The responsiveness of different APTT reagents to mild factor VIII,IX and XI deficiencies

Introduction: The sensitivity of APTT reagents to deficiencies of factors VIII, IX, XI and XII varies because of their composition. The APTT is used as a screening test for these factors, and a deficiency should manifest with a prolongation to the APTT, which may trigger the need for specific factor assays to be performed. Methods: The suitability of APTT reagents to detect mild deficiencies can be assessed by the analysis of the APTT of plasma, which has an increasing concentration of the factor in question. The APTT responsiveness can be determined from the intersection of the curve and the upper limit of the APTT normal reference range for that APTT reagent. We assessed the APTT responsiveness (in U/dl) to factors VIII, IX and XI of four APTT reagents; Actin FS (Siemens), Synthasil (IL), STA‐PTTA (Stago) and Dapttin (Technoclone). Results: Actin FS was the most sensitive reagent to mild reductions of factors VIII, IX and XI [Correction added on 26 October 2010, after first online publication: Synthasil was corrected to Actin FS]. STA‐PTTA showed less sensitivity than Synthasil and Actin FS; Dapttin was insensitive to mild deficiencies of factors IX and XI and should not be used as a screening test. Conclusion: Both Synthasil and Actin FS are acceptable reagents to screen for reduced factors VIII, IX and XI, and the number of mildly reduced factors not diagnosed will be limited.     

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.     

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.     

A comparison of two APTT reagents which use silica activators

The Ortho activated partial thromboplastin time (APTT) reagent, Thrombosil 1 (TS), was compared to the General Diagnostics automated APTT reagent (GD). TS produced more precise results over a 38-day period of testing a normal control plasma, indicating that the upper limit of the normal range could be more precisely set with TS. This normal range was better represented if the normal values with both reagents were logarithmically transformed before calculating the mean +/- 2 SD. TS was more sensitive to plasma which had been heparinized in vitro. This was also demonstrated in vivo by the testing of 100 plasmas from heparinized patients. On testing of in-vitro dilutions of normal plasma with factor-deficient plasmas, TS was more sensitive to decreasing levels of factors VIII, IX and XI but less sensitive to decreasing factor XII. This was demonstrable in vivo in 71% of cases with plasmas from factor-deficient patients. GD was more sensitive to the lupus anticoagulant in most cases.     

Activation of Hageman factor in the nephrotic syndrome

The patient described had the nephrotic syndrome associated with decreased levels of plasma coagulation factors XI (35 per cent) and XII (15 per cent). The patient also had a decrease in concentration of prekallikrein and kallikrein inhibitor, suggesting that the kallikrein system was activated. Addition of purified factor XII did not correct this defect. The fibrinolytic system was activated as indicated by an increase in fibrinogen split products. Thus, it seems that three Hageman-dependent proteolytic pathways (coagulation, fibrinolysis and kallikrein) were activated in this patient with the nephrotic syndrome.Another possible cause of decreased factors XI and XII is urinary loss of these proteins. The urine did contain apparent activities of factors XI and XII. The finding of factor VIII in the urine in higher concentrations than XI or XII, however, as well as the inability to adsorb the activity with Celite®, suggested that the activity was due to a nonspecific urinary procoagulant. This hypothesis was confirmed by removal of the activity via adsorbtion of the urine with barium citrate.     

Acquired inhibitors to the coagulation factor XII associated with liver disease

Three patients with liver disease and prolonged activated partial thromboplastine time (APTT) on routine tests are presented. One woman had metastatic liver disease from gastric carcinoma, a second woman had autoimmune hepatitis, and one man had severe chronic hepatitis B. APTT was not corrected after mixing experiments with 25%, 50%, and 75% of normal pool plasma, indicating the presence of an acquired inhibitor. In all three cases, factor XII coagulant activity was reduced: <1%, <1%, and 3%, respectively, while all of the other coagulation factors were normal. In all three cases no other auto-antibody was detected. In the first patient, APTT was normalized after a left liver lobectomy, whereas the primary lesion remained unresected. In the second patient, the FXII activity was improved after corticosteroid therapy but never returned to normal values. In the third patient, the APTT was improved after hydroxychloroquine therapy. None of the patients had hemorrhagic or thrombotic phenomena.     

