VIIaAT complexes, procoagulant phospholipids, and thrombin generation during postprandial lipemia.

INTRODUCTION: Factor VII activation occurs postprandially. A proportion of activated factor VII (VIIa) circulates in complex with antithrombin (VIIaAT). Our primary objective was to assess the effects of postprandial lipemia on circulating VIIaAT, procoagulant phospholipid (PPL) activity, and thrombin generation. METHODS: Plasma samples from postmyocardial infarction patients (n = 40) and controls (n = 39) were taken before and at 3 and 6 hours during a standardized oral fat tolerance test (OFTT). Fasting PPL activity measurements were also made in a second cohort of 108 postinfarction patients and 109 controls. VIIaAT was analyzed with the Asserachrom VIIaAT ELISA, PPL activity with the STA-Procoag-PPL kit, and thrombin generation with calibrated automated thrombogram with PRP-Reagent as trigger (all Diagnostica Stago products). RESULTS: Postprandially, VIIaAT increased in all samples without significant case-control differences in the overall response during the OFTT. Thrombin generation measures peak height and velocity, and PPL activity, were marginally affected by the test meal in the controls. Levels of all patient baseline measures were significantly different from controls, indicating a more hypercoagulable state, and these differences were maintained throughout the OFTT. Fasting samples from cases showed higher PPL activity than control samples. CONCLUSION: Viewing VIIaAT quantitation as a surrogate for TF activity measurement, postprandial increase in VIIaAT may reflect a mechanism that adds to the cardiovascular risk associated with postprandial lipemia. On the other hand, the impact of postprandial lipemia on PPL activity and thrombin generation seems to be minor.

Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation?

BACKGROUND: Tumor-derived tissue factor (TF) activates coagulation in vitro and in vivo in an orthotopic model of human pancreatic cancer. Here, we further characterized tumor-derived TF in this model. METHODS: Conditioned medium (CM) of L3.6pl human pancreatic tumor cells and plasma from nude mice bearing L3.6pl tumors were ultracentrifuged, and the pellets were filtered through membranes with different pore sizes. The size distribution of particles was analyzed in CM or plasma fractions with nanoparticle tracking and dynamic light scattering. Human TF antigen and activity were measured in pellets and supernatants with ELISA and clotting or thrombin generation assays, respectively. Human alternatively spliced TF (asTF) was measured with ELISA. Human TF and thrombin-antithrombin complex (TAT) concentrations were assessed in plasma of mice injected with filtered fractions of CM. RESULTS: Particles in both CM and plasma were < 0.4 µm. TF antigen and activity in the CM were mainly associated with microparticles (MP). Approximately 50% of antigen and 20% of activity were associated with particles of < 0.1 µm. Injection of < 0.1 µm particles into mice caused a 30% drop in platelet counts and an increase in TAT levels. In contrast, ~ 90% of TF antigen in tumor-bearing mice plasmas was non-sedimentable, whereas TF activity was exclusively associated with MP. Particles of < 0.1 µm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. Although asTF was found in MP-free CM supernatants, it was also present in CM and plasma pellets. CONCLUSIONS: Tumor-derived particles of < 0.1 µm and non-sedimentable TF are or can become procoagulant in the presence of phospholipids, and may contribute to the procoagulant potential of circulating TF.

The evaluation of two automated soluble fibrin assays for use in the routine hospital laboratory.

INTRODUCTION: The soluble fibrin monomer (sFM) assay, like the D-dimer (DDi) assay, has the potential to be used both as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and as a thrombotic marker. It differs from DDi in that it is a much earlier produced fragment produced only by thrombin action on fibrinogen, whereas DDi is a much later produced fragment formed by plasmin cleavage of cross-linked fibrin. METHODS: In our study, we compared two commercially available automated sFM assays in the routine hospital setting using samples obtained from the general hospital ward and the emergency room. The results obtained with the two automated assays (Stago LIA sFM assays and the LPIA-Iatro SF assay) were compared with each other and with the results obtained using the routine semiquantitative hemagglutination assay. RESULTS: The study showed that both automated assays were comparable with each other. No patient sample previously classified as positive would be missed, but with the higher sensitivity in the automated tests, more samples are positive. CONCLUSION: In conclusion, we suggest that both automated tests are suitable for routine laboratory use. Both assays had the advantage over the hemagglutination assay in that previously frozen samples could be used, and the assays are easier and quicker to perform. The LIA sFM Stago has slightly better sensitivity but has a tendency to lower specificity than the Iatro SF test.

