ISSN:
0730-2312
Keywords:
platelets
;
receptors
;
cytoskeleton
;
actin
;
membranes
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
When intact platelets are incubated at 37°C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton [4]. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37°C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymcrizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3′-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these crosslinked species were analyzed by SDS-PAGE in the presence of β-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction.Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly ( 〈 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III ( ∼ 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240270309
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