Rapid purification and partial characterization of human platelet glycoprotein IIIb. Interaction with thrombospondin and its role in platelet aggregation

Glycoprotein IIIb (also known as glycoprotein IV) is a major glycoprotein present on the surface of human platelets. Recent studies suggest that glycoprotein IIIb may be a receptor site for thrombospondin. Thrombospondin, a multifunctional adhesive glycoprotein released from stimulated platelets, plays an important role in the stabilization of platelet aggregates. In this study, a new method for the purification of glycoprotein IIIb is described. Glycoprotein IIIb was isolated from Triton X-114 platelet membrane extracts, under nondenaturing conditions, by tandem anion-exchange and size exclusion fast protein liquid chromatography. The purified glycoprotein had the same apparent molecular mass (88 kDa) under nonreducing or reducing conditions. The tryptic peptide map of the purified protein was identical to that of bona fide glycoprotein IIIb as isolated from two-dimensional polyacrylamide gels of platelet membrane proteins. In addition, the purified glycoprotein was recognized by an anti-GPIIIb monoclonal antibody (OKM5). The purified glycoprotein specifically bound to thrombospondin in the presence of calcium. Monospecific anti-GPIIIb antibodies interfered with the expression of endogenous thrombospondin on thrombin-activated platelets and partially inhibited collagen- and thrombin-induced platelet aggregation without a significant effect on platelet secretion. Glycoprotein IIIb, by interacting with thrombospondin on the activated platelet surface, may play an important role in the platelet aggregation process.     

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is thought to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 mM calcium was immediately treated with EDTA, labelled with 125I and incubated with monoclonal antibodies P10 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with 125I-labelled platelet releasates treated for 48 h at 4 degrees C with 10mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 125I-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas 125I-labelled P12 only recognized a 400-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trypsinized, formaldehyde-fixed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calcium-treated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies P10 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.     

The domain of platelet glycoprotein I as recognized by various antibodies, both monoclonal and polyspecific

Platelet membrane glycoprotein Ib plays a major role in the binding of factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium. We have used polyspecific and monoclonal antibodies to glycoprotein Ib and have demonstrated that both antibodies were directed to glycoprotein Is, a soluble fragment of glycoprotein Ib. By showing an inhibition of the binding of factor VIII/von Willebrand factor to control platelets in presence of the antibodies, it can be concluded that glycoprotein Is is involved in these binding sites.     

Binding of fibronectin to alpha-granule-deficient platelets

Most of the proposed functions for fibronectin involve its interaction with cells, yet the molecular nature of cellular fibronectin binding site(s) has remained obscure. Thrombin induces saturable platelet binding sites for plasma fibronectin and concurrently stimulates surface expression of a number of platelet alpha-granule constituents including thrombospondin and fibrin which are known to interact with fibronectin. To test the hypothesis that these (or other alpha-granule proteins) mediate plasma fibronectin binding, we used platelets of patients with the Gray Platelet Syndrome. These cells were deficient in thrombospondin, beta-thromboglobulin, platelet factor 4, fibronectin, and fibrinogen as measured in radioimmunoassay. They also had reduced von Willebrand factor content as judged by immunofluorescence. At plasma fibronectin inputs from 0.03 to 3 times the apparent kilodalton, these Gray platelets bound virtually identical quantities of fibronectin as normal cells. Thus, platelets containing 1,500 molecules of thrombospondin per platelet could bind more than 100,000 molecules of plasma fibronectin per cell following thrombin stimulation. These data preclude any simple model in which newly surface expressed thrombospondin (or other alpha-granule protein) functions as the major thrombin-stimulated plasma fibronectin receptor in this cell type.     

Characterization of thrombospondin as a substrate for factor XIII transglutaminase

Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.     

Immunoelectron-microscopic studies of human platelet thrombospondin, Von Willebrand factor, and fibrinogen redistribution during clot formation

Summary The relative distributions of the human platelet -granule proteins fibrinogen, thrombospondin, and von Willebrand factor were mapped by immunoelectron microscopy in thin cryosections of activated platelets, platelet aggregates, and clots during the first 24 h ofin vitro clot formation. In early activated platelets, the results suggest that the canalicular system constitutes a significant component of the external platelet surface, and may act as a compartment for biochemical reactions occurring during granule relase. Further, detection of coagulation proteins by various non-morphological procedures may reflect protein contained within canalicular elements. Later in the release process, von Willebrand factor was detected as a major antigen on the platelet canalicular and plasma membranes; thrombospondin, on the other hand, showed minimal binding to platelets and only limited binding to the extensive fibrin network. Comparison of radioimmunoassays of supernatants of thrombin-stimulated platelets in plasma, clotted whole blood, and Triton X-100 platelet releasates indicated that virtually all of the platelet thrombospondin appears in serum. These data confirm the immunocytochemical results indicating that very little platelet thrombospondin binds to the platelet surface, compared with von Willebrand factor, studied here under the same conditions, which binds extensively to the platelet membrane following release and clot formation.     

