A new global assay of coagulation and fibrinolysis.

INTRODUCTION: Global clotting assays may reflect an individual's net hemostatic balance and could contribute to prothrombotic and hemorrhagic risk assessment. In this research, a global assay that measures both coagulation and fibrinolytic capacities was developed and investigated. MATERIALS AND METHODS: In the Clot Formation and Lysis (CloFAL) assay, a buffered reactant solution containing trace amounts of calcium, tissue factor, and tissue-type plasminogen activator is added to plasma samples on a 96-well microplate in an automated, thermoregulated (37 degrees C) spectrophotometer. Clot formation and lysis are monitored as continuous changes in absorbance over the course of 3 h. Measurements include maximum amplitude (MA), times to maximum absorbance (T1) and completion of the first phase of decline in absorbance (T2), and area under the curve (AUC), from which a coagulation index (CI) and various fibrinolytic indices (FI) may be calculated. RESULTS AND CONCLUSIONS: MA, T1, and CI were principally influenced by fibrinogen and procoagulant factors. FI was found to be altered by inhibiting activation of plasminogen or thrombin activatable fibrinolytic inhibitor. Median CI was significantly decreased, while FI was markedly increased, in term neonates as compared to healthy adults (CI: 58% vs. 115%, FI: 210% vs. 90%; P<0.001 for each). By contrast, median CI was notably increased, and FI decreased, in healthy pregnant women when compared to adults (CI: 239% vs. 115%, FI: 59% vs. 90%; P<0.001 for each). The CloFAL global assay is analytically sensitive to several key components in the coagulation and fibrinolytic systems, as well as to physiologic alterations in hemostasis.

A new euglobulin clot lysis assay for global fibrinolysis.

Plasma fibrinolytic activity has been measured by the euglobulin clot lysis time (ELT) since the late 1950s. The euglobulin clot lysis assay (ECLA) method has been modified using a computerized kinetic spectrophotometric microtiter plate reader and measures optical density changes of recalcified euglobulin fraction of plasma samples over time. This method has been applied to normal healthy adults, children, pregnant women and newborn infants, which represent physiologic extremes of the ELT. The ECLA method adds measurements of maximum absorbance (Max Abs), area under the curve (AUC) and mean velocity to the standard clot lysis time. The resulting curves are unique to this method and have been analyzed and compared in order to establish normal ranges. Fibrinogen levels, plasminogen activator inhibitor-1 (PAI-1) antigen, PAI-1 activity and thrombin activatable fibrinolytic inhibitor (TAFI) antigen levels were measured in each individual of the four groups. Each protein measured within each study group except TAFI correlated with the lysis time, maximum absorbance and area under the curve. Considering all four groups together, PAI correlates most highly with lysis time, fibrinogen correlates the highest with Max Abs; fibrinogen and PAI-1 antigen have equally high correlations to AUC. Area under the curve is highly correlated with all coagulation parameters measured; the most significant contributor is fibrinogen. These observations are interesting, but at this time, it cannot be said that any of the test parameters are better than lysis time in distinguishing between these normal physiologic states.

Monitoring hypercoagulability and hypofibrinolysis following acute venous Thromboembolism in children: application of the CloFAL assay in a prospective inception cohort study.

Although individual thrombophilia tests are frequently performed in children with venous thromboembolism (VTE), global assays provide the opportunity to fill the gap in knowledge regarding their net impact on overall coagulative (and in some cases fibrinolytic) function. We first evaluated analytic sensitivity of the Clot Formation and Lysis (CloFAL) global assay to hypercoagulability and alterations in fibrinolysis, and then characterized changes in plasma coagulative and fibrinolytic capacities over time in children with acute VTE. In plasma ex vivo and in vitro experiments, the CloFAL assay area-under-the-curve (AUC) was analytically sensitive to hypercoagulable states, and its modified fibrinolytic index (FI2) was sensitive to both hyper- and hypofibrinolytic conditions. Clinical data and plasma samples for assay were collected during follow-up of 50 children enrolled in a prospective inception cohort study of VTE from May 2006 to June 2010. Follow-up periods were designated as follows: acute (<1 month post-event), sub-acute (1-3 months), early chronic (3-12 months), and late chronic (>12 months). Since most children were sampled at fewer than three pre-defined follow-up periods, study population findings were grouped by timepoint. AUC was significantly increased, and FI(2) significantly decreased, in the acute period of VTE when compared to healthy controls, indicating hypercoagulability and hypofibrinolysis, respectively. One-third of patients were hypercoagulable, and 23% were hypofibrinolytic, in the late chronic phase. AUC and FI(2) were strongly correlated with functional fibrinogen levels. These findings indicate the utility of the CloFAL assay in monitoring plasma coagulative and fibrinolytic capacities in children with VTE. Studies of its potential role in outcome prediction are ongoing.

Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults.

INTRODUCTION: Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation. MATERIALS AND METHODS: An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated. RESULTS: STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (p<0.001) and the maximum amplitudes of thrombin and plasmin generation were less in children than adults (p<0.01). No significant differences were measured among pediatric age groups. The most profound impairments in thrombin generation were observed for extrinsic and common pathway factor deficiencies, with the exception of afibrinogenemia. Plasmin generation was severely impaired in deficiencies of fibrinogen and plasminogen as well as with decreased tPA reagent concentration and addition of aminocaproic acid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent. CONCLUSION: Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults.