Twinfilin 2a regulates platelet reactivity and turnover in mice

Abstract

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice () display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Profilin 1–mediated cytoskeletal rearrangements regulate integrin function in mouse platelets

“…13,21 Similarly, Pfn1 fl/fl-Pf4Cre platelets showed severely delayed and less effective clot retraction ( Figure 1H), further reflecting impaired integrin function and actin dynamics. 10,13 Because Pfn1 is an effector of the RhoA/ Rho-associated kinase (ROCK) pathway, these findings are in agreement with reports on RhoA-deficient platelets that show abolished clot retraction. 22 Furthermore, the strongly impaired filopodia and lamellipodia formation of Pfn1 fl/fl-Pf4Cre platelets suggests that Pfn1 acts as a key effector in actin dynamics and integrin function downstream of Rho guanosine triphosphatases (GTPases).…”

“…[5][6][7][8] Tln1 and kindlins connect integrins to the actin cytoskeleton, thereby enabling the sensing and exertion of mechanical forces as well as regulating adhesion formation and turnover. [9][10][11][12] Consistently, defects in actin-regulating proteins result in altered platelet and megakaryocyte integrin function. [13][14][15][16][17][18][19] Furthermore, we have recently shown a critical role of twinfilin 2a (Twf2a) and the cortical cytoskeleton in regulating platelet integrin turnover in a profilin 1 (Pfn1)-dependent manner.…”

“…10 Based on this, together with the disrupted cortical actin-cytoskeleton and the enhanced Ca 21 signaling in Pfn1 fl/fl-Pf4Cre platelets, we statistical differences between the groups: ***P , .001; **P , .01; *P , .05. , 65.6% 6 4.3%; **P , .01) upon stimulation with thrombin and CRP (Figure 2E-F; supplemental Figure 12A,C). Similar observations were made upon stimulation with CRP alone, but not when using the A23187 ionophore that efficiently induces phosphatidylserine exposure without integrin activation ( Figure 2G; supplemental Figure 12A-B,D-F).…”

“…[13][14][15][16][17][18][19] Furthermore, we have recently shown a critical role of twinfilin 2a (Twf2a) and the cortical cytoskeleton in regulating platelet integrin turnover in a profilin 1 (Pfn1)-dependent manner. 10 The small actin-binding protein Pfn1 is central for actin dynamics by mediating the nucleotide exchange on G-actin monomers, thereby promoting filament assembly with implications for platelet biogenesis. 13,20 Megakaryocyte-specific Pfn1 deficiency resulted in microthrombocytopenia because of cytoskeletal alterations and accelerated platelet clearance (supplemental Figure 1).…”

Molecular mechanism for inhibition of twinfilin by phosphoinositides

“…All twinfilins consist of two actin depolymerizing factor homology (ADF-H) domains separated by a short linker and followed by a short carboxy-terminal (C-terminal) tail region. Twinfilins are involved in several actin-dependent cellular and developmental processes, such as cell migration (26,27), endocytosis (28,29), epithelialto-mesenchymal transition (30), morphology of inner ear stereocilia (31), axonal growth of neurons (27), and platelet activation (32). Moreover, human twinfilin-1 has been shown to facilitate resistance to chemotherapy agents in breast cancer and lymphoma (26,30).…”