Coagulation abnormalities in type 1 Gaucher disease are due to low-grade activation and can be partly restored by enzyme supplementation therapy

In type 1 Gaucher disease a bleeding tendency occurs which is partly caused by thrombocytopenia due to massive splenomegaly. In addition, low levels of factors IX and XI have been described. The mechanism responsible for these clotting factor abnormalities is unknown.
We performed a detailed study of parameters of coagulation and fibrinolysis in 30 type 1 Gaucher disease patients (14 splenectomized) before and after treatment with enzyme supplementation therapy. Pre-treatment aPTT and PT were prolonged in 42% and 38% of patients, respectively. In 30–60% serious deficiencies (>50%) of coagulation factors XI, XII, VII, X, V and II were observed. The low levels of factor V correlated with platelet count and were inversely associated with splenic volume. Levels of inhibitors were mildly decreased. Markers for activation of coagulation (thrombin–antithrombin (TAT) complex) and fibrinolysis (PAP complex, fibrin cleavage product D-dimer) were significantly elevated, especially in the splenectomized patients, indicating ongoing activation of these processes. After 12 months of enzyme supplementation therapy partial correction occurred.
Thus, severe disorders of the coagulation system occur in Gaucher disease, contributing to the bleeding tendency. The deficiencies may be the result of consumption of coagulation factors caused by ongoing low-level coagulation activation, possibly due to mononuclear cell activation.     

Lupus anticoagulant-hypoprothrombinemia syndrome with a false-positive test for coagulation factor inhibitors

We report a case of a 1-year-old boy diagnosed with lupus anticoagulant-hypoprothrombinemia syndrome (LA-HPS), which is a rare disorder. His initial presentation of sinusitis was accompanied by hemorrhagic episodes including ecchymoses and epistaxis 6 months after antibiotic therapy. Laboratory results revealed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) that did not correct with mixing studies. Factors II, VIII, IX, X, XI, and XII activities were 20%, 44%, 42.5%, 59%, 4%, and 10%, respectively. The Bethesda inhibitor assay showed inhibitors against multiple coagulation factor. APTT, mixing studies, diluted Russell's viper venom time, and the Bethesda inhibitor assay detected LA. LA-HPS with a suspected false-positive test for coagulation factor inhibitors was diagnosed. Bleeding stopped and results of coagulation studies returned to normal without therapy 2 months after onset of the disease.     

Fibrin formation, fibrinopeptide A release, and platelet thrombus dimensions on subendothelium exposed to flowing native blood: greater in factor XII and XI than in factor VIII and IX deficiency

Fibrin deposition and platelet thrombus dimensions on subendothelium were studied in four groups of patients with coagulation factor deficiencies. Five patients with factor VIII deficiency (APTT 120 +/- 8 sec) and three patients with factor IX deficiency (APTT 125 +/- 11 sec) were severe bleeders, whereas four patients with factor XII deficiency and seven with factor XI deficiency were either asymptomatic or only mild bleeders despite APTT values of 439 +/- 49 and 153 +/- 13 sec, respectively. Everted segments of deendothelialized rabbit aorta were exposed at a shear rate of 650 sec(-1) for 5 and 10 min to directly sampled venous blood in an annular chamber. Blood coagulation was evaluated by measuring fibrin deposition (percent surface coverage) on the subendothelium and post-chamber fibrinopeptide A levels; platelet thrombus dimensions on the subendothelium were evaluated by determining the total thrombus volume per surface area (using an optical scanning technique) and the average height of the three tallest thrombi. Consistent differences were observed among the patient groups for both the 5-min and 10-min exposure times. The larger of the 5- and 10-min exposure-time values was used to calculate group averages. Fibrin deposition in normal subjects was 81% +/- 5% surface coverage, and post- chamber fibrinopeptide A values were 712 +/- 64 ng/ml. Markedly decreased fibrin deposition and fibrinopeptide A levels were observed in factor VIII deficiency (2% +/- 1% and 102 +/- 19 ng/ml) and factor IX deficiency (11% +/- 7% and 69 +/- 11 ng/ml). In contrast, significantly higher values were obtained in patients deficient in factor XI (33% +/- 5% and 201 +/- 57 ng/ml) and factor XII (66% +/- 12% and 306 +/- 72 ng/ml). Differences in thrombus dimensions were also observed. In normal subjects, the value for thrombus volume and average height of the tallest thrombi were 8.3 +/- 1.3 cu micron/sq micron and 145 +/- 11 micron, respectively, and in patients were as follows: FVIII, 2.7 +/- 0.6 and 71 +/- 7; FIX, 4.5 +/- 1.8 and 88 +/- 14; FXI, 11.8 +/- 1.9 and 125 +/- 10; and FXII, 7.9 +/- 3.1 and 130 +/- 25. Platelet thrombus dimensions were normal in a patient with fibrinogen deficiency, indicating that the smaller thrombi in factor VIII and factor IX deficiencies were probably due to impaired evolution of thrombin rather than diminished fibrin formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel congenital haemostatic defect: combined factor VII and factor XI deficiency.

