# Macromolecular Recognition in Coagulation > **NIH NIH P01** · CHILDREN'S HOSP OF PHILADELPHIA · 2024 · $575,812 ## Abstract Project 1 Abstract We employ the prothrombinase complex, its substrate, prothrombin and its product thrombin as paradigms for the enzyme complexes, zymogens, substrates and cofactors of coagulation to investigate generalizable features establishing the molecular bases for their function. An emphasis is placed on addressing major unanswered questions relating to allostery or how interactions or ligand binding to one site can affect function at distant sites by combining biophysical studies with structural and functional measurements based on a foundation of established mechanism to provide new mechanistic insights. The first aim extends previous observations that despite irreversible cleavage of prothrombin to yield thrombin, the proteinase can exist in an equilibrium between zymogen-like and proteinase-like states. We extend these findings in a direct thermodynamic approach to understand how ligand binding at exosite 1 favors proteinase-like forms and exactly counteracts liganding at exosite 2 on the opposite face of the molecule. The second aim expands to mechanisms related to allosteric changes in factor Xa using a class of small molecules with previously unseen behavior; they inhibit Xa function in solution but become poor and partial inhibitors of Xa when it is incorporated into prothrombinase. Kinetic studies combined with structural work will seek to establish the mechanisms underlying the putative allosteric modulation of Xa by this class of molecules. The third aim seeks to provide an ultimate functional test of how proteolytic activation of factor V imparts Xa binding and cofactor function by disrupting a tripartite inhibitory motif comprising two acidic regions (AR1 and AR2) flanking the B domain interact with a centrally located basic region (BR). We propose to identify tight binding nanobody probes to AR2 and BR. Probes that destabilize or stabilize the inhibitory structure are proposed to allow cofactor function without proteolysis or prevent the development of factor function after proteolysis. The last aim extends studies to factors IXa and VIII/VIIIa of intrinsic Xase to address peculiarities that set the proteinase and cofactor of this complex apart from those of prothrombinase. With IXa, we pursue structural evidence that suggests that the proteinase may distribute between forms with a distorted and open active site. In factor VIII, we focus on a thrombin cleavage site at the junction of the A1 and A2 domains, that is essential for cofactor activation but without a counterpart in factor V. We pursue the hypothesis that loss of cofactor function associated with the removal of the cleavage site can be rescued by the insertion of a polypeptide spacer to mimic the putative repositioning of the A1 and A2 domains that might follow cleavage. These strategies, utilizing novel concepts, are all directed towards how ligand binding or other effects modulate conformation and dictate function. The proposed approaches will shed new light on i... ## Key facts - **NIH application ID:** 10768829 - **Project number:** 2P01HL139420-06 - **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA - **Principal Investigator:** Sriram Krishnaswamy - **Activity code:** P01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $575,812 - **Award type:** 2 - **Project period:** 2018-09-01 → 2029-06-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10768829 ## Citation > US National Institutes of Health, RePORTER application 10768829, Macromolecular Recognition in Coagulation (2P01HL139420-06). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10768829. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*