Distinct but critical roles for integrin aIIbb3 in platelet lamellipodia formation on fibrinogen, collagen-related peptide and thrombin Kelly Thornber1, Owen J. T. McCarty2,3, Steve P. Watson2 and Catherine J. Pears1 1 Department of Biochemistry, University of Oxford, UK 2 Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, UK 3 Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA Keywords Integrins are the major receptor type known to facilitate cell adhesion and aIIbb3; adhesion; integrins; lamellipodia; lamellipodia formation on extracellular matrix proteins. However, collagen- platelets related peptide and thrombin have recently been shown to mediate platelet lamellipodia formation when presented as immobilized surfaces. The aims Correspondence C. Pears, Department of Biochemistry, of this study were to establish if there exists a role for the platelet integrin South Parks Road, University of Oxford, aIIbb3 in this response; and if so, whether signalling from the integrin is Oxford, OX1 3QU, UK required for lamellipodia formation on these surfaces. Real-time analysis Fax: +44 1865 275259 was used to compare platelet morphological changes on surfaces of fibrino- Tel: +44 1865 275737 gen, collagen-related peptide or thrombin in the presence of various E-mail: [email protected] pharmacological inhibitors and platelets from ‘knockout’ mice. We demon- Website: http://www.bioch.ox.ac.uk strate that collagen-related peptide and thrombin stimulate distinct patterns 2+ (Received 11 July 2006, revised 22 August of platelet lamellipodia formation and elevation of intracellular Ca to 2006, accepted 12 September 2006) that induced by the integrin aIIbb3 ligand, fibrinogen. Nevertheless, lamelli- podia formation on collagen-related peptide and thrombin is dependent doi:10.1111/j.1742-4658.2006.05500.x upon engagement of aIIbb3, consistent with release of aIIbb3 ligand(s) from platelet granules. However, the requirement for signalling by the integrin on fibrinogen can be bypassed by the addition of thrombin to the solution. These observations reveal a critical role for aIIbb3 in forming lamellipodia on collagen-related peptide and thrombin which is dependent on its ability to function as an adhesive receptor but not necessarily on its ability to sig- nal. These results suggest that integrins may play an important role in lamellipodia formation triggered by nonintegrin ligands in platelets and possibly in other cell types. Platelets play an essential role in the formation of a series of morphological changes that include round- haemostatic plug at the site of vascular injury. This ing, generation of filopodia and lamellipodia, and for- process requires adhesion of the platelet to the mation of actin stress fibres [1]. These events serve to exposed subendothelial matrix, followed by powerful stabilize the thrombus, thereby enabling it to with- intracellular signalling events that lead to platelet– stand the high shear forces found in arteries and arte- platelet interactions and thrombus formation. One rioles. critical feature in this scheme is the dramatic alter- Stable adhesion of platelets to the subendothelial ation in platelet morphology in response to activa- matrix is dependent upon sustained activation of tion. Thus, the resting, discoid platelet undergoes a integrins [2]. Integrins are glycoprotein heterodimers Abbreviations CRP, collagen-related peptide. 5032 FEBS Journal 273 (2006) 5032–5043 ª 2006 The Authors Journal compilation ª 2006 FEBS K. Thornber et al. Platelet lamellipodia formation via aIIbb3 composed of a-subunits and b-subunits that exist in an formation but demonstrate that signalling by aIIbb3 is inactive or low-affinity conformation in nonactivated not essential for this response. cells. Intracellular signals within the platelet (known as ‘inside-out’ signalling) promote a conformational Results change in the extracellular domain, leading to an increase in affinity, thereby promoting integrin–ligand Morphological changes of human platelets on interactions [2]. In turn, clustering of integrins gene- fibrinogen, CRP and thrombin rates a series of intracellular signals (‘outside-in’ signal- ling) that serve to reinforce platelet activation [3]. The three ligands fibrinogen, CRP and thrombin sup- The major platelet integrin aIIbb3 is a receptor for port platelet adhesion and lamellipodia formation when fibrinogen, von Willebrand factor, vitronectin, CD40 presented as a monolayer, even though they bind to ligand and fibronectin. Integrin aIIbb3 plays a vital role distinct classes of surface receptor. This raises the ques- in supporting platelet adhesion to the extracellular tion of the molecular basis of adhesion to these ligands matrix and promoting platelet–platelet