Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.

Abstract

Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3Hjborohydride method and 1ysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) lb. Approximately 70% of the platelet GP lb was present in this skeleton. Several other minor glycoproteins, including >50% of the GP Ia and small amounts of three unidentified glycoproteins of M, > 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actinbinding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and coisolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP lb were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 M, on the plasma membrane.

Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite outgrowth

“…A measure of 2 mg/ml DNaseI was added to one-half of the lysate to depolymerize Factin. 19,20 After shaking in an ice bath for 15 min, the lysates were centrifuged at 16 000 g for 2 min to remove triton-insoluble cytoskeleton fibers, and the resulting supernatants were further centrifuged at 100000 g for 20 min. The pellets contain Tritonsoluble F-actin, whereas the supernatants contain soluble G-actin.…”

“…18 FEZ1 was found in P fraction as well as S fraction (Figure 4q, lanes marked DNaseIÀ). When the lysate was treated with DNaseI to depolymerize Factin 19,20 before the fractionation, FEZ1 was not detectable in P fraction (Figure 4q, lanes marked DNaseI þ ). The depolymerization of F-actin was confirmed by the change of the actin population.…”

Cytoskeletal domains in the activated platelet

“…3). Glycoprotein IB (150 kD) which binds to actin via filamin [34,38] is detected in blots (Fig. 3) but is not readily visualized by Coomassie staining of protein gels and thus appears to be retained at low levels.…”

Naturally occurring human IgG antibodies to intracellular and cytoskeletal components of human platelets

“…In Ihe experiment shown in Fig. 2, proteins identified as actin and myosin (Fox, 1985) did not bind IgG while strong binding was seen at the position of talin, previously termed P235 by Fox (1985), and some in the region of actin-binding protein (ABP). Strong IgG binding at 140 kD occurred in an area of very weak Coomassie blue staining.…”