Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts

Abstract

Key Points• Clot retraction of sphingomyelin-rich raftdepleted platelets from sphingomyelin synthase knockout mouse is delayed.• Translocation of fibrin to sphingomyelin-rich rafts in platelet membrane is induced by thrombin in the presence of FXIII crosslinking activity.Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen g-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunitdeficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-b-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-aIIbb3-myosin complex is formed as a primary axis to promote platelet contraction. (Blood. 2013;122(19):3340-3348) IntroductionMembrane rafts are dynamic assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms involved in the regulation of a number of vital cellular processes. 1 The important role of rafts at the cell surface may be their function in signal transduction. A number of studies provide considerable evidence that rafts are integral to the regulation of immune and neuronal signaling. Membrane rafts are also involved in hemostasis and thrombosis. Among blood cells, platelets are critical for maintaining the integrity of the blood coagulation system. Platelet rafts are critical membrane domains in physiological responses such as adhesion and aggregation. 2 The localization of the adhesion receptor glycoprotein (GP)Ib-IX-V complex to membrane rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor. 3 Membrane rafts are also required for platelet aggregation via the collagen receptor GPVI, 4 the adenosine 59-diphosphate (ADP) receptor P2Y12, 5 the Fcg receptor FcgRIIa, 6 and the C-type lectinlike receptor CLEC-2.7 Detergent-resistant membrane (DRM) rafts of platelets show round vesicles of heterogeneous sizes ranging from 20 to 500 nm, which are enriched in CD36 (GPIV). 8,9 Recent reports have demonstrated that membrane rafts are ...

Transbilayer lipid distribution in nano scale

“…The isolated cells from freshly drawn blood were sandwiched between two carriers. Platelets were prepared from freshly drawn blood as previously reported (Kasahara et al, 2013). The platelet-rich plasma was activated with 0.5 units/ml thrombin (Sigma-Aldrich) for 3 min at 37˚C.…”

Lipid Rafts and Anti-Glycolipid Antibodies

“…Sphingomyelin-rich rafts localize in the central region of adhering platelets with thrombin, whereas cholesterol-rich rafts localize evenly on the membrane. Sphingomyelin-rich rafts act as platforms of fibrin-mediated outside-in signals, leading to clot retraction (19). These observations suggest that lipid rafts are compositionally and functionally heterogeneous (20).…”

Translational Approaches In Cardiovascular Diseases by Omics

“…Lipid rafts are about 10-200 nm in lipidordered microdomains enriched with sphingomyelin, glycosphingolipids and cholesterol (Rabani, Davani, Gambert-Nicot, Meneveau and Montange, 2016;Thomas et al, 2014). It is now established that some platelet proteins, such as P2Y12 (Savi et al, 2006) and FXIII (Kasahara et al, 2013)need to be on lipid rafts to be functional.…”

“…Lipidomics provided a good tool for identification of plasma membrane fractions and characterization of these microdomains (Figure 4) by establishing the ratio of cholesterol/sphingomyelin in lipid raft microdomains (Koumanov et al, 2005;Wolf and Quinn, 2008). These microdomains are the focus of attention in membrane biological research and are implicated in cardiovascular diseases through their participation in platelet activation (Kasahara et al, 2013;Savi et al, 2006), monocyte membrane reorganization (Salvary et al, 2012) and in the mechanisms mediating drug effects (García and Senis, 2011;Zinzalla and Thurston, 2009 ! 8 Figure 4.…”