Isolated deficiencies of factors VII and XI are both rare. Not surprisingly, therefore, combined factor VII and XI deficiency has not been reported previously. We report here a kindred with a combined heterozygous deficiency for both factors VII and XI. The proposita is a 28-year-old woman who had both a prolonged prothrombin time (PT) and a prolonged activated partial prothrombin time (APTT) associated with a mild bleeding tendency. Coagulation studies were performed on the six available members of this kindred. The PT and APTT were normal or mildly abnormal in five of these individuals. Factor VII coagulant activity (VII:C) varied from 0.33 to 0.77 units/ml in affected subjects. In contrast, the concentration of factor VII-related antigen for the six individuals ranged from 0.68 to 2.10 units/ml. Comparable factor VII:C levels were obtained when each subject's plasma was tested with either a rabbit or a human thromboplastin reagent. Factor XI coagulant activity was less than 0.5 units/ml in three of the six subjects and normal (approximately 1.0 units/ml) in the other three. The concentrations of thrombin-antithrombin-III and prothrombin fragment 1.2 were within normal limits for all individuals. In addition to being associated with heterozygous factor XI deficiency, the abnormal factor VII molecule in the plasma of affected individuals in this kindred appears to represent a newly described mutation. This is suggested by the pattern of reactivity with thromboplastin from different species, the normal tissue factor binding and the bleeding tendency in heterozygous individuals in this kindred.     

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.     

Lupus anticoagulant in myasthenia gravis associated with IgM gammopathy

The plasma of a patient with myasthenia gravis had strong lupus anticoagulant activity and his IgM paraprotein displayed non-specific inhibition to coagulation factors IX, XI, XII, prekallikrein, and high molecular weight kininogen. He was placed on prednisolone, which resulted in improvement in his myasthenic symptoms, but the prolongation of APTT and macroglobulinemia remained. Double filtration plasmapheresis successfully decreased the serum IgM level from 1,190 mg/dl to 375 mg/dl and APTT improved from 58 s to 38 s. Myasthenia gravis is frequently associated with other autoimmune diseases, but the association with lupus anticoagulant and IgM gammopathy is rare.     

Relationship between short activated partial thromboplastin times, thrombin generation, procoagulant factors and procoagulant phospholipid activity

Short activated partial thromboplastin times (APTTs) are associated with thrombosis. However, what short APTTs actually represent in terms of possible mechanistic pathways is not well characterized. We have assessed thrombin generation as compared with levels of procoagulant factor (fibrinogen, V, VIII, IX, XI and XII) activities, von Willebrand factor level and activity using collagen binding, as well as procoagulant phospholipid activity, in 113 consecutive samples exhibiting a short APTT compared with an equal number of age-matched and sex-matched samples yielding a normal APTT. We found a significant difference in peak thrombin generation, velocity index and area under the curve between the two groups, and that thrombin generation markers correlated with the APTT, procoagulant phospholipid activity and several procoagulant clotting factors. We conclude that short APTTs represent a procoagulant milieu, as represented by heightened thrombin generation and several other heightened procoagulant activities, which may help explain the association with thrombosis.     

