Platelet Lamellipodia Formation is not Required for Thrombus Formation and Stability

Background: During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. The latter structure displays a circumferential zone with orthogonally arrayed short actin filaments. Controversial data have been published on the role of lamellipodia in thrombus formation and stability, mainly based on inhibitor studies or analyzing platelets with additional functional defects. One study showed that Rac1 deficiency leads to defective lamellipodia formation and a specific ITAM signaling defect, resulting in impaired platelet adhesion and defective thrombus formation on collagen under flow. Supplementation with ADP and a thromboxane A2 analogue resuced this defect (Pleines et al, 2009), thus providing indirect evidence that lamellipodia formation might be dispensable for thrombus formation and stability. The WAVE complex, which has been shown in other cells to drive lamellipodia formation by enhancing actin nucleation via the Arp2/3 complex, is activated by Rac1 interaction with the WAVE complex subunit Cyfip1. We suggest that Cyfip1 functions as an important regulator of platelet lamellipodia formation. However, its role in platelet function is unknown.

Results: We used Cyfip1 deficient mice (PF4-Cre system) to study the impact on platelet physiology, and the role of platelet lamellipodia in thrombus formation and stability. Cyfip1^{− / −} mice displayed normal platelet counts and a slight reduction in platelet volume, but unaltered glycoprotein surface expression. Activation of mutant platelets was moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-Selectin surface exposure. Cyfip1^{− / −} platelets adhered to multiple adhesive surfaces including fibrinogen under static conditions but, strikingly, lamellipodia formation was completely abolished and the platelets rather exhibited an increased length and number of filopodia. Despite defective lamellipodia formation, Cyfip1^{− / −} platelets formed stable thrombi, when perfused ex vivo over collagen fibers at low and intermediate shear rates, and in vivo in two models of occlusive arterial thrombosis. Similarly, the hemostatic function was unaltered in the mutant mice. Investigation of control and Cyfip1^{− / −} platelet morphology in an induced thrombus under flow by scanning electron microscopy revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodia-like structures when in direct contact to collagen fibers at the bottom of the thrombus.

Conclusions: Here, we provide for the first time direct evidence by analyzing Cyfip1-deficient mice that surprisingly platelet lamellipodia formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow. We are currently investigating, whether platelet lamellipodia formation plays a role in other platelet-dependent processes in vivo.