Comparison of secretion and subcellular localization of von Willebrand protein with that of thrombospondin and fibronectin in cultured human vascular endothelial cells

Cultured human vascular endothelial cells synthesize von Willebrand protein, thrombospondin and fibronectin. These proteins are secreted in the culture medium and incorporated into the extracellular matrix. We have compared the subcellular localization and the secretion of these proteins in response to stimulants in cultured human umbilical vein endothelial cells. Density gradient centrifugation using colloidal silica showed that the storage and secretion organelle with von Willebrand protein did not contain thrombospondin or fibronectin. Indirect immunofluorescence microscopy indicated that thrombospondin and fibronectin are not located in the rod-shaped organelles containing von Willebrand protein. Thrombin, ionophore A23187 and phorbol myristate acetate did not affect secretion of thrombospondin and fibronectin, while von Willebrand protein secretion was stimulated upon incubation of cells with these agents for 30 min. Prolonged incubation of cultured endothelial cells after a 1-h treatment with phorbol myristate acetate resulted in an increased secretion of von Willebrand protein into the conditioned medium; in contrast, accumulation of thrombospondin and fibronectin in endothelial cell-conditioned medium was decreased. These findings indicate that, unlike in platelets, these major endothelial proteins are not located in the same subcellular compartments. Von Willebrand protein is distinguished from thrombospondin and fibronectin both by its unique subcellular localization and its secretion rate in response to stimuli.     

Interaction of thrombospondin with resting and stimulated human platelets

The interaction of isolated and radioiodinated thrombospondin with washed human platelets has been characterized. The ligand bound to nonstimulated and thrombin-stimulated platelets in a time-dependent manner, and apparent steady state was reached within 25 min. Binding was not due to iodination of the ligand and was inhibited by nonlabeled thrombospondin but not by unrelated proteins, and bound ligand was identical with thrombospondin in terms of subunit structure. Nonlinear curve-fitting analyses of binding to resting platelets suggested the presence of a single class of sites which bound 3,100 +/- 1,000 molecules/platelet with an apparent Kd of 50 +/- 20 nM. This interaction was not attributable to contaminating cells or inadvertant platelet activation. Binding to thrombin-stimulated platelets had a lower apparent affinity (Kd = 250 +/- 100 nM) and higher apparent capacity (35,600 +/- 9,600 molecules/platelet). Thrombin-enhanced binding was dependent upon agonist dose and platelet stimulation. Fibrinogen, a monoclonal antibody to GPIIb-IIIa, temperature, and divalent ions had differential effects upon thrombospondin binding to resting and stimulated platelets, suggesting the presence of two distinct mechanisms of thrombospondin binding to platelets. While thrombospondin binding to thrombin-stimulated platelets occurs with characteristics similar to those observed for fibrinogen, fibronectin, and von Willebrand Factor, its high affinity interaction with resting platelets is unique to this adhesive glycoprotein.     

The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.     

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.     

Signalling through the platelet glycoprotein Ib-V-IX complex

The glycoprotein Ib-V–IX is one of the major adhesive receptors expressed on the surface of circulating platelets. It is composed of four different polypeptides—GPIb, GPIbβ, GPIX, and GPV—and represents a multifunctional receptor able to interact with a number of ligands, including the adhesive protein von Willebrand factor, the coagulation factors thrombin, factors XI and XII, and the membrane glycoproteins P-selectin and Mac-1. Interaction of GPIb-V–IX with the subendothelial von Willebrand factor is essential for primary haemostasis, as it initiates platelet adhesion to the subendothelial matrix at the sites of vascular injury even under high flow conditions. Upon interaction with von Willebrand factor, GPIb-V–IX initiates transmembrane signalling events for platelet activation, which eventually result in integrin _IIbβ₃ stimulation and platelet aggregation. The investigation of the biochemical mechanisms for platelet activation by GPIb-V–IX has attracted increasing attention during the last years. This review will describe and discuss recent findings that have provided new insights into the events underlying GPIb-V–IX transmembrane signalling. In particular, it will summarise basic concepts on the structure of this receptor, extracellular ligands, and intracellular interactors potentially involved in transmembrane signalling. The recently suggested role of membrane Fc receptors in GPIb-V–IX-initiated platelet activation will also be discussed, along with the involvement of lipid metabolising enzymes, tyrosine kinases, and the cytoskeleton in the crosstalk between GPIb-V–IX and integrin _IIbβ₃.     

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.     

