Abstract Number: PB0755
Meeting: ISTH 2020 Congress
Background: Plasmin, the central fibrinolytic enzyme, is formed upon cleavage of plasminogen by tissue plasminogen activator (tPA) or urokinase (uPA). We have shown enhanced binding and plasmin generation on the activated platelet membrane. Development of a plasmin-specific biosensor using technology previously described for thrombin, will permit study of real-time plasmin generation on the platelet surface and help reveal the dynamics of plasmin formation within the clot.
Aims: To develop a novel plasmin biosensor (PlnS-Ab) targeted to the platelet surface.
Methods: A plasmin-specific biosensor (PlnS-Ab) was constructed by click chemistry consisting of an anti-human CD41 antibody, dibenzocyclooctyne and a plasmin-sensitive peptide (PlnS-P) carrying a fluorescent reporter system. Cleavage of PlnS-P or PlnS-Ab by purified plasmin and on the surface of human platelets ± thrombin (100 nM) + convulxin (100 ng/ml) was quantified as fluorescence release. Collagen-induced aggregation in platelet rich plasma (PRP) preincubated with anti-CD41 antibody (1-10 µg/ml) was measured by light aggregometry. Lysis by tPA of PRP clots was measured as change in absorbance at 405 nm.
Results: PlnS-P was rapidly and selectively cleaved by plasmin (Km of 28.18 µM and Kcat of 4.5 s-1) with negligible affinity for thrombin and no cleavage by tPA or uPA. Endogenous plasmin activity was detected on stimulated platelets using PlnS-P and was significantly augmented by addition of tPA or uPA (10 nM). Plasmin activity was inhibited by inclusion of aprotinin (1 mM). Preincubation of PRP with anti-CD41 had no effect on platelet aggregation, clot formation or lysis. Plasmin activity in solution and on the platelet surface was successfully detected after linkage of PlnS-P to anti-CD41 antibody.
Conclusions: We have developed a PlnS-Ab with a plasmin-releasable quencher which is targeted directly to platelets. Use of this biosensor in flow-based and in vivo models will permit characterisation of novel profibrinolytic platelets functions in real-time within the dynamic thrombus environment.
To cite this abstract in AMA style:Simpson M, Veuskens B, Whyte CS, Mutch NJ. Targeted Measurement of Plasmin Kinetics on the Activated Platelet Membrane [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/targeted-measurement-of-plasmin-kinetics-on-the-activated-platelet-membrane/. Accessed January 28, 2022.
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