HKH20 interferes with the intrinsic pathway of coagulation. (A) Human plasma or BALB/c mouse plasma was incubated with 50 μM HKH20, GCP28, or buffer alone (control) for 60 seconds and analyzed by the aPTT test. (B) Human plasma was incubated with increasing amounts of HKH20 or GCP28, and the aPTT was measured. (C) Normal human plasma was incubated with 50 μM HKH20, GCP28, or buffer alone (control) for 60 seconds and analyzed by the PT and the TCT tests. (D) Human plasma was incubated with kaolin in the presence of 50 μM HKH20, GCP28, or buffer alone (control) for 15 minutes. Plasma was removed by centrifugation and pelleted kaolin was washed and resuspended in substrate buffer. After 15 minutes of incubation, plasma kallikrein activity was measured in a substrate assay. Data are presented as percentage activity compared with the control; values are mean ± SD (n = 3). (E) Human plasma was incubated with buffer (lane 1), kaolin (lane 2), or kaolin and 50 μM HKH20 (lane 3) for 15 minutes. Samples were analyzed by Western blotting with antibodies identifying HK and LK.