Identification of membrane proteins mediating the interaction of human platelets

Abstract

Membrane glycoproteins that mediate platelet-platelet interaction were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets . The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombinaggregated platelets (platelets activated in the presence of mM Ca") were prepared by first treating platelet suspensions with 1% Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation . The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins : actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties identical to actin-binding protein (filamin) . When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction . Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material -1 Am in diameter . SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides . Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction . The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombinactivated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombinaggregated platelets contained membrane glycoproteins Ilb (26% of the total in pre-extracted platelets) and III (14%), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation .In 1886, Eberth and Schimmelbusch (l2) showed that blood platelets had the ability to "sort out" or aggregate to the exclusion of other blood cells. This aggregation reaction can occur in vivo at a site of vascular injury (which occurs during hemostas...

Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

“…We further demonstrated that these proteins can be crosslinked to one another in intact and detergent-extracted membranes by protein crosslinking agents (29). In addition, other work indicates that stimuli such as concanavalin A or thrombin can under the appropriate conditions induce a large increase in the total surface GPIIb and III (which bind concanavalin A [24]) recovered in the Tritoninsoluble cytoskeleton (30,35). These results indicated that even in the resting platelet membrane, a subfraction of GPIIb and III that accounted for -10 to 15 % of the total was already associated with the Triton-insoluble fraction of isolated membranes.…”

“…Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPllb and III exist: one that binds directly to endogenous membrane actin and one that does not.Numerous studies have provided largely indirect evidence for transmembrane interactions among surface proteins and receptors of cells and an internal membrane matrix composed of actin and other cytoskeletal proteins (3,5,7,10,14,16,17,19,23,25,27,31,35,40,45). Indeed, a number of studies have shown that the surface topography and lateral mobility of receptors can be modulated by the underlying cytoskeleton and its associated contractile and motile elements (20,28,30,31,36,37,39).…”

Autoregulation of Actin Synthesis by Physiological Alterations of the G-Actin Level in Hepatocytes

“…In addition, the ratio of filamentous to non-filamentous actin was determined by separation of proteins insoluble of soluble in Triton X-100, respectively (22). After treatment of hepatocytes with or without osmotic stress or with toxins, respectively, the cells were lysed by addition of an ice cold Triton solution containing 20 g/1 Triton X-100, 160 mmol/1 KC1, 20 mmol/1 EGTA, 8 mmol/1 sodium azide, and 40 mmol/1 imidazole HC1, pH 7.O.…”

The association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton of aggregated platelets is a reversible process regulated by calcium

“…Cytoskeletal fractions were prepared by using a modification of the method described by Phillips et al [2]. Briefly, platelet suspensions (1.5 ml) were lysed before or at various intervals after agonist addition by adding equal volumes of ice-cold 2 x Triton lysis buffer (pH 7.5) containing 100 mM Tris-HCl, 2% Triton X-100, 10 mM EGTA, 10 mM EDTA, 2 mM sodium orthovanadate, 20 mM leupeptin, 2 rnM phenylmethylsulfonyl fluoride, 20,uM pepstatin A and 0.56 trypsin inhibitor unit (t.i.u.…”

“…Several regulatory proteins in platelets are known to associate with the actin-rich cytoskeleton in a manner dependent on activation and cross-linking of the integrin e.uB3. These include the integrin %tf13 itself [2][3][4][5], protein tyrosine kinases such as pp60 st" [6--12] and pp60 c-v~s [12], the protein tyrosine pbosphatase SH-PTP1 [13], small molecular weight GTP-binding proteins like Rho A [14], RaplB [15,16], Rap2B [17] and CDC42Hs (submitted for publication) and other signalling proteins [6,7]. This complex of specific signalling proteins might regulate integrinactin interaction in aggregating platelets.…”