Actin dynamics in platelets

Abstract

The human blood platelet circulates in the blood as a non-adherent disk. Upon receiving signals of blood vessel damage, the platelet reorganizes its actin cytoskeleton which transforms it into a spiky dynamic adherent glue. This transformation involves a temporal sequence of four morphologically distinct steps which is reproducible in vitro. The actin dynamics underlying these shape changes depend on a large number of actin-binding proteins. Maintenance of the discoid shape requires actin-binding proteins that inhibit these reorganizations, whereas transformation involves other proteins, some to disassemble old filaments and others to polymerize new ones. F-Actin-affinity chromatography identified a large set of actin-binding proteins including VASP, Arp2 and 2E4/kaptin. Recent discoveries show that VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments. Morphological analysis of the distribution of these actin-binding proteins in spread platelets together with biochemical measurements of their interactions with actin lead to a model of interactions with actin that mediate shape change.

FIG. 1

Electron microscopy of a resting platelet. (Bearer, unpublished)

FIG. 2

Video microscopy of the same platelet as it spreads on glass, transforming from spheroid (a) to fully spread (h). Photographs were taken at 1-min intervals until the last, which was after a 5-min interval. Structures visible include pseudopodia (Ps), dense bodies (DB), and crater (Cr). Scale bar = 0.5 μm. (Reproduced from Allen et al., 1979, by permission.)

FIG. 3

Actin structures by fluorescence. (A) An example of a platelet 15 min after spreading on glass imaged by fluorescence microscopy of F-actin as stained with phalloidin. Arrow indicates the position of a former filopodia. (B) Diagram of the actin structures in the spread platelets: (1) leading edge of the lamellipodium; (2) filopodia; (3) lamellipodium; (4) contractile ring; (5) stress fibers. (Reproduced from Bearer, 1995, by permission.)

FIG. 4

Direct observation of actin filament severing. Rhodamine-phalloidin allows individual actin filaments to be imaged by fluorescence microscopy. Such imaging revealed for the first time that gelsolin severed filaments and was phalloidin-blind. In addition, microscopy of the effects of proteins on actin filaments can be used as a biochemical assay to follow proteins through purification strategies. (Reproduced from Bearer, 1991, by permission.)

FIG. 5

F-actin affinity chromatography identifies a large number of F-actin-binding proteins in platelets. Four individual experiments are shown (lanes labeled 1–4). Platelets were activated for 1 min with ADP, protein solubilized by sonication in a low ionic strength buffer containing detergent, and the lysate clarified by high-speed centrifugation. Extracts (lanes labeled X) were loaded on filamentous actin affinity columns and eluted sequentially with 5 mM ATP (lanes labeled ATP) and 1.0 M KCl (lanes labeled KCl). Note that a large number of different protein species reproducibly elute with each of the elution buffers. (Reproduced from Bearer, 1995, by permission).

FIG. 6

Immunofluorescence of actin-binding proteins. Two examples of patterns obtained using antibodies against platelet proteins eluting from the actin columns. Ab #32 (2E4/kaptin) stains lamellipodia and the cytoplasm, whereas Ab #21 (Arp2/3) stains the filopodia at this stage of spreading. (Adapted from Bearer, 1995, by permission.)

FIG. 7

The platelet membrane skeleton is composed of three layers. By thin section, the platelet membrane is lined by a periodic array of filaments that appear as dots when transected in sectioning for thin-section electron microscopy (A and A′) and as a linear array in quick-freeze deep-etch replicas of living platelets frozen in suspension (B and C). In A′ the cytoplasmic surface is to the left of the membrane. Note the rough texture of the P-face of the plasma membrane (PF) which is embossed by the underlying net of the membrane skeleton to which the sheet of actin filaments attaches. The three layers are best appreciated in (C), where the extracellular surface (EF), the membrane skeleton (PF) and the filaments (arrows) are shown. A granule is seen lying beneath the membrane (G). (Adapted from Bearer, 1990, with permission.)