The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++- dependent adhesion of platelets to collagen

We have purified the platelet membrane glycoprotein Ia-IIa complex by detergent solubilization and sequential affinity chromatography on Concanavalin A-Sepharose and collagen-Sepharose. The complex, which is identical to the VLA-2 complex of lymphocytes and other cells and contains subunits of 160 and 130 kD on SDS-PAGE, was labeled with 125I and incorporated into phosphatidyl choline liposomes. The liposomes, like intact platelets, adhered to collagenous substrates in an Mg++-dependent manner with a K'a(Mg++) of 3.5 mM. Little adhesion of the liposomes to collagen occurred when Mg++ was replaced by Ca++ or EDTA. Calcium ions inhibited the Mg++-dependent adhesion with a K'i(Ca++) of 5.5 mM. Liposomes containing the Ia-IIa complex adhered to substrates composed of types I, II, III, and IV collagen, but did not effectively adhere to substrates composed of type V collagen or gelatin. Adhesion to collagen was specific. The liposomes did not adhere to fibronectin, vitronectin, laminin, thrombospondin, fibrinogen, or von Willebrand factor substrates. The monoclonal antibody P1H5, which specifically immunoprecipitated the Ia-IIa complex, also specifically inhibited the Mg++-dependent adhesion of both platelets and Ia-IIa-containing liposomes to collagen substrates. These findings provide additional evidence that the platelet membrane Ia-IIa complex is the mediator of Mg++-dependent platelet adhesion to collagen and suggest that the VLA-2 complex may also function as an Mg++-dependent collagen receptor in other cells.     

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.     

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm² and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.     

Analysis of the human CD36 leucocyte differentiation antigen by means of the monoclonal antibody NL07.

The murine monoclonal antibody (MoAb) NL07 was generated by immunization with human platelet extracts. NL07 MoAb recognized a molecule expressed by human platelets, monocytes, and endothelial cells, as well as by the myelomonocytic line U937 and by some melanoma cells or lines. Normal endothelial cells and the melanoma cells express the NL07 epitope only while adhering to a substrate. SDS-polyacrylamide gel electrophoresis and two-dimensional gel analysis indicate that the molecule recognized by NL07 MoAb on platelets is a single chain structure featuring a molecular weight of 85 kDa under reducing conditions, with an acidic isoelectric point ranging from 5.2 to 5.5. The specific phenotype distribution and the biochemical structure indicate that NL07 MoAb recognizes the platelet GPIV (CD36) molecule, a surface glycoprotein with a functional role of thrombospondin receptor. The results of competition tests with OKM5 MoAb (specific for the CD36 molecule) confirm the molecular specificity and epitope coincidence. Furthermore, upon binding to the platelets, NL07 MoAb is able to transmit via CD36 an activation signal which is followed by a potent aggregation. On the contrary, there is lack of evidence concerning the ability of the CD36 molecule of transmitting signal(s) on the U937 cells.     

Characterization of an antibody directed against a 128 kDa glycoprotein involved in the thrombogenicity of the elastin-associated microfibrils.

We have developed a monospecific antiserum directed against a major glycoprotein in the elastin-associated microfibrils with an apparent molecular mass of 128 kDa (GP 128). When immunoblotting or enzyme-linked immunosorbent microassay was used, its IgGs recognized thrombospondin in a platelet lysate, but did not react with several basement-membrane-derived macromolecules, nor with plasma fibronectin. Similar patterns of immunofluorescence and immunoperoxidase were found after incubation of endothelial cells with either anti-GP 128 or anti-(platelet thrombospondin) IgGs. Both antibodies inhibited the microfibrils- and thrombin-induced platelet aggregation, and were without effect on the aggregation by other inducers. These results confirm that there is an antigenic homology between GP 128 and thrombospondin.     

Platelet membrane glycoproteins and their function: an overview

The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.     

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.     

Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein.

Platelet membrane glycoprotein IIb-IIIa has been widely studied in the last years because of its role as an activation-dependent, adhesive protein receptor. Recently we demonstrated that occupancy of glycoprotein IIb-IIIa-receptor sites by specific ligands exerts an inhibitory effect on platelet responses induced by mild stimulation, leading us to suppose that this event may interact with activation pathways. Although the mechanisms of signal transduction in human platelets are not completely elucidated, the hypothesis that GTP-binding proteins are involved is generally accepted. Our results demonstrate that platelet ConA receptors, known to be located mainly on GP IIb-IIIa, are able to bind [35S]GTP gamma S; the GTP-binding activity is specific and is due to the association with the receptors of two G-proteins, with apparent molecular masses of 25 and 21 kDa, respectively. After the purification of GP IIb-IIIa, a glycoprotein complex electrophoretically pure was obtained that was still associated with a GTP-binding activity, migrating in SDS-polyacrylamide gel electrophoresis as a narrow band of about 21 kDa.