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Spectrin is associated with membrane-bound actin filaments in platelets and
is hydrolyzed by the Ca2+-dependent protease during platelet activation
JE Fox, CC Reynolds, JS Morrow and DR Phillips
We recently showed that platelets contain submembranous actin filaments
that are linked to glycoprotein (GP) Ib on the plasma membrane. In the
present study, experiments were performed to determine whether spectrin was
associated with these filaments. The membrane-bound filaments were isolated
from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by
differential centrifugation. Platelet spectrin was detected immunologically
by using antibodies against human brain and RBC spectrin. Immunoblots
showed that platelet spectrin consisted of two polypeptides (mol wt 240,000
and 235,000) that were similar in apparent mol wt to those of the alpha and
beta chains of brain spectrin but differed slightly from those of RBC
spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments
identified platelet spectrin as two minor polypeptides migrating on sodium
dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein
(mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000).
Immunoblots of fractions isolated from Triton X-100-lysed platelets
revealed that the alpha and beta chains of platelet spectrin were
associated almost entirely with the actin filaments that were linked to the
plasma membrane. Little spectrin was recovered in the Triton X-100-soluble
fraction or with the actin filaments that were not membrane bound. During
activation of platelets with thrombin or ionophore A23187, the alpha and
beta chains of spectrin were hydrolyzed, generating a major degradation
product of mol wt 160,000 and a minor one of mol wt 170,000. These two
hydrolytic products were also generated in Triton X- 100 lysates incubated
in the presence of Ca2+ but were not produced when lysates were treated
with leupeptin, ethylene glycol bis(beta- aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), or N- ethylmaleimide, known
inhibitors of the Ca2+-dependent protease. These experiments show that
spectrin is a previously unidentified component of the membrane-bound actin
filament network and that hydrolysis of spectrin by the Ca2+-dependent
protease may regulate the interactions of the filaments during platelet
activation.
Volume 69,
Issue 2,
pp. 537-545,
02/01/1987
Copyright © 1987 by The American Society of Hematology
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