Fibrin clot lysis analysis; tissue factor versus thrombin as activator
ISTH Academy. Veirup M. Jul 9, 2019; 264533; PB1343 Topic: Fibrinolytic Factors & Inhibitors
Mai Stenulm Veirup
Mai Stenulm Veirup
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Fibrin Clot Lysis Analysis: Tissue Factor versus Thrombin as Activator

M.S. Veirup, V.B. Mogensen, S. Neergaard-Petersen, A.-M. Hvas
Aarhus University Hospital, Thrombosis & Haemostasis Research Unit, Department of Clinical Biochemistry, Aarhus, Denmark

Main Topic: Fibrinolysis and Proteolysis
Category: Fibrinolytic Factors & Inhibitors

Background: Fibrin clot formation and fibrinolysis can be evaluated using a dynamic turbidimetric clot lysis assay. However, the assay is not standardized across laboratories; some of the most essential differences are the use of different activators and concentrations of the lysis agent tissue plasminogen activator (tPA).
Aims: Firstly, to compare tissue factor and thrombin as activators in an in-house clot lysis assay. Secondly, to compare two different concentrations of tPA in an assay employing tissue factor as activator.
Methods: We included 538 patients with stable coronary artery disease receiving a low-dose aspirin 75 mg. Blood samples were obtained in tubes containing 3.2% sodium citrate, and platelet-poor plasma was separated. Two published protocols were used employing different activators; a) tissue factor (final dilution 1:5000) in combination with phospholipid or b) thrombin (final concentration 0.03 U/mL). For both assays, calcium was added in excess beyond tPA (final concentration 83 ng/ml). In samples from a subset of 417 patients, clot lysis was compared using a final tPA concentration at 83 ng/ml with 116 ng/ml employing tissue factor as activator.
Results: The protocol employing tissue factor as activator provided both higher maximum amplitude and area under the curve as well as longer clot lysis time, than the protocol using thrombin, Table 1.

Parameter: Thrombin 0.03 U/mL Tissue factor diluted at 1:5000 P Value
Max amplitude, au 0.32 (0.26;0.40) 0.68 (0.59;0.75) < 0.0001
Area under the curve 408 (289;585) 1381 (1083;1733) < 0.0001
50% lysis time, s 726 (570;912) 1483 (1154;1828) < 0.0001
[Table 1: Fibrin clot properties, medians (25% ;75% percentiles) are indicated, n=538, tissue plasminogen activator in final concentration 83 ng/ml.]

The fibrinolytic potential evaluated by area under the curve and lysis time was approximately halved at tPA 83 ng/ml relative to 116 ng/ml, Table 2.

Parameter: Tissue plasminogen activator 83 ng/ml Tissue plasminogen activator 116 ng/ml P Value
Max amplitude, au 0.69 (0.61;076) 0.68 (0.58;0.75) < 0.0001
Area under the curve 1410 (1111;1748) 826 (665;1025) < 0.0001
50% lysis time, s 1509 (1166;1830) 802 (653;1027) < 0.0001
[Table 2: Fibrin clot properties, medians (25% ;75% percentiles) are indicated, n=417, tissue factor in final dilution 1:5000.]

Conclusions: The balance between fibrin clot formation and lysis time is substantially influenced by the use of activator and concentration of the lysis agent. The two protocols are targeted at different patient populations; hypercoagulant or hypocoagulant patients. Beyond the importance of standardization of the analysis across laboratories, it is essential to design the optimal protocol according to the patient population.

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