Photometric assay of platelet factor 4 with a chromogenic substrate

A photometric assay procedure for platelet factor 4 is described. The synthetic oligopeptide benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) is used as a substrate. By the action of factor Xa, p-nitroaniline (pNA) is split form the peptide bond. The amount of pNA liberated from S-2222 per minute is in direct relation to the activity of factor Xa. This reaction permits a photometric assay. Addition of heparin to an activation system consisting of plasma, thromboplastin and calcium chloride inhibits development of Xa activity. Since platelet factor 4 neutralizes heparin, its activity can be measured in such a system when all other components are kept at a constant level. Experimental details of the reactions involved and clinical results of the assay in comparison to a clotting method are described.