FIG. 8

VASP bundles actin filaments and nucleates polymerization. (A) Fluorescently labeled filaments (top panel) mixed with VASP (lower panel) display thick bundles proportional to the amount of VASP added. (B) Polymerization of pyrene-actin(2 μM) is accelerated in the presence of VASP. Calcium-actin is more sensitive to VASP (inset, open squares) than magnesium-actin (inset closed squares). (Reproduced by Bearer et al., 2000, by permission.)

FIG. 9

Gelsolin binds but does not sever VASP-bundled filaments. (A) By fluorescence microscopy, VASP bundles filaments even at low concentrations. Only the bundles resist the severing effects of gelsolin. The shower of dots in the background (top panel) indicates short pieces left after gelsolin severing of individual filaments not part of a bundle. At higher VASP concentrations, all filaments are grouped into bundles, and no severing is detected (lower panel). (B) Sedimentation of VASP-actin bundles demonstrates that VASP protects filaments from solubilization by gelsolin, but gelsolin binds the filaments. Increasing amounts of gelsolin are found in the sediment, directly proportional to the amount of VASP. (Reproduced from Bearer et al., 2000, by permission.)

FIG. 10

Immunofluorescence of VASP and gelsolin in a spread platelet. VASP and gelsolin colocalize in the lamellipodia and filopodia (arrowheads) but not in stress fibers, where only VASP is found (arrows). (See also color insert.)

FIG. 11

Kaptin/2E4 localizes to the leading edge of platelets. Platelets spread on glass were stained with antibodies generated against the recombinant 2E4/kaptin protein. As seen with the original antibody (#32) generate against the human protein isolated by F-actin-affinity chromatography, staining was throughout the lamellipodia and in the dome of cytoplasm (the hyalomere). (Reproduced from Bearer and Abraham, 1999, by permission.)

FIG 12

Proposed model of actin reorganization in platelet activation and spreading. The discoid platelet has a trilaminar membrane shield composed of the outer membrane, the spectrin-ABP-rich inner membrane skeleton and a cage of closely aligned actin filaments that connect to the central core via radiating spokes. VASP bundles the filaments radiating from the central core. Most filaments are long and their barbed ends are capped. Monomer is sequestered by thymosin β4. Upon activation, the platelet rounds and contracts, the membrane skeleton is dissociated, at least in part through proteolysis. Side-binding proteins such as VASP, are released from the radiating bundles and gelsolin is activated by a calcium influx to sever these filaments and the membrane-associated cage. Activation of myosin results in condensation of the radiating filaments, severed from their membrane anchors, into the central core and formation of the contractile ring. Disassociation and severing are rapidly followed by rounds of polymerization of new actin from activated nucleators like Arp2/3 and 2E4/kaptin and from uncapping of severed barbed ends. Arp2/3 is activated at the membrane surface and on the sides of the severed filaments. Cofilin regulates filament length and recycles monomers that are recharged by profilin with ATP. Capping protein regulates which filaments are free to elongate, and hence the site of lamellipodia formation. VASP and other side-binding proteins restructure the new filaments into bundles. (See also color insert.)

FIG. 13

Arp2/3 is required for actin polymerization and for shape change in platelets. (A) Pyrene-actin assay showing the effects of anti-Arp2 on actin polymerization in TRAP-stimulated permeabilized platelets. (B) Histogram of the morphological effects of Arp2/3 inhibition on platelets loaded with pre-immune-antibodies (Pre), anti-Arp2 antibody (αArp) or its Fab fragments (Fab). Anti-Arp2 decreases filopodial formation, and freezes platelets at the rounded. Recombinant Arp2 (rArp) blocks this effect. (C) Arp2/3 is located at the tips of filopodia (arrows) and in the lamellipodia in two platelets spread on glass and double labeled for Arp2 (top) and actin (bottom) (Modified from Li, Kim and Bearer, 2002, with permission).