Ca2+-dependent protease in human platelets. Specific cleavage of platelet polypeptides in the presence of added Ca2+.

386

190

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...

Section: Identification Of Actin In the Triton Residuessupporting

confidence: 88%

Section: Preparation Of Cytoskeletal Structuresmentioning

confidence: 99%

Section: Thrombin-induced Actin Polymerizationmentioning

confidence: 99%

See 1 more Smart Citation

Identification of membrane proteins mediating the interaction of human platelets

Dr¹,

Jennings²,

Hh³

1980

386

190

“…Our ability to detect an aggregation independent interaction between intracellular GPl/b-lIIa and the actin cytoskeleton contrasts with previous findings reported by other groups (Phillips et al, 1980;Painter and Ginsberg, 1982;Wheeler et al, 1984) and absolutely depends on (a) the addition of divalent cation chelators at least 1 min before detergent lysis of the platelets, and (b) the use of Western immunoblot analysis to detect cytoskeleton-associated GPIIb-ma. We believe that chelation of divalent cations contributes to the stability of the cytoskeletal complexes by reducing the activity of calcium-dependent proteases (Phillips and Jakabova, 1977;Fox et al, 1985) and actin severing proteins such as gelsolin (Lind et al, 1982). In the absence of these chelators, our cytoskeletal pellets are smaller and contain significantly less actin, myosin, and GPIIb-IHa.…”

Section: Discussionmentioning

confidence: 99%

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets.

Bertagnolli

Beckerle

1993

Abstract. Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IHa (GPIlb-HIa) (also referred to as ottr~3 integrin), becomes incorporated into the detergent-insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-lIla is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, ~,,22 % of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, ot-actinin, or vinculin in the complex. We found that the cytoskeleton-associated GPllb-llla is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPl/b-IIIa associated with the a-granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, a-granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPHb-INa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPllb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-INa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.

“…Activation of the endogenous CANP in intact platelets with the ionophore A23187 in the presence of calcium has also been shown to result in the proteolysis of GPIb with release of glycocalicin (10). An additional substrate of CANP, ABP, has also been shown to be cleaved concomitant with platelet activation by thrombin or A23187 (7,16,24). Thus, it has been postulated that proteolysis of selected substrates, perhaps GPIb and/or ABP, by CANP may be a requisite step in platelet activation.…”

Section: Discussionmentioning

confidence: 99%

On the association of glycoprotein Ib and actin-binding protein in human platelets.

Okita¹,

Pidard²,

Newman³

et al. 1985

159

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.