The activity “blind spot”: why understanding which proteinases are active, not merely present, is essential for rigorous osteoarthritis research

Proteinases are the critical drivers of articular cartilage breakdown in osteoarthritis (OA). Most studies infer proteinase involvement by measuring RNA or protein abundance, although proteinases can exist in multiple inactive states in addition to their active forms. This review addresses a critical “blind spot” in OA research, the frequent assumption that proteinase presence equates to proteolytic activity. We highlight why understanding which proteinases are active, when and where, is essential for rigorous mechanistic insight and translational progress. This narrative review synthesises evidence from biochemical, functional and proteomic studies of cartilage and synovial fluid to examine regulation of proteinase activity in OA. We summarise the molecular states of proteinases that confound interpretation of abundance-based measurements and critically evaluate established and emerging methodologies used to assess historical and current proteolytic activity. These include neo-epitope antibodies, degradomics, fluorescent peptide substrates, zymography, and activity-based probes, with emphasis on their strengths, limitations and applications. Accumulating evidence demonstrates a frequent dissociation between proteinase abundance and proteolytic activity in OA. Proteinase activation, inhibition by endogenous inhibitors, endocytic clearance, and proteolytic inactivation, each determine net proteolytic activity but are largely invisible to conventional transcriptomic, antibody-based or proteomic approaches. Activity-focused studies reveal a more extensive and heterogeneous proteolytic landscape in OA joints than previously appreciated. Joint destruction in OA is governed not by proteinase expression alone, but by tightly regulated spatial and temporal activation. Hence, abundance-based measurements can substantially overestimate functional proteolysis. Greater emphasis on direct or proxy measures of activity - conceptualized as the proteinase “activome” - will strengthen experimental interpretation, improve disease stratification, and support discovery of activity-based biomarkers and therapeutic strategies.