WASP plays a novel role in regulating platelet responses dependent on αIIbβ3 integrin outside‐in signalling

Abstract

Summary The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent –yet– actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by αIIbβ3 integrin outside‐in signalling, we questioned whether its function might be linked to integrin. Agonist‐induced αIIbβ3 activation (PAC‐1 binding) was normal for patient platelets, indicating normal integrin inside‐out signalling. Inside‐out signalling (fibrinogen, JON/A binding) was also normal for wasp‐deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp‐deficient murine platelets, indicating decreased integrin outside‐in responses. Another integrin outside‐in dependent response, fibrin clot retraction, involving contraction of the post‐aggregation actin cytoskeleton, was also decreased for patient platelets and wasp‐deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp‐deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro‐coagulant response, was enhanced for WASP‐deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro‐aggregatory and pro‐coagulant responses downstream of integrin outside‐in signalling.

Effects of eltrombopag on platelet count and platelet activation in Wiskott-Aldrich syndrome/X-linked thrombocytopenia

“…by guest www.bloodjournal.org From a deficiency of WASP could result in impairments of actin filaments and therefore an increase in PS-positive microparticles. 18 This is in agreement with the results observed in our study, although variations between patients were too large to observe statistically significant increases in the percentage of PS-positive microparticles and mean fluorescence ( Figure 1F).…”

“…A recent study proposed a novel role for WASP downstream of integrin outside-in signaling. 18 Agonist-induced GPIIb-IIIa activation measured by PAC1 binding was normal in WAS patients, whereas adherence and spreading on immobilized fibrinogen was decreased, suggesting that WASP does not play a role in the initial platelet response but does play a role with GPIIb-IIIa outside-in responses such as platelet spreading, clot retraction, and platelet plug stability. Also, since WASP might regulate actin filament linkages between the plasma membrane and the cortical cytoskeleton, 29 Epag, eltrombopag; ICH, intracranial hemorrhage; n/a, not available; Pt, patient; tx, treatment with eltrombopag.…”

“…16 Two studies by Shcherbina et al showed increased expression of phosphatidylserine (PS) on the surface of circulating platelets and an increase in platelet-derived microparticles in the plasma of WAS/XLT patients, raising the possibility that these features explain or at least contribute to the reduced platelet lifespan and size in this disease. 17,18 To better understand the pathogenesis of bleeding in WAS/XLT, the present study examined platelet activation by whole blood flow cytometry in WAS/XLT patients compared with healthy age-matched controls. Platelet activation markers were corrected for platelet size difference because of the microthrombocytopenia in WAS/XLT.…”

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition

“…33 WAS platelets are smaller than healthy control platelets and have impaired outside-in a IIb b 3 signaling and abnormal a-granule distributions. 24,34 When adhered to protein microdots, WAS platelets exhibited less spreading beyond the microdot boundaries compared with healthy wild-type platelets ( Figure 6D). As expected, on non-geometrically constrained areas of collagen, spread areas of WAS and normal platelets were 12.7 6 1.3 mm 2 vs 17.3 6 1.1 mm…”

Integrin αIIbβ3 outside-in signaling

“…77 Of the many effector molecules downstream of the Rho GTPases, WASP is a major component of a IIb b 3 outside-in signaling, with platelets from WAS patients and WASP-deficient mice showing defects in platelet spreading on fibrinogen, clot retraction, and primary plug stabilization. 117 WASP is activated by Cdc42 and partners with and activates actin-related protein complex 2/3 (Arp2/3), supporting the nucleation of actin filaments to initiate formation of the branched actin network, which is important for events like filopodia and lamellipodia formation during platelet spreading. Rac can also activate Arp2/3 through the WASP-family verprolin-homologous (WAVE) 1-3 proteins, although loss of WAVE-1 in platelets did not lead to a defect in lamellipodia formation on fibrinogen.…”