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NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.

Agonist-generated inside-out signals enable the platelet integrin ␣ IIb ␤ 3 to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to ␣ IIb ␤ 3 . By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D-and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating ␣ IIb ␤ 3 function, cytochalasin D-and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains ␣ IIb ␤ 3 in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on ␣ IIb ␤ 3 , allowing it to assume the high affinity conformation required for soluble ligand binding.The function of integrins in circulating blood cells is regulated by limiting their access to ligands. The prototypical example of this form of regulation is the platelet integrin ␣ IIb ␤ 3 since agonist-generated "inside-out" signals are required to enable ␣ IIb ␤ 3 to bind soluble ligands such as fibrinogen (1). Because the binding of soluble fibrinogen to ␣ IIb ␤ 3 is a prerequisite for platelet aggregation (2), regulating the affinity of ␣ IIb ␤ 3 for fibrinogen in this way assures that only stimulated platelets aggregate. It is likely that intracellular molecules regulate the function of ␣ IIb ␤ 3 by interacting with its cytoplasmic domains (3), but the identity of these molecules and how they interact with ␣ IIb ␤ 3 are not known.The actin cytoskeleton appears to play a role in regulating ␣ IIb ␤ 3 function. Thus, micromolar concentrations of cytochalasins, fungal metabolites that impair actin polymerization by binding to the barbed end of actin filaments (4), inhibit agonistinduced fibrinogen binding to ␣ IIb ␤ 3 on platelets (5-7), whereas nanomolar concentrations induce fibrinogen binding to recombinant ␣ IIb ␤ 3 expressed on the surface of B lymphocytes (8). In the work described in this paper, we used cytochalasin D (Cyto-D), 1 latrunculin A (Lat-A; another inhibitor of actin polymerization (9)), and jasplakinolide (a compound that stabilizes actin filaments (10)) to examine the role of actin filament turnover in...