The present invention disclosed the cloning and the purification of a mutant of prethrombin-2, which contains a thrombin cleavage site instead of a factor Xa cleavage site. The stable mutant prethrombin-2 is able to convert itself autocatalytically into active α-thrombin in the absence of ecarin or factor Xa. The new concept of signal amplification using self-replicating enzymes can be applied to improve sensitivity of α-thrombin assays and also for the preparation of different enzymes.