At sites of vessel injury, thrombin mediates coagulation by catalyzing fibrin clot formation and platelet activation. Conversely, plasmin facilitates fibrinolysis by catalyzing the degradation of fibrin clots.
Thrombin generation is most frequently measured in plasma samples using small molecule substrates, these substrates have low free thrombin efficiency and specificity, cannot measure thrombin’s exosite interactions and have limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells, require anticoagulation and the addition of supraphysiological concentrations of calcium. To overcome these limitations, we have designed and characterized a fluorescence resonance energy quenching (FREQ)-based thrombin sensor (FTS) protein. Compared to small molecule substrates, the FTS demonstrated high specificity for thrombin because it is not cleaved by thrombin inhibited by α2-macroglobulin and interacts with thrombin’s anion binding exosite I. The FTS can effectively measure thrombin generation in plasma and in whole blood. The FTS does not inhibit standard thrombin generation assays. Lastly, FTS-based thrombin generation in non-anticoagulated finger-prick blood is delayed but enhanced compared to citrated plasma.
Similarly, plasmin generation is also restricted to plasma samples and measured using the fluorogenic Boc-Glu-Lys-Lys-AMC, which have low free plasmin efficiency and specificity, cannot measure plasmin’s kringle domain interactions and are insensitive to the effects of plasminogen activator inhibitor-1 (PAI-1). Here, we describe the design and characterization of a (FREQ)-based plasmin sensor (FPS) protein that demonstrated high efficiency for plasmin that is not inhibited by α2-macroglobulin when compared to Boc-Glu-Lys-Lys-AMC and interacts with plasmin’s kringle domain 5. The FPS measures plasmin generation in plasma, where it demonstrated greater sensitivity to tranexamic acid compared to Boc-Glu-Lys-Lys-AMC as well as sensitivity to PAI-1 and the effects of fibrin.
Therefore, the FTS and FPS will broaden our understanding of thrombin and plasmin generation in ways that are not attainable with current methods. / Thesis / Doctor of Philosophy (PhD) / Increased blood clot formation leads to strokes and heart attacks while the inability to form blood clots when needed leads to bleeding disorders. In the body, thrombin makes blood clots while plasmin breaks them down. Therefore, clotting disorders occur in the absence of proper thrombin and plasmin function and appropriate diagnostics of these processes help to determine appropriate treatment. Currently available tests of thrombin and plasmin do not just measure their active forms and are restricted to plasma assays that do not reflect physiological settings and are not useful for quick diagnosis. Here, we describe the production of novel sensors for thrombin and plasmin that are specific for thrombin and plasmin’s active forms. These sensors have potential to be developed into portable diagnostic tests of thrombin and plasmin activity and to be valuable research tools for the development of better treatment options of blood clotting diseases.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/29385 |
| Date | January 2024 |
| Creators | Dai, Ying |
| Contributors | Gross, Peter, Medical Sciences (Blood and Cardiovascular) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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