Filamin A (FLNa), a dimeric actin crosslinking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIb␣ binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIb␣ cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIb␣ binds in a groove formed between the C and D ␤ strands of FLNa domain 17. The interaction is strikingly similar to that between the ␤7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data. (Blood. 2006;107:1925-1932

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1−/−) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1−/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca ؉؉ -dependent activation of ␣IIb␤3, resulting in (patho)physiological thrombus formation. It is demonstrated here that the cytoplasmic domain of GPIb-IX-V associates with cytosolic calmodulin. First, an anti-GPIb␣ antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IXassociated proteins 14-3-3 and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIb␤, R149-L167 (single-letter amino acid codes), affinity-isolated calmodulin from platelet cytosol in the presence of Ca ؉؉ as confirmed by comigration with bovine calmodulin on sodium dodecyl sulfate-polyacrylamide gels, by sequence analysis, and by immunoreactivity with the use of an anticalmodulin antibody. The membraneproximal GPIb␤ sequence was analogous to a previously reported calmodulinbinding sequence in the leukocyte adhesion receptor, L-selectin. In addition, the cytoplasmic sequence of GPV, K529-G544, was analogous to a calmodulinbinding IQ motif within the ␣1c subunit of L-type Ca ؉؉ channels. Calmodulin coimmunoprecipitated with GPV from resting platelet lysates, but was dissociated in stimulated platelets. A GPV-related synthetic peptide also bound calmodulin and induced a Ca ؉؉ -dependent shift on nondenaturing gels. Together, these results suggest separate regions of GPIb-IX-V can directly bind calmodulin, and this novel interaction potentially regulates aspects of GPIb-IX-V-dependent platelet activation. IntroductionThe platelet membrane glycoprotein (GP) Ib-IX-V complex binds to the adhesive ligand von Willebrand factor (vWF), an interaction that initiates platelet adhesion and aggregation at high-shear stress. [1][2][3][4] Adhered platelets become activated, with elevation of cytosolic Ca ϩϩ and triggering of signaling pathways, leading to cytoskeletal rearrangements and activation of ␣IIb␤3 integrin (GPIIb-IIIa), which binds to fibrinogen or vWF, resulting in platelet aggregation. [5][6][7] The pathway by which ligand binding to GPIb-IX-V leads to ␣IIb␤3 activation and thrombus formation is poorly understood, although delineation of early signaling events mediated by GPIb-IX-V offers potential targets for therapeutic intervention.The GPIb-IX-V complex consists of 4 membrane-spanning glycoproteins. GPIb␣ is disulfide-linked to GPIb␤, forming GPIb, which is noncovalently associated with GPIX in a 1:1 complex. GPIb-IX-V is composed of GPIb-IX and GPV in a 2:1 ratio. 3 In extracts of platelets in the presence of Triton X-100 detergent, GPIb-IX remains a complex, while GPV is dissociated. 8,9 The extracellular, N-terminal 282 residues of GPIb␣ contain the vWF binding site. 3,10 Recent studies have identified signaling molecules associated with the cytoplasmic domain of GPIb-IX-V that appear to be important for the function of the receptor. One of these molecules is 14-3-3, which direc...