V. VII, VIII, fibrinogen and D-dimer levels were unchanged with IL-2 treatment. This pattern of coagulopathy has not previously been reported.     

Stability of coagulation factors in thawed, solvent/detergent-treated plasma during storage at 4 degrees C for 6 days

BACKGROUND AND OBJECTIVES: Transfusion of fresh-frozen plasma is still a pillar in emergency medicine for using to prevent dilutional coagulopathy or disseminated intravascular coagulation after severe blood loss, but thawing procedures can delay its availability. On the other hand, the wastage of plasma, once thawed and not transfused within a defined time-period, represents an inefficient handling of economic resources and is contradictory to blood donor intentions. In this study we investigated the stability of coagulation factor activities and plasma protein levels during 6 days of storage of thawed solvent/detergent (S/D)-treated plasma at +4 degrees C. Our results may form the basis for reconsideration of expiry times of thawed S/D-treated plasma. MATERIALS AND METHODS: Five units of S/D-treated plasma (Octaplas) were thawed and warmed to 20 degrees C, then recooled and stored at +4 degrees C for 6 days. The activities of coagulation factors II, V, VII, VIII, IX, X, XI and XII, fibrinogen, antithrombin (AT), protein C, protein S and von Willebrand factor antigen (vWF:Ag) were measured on days 0, 1, 2, 3 and 6. RESULTS: Except for protein S, the activities of all coagulation factors and inhibitors were at least 0.5 U/ml during storage at 4 degrees C for 6 days. The mean levels, during storage, of factors IX, X, XI and XII, vWF:Ag, fibrinogen and protein C were at least 94%, and of factors II, V and VIII, and AT at least 78%, of the levels immediately after thawing; the activity of factor VII decreased to 83% and of protein S to 43% of the baseline values. CONCLUSIONS: Thawed S/D-treated plasma stored at +4 degrees C for up to 6 days still contains sufficient coagulation activities and plasma proteins to be regarded as suitable for transfusion in the established indications.     

Agarose gel method: its usefulness in assaying factor VIII inhibitors, evaluating treatment and suggesting a mechanism of action for factor IX concentrates

Plasmas from 14 patients with factor VIII inhibitors, 10 haemophiliacs and four non-haemophiliacs, were assayed by both the agarose gel and Bethesda methods. Good correlation was observed in 34 samples from 13 patients, but there was poor correlation in three samples from a single haemophilic patient. The sensitivity of the method was increased by diluting normal platelet rich plasma (PRP) with congenital factor VIII deficient plasma. With this modification, as little as 0.4 of a Bethesda unit (Bu) could be measured accurately. The agarose method is easier to perform and requires much less technician time than the Bethesda assay (Ba). Inhibitory activity can be measured even in the presence of large amounts of transfused factor VIII or factor IX concentrates. To study the effect of factor IX concentrates on the inhibitor, the method was modified by incorporating into the gels plasmas specifically deficient in either factor VII, IX or X. Our data suggest that factor VII is probably responsible for the 'bypassing' activity of factor IX concentrates.     

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.