Assay of unfractionated and LMW heparin with chromogenic substrates: twin methods with factor Xa and thrombin

Utilizing two newly synthesized chromogenic substrates (CS), two different assay methods for heparin in plasma have been developed. The assay with bovine factor Xa and the highly reactive "Substrate FXa-1" (CH3 OCO-D-CHA-Gly-Arg-pNA X AcOH) measures both unfractionated (UF) heparin and low molecular weight (LMW) heparin within a single standard curve in the 0.05-1.5 U/ml plasma range. The very similar (and less expensive) assay with bovine thrombin and "Substrate Th-1" (2AcOH X H-D-CHG-Ala-Arg-pNA), measures UF heparin, but not LMW heparin. The standard curves are highly reproducible (CV 3.5-4.7%). For clinical work, a linear standard curve is obtained with three standards and lin-log plot. The "within run" SD was 0.007-0.026 U/ml. Mean recovery of 0.5 U/ml heparin added to 10 pathological plasma samples ranged 0.46-0.53 U/ml (SD 0.034-0.040). Activities of three UF heparin and three LMW heparin preparations are reported.