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Venous thromboses have been reported following the clinical use of some factor IX preparations. This may be due to several causes as for instance traces of FXa or thrombin formed during processing or presence of surface activation products involved in the early stages of the coagulation pathway. There is an urgent need for simple and reliable thrombogenecity tests. Activation by Ca2+ should preferentially be avoided, as Ca2+ by itself is capable of activating the prothrombin complex. All clotting methods are affected by the heparin content (4-5 u/ml) and excess amount of sodium citrate present in the F IX concentrates. We have therefore developed a colourimetric method using synthetic substrates. The assay is performed in the absence of precence of known proteolytic inhibitors in order to improve the specificity of the test system and identify the nature of the thrombogenicity. Using this method, various concentrates can be classified in three groups according to the rate of cleavage of the substrates. Hot materials have been titrated with several inhibitors. The contaminating enzymes have also been isolated and characterized. The results clearly indicate the presence of two entirely different types of hot material. One group contains mainly FXa and thrombin and can be neutralized by AT III. The other group probably contains large amounts of surface products. The “thrombogenicity” of this group can be mimiced by kaolin activation of plasma and inhibited by Trasylol.

In Vitro Thrombogenicity Test for Clinical Factor IX Concentrates Using Synthetic Substrates