On-demand but not prophylactic treatment with FVIII concentrate increase thrombin activatable fibrinolysis inhibitor activation in severe haemophilia A patients.

INTRODUCTION: Thrombin activatable fibrinolysis inhibitor (TAFI) down-regulates fibrinolysis after activation by thrombin/thrombomodulin. We investigated the effect of treatment with FVIII concentrate on plasma levels of pro-TAFI and activated TAFI in haemophilia A patients. METHODS: Samples were collected pre and posttreatment from patients treated prophylactically or on-demand. Pro-TAFI, TAFI/TAFIi and FVIII levels were measured in all samples. RESULTS: Treatment had no effect on pro-TAFI levels. Pro-TAFI was similar in both patient groups but higher than in controls. Patients from the prophylactic treatment group had measurable FVIII levels pretreatment while in the treatment-on-demand group FVIII levels were ≤0.01 IU/mL. In the prophylactic treatment group, the levels of TAFI/TAFIi were significantly lower pre- and posttreatment (4.31 ± 3.14 and 3.48 ± 2.65 ng/mL respectively) than in the on-demand group (13.02 ± 3.47 and 14.87 ± 3.47 ng/mL respectively). This difference may be due to release of tissue factor at the injury site in the on-demand group. This could induce thrombin and TAFI activation within the clot counterbalancing fibrinolysis in these patients. In the prophylactic group, no injury existed, thus there was insufficient thrombin generation within the clot to activate TAFI. CONCLUSION: These findings suggest that in patients to whom FVIII is administered on demand the fibrinolysis activity is more down regulated than in patients following a prophylactic treatment regime.

An international multicentre-laboratory evaluation of a new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta2-glycoprotein autoantibodies.

BACKGROUND: Antiphospholipid syndrome (APS) is diagnosed by the simultaneous presence of vascular thrombosis and/or pregnancy morbidity and detection of antiphospholipid antibodies in plasma. OBJECTIVES: We have shown that prolongation of clotting time by anti-beta2-glycoprotein I (beta2GPI) antibodies correlates better with thrombosis than a positive classic lupus anticoagulant (LAC) assay in a single center study. To confirm or falsify this finding we have conducted a multicenter study. METHODS AND RESULTS: In 325 LAC-positive samples, we found that the beta2GPI-dependent LAC correlated 2.0 times better with thrombosis than the classic LAC assay. Although significant, this was a minimal improvement compared with the 'classic' LAC. It was published that calcium influences the behavior of anti-beta2GPI antibodies in coagulation assays. To investigate whether calcium plays a role in the present study, we divided the patient population into two groups: (i) blood was collected in 0.109 m sodium citrate and (ii) blood was drawn in 0.129 m sodium citrate as anticoagulant. We found that a positive result with the beta2GPI-dependent LAC assay correlated better with thrombosis [odds ratio (OR): 3.3, 95% confidence interval (CI) 1.9-5.8] when 0.109 m sodium citrate was used compared with 0.129 m sodium citrate (OR: 0.4, 95% CI 0.1-1.1). CONCLUSION: We were able to confirm in an international multicenter study that a positive result in a beta2GPI-dependent LAC assay correlates better with thrombosis than the classic LAC assay, but that the assay needs further study as it is sensitive to external factors such as the sodium citrate concentration used as anticoagulant in the test sample.

Annexin A5 polymorphism (-1C-->T) and the presence of anti-annexin A5 antibodies in the antiphospholipid syndrome.

BACKGROUND: Annexin A5 is thought to have a role in the pathophysiology of the antiphospholipid syndrome (APS)-a syndrome characterised by recurrent thrombosis and pregnancy morbidity. OBJECTIVE: To investigate whether anti-annexin A5 immunoglobulin (Ig)M or IgG antibodies, or the -1C-->T polymorphism of annexin A5, is a risk factor for thrombosis or miscarriage, and whether the -1C-->T polymorphism is correlated with APS. METHODS: A cohort study was carried out with a population of 198 patients with primary APS, systemic lupus erythematosus or lupus-like disease. For the detection of anti-annexin A5 antibodies and the measurement of annexin A5 plasma levels, ELISA-type methods were used. The annexin A5 -1C-->T mutation was detected by restriction fragment length polymorphism. RESULTS: 71 patients were positive for annexin A5 IgM or IgG antibodies, of whom 53 patients were positive for anti-annexin A5 IgG antibodies and 27 of 198 patients were positive for anti-annexin A5 IgM antibodies. The prevalence of IgM or IgG anti-annexin A5 antibodies was not significantly associated with thrombosis or miscarriage on multivariate analysis. The prevalence of the -1C-->T mutation in the annexin A5 gene (46/198 patients) was significantly associated with miscarriage (odds ratio 2.7, 95% confidence interval 1.1 to 6.7, independent risk factor). CONCLUSION: The detection of anti-annexin A5 antibodies does not seem relevant for estimating the risk for thrombosis or miscarriage in APS. The -1C-->T mutation was an independent risk factor for miscarriage, which is independent of APS.

