Dystrophin-related Protein in the Platelet Membrane Skeleton

Abstract

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 ؋ g) but the membrane skeleton requires 100,000 ؋ g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin ␣ IIb ␤ 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following ␣ IIb ␤ 3 -ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate a ϳ140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.Duchenne muscular dystrophy is one of the most common inherited human diseases. It is caused by a defective gene that codes for a 427-kDa protein, dystrophin (1-5). The deduced amino acid sequence of dystrophin shows that it consists of four domains and suggests that it is a cytoskeletal protein (6). The major rod-shaped domain contains 24 spectrin-like repeats. This domain is flanked on the amino terminus by a domain that has a high degree of homology to the actin-binding domains of spectrin and ␣-actinin, and on the carboxyl terminus by a cysteine-rich domain that shows some homology to a Ca 2ϩ -binding region in ␣-actinin. The most carboxyl-terminal end of dystrophin consists of a short domain that has no homology to any known protein and appears to play a role in linking the molecule to the plasma membrane (7,8). Recent studies using purified protein or recombinant fragments containing the putative actin-binding domain (9 -11) have shown that the protein can bind to actin filaments in vitro, supporting the idea that this molecule functions as a cytoskeletal protein. The finding that dystrophin exists in a submembranous location (8,12,13) and that the carboxyl-terminal end of the molecule associates tightly with a complex of membrane glycoproteins (termed dystroglycan) (14 -17) suggests that dystrophin is a component of a submembranous cytoskeleton.Although there is now considerable inform...

Identification of Dp71 Isoforms in the Platelet Membrane Cytoskeleton

“…1D). Consistent with our previous findings (27), utrophin was identified in platelets using a monoclonal antibody (MAN-CHO3) against the C-terminal domain of utrophin (Fig. 1E).…”

“…8A). Furthermore, sedimentation of syntrophin was accelerated in stirred platelet suspensions in which cell contact and aggregation were maximized (data not shown), a result consistent with utrophin, GPIIbIIIa, and pp60 c-src (27,44). Platelets from patients with Glanzmann's thrombasthenia are deficient in ␣ IIb ␤ 3 and do not undergo ␣ IIb ␤ 3 -mediated transmembrane signaling (44).…”

“…In contrast to normal platelets, syntrophin failed to redistribute to the low speed detergentinsoluble pellet of thrombasthenic platelet that were activated in the same way (Fig. 8B), a result consistent for utrophin, GPIIb-IIIa, and pp60 c-src (27,44). These findings indicate that syntrophin, like Dp71⌬ 110 and utrophin, is part of the platelet membrane cytoskeleton that is incorporated into integrin-rich cytoskeletal complexes as a result of integrin-mediated transmembrane signaling.…”

“…4B, lanes 3 and 4). Consistent with our previous findings (27), utrophin was recovered in the detergent-insoluble fraction, with a minor amount in the detergent- First-strand cDNA was synthesized by using total RNA from human platelets and a primer complimentary to a sequence in the 3Ј-untranslated region of the Dp71 mRNA (designated primer 513). The cDNA was amplified by PCR with primers specific for the 3Ј-end of Dp71 (designated primers 837 and 2296) that flank exon 78.…”

“…Our previous studies have identified utrophin as a component of the platelet membrane cytoskeleton and indicate that it participates in cytoskeletal reorganization during platelet activation (27). Although platelets do not contain full-length dystrophin, the presence of Cterminal dystrophin isoforms was not examined.…”