Evaluation of primary haemostasis in people with neurofibromatosis type 1

Assessment of haemostasis in people with neurofibromatosis type 1 (NF-1) is essentially lacking, despite case reports of an association with von Willebrand disorder (VWD) and reported excessive bleeding post-surgery. We assessed routine blood haematology, routine coagulation parameters [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen], coagulation factors II, V, VII, VIII, IX, X, XI and XII, von Willebrand (VWF) factor antigen and activity, and platelet function [using the platelet function analyser (PFA-100)] in a group of individuals with NF-1 (n = 30). Their perceived haemorrhagic bleeding risk was also graded by means of a structured clinical assessment and physical examination. Routine blood assessments including platelet counts were generally normal, as were the routine coagulation tests PT, TT and fibrinogen, and most coagulation factors. Elevated APTTs were detected in 11 individuals, reduced factor XII levels in three, reduced VWF levels in four, and elevated PFA closure times (CTs) in 13. Laboratory results correlated with each other in some but not all cases. For example, elevated APTTs were identified in two of three individuals with a reduced factor XII level and prolonged CTs were identified in three individuals who also showed reduced aggregation responses in classical platelet function studies. Moreover, all individuals with VWF results below the normal reference range showed elevated CTs with both PFA test cartridges, and those with VWF results identified as borderline normal (i.e. 50-65%) also showed elevated CTs with both PFA test cartridges in three of five cases. The relationship between VWF and CTs was also identified by linear regression analysis (P-values of <0.05, for all comparisons). However, as clinically perceived bleeding risk did not appear to be correlated with laboratory test results in most cases, blanket screening of NF-1 individuals for evaluation of laboratory haemostasis may not be warranted.     

Splenic marginal zone lymphoma associated with antiphospholipid antibodies

A 61-year-old woman experienced a high fever with anemia and APTT prolongation after suffering a herpes zoster virus infection. Physical examination revealed a large splenomegaly without lymphadenopathy. Laboratory evaluations were positive for lupus anticoagulant (LA) and monoclonal IgM-kappa protein. LA was associated with the presence of anti-beta2GPI antibody, anti-cardiolipin antibody, and anti-prothrombin antibody. Moreover, the results of factors IX, XI, and XII assays and CRP and FDP-E were disturbed. A splenectomy was performed, and a splenic marginal zone lymphoma (SMZL) was diagnosed. All hematological findings rapidly recovered after the splenectomy. No thrombotic events occurred after the splenectomy even though thrombosis prophylaxis was not performed. The clinical course suggested that the SMZL-producing antibody induced immunological abnormalities in the labolatory tests. Since the patient suffered disease progression soon after the splenectomy, an autologous peripheral stem cell transplantation with rituximab administration was performed.     

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.     

Assessment of a new instrument (Coag-A-Mate X2) for performing global clotting tests and specific factor assays

Coag-A-Mate X2 (General Diagnostics) is an automated photo-optical system for detection of clots, which can be used for measuring prothrombin time (PT) and activated partial thromboplastin time (APTT) and assaying coagulation factors. We have compared results obtained with this instrument with those obtained by the manual method used routinely in our specialized laboratory. The instrumental results showed good precision (PT: between-assay CV from 1.3 to 3.0, APTT: from 2.2 to 3.1), and high degree of correlation with the manual results (for PT r = 0.99, for APTT r = 0.95). There was also good correlation between the two methods (0.98-0.99) for factor assays (V, VII, VIII, IX, X, XI, and XII). High output, reproducibility, comparability and flexibility make this instrument suitable for both clinical and research 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.     

John Hageman's factor and deep-vein thrombosis: Leiden Thrombophilia Study

SUMMARY. Because the relationship between factor XII deficiency and venous thrombosis in unclear and study results seem contradictory, we undertook a populationbased case-control study.
Among 350 unselected patients younger than 70 years, with a first, objectively confirmed, episode of deep-vein thrombosis and without underlying malignant disease we detected a 6% frequency (21/350 patients) of factor XII deficiency (activity level 57%). Among 350 healthy control subjects, matched for age and sex, the frequency was 5% (18 subjects). Thus there is no increase in prevalence of factor XII deficiency among thrombosis patients and no increase in thrombosis risk for subjects with low factor XII levels (mathced odds ratio 1.2 (95% CI 0.6-2.4)). In addition, there was no relation between strata of factor XII levels and thrombosis risk. In conclusion, we do not consider factor XII to be a determinant of deep-vein thrombosis.     

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.