The clinical relevance of the citrate effect on International Normalized Ratio determinations depends on the reagent and instrument combination used.

Multiple studies have shown that the two different citrate concentrations in common use as the anticoagulant in blood collection for haemostasis assays can affect the results obtained with the prothrombin time assay. It is clear from the literature that there is considerable variability in the results obtained using different instrument-reagents combinations, but the clinical relevance of these differences is unclear. Most of the studies have used an optical system for end-point detection. This study reports on the citrate sensitivity using mechanical end-point detection. Using two different reagents, one previously shown to be citrate sensitive on optical systems (Neoplastin CI plus) and a citrate-insensitive reagent (Neoplastin CI), we demonstrate that the effect of using different citrate concentrations (0.105 or 0.129 mol/l) has statistically significant but clinically irrelevant effects on the International Normalized Ratio using a mechanical instrument (STA)-reagent combination (mean percentage difference in results, 1.9 and 3.8% respectively). This demonstrates that the citrate effect is both instrument type and reagent dependent. Every reagent and instrument combination needs to be tested to see whether any citrate effect exists. In a secondary study, it was shown that the international reference rabbit thromboplastin (CRM 149(s)) was not citrate-concentration sensitive.

A study of the variability seen in the international normalized ratio obtained using different sensitivity thromboplastin reagents on different instrument types.

We performed an extensive study to check international normalized ratio (INR) recovery using three different rabbit thromboplastin reagents of different sensitivities [international sensitivity index (ISI) circa 1.1, 1.3 and 2.0] on two different instrument types. Both instrument types used an optical end point: one nephelometric at 660 nm, one photometric at 880 nm. Each thromboplastin reagent in the study had an instrument-specific ISI value assigned to it by the reagent manufacturer. Samples obtained from stable orally anticoagulated patients were tested on four different instruments of each type with the three different sensitivity reagents. Excellent correlation between INR values was seen between all results (r values between 0.97 and 0.99). Regression analysis gave slopes between 0.92 and 1.07, and a maximum deviation of intercept from zero of 0.21. Variations in CV of less than 2% were seen within each instrument type. This study shows that if instrument-specific ISI values are used, comparable INR values can be obtained using reagents of different sensitivity on multiple instruments of the same or different types. This study demonstrates the validity of the INR/ISI system.

von Willebrand factor activity detected in a monoclonal antibody-based ELISA: an alternative to the ristocetin cofactor platelet agglutination assay for diagnostic use.

The monoclonal antibody RFF-VIII:R/1 recognises an epitope on von Willebrand factor involved in its interaction with GPIb alpha. A two-site, solid phase ELISA has been established using RFF-VIII:R/1 as the solid-phase, capture antibody and an enzyme-conjugated, polyclonal antibody to human VWF, which provides an assay for VWF functional activity with a detection limit of 0.5 U/dl VWF and an interassay %CV < 10. Plasma from 192 VWD patients (48 studied retrospectively; 144 prospectively) showed VWF levels of < 50 U/dl in type 1 patients (n = 156), < 25 U/dl in type 2A (n = 26) and < 35 U/dl in type 2B (n = 8) which, in type 1 and 2A patients, correlated with RiCoF activity (r > or = 0.82). In plasma from patients with type 1 VWD values of VWF in the Mab-based ELISA were similar to levels of VWF:Ag measured in a polyclonal antibody-based ELISA (r > or = 0.87) but were significantly lower than VWF:Ag in type 2A and 2B plasmas (p < or = 0.0005), allowing discrimination of variant VWD. The Mab-based ELISA has advantages of sensitivity and reproducibility over the RiCoF assay to measure VWF activity and can be used to analyse stored samples. In conjunction with an ELISA for VWF:Ag and VWF multimer analysis, it provides a reliable method for the laboratory diagnosis of VWD.

