Determination of the overall haemostasis potential and fibrin gel permeability : method development and application in research and in clinical materials

Abstract

We have previously developed a laboratory method that may screen the Overall Haemostasis Potential (OHP) in plasma based on spectophotometric measurement of an area under the fibrin aggregation curve in citrated plasma samples to which tiny amounts of exogenous thrombin and tissue-plasminogen activator (t-PA) are added. When the fibrin aggregation curve is created, the fibrinogen originally present in plasma is gradually converted to fibrin by the generated thrombin. At the same time, plasminogen activation produces plasmin that in turn digests fibrin. Each absorbance value (Abs) represents the fibrin level at the corresponding time point, and the area under the curve, should reflect a balance between the generation and proteolysis of fibrin throughout the measurement period.

To improve the assay sensitivity for the application in clinic or research work, various modifications were introduced. Thrombin in a decreased dose (0.04 IU/mL, compared to 0.2 IU/mL previously used) with or without t-PA was added to plasma. Areas under the two fibrin-aggregation curves i.e OHP and Overall Coagulation Potential (OCP) were thus created. A difference between the two parameters reflects the Overall Fibrinolysis Potential (OFP), calculated by (OCP-OHP)/OCP) x 100%.

The modified method has shown its usefulness in detecting hypercoagulation in normal pregnancy, preeclampsia and coronary heart disease. Increased levels of OHP were also found in women with previous thromboembolim especially related to the presence of FV Leiden mutation. Moreover, assay of this parameter can screen immediate changes in the haemostatic system after the injection of low molecular mass heparin (dalteparin) and may be used for monitoring the anticoagulant effects.

To ensure the sensitivity of the assay for determining different severity of hypocoagulation, further modifications were performed by introducing tissue factor and phospholipids to the reaction system. All the factors belonging to the two pathways of coagulation cascade, apart from FXII, affected the OHP outcome. This indicates that the modified assay system is similar to the haemostasis balance in circulating blood, and may thus become a laboratory too] to estimate bleeding tendency in haemophilic patients and distinguish prothrombotic cases among patients with FXII deficiency.

OHP assay is thus a quantitative method to determine the fibrin level associated with combined potential of coagulation and fibrinolysis. However studies on fibrin gel porosity may give information about quality of the fibrin network which is important e.g. in atherosclerosis. We made modifications in a flow measurement previously established by B Blombäck et al and evaluated the resultant advantages. The essential equipment was simplified and the sample volume minimized which rendered the assay easier to apply in any clinical or research laboratory settings. By using different concentrations of thrombin with, or without phospholipids, it was possible to asses whether the fibrin gel porosity depends on both thrombin generation potential and fibrinogen clotting properties or only on the latter respectively.

The modified flow measurement technique was used to determine the effects of acetylsalicylic acid (ASA, aspirin) effects on haemostasis. Fibrin gel porosity was more markedly increased during treatment with lower doses of ASA, compared to medium- / high-doses. These results are further confirmed by the findings in three-dimensional confocal microscopy where thicker fibrin fibers and larger network pores with irregular structure were observed during the low dose treatment. The greater increase in fibrin gel permeability and alterations in the structure of the fibrin network support the clinical findings of better prevention of arterial thrombosis such as in stroke and cardiovascular disease during treatment with low ASA doses such as 75mg daily, than with the higher doses.