Gunnink, Susanna F. (2012) Allosteric inhibition of protease activated receptor 1: a new antiplatelet therapy. thesis, Medicine.
Full text available on request.Abstract
Introduction: Arterial thrombosis is the leading cause of death in the world. A critical pathway for thrombus formation is the interaction between thrombin and the platelet protease activated receptor 1 (Par1), a G-protein coupled receptor (GPCR). Recently developed Par1 inhibitors act on the thrombin binding site (orthosteric inhibition), blocking all Par1 mediated responses. However, they are associated with an increased bleeding risk in clinical trials. ML161 is a new Par1 inhibitor that targets a Par1 binding site different from the thrombin binding site (allosteric inhibition). Allosteric inhibitors selectively block part of the receptor mediated response. In this study we assessed: 1) whether ML161 inhibited platelet thrombus formation in vivo, 2) whether ML161 inhibited platelet aggregation via different GPCRs that might share a similar allosteric binding site, and if it was able to inhibit platelet shape change mediated via one of the Par1 coupled G-proteins (Gα12/13), and 3) whether ML161 caused prolongation of bleeding time. Material and methods: 1) Intravital microscopy for assessing thrombus formation in vivo: wild type mice were anesthetized and an arteriole in their cremaster muscle exposed. Infused fluorescently labeled antiplatelet antibodies were visualized using intravital microscopy and their fluorescence intensity recorded during laser induced thrombus formation. Thrombi before and after infusion of ML161 were compared. 2) Platelet aggregometry: washed platelets were activated with different agonists in the presence or absence of ML161, to assess different GPCRs. Platelet shape change is visible as an initial decrease in light transmission in in the aggregation curve, this was assessed in the presence or absence of ML16. 3) Tail tip bleeding assay for assessing bleeding time: the distal 5 mm of the tail of anesthetized mice was cut off after infusion of ML161 or no infusion and time to cessation of bleeding recorded. Hemoglobin loss was measured spectophotometrically. Results: ML161 significantly inhibited platelet thrombus formation in vivo with a 73% decrease in platelet thrombus size (p=0.004). The control group showed a non-significant trend towards inhibition of platelet thrombus size. (32%, p=0.23). ML161 inhibited platelet aggregation via Par1 and the α2A-adrenergic receptor, but not via the thromboxane receptor, Par4 and the P2Y12 receptor. ML161 does not inhibit Par1 mediated Gα12/13 signaling. Bleeding times were not increased in ML161 treated mice versus control mice. (69.5 seconds versus 79.5 seconds, p=0.36). Conclusion: ML161 is a potent allosteric inhibitor of Par1, inhibiting platelet aggregation in vitro and platelet thrombus formation in vivo. It selectively inhibits part of the Par1 mediated signaling pathways, and does not prolong bleeding time. Allosteric Par1 inhibition is a promising and potentially safer alternative for orthosteric inhibition in the treatment of arterial thrombosis.
| Item Type: | Thesis (Thesis) |
|---|---|
| Supervisor name: | Faculty supervisor: and Tamminga, R.Y.J. MD |
| Supervisor name: | External supervisor: and Flaumenhaft, R. MD and Beth Israel Deaconess Medical Center and Boston, MA 02215 |
| Faculty: | Medical Sciences |
| Date Deposited: | 25 Jun 2020 11:05 |
| Last Modified: | 25 Jun 2020 11:05 |
| URI: | https://umcg.studenttheses.ub.rug.nl/id/eprint/2462 |
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