interaction and whether they induce distinct patterns of change in (aggregation). In addition, integrin aIIbb3 generates morphology. To address this, real-time imaging of outside-in signals that mediate platelet activation. It is platelets adhering to each surface was undertaken using now established that engagement of aIIbb3 activates a coating of ligands that induces maximal platelet adhe- Src family kinases, leading to activation of Syk [4], sion. These experiments were performed in the presence SLP-76 [5], Vav1 ⁄ 3 [4] and phospholipase Cc2 (PLCc2) of concentrations of apyrase and indomethacin shown [6], and thereby to activation of several second messen- to fully block the effect of the feedback agonists ADP 2+ ger pathways, including protein kinase C [7] and Ca and thromboxane A2, respectively, in order to directly [8]. These signalling events promote actin assembly, monitor the ability of each ligand to support adhesion leading to formation of filopodia, lamellipodia and and lamellipodia formation. stress fibres [9]. Consistent with previous reports, our results dem- In addition to integrin ligands, formation of filo- onstrate that platelets exposed to immobilized fibrin- podia and lamellipodia has been described on a ogen go through sequential formation of filopodia monolayer of collagen-related peptide (CRP), which and lamellipodia over a period of 30 min (Fig. 1 and selectively activates the immunoglobulin receptor glyco- supplementary Video S1). Both structures were stable protein VI (GPVI) [10,11], and on thrombin that has and did not retract once formed, although discrete been immobilized by fibrin. In platelets, thrombin binds movements could still be seen around the periphery to and signals via GPIba and the G protein-coupled of the cell. Fluorescent labelling of the actin cytoske- protease-activated receptor (PAR) receptors (PAR 1 leton revealed that stress fibres were formed within and 4) [12,13]. Immobilized thrombin that has become platelets that had undergone full lamellipodia forma- trapped by fibrin is able to promote platelet adhesion tion (Fig. 1B). In contrast, a distinct pattern of and aggregate formation at intermediate rates of flow, platelet morphological changes was observed on the leading to the speculation that it may function as an GPVI-specific agonist CRP (Fig. 1A and supplement- adhesion ligand in vivo [13]. In epithelial cells, it has ary Video S2). Limited small filopodia were seen, been suggested that immobilized thrombin can bind to with wave-like lamellipodia appearing before filopo- integrins through an RGD site, raising the possibility dia formation was complete, in contrast to the that is may bind directly to integrins in platelets [14]. spherically synchronized growth on fibrinogen. Full The present study was undertaken to investigate the lamellipodia formation was reached within 5–9 min, mechanism by which CRP and thrombin are able to three times more rapidly than on fibrinogen support platelet lamellipodia formation in comparison (Fig. 1C). Strikingly, even after platelets had reached to that induced by fibrinogen. It was of particular 90% of their final surface area, the lamellipodia were interest to discern whether thrombin and CRP stimu- very dynamic (supplementary Video S2), even though late lamellipodia formation directly, or whether they they were accompanied by formation of stress fibres require aIIbb3. The results demonstrate a critical role (Fig. 1B). A similar pattern of rapid yet unstable for aIIbb3 in promoting platelet lamellipodia formation lamellipodia formation and stress fibre formation on CRP, thrombin and on fibrinogen, but that out- was observed for platelets adhering to immobilized side-in signalling by the integrin is not required for thrombin (Fig. 1A,B and supplementary Video S3). lamellipodia formation on fibrinogen in the presence Significantly, lamellipodia formation on thrombin of thrombin. These results further emphasize the was not altered in the presence of the fibrin poly- importance of integrin engagement in lamellipodia merization inhibitor Gly-Pro-Arg-Pro (GPRP) (data FEBS Journal 273 (2006) 5032–5043 ª 2006 The Authors Journal compilation ª 2006 FEBS 5033 Platelet lamellipodia formation via aIIbb3 K. Thornber et al. A B C 120 100 80 60 FG CRP 40 THR % of maximum surface area 20 0 0 200 400 600 800 1000 1200 1400 1600 Time (sec) Fig. 1. Distinct morphological changes in human washed platelets exposed to fibrinogen, collagen-related peptide (CRP) and thrombin. (A) Human washed platelets were exposed to surfaces of fibrinogen (FG), CRP or thrombin (THR). Representative morphology of a single plate- let on each surface at the time points shown (s). (B) Rhodamine–phalloidin staining to show actin stress fibres of