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 PGI2 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.

Prostacyclin reverses platelet stress fibre formation causing platelet aggregate instability.

Prostacyclin (PGI2) modulates platelet activation to regulate haemostasis. Evidence has emerged to suggest that thrombi are dynamic structures with distinct areas of differing platelet activation. It was hypothesised that PGI2 could reverse platelet spreading by actin cytoskeletal modulation, leading to reduced capability of platelet aggregates to withstand a high shear environment. Our data demonstrates that post-flow of PGI2 over activated and spread platelets on fibrinogen, identified a significant reduction in platelet surface area under high shear. Exploration of the molecular mechanisms underpinning this effect revealed that PGI2 reversed stress fibre formation in adherent platelets, reduced platelet spreading, whilst simultaneously promoting actin nodule formation. The effects of PGI2 on stress fibres were mimicked by the adenylyl cyclase activator forskolin and prevented by inhibitors of protein kinase A (PKA). Stress fibre formation is a RhoA dependent process and we found that treatment of adherent platelets with PGI2 caused inhibitory phosphorylation of RhoA, reduced RhoA GTP-loading and reversal of myosin light chain phosphorylation. Phospho-RhoA was localised in actin nodules with PKA type II and a number of other phosphorylated PKA substrates. This study demonstrates that PGI2 can reverse key platelet functions after their initial activation and identifies a novel mechanism for controlling thrombosis.

Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading.

BACKGROUND: During platelet spreading, the actin cytoskeleton undergoes marked changes, forming filopodia, lamellipodia and stress fibres. In the present study, we report the identification of a novel actin-rich structure, termed an actin nodule, which appears prior to lamellipodia and stress fibre formation. METHODS: Platelet spreading was monitored using human platelets and mouse GFP-actin platelets using real-time and end-point DIC, and fluorescent and electron microscopy (EM). RESULTS: We identified a small, novel actin structure, the actin nodule, in the early stages of adhesion and spreading, which we hypothesize to be a precursor of lamellipodia and stress fibres. Nodule formation shows an inverse correlation to Rho kinase and myosin-II activity, is independent of PI3-kinase, but dependent on Src kinase activity. Actin nodules contain multiple proteins, including Arp2/3, Fyn, Rac, and beta1- and beta3- integrins, but not Src. EM analysis revealed that actin filaments extend in all directions from the nodules. Actin nodules are present on multiple matrices, including fibrinogen, laminin and VWF + botrocetin. CONCLUSION: This work identifies a novel platelet actin structure, which we propose is a precursor to both lamellipodia and stress fibres and acts to drive platelet spreading.

MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

BACKGROUND: MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cell's contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation. OBJECTIVE: To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin). RESULTS: Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model. CONCLUSIONS: MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

A major role for Scar/WAVE-1 downstream of GPVI in platelets.

BACKGROUND: The small GTPase Rac1 plays a critical role in lamellipodia assembly in platelets on matrix proteins in the absence or presence of G protein-coupled receptor (GPCR) agonists. Rac mediates actin assembly via Scar/WAVE, a family of scaffolding proteins that direct actin reorganization by relaying signals from Rac to the Arp2/3 complex. OBJECTIVE: To evaluate the role of Scar/WAVE-1 in mediating platelet activation and cytoskeletal reorganization. METHODS AND RESULTS: Using specific antibodies, we demonstrate that murine platelets, like human platelets, express Scar/WAVE-1 and Scar/WAVE-2. Lamellipodia formation in Scar/WAVE-1(-/-) platelets is markedly inhibited on immobilized collagen-related peptide (CRP) and on laminin, both of which signal through the collagen receptor GPVI. In contrast, lamellipodia formation on collagen, which requires release of the GPCR agonists ADP and thromboxane A(2), is not altered. Immobilized fibrinogen supports limited formation of lamellipodia in murine platelets, which is not altered in Scar/WAVE-1(-/-) platelets. As with Rac1(-/-) platelets, Scar/WAVE-1(-/-) platelets exhibit a marked inhibition of aggregation in response to CRP, whereas the response to the GPCR agonist thrombin is not altered. Platelet aggregation on immobilized collagen under shear, which is dependent on signaling by matrix and GPCR agonists, was unaltered in the absence of Scar/WAVE-1. CONCLUSION: This study demonstrates a major role for Scar/WAVE-1 in mediating platelet cytoskeletal reorganization and aggregate formation downstream of activation by GPVI but not by GPCR agonists.

von Willebrand factor mediates platelet spreading through glycoprotein Ib and alpha(IIb)beta3 in the presence of botrocetin and ristocetin, respectively.

BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.