Clathrin-mediated integrin αIIbß3 trafficking controls platelet spreading.

Dynamic endocytic and exocytic trafficking of integrins is an important mechanism for cell migration, invasion, and cytokinesis. Endocytosis of integrin can be classified as clathrin dependent and clathrin independent manners. And rapid delivery of endocytic integrins back to the plasma membrane is key intracellular signals and is indispensable for cell movement. Integrin αIIbß3 plays a critical role in thrombosis and hemostasis. Although previous studies have demonstrated that internalization of fibrinogen-bound αIIbß3 may regulate platelet activation, the roles of endocytic and exocytic trafficking of integrin αIIbß3 in platelet activation are unclear. In this study, we found that a selective inhibitor of clathrin-mediated endocytosis pitstop 2 inhibited human platelet spreading on immobilized fibrinogen (Fg). Mechanism studies revealed that pitstop 2 did not block the endocytosis of αIIbß3 and Fg uptake, but inhibit the recycling of αIIbß3 to plasma membrane during platelet or CHO cells bearing αIIbß3 spreading on immobilized Fg. And pitstop 2 enhanced the association of αIIbß3 with clathrin, and AP2 indicated that pitstop 2 inhibit platelet activation is probably due to disturbance of the dynamic dissociation of αIIbß3 from clathrin and AP2. Further study demonstrated that Src/PLC/PKC was the key pathway to trigger the endocytosis of αIIbß3 during platelet activation. Pitstop 2 also inhibited platelet aggregation and secretion. Our findings suggest integrin αIIbß3 trafficking is clathrin dependent and plays a critical role in platelet spreading, and pitstop 2 may serve as an effective tool to address clathrin-mediated trafficking in platelets.

Involvement of cellular cytoskeleton components in antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and this may interfere with antibody-dependent cell lysis. We investigated the role of actin, microtubules, clathrin, and dynein, the major cellular components involved in physiological endocytosis during this virological internalization. Porcine monocytes were infected in vitro for 13 h and afterward treated with different concentrations of colchicine, cytochalasin D, latrunculin B, and amantadine-HCl, which inhibit polymerization of microtubules, actin/clathrin, actin, and clathrin, respectively. This resulted in a significant reduction of internalization compared to the nontreated control, indicating that these components are involved in the process. A double labeling was performed during the internalization process and a clear colocalization of actin, microtubules, clathrin, and dynein with the viral glycoproteins was observed at different stages during the internalization process. We conclude that these cellular components are used by PrV to generate the antibody-induced internalization of viral glycoproteins.

Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor, fibrinogen and fibronectin.

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of alpha-granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the alpha-granules.

Human erythrocyte clathrin and clathrin-uncoating protein.

Clathrin, a Mr = 72,000 clathrin-associated protein, and myosin were purified in milligram quantities from the same erythrocyte hemolysate fraction. Erythrocyte clathrin closely resembled brain clathrin in several respects: (a) both are triskelions as visualized by electron microscopy with arms 40 nm in length with globular ends and a flexible hinge region in the middle of each arm, and these triskelions assemble into polyhedral "cages" at appropriate pH and ionic strength; (b) both molecules contain heavy chains of Mr = 170,000 that are indistinguishable by two-dimensional maps of 125I-labeled peptides; and (c) both molecules contain light chains of Mr approximately 40,000 in a 1:1 molar ratio with the heavy chain. Erythrocyte clathrin is not identical to brain clathrin since antibody raised against the erythrocyte protein reacts better with erythrocyte clathrin than with brain clathrin and since brain clathrin contains two light chains resolved on sodium dodecyl sulfate gels while the light chain of erythrocyte clathrin migrates as a single band. The erythrocyte Mr = 72,000 clathrin-associated protein is closely related to a protein in brain that mediates ATP-dependent disassembly of clathrin from coated vesicles and binds tightly to clathrin triskelions (Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733). The erythrocyte and brain proteins have identical Mr on sodium dodecyl sulfate gels and identical maps of 125I-labeled peptides, share antigenic sites, and bind tightly to ATP immobilized on agarose. Clathrin and the uncoating protein are not restricted to reticulocytes since equivalent amounts of these proteins are present in whole erythrocyte populations and reticulocyte-depleted erythrocytes. Clathrin is present at 6,000 triskelions/cells, while the uncoating protein is in substantial excess at 250,000 copies/cell.

Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes.

We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20-50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.