Actin filament content and organization in unstimulated platelets.

Abstract

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37°C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin . About 40% of the actin in the discoid platelets obtained by either method existed as filaments . These filaments could be visualized by electron microscopy of thin sections . Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min) . However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked . Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia . These platelets also contained -40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37°C, which resulted in the reversal of filament organization . High g forces were then required for the sedimentation of the actin . Approximately 80-90% of the actin in platelets washed at 4°C was filamentous ; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures . These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization .

Transcript profiling of human platelets using microarray and serial analysis of gene expression

“…Previous estimates suggest that 20% to 30% of the total platelet proteome is comprised of actin with other components such as actin-binding protein, mysosin, and talin accounting for an additional 2% to 5% of the total protein. 1,22 The mRNAs encoding the actin-related machinery are overrepresented in our microarray analysis, with 8 such transcripts found among the 50 highest platelet-expressed genes. Interestingly thymosin ␤4 demonstrated the highest expression pattern.…”

“…Interestingly thymosin ␤4 demonstrated the highest expression pattern. In unstimulated platelets, 30% to 40% of actin is polymerized as F-actin, 22 whereas the balance of actin monomers (G-actin) are polymerization inhibited by sequestering proteins such as profilin (100 M) and thymosin ␤4 (600 M). 23 The high thymosin ␤4 transcript expression not only correlates with its known abundance in platelets but also supports the importance of actin inhibitory proteins in maintaining the nonstimulated state of circulating platelets.…”

Platelet activation.

“…16 ' 17 The amorphous component could be composed of actin complexed with profilin, a protein present in platelets in amounts sufficient to complex part, but not all, of the actin (see Table 1J. 18 - 50 However, some investigators, using different methods, 49 .…”

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

“…The signals that initiate actin polymerization are not known, but are important to determine because of the central role of actin in neutrophil functions (Southwick and Stossel, 1983; Yassin et al, 1985;Fox et al ., 1984). Under conditions similar to those under which actin gets polymerized, chemotactic factors also in-'Abbreviations used in this paper: fMet-Leu-Phe, formylMethionyl-LeucylPhenylalanine ; IP3, inositol, 1,4,5-trisphosphate; PAF, platelet-activating factor; PIP2, phosphatidylinositol 4,5-bis-phosphate ; PMA, phorbol 12-myristate, 13-acetate.…”