Waveform Analysis Extends the Capabilities of aPTT and PT Clotting Assays for Detecting Factor Deficiency and Monitoring Procoagulant Drug Therapy

Abstract 1205

Coagulation assays, such as activated partial thromboplastin time (aPTT) and prothrombin time (PT), are important for the diagnosis and treatment of bleeding disorders. Waveform analysis (WA) of the optical data generated in performing coagulation assays may provide additional information that will allow further understanding of coagulation disorders and their treatment (Hemophilia. 2006;12(suppl 3):76). Since WA employs assays currently in use in the clinical laboratory, it has the potential to be easily adapted to the clinical setting. To assess the utility of WA in procoagulant drug discovery for hemophilia, we determined its ability to: 1) discriminate between different factor deficiencies by factor-specific coagulation assays and by standard aPTT; and 2) discriminate between hemophilic plasma (with and without inhibitors) spiked with therapeutic proteins used to treat hemophilia. The ultimate goal of the study was to determine the feasibility of WA in monitoring hemophilia therapy in the clinic.

WA of plasma from factor (F)-deficient (FVIII, von Willebrand factor [type III vWD], FIX, and FVII) donors showed both absorbance and first-derivative waveform differences. Differences in first-derivative curves for severe hemophilia A plasma relative to vWD type III plasma were observed. The hemophilic plasma had a longer lag time (113 vs 93 seconds, respectively) and lower peak amplitude (29.7 vs 54.7 AU) than the vWD type III plasma. To determine whether WA might be useful for assessing the procoagulant activity of FVIII and FVIII bypass therapeutic agents, we compared waveforms of plasma-derived FVIII (WHO 8^th) with pegylated FVIII (BAY 94–9027) in hemophilia A plasma or of rFVIIa variant and BAY 86–6150 in hemophilia A plasma containing 112 BU inhibitor. The waveforms obtained with WHO 8^th and BAY 94–9027 were almost identical by ellagic acid aPTT with the first derivative giving lag times of approximately 31 seconds for both 5% WHO 8^th and 5% BAY 94–9027. WA was able to detect rFVIIa and BAY 86–6150–mediated coagulation at 1.4 nM by first-derivative waveform in silica aPTT. Comparison of rFVIIa with BAY 86–6150 at this concentration indicated that BAY 86–6150 was more procoagulant than rFVIIa, with a shorter lag time of 75.9 seconds vs 97.9 seconds (measured by taking the first derivative of the waveform). Therefore, in addition to clot time or activity levels currently available by standard aPTT/PT, WA allows further insight into the kinetics of coagulation. In conclusion, WA holds promise as a global hemostasis assay that will extend the current utility of optical coagulation analyzers used in the clinic.