Artikel
Hydrogen sulfide promotes microvascular thrombolysis through an alteration of thrombus stability
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Autoren
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Eberhard Grambow
- Department of General, Thoracic, Vascular and Transplantation Surgery, University of Rostock; Institute for Experimental Surgery, University of Rostock
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Christian Leppin
- Institute for Experimental Surgery, University of Rostock
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Marcus Frank
- Electron Microscopy Centre, University of Rostock
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Ernst Klar
- Department of General, Thoracic, Vascular and Transplantation Surgery, University of Rostock
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Brigitte Vollmar
- Institute for Experimental Surgery, University of Rostock
| Veröffentlicht: | 28. September 2015 |
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Gliederung
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Background: The volatile transmitter hydrogen sulfide (H2S) is known for its various functions in vascular biology. This study evaluates the effect of the H2S-donor GYY4137 (GYY) on thrombus stability and microvascular thrombolysis.
Material and methods: Human whole blood served for all in vitro studies and was analysed in a resting state, after stimulation with thrombin-receptor activating peptide (TRAP) and after incubation with 10mM or 30mM GYY or its vehicle DMSO following TRAP-activation, respectively. As a marker for thrombus stability, platelet-leukocyte aggregation was assessed using flow cytometry after staining of human whole blood with FITC- or PE-coupled antibodies against CD62P and CD45 respectively. Further more, morphology and quantity of platelet-leukocyte aggregation were studied by means of scanning electron microscopy. Therefore, platelets were stained for CD62P followed by gold labeling.
In vivo, the dorsal skinfold chamber preparation was performed in C57Bl6/J mice to study light/dye induced thrombus formation in arterioles and venules using intravital fluorescence microscopy. Thrombolysis was assessed 10h and 22h after thrombus induction and intraperitoneal treatment with GYY, DMSO or intravenous injection of recombinant tissue plasminogen activator (rtPA). Means±SEM. One way- or repeated measurements ANOVA respectively, followed by appropriate post-hoc comparison tests.
Results: Flow cytometry revealed an increase of CD62P/CD45 positive aggregates after TRAP stimulation of human whole blood witch was significantly reduced by pre-incubation with 30mM GYY (TRAP: 100±0% p<0.05 vs 30mM GYY following TRAP: 51±4%). Preliminary data gained by scanning electron microscopy additionally showed a reduced platelet-leukocyte aggregation after pre-incubation with GYY compared to TRAP stimulation alone. Further on, morphological signs of platelet activation were found markedly reduced upon treatment with GYY.
In mice, both GYY or rtPA significantly accelerated arteriolar thrombolysis compared to DMSO at 10h (GYY: 80±13%; rtPA: 76±12% each p<0.05 vs DMSO: 5±11%) and 22h (GYY: 85±11%; rtPA: 78±14% each p<0.05 vs DMSO: 4±18%). In venules, rtPA but not GYY significantly facilitated thrombolysis compared to DMSO after 10h (rtPA: 84±7% p<0.05 vs DMSO: 43±12%) and 22h (rtPA: 80±10% p<0.05 vs DMSO: 39±13%).
Conclusion: GYY impairs thrombus stability by reducing platelet-leukocyte aggregation, thereby facilitating endogenous thrombolysis in arterioles.
