Rac1 Is Essential for Platelet Lamellipodia Formation and Aggregate Stability under Flow

Abstract

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.

The Actin Polymerization Regulator WAVE2 Is Required for Early Bone Marrow Repopulation by Hematopoietic Stem Cells

“…In platelets, Rac1 is essential for lamellipodium formation, leading to stable thrombus formation in vivo [29]. Our previous studies on megakaryocytes and platelets suggest that WAVE2 but not WAVE1 is a primarily responsible target downstream of Rac1 [20].…”

Intracellular cyclophilin A is an important Ca2+ regulator in platelets and critically involved in arterial thrombus formation

“…To determine the impact of CyPA on cytoskeletal reorganization, Cypa ϩ/ϩ and Cypa Ϫ/Ϫ platelets were allowed to adhere and spread on a fibrinogen matrix using thrombin stimulation to induce full spreading of murine platelets. 33 Spreading of platelets was analyzed by the development of filopodia and lamellipodia at different time points. Subsequently, platelets were stained with rhodamine phalloidin and analyzed by confocal microscopy ( Figure 1C).…”

S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation

“…4 As platelets adhere to an immobilized surface of fibrinogen, the platelet integrin ␣ IIb ␤ 3 recruits and activates tyrosine kinases such as Src and Syk at its intracellular domains to ultimately lead to the activation of Rac1. 4,32 To better understand how these tyrosine kinase pathways activate Rac1 to mediate platelet spreading, we first examined the effects of Src and Syk kinase inhibition on platelet lamellipodia formation. Human platelets were purified from freshly drawn blood and allowed to bind to an immobilized surface of fibrinogen.…”

“…3 On the engagement of the adhesive proteins fibrinogen and fibronectin, platelet tyrosine kinases such as Src, Syk and FAK are recruited to the platelet cytosolic cell surface to initiate signaling pathways to drive platelet cytoskeletal reorganization through the Rho family small GTPase Rac1. [3][4][5] Rac1 regulates actin polymerization at the cell membrane to drive the growth and extension of platelet lamellipodiae that form the basis for platelet spreading. 4 The molecular mechanisms by which tyrosine kinases ultimately activate Rac1 remain ill-defined.…”

“…[3][4][5] Rac1 regulates actin polymerization at the cell membrane to drive the growth and extension of platelet lamellipodiae that form the basis for platelet spreading. 4 The molecular mechanisms by which tyrosine kinases ultimately activate Rac1 remain ill-defined.The 70 kDa ribosome S6 protein kinase (S6K1) regulates the ribosome S6 protein to integrate processes of protein translation with cell growth and cell proliferation. 6 In cultured cells as well as in vivo, mitogenic signals triggered by nutrients and growth factors initiate a complex sequence of signaling events to activate the mammalian target of rapamycin (mTOR), a serine/threonine kinase which regulates S6K1 phosphorylation and activation.…”