The prothrombinase complex catalyzes the conversion of Prothrombin (Factor II), an inactive zymogen, to Thrombin (Factor IIa), an active serine protease. The complex consists of the serine protease, Factor Xa, and the protein cofactor, Factor Va. The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions.

To produce thrombin, the prothrombinase complex cleaves two peptide bonds in prothrombin, one after Arg271 and the other after Arg320.[1] Although it has been shown that Factor Xa can activate prothrombin when unassociated with the prothrombinase complex, the rate of thrombin formation is severely decreased under such circumstances. The prothrombinase complex can catalyze the activation of prothrombin at a rate 3 x 105-fold faster than can Factor Xa alone. Thus, the prothrombinase complex is required for the efficient production of activated thrombin and also for adequate hemostasis.