Differences between the venoms of two sub-species of Russell's viper: Vipera russelli pulchella and Vipera russelli siamensis.

Ion exchange chromatography was carried out using venoms obtained from two sub-species of Russell's viper; V. russelli siamensis from Burma and V. russelli pulchella from Sri Lanka. Differences were observed in the elution position of venom components having haemolytic and procoagulant activity but not those causing fibrinolysis. Only the V. russelli siamensis venom exhibited any platelet aggregating activity. The Indian (Haffkine) polyspecific and the Burmese (Burma Pharmaceutical Industries) monospecific antivenoms, when used in cross immunoelectrophoresis against the two venoms, revealed differences in the number and/or intensity of the precipitin bands present. An important functional consequence of this was that the Burmese antivenom did not neutralize the haemolytic activity of the V. russelli pulchella venom in vitro and would thus probably not be effective in treating this consequence of envenoming by Russell's viper in Sri Lanka. Differences in the composition and the clinical effects of the two venoms emphasizes the importance of using venom from the local snake for antivenom production if optimal clinical efficacy is to be achieved.

Changes in coagulation and fibrinolysis during pregnancy: evidence of activation of coagulation preceding spontaneous abortion.

In order to monitor physiological changes in coagulation and fibrinolysis that occur during normal pregnancy, blood samples were collected in each trimester of pregnancy from 17 volunteers. Control samples were collected from 12 non-pregnant female volunteers. As pregnancy advanced there was a rise in the basal levels of fibrinopeptide A, cross linked D-dimer fragment and the B beta 15-42 fragment and an increase in the in vitro rate of fibrinopeptide A generation. These results were consistent with an increased activation of coagulation during normal pregnancy, compensated for by a concomitant rise in fibrinolytic activity. In two patients who spontaneously aborted, evidence of uncompensated activation of coagulation could be detected before the manifestation of any clinical signs. In a second pregnancy in one of these patients similar changes were observed, but were reversed by heparin treatment and the pregnancy progressed to full-term delivery of a normal infant.

The action of Russell's viper venom on fibrin formation and fibrinolysis in vivo.

Envenoming by Russell's viper caused a marked rise in FPA, B beta 15-42 fragment and fibrin derived cross-linked D-dimer fragment indicative of a consumptive coagulopathy with hyperfibrinolysis. There was no increase in tPA or tPA-I levels post envenoming, which suggests that the increase in fibrinolytic activity was not due to venom-induced release of tPA from the vessel walls but may have been attributable to a direct effect of the venom or to a secondary physiological response to fibrin deposition. The effectiveness of the antivenom is demonstrated by its ability to prevent further cleavage of fibrinogen and the return to normal fibrinogen levels by 24 h. A secondary rise in FPA at this time indicates that the initial dose of antivenom may have been too small. The antivenom alone or in combination with the venom causes the release of tPA, tPA-I and vWF by the vessel walls. This may be a consequence of the severe anaphylactic reactions seen in some patients.

The simultaneous measurement of total and free protein S by ELISA.

An ELISA for the simultaneous measurement of total and free protein S is described. Polyethylene glycol precipitation was used to remove the C4b -binding protein/protein S complex from plasma. This allows free protein S to be measured. The total protein S was measured directly on plasma samples that had been diluted to dissociate the C4b binding protein/protein S complex. The concentration of total and free protein S found in normal subjects (n = 24) was 92.9 (SD = 10) u/dl and 40.3 (SD = 8.8) u/dl respectively. The within assay coefficient of variation was 5.1%. The between assay coefficient of variation was 5.7%. Warfarin therapy caused a reduction in the level of both total and free protein S but this reduction correlated poorly with the INR value.

The application of polyethylene glycol to radioimmunoassays used in haemostasis.

Use of polyethylene glycol 6000 in the second stages of double antibody radioimmunoassays for fibrinopeptide A, B-thromboglobulin and platelet factor 4 facilitates the more rapid separation of free from bound antigen, and allows precipitating antiserum to be used in greater dilution. At a final PEG 6000 concentration of 5%, separation of free from bound antigen was complete within 1 hour, and antisera could be used in dilutions 3-9 times greater than those recommended by the manufacturers. PEG 6000 had a negligible effect on the affinity of first stage antibodies for their respective antigens. Radioimmunoassays using PEG 6000 were sensitive to protein concentration, failure to adjust standards to similar protein concentrations as those of test samples causing artefactually low results.