HDX-MS Unveils Structure-Function Relationships in Engineered Thrombolytic Drug: Staphylokinas

Ischemic stroke, the second leading cause of death worldwide, accounts for ˜65% of incident strokes and kills over 3.6 million annually. Despite this burden, thrombolytic options remain limited. Since its FDA approval for acute myocardial infarction in 1987 and later for ischemic stroke treatment, alteplase has remained the standard thrombolytic agent. Yet, it carries severe side efects, including intracranial bleeding (occurring in up to 8% of strokepatients in recent trials) and documented neurotoxic efects (1,2).Staphylokinase (SAK) is emerging as a compelling alternative that demonstrates high fbrinspecifcity, meaning it selectively targets blood clots without degrading circulating fbrinogenor other systemic proteins, which is a critical advantage that distinguishes it from conven-tional thrombolytic agents. The FRIDA clinical trial (1) confrmed its non-inferiority toalteplase, making SAK a promising candidate for next-generation thrombolytic therapy.To support rational design of safer and more efective SAK variants, we applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate conformational dynam-ics of wild-type and engineered non-immunogenic SAK variants. We employed automatedHDX-MS with a LEAP HDX robotic workstation (Trajan Scientifc and Medical). Quenchedprotein samples were digested online using the AnPep/Pepsin enzymatic column (AfPro).The peptides were analyzed using a timsTOF SCP mass spectrometer (Bruker Daltonics).Peptide coverage, redundancy, and back-exchange were validated prior to uptake analysisusing HDExaminer (Sierra Analytics). Residue-level protection factors and dynamic profles were further interpreted using pyHDX (3).We performed comparative HDX-MS of SAK mutants containing 3, 8, and 12 mutations,identifying site-specifc diferences in deuterium incorporation patterns. Two functionally im-portant regions exhibited signifcant conformational variations: (a) the bulge region (residuesLeu40-Glu46), which comprises part of the plasminogen activation epitope, and (b) the C-terminal domain containing lysine residues implicated in plasmin binding. We integrated ourdata with molecular dynamics (MD) simulations and NMR spectroscopy. MD simulationsconfrmed that specifc mutations induced measurable changes in local fexibility, and sol-vent exposure was characterized experimentally. These alterations suggest that engineeredmutations modulate interaction dynamics with plasminogen and plasmin substrates throughan induced-ft mechanism (4). The distinct conformational signatures among SAK variantscorrelated with biochemical properties, suggesting that HDX-MS can guide structure-basedthrombolytic engineering.We are currently extending this approach to characterize SAK-plasmin/plasminogen interac-tions, combining HDX-MS, MD simulations, and biochemical assays to reveal how mutation-induced conformational changes infuence thrombolytic function.