The therapeutic action of tissue plasminogen activator (t-PA) is a two-step process: (1) binding to lysinerich fibrin (K-m event) and (2) converting local plasminogen into plasmin (K-cat, event). Overcoming limitations of other structural biophysics methods, we wanted to employ small-angle X-ray scattering (SAXS) to visualize what shape changes occur/accompany t-PA activation, but the prime hurdle was the polydisperse nature of the fibrin, which occluded scattering Groove Open information from 'c-PA. Earlier, larger polylysine peptides have been used to potentiate activation of t-PA, so while screening short polylysine peptides as alternatives to fibrin or larger peptides, we found that penta-polylysine (PS) specifically activates t-PA in a dose-dependent manner, averaging to almost 3-fold more than in the absence of any peptide. SAXS data analysis confirmed that PS does not induce association of t-PA molecules, and a narrower peak profile of the Kratky plot indicated that PS binding quenches inherent motion in t-PA. Shape reconstruction of the PA -/+ P5 revealed that P5 closes the "gap" between the two gross domains of t-PA, viz. fused F/E, K1 and K2 domains, and the P domain. Docking experiments suggested that, while other polylysine peptides preferentially interacted with the surfaces of kringle domains, PS "slipped into" the gap/groove between 1(2 and P domains, thereby mediating a substantial increase in the number of long-range interactions between the 1(2 domain and exosites in the P domain. We report here dissection of shape events involved in between K-m/K-cat steps of t-PA activation, which can pave the way toward the search for small molecule function regulator(s) of t-PA.