University of Birmingham Reversal of stress fibre formation by Nitric Oxide mediated RhoA inhibition leads to reduction in the height of preformed thrombi.

Evidence has emerged to suggest that thrombi are dynamic structures with distinct areas of differing platelet activation and inhibition. We hypothesised that Nitric oxide (NO), a platelet inhibitor, can modulate the actin cytoskeleton reversing platelet spreading, and therefore reduce the capability of thrombi to withstand a high shear environment. Our data demonstrates that GSNO, DEANONOate, and a PKG-activating cGMP analogue reversed stress fibre formation and increased actin nodule formation in adherent platelets. This effect is sGC dependent and independent of ADP and thromboxanes. Stress fibre formation is a RhoA dependent process and NO induced RhoA inhibition, however, it did not phosphorylate RhoA at ser188 in spread platelets. Interestingly NO and PGI 2 synergise to reverse stress fibre formation at physiologically relevant concentrations. Analysis of high shear conditions indicated that platelets activated on fibrinogen, induced stress fibre formation, which was reversed by GSNO treatment. Furthermore, preformed thrombi on collagen post perfused with GSNO had a 30% reduction in thrombus height in comparison to the control. This study demonstrates that NO can reverse key platelet functions after their initial activation and identifies a novel mechanism for controlling excessive thrombosis. discoid platelets can also contribute to thrombus under certain conditions 28 We have recently identified that the actin cytoskeleton in activated platelets is a dynamic structure which can undergo reorganisation following treatment with PGI 2 , leading to a reduction in preformed microthrombi coverage 18 . In agreement with this post perfusion of Rac inhibitors on preformed thrombi also leads to thrombus instability 29 In this present study, we examined the effect of NO signalling on spread platelets. We demonstrate that (i) NO reduces the thrombus height of preformed thrombi on collagen, (ii) NO reduces surface area coverage of platelets under high shear on fibrinogen, (iii) NO reverses stress fibre formation in activated platelets, leading to actin nodule formation in a GMP-dependent manner, (iv) NO targets inhibition of RhoA but does not cause phosphorylation at serine 188 and (v) NO and PGI 2 can work synergistically to reverse stress fibre formation using physiologically relevant doses of both platelet inhibitors. This work has important implications on the current understanding of the thrombus microenvironment and for understanding the mechanisms by which thrombus growth is controlled to prevent excessive thrombus formation in response to vascular injury and atherosclerotic plaque rupture. of ADP TXA 2 signalling. Part of the mechanism by which thrombus growth is regulated is via a controlled actin cytoskeletal response. As we had previously identified that PGI 2 /PKA signalling reverses stress fibre formation 18 , we hypothesised that NO would induce a similar response. Here we demonstrate that NO induced reversal of stress fibre forma- tion in fully spread platelets causing the formation of actin nodules, although the effects were not as strong as that observed with PGI 2 . The NO driven reversal of stress fibres was dependent on the formation of cGMP and PKG activity and is in agreement with observations in other cell types 35 . Furthermore, addition of a Rho Kinase (ROCK) inhibitor to platelets produced a similar response to that seen with NO, allowing us to speculate that the mechanism of reversal was via inhibition of the Rho/ROCK pathway. Regulation of RhoA through phosphorylation of serine188 has previously been observed in platelets and other cell types in response to cGMP/PKG signalling 30,36 . However, although NO induced a clear inhibition of Rho activation downstream of sGC, this was not via phosphorylation of Ser188. Although, previous studies have shown that RhoA can be phosphorylated at serine 188 downstream of NO by PKG, these studies were carried out at higher doses of NO and in platelets in suspension rather than in spread platelets 30,37 . The lack of Ser188 phosphorylation in our experiments indicates that rather than targeting RhoA interaction with a RhoGDI to prevent its activation 38 , NO is possibly targeting the RhoGEFs or RhoGAPS that control RhoA activity levels. It is of interest that NO and PGI 2 both induce reversal of stress fibre formation, and yet the data suggests their mechanism of action maybe distinct. This is an area that needs further investigation to understand the distinct mechanism by which NO and PGI 2 signalling modulate RhoA activity. Interestingly, studies have previously demonstrated the ability for NO to affect RhoA function in vitro through PKG-independent s-nitrosylation in are affected by cyclic nucleotides in activated platelets. Post treatment of GSNO on spread platelets does not reduce surface area coverage of spread platelets. This indicates that Rac activation is likely not inhibited by GSNO. is seen with PGI 2 , showed a consistent and marked surface area reduction . , small of platelets to NO to varia-ble levels of signalling, addition of apyrase a significant reduction in average surface area induced by GSNO. Kirkby et al . , show a potentiation of GSNO signalling in the presence of P 2 Y 12 inhibitors 40 the pathways that modulate RhoGTPase activity in platelets downstream of cyclic nucleotides, we how best to target thrombus formation to therapeutic benefit.Given that NO and PGI 2 can synergise 16 we investigated if this was the case in activated spread platelets. of their labile nature and poor methods for accurate concentration determination, the physi- ological levels of both NO and PGI 2 in the are physiological ranges of 0.1–5 and 100 pg/ml (27 nM) for PGI 2 have been postulated/measured 41,42 Our data that NO and PGI 2 which are close to the levels postulated in vivo , singularly do not reverse stress fibre formation, yet synergise to induce stress fibre reversal. This data is therefore in agreement with the Moncada group and others, demonstrating synergy between NO and PGI 2 signaling 13,16 . Interestingly, platelets have been reported to and an in vitro assay to show the effect of NO on thrombus disaggregation. Post perfusion of GSNO on preformed thrombi on collagen demonstrated a significant reduction in thrombus height, whilst thrombus surface area was unaffected. This data suggested that it is the shell region of the thrombus which is affected by post-perfusion of NO. This region is lacking thrombin, and therefore fibrin, with fibrinogen acting as the bridge between platelets 37 . Therefore, to identify if platelets adhered on fibrinogen at high shear were also affected by NO, whole blood was flowed over fibrinogen coated channels, and then post perfused with GSNO. Firstly, this clearly identified that platelets on fibrinogen in high shear conditions spread effectively, and form actin nodules and ultimately stress fibres. Secondly it was seen that GSNO treatment of these platelets did not affect their adhesion, but did reverse stress fibre formation leading to reduction in surface area of the platelets. It is not clear why the high shear rate induces a reduction in surface area, which is not present in static conditions. However, we hypothesise that the lack of stress fibres means the cell is unable to withstand hydrodynamic forces and therefore this causes the reduction in size. Furthermore, it would be interesting to increase the shear stress to pathophysiological levels to identify if platelet adhesion is then affected by post-perfusion of GSNO. The in vitro flow data suggests that the shell region of the thrombus is susceptible to the effect of NO, whilst the core region is unaffected. This data indicates that NO can potentially reverse thrombus formation and prevent occlusive thrombi. In agreement with this, knockouts of the prostacyclin receptor or sGC in platelets show no resting haemostatic abnormalities until vascular injury, under which conditions the formed thrombi are larger and more occlusive. This suggests that lack of PKG or PKA signalling alone is not enough to cause spontaneous the of that of of and that inhibition of RhoA, causes a reversal of stress fibre formation in spread platelets. Furthermore, NO can synergise with PGI 2 at very low, physiologically relevant concentrations to reverse platelet stress formation and so demonstrate that they act as synergistic partners. This reversal of stress fibre formation could have a significant impact on regulating thrombus size and preventing occlusive thrombi in vivo and represent a mechanism for targeting pathological thrombus formation.