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1.
Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule. 相似文献
2.
Stimulated human blood platelets release thrombospondin, an alpha-granule glycoprotein of 450 kDa. The aim of this work was to characterize an anti-thrombospondin monoclonal antibody (P8) in order to study the role of thrombospondin in platelet functions. The presence of thrombospondin receptor sites on resting and thrombin-stimulated platelets of three Glanzmann's thrombasthenia patients and normal donors was investigated using the P8 monoclonal antibody. Monoclonal antibody P8 was extensively characterized using ELISA, immunoprecipitation, immunoadsorbent affinity chromatography combined with tryptic peptide map analysis and crossed immunoelectrophoretic techniques. Labelled P8 bound strongly to thrombin-stimulated normal platelets (n = 14917 +/- 420, mean +/- SD) (Kd = 9.2 +/- 3.0 nM) and poorly to resting platelets (n = 2697 +/- 1278) (Kd = 24.8 +/- 18.6 nM). Moreover, the number of binding sites for P8 on thrombin-stimulated platelets from three Glanzmann's thrombasthenia patients, lacking the IIb-IIIa glycoprotein complex, were found similar to normal samples. F(ab')2 fragments of P8 inhibited aggregation of, and reduced secretion from, washed platelets stimulated by low concentrations of thrombin (0.05-0.06 U/ml) and collagen (0.5-0.6 microgram/ml). F(ab')2 fragments of P8 inhibited thrombin-induced platelet aggregation, but did not reduce fibrinogen binding (n) nor affect its dissociation constant (Kd). Inhibition of platelet aggregation by P8 suggests that thrombospondin plays an active role in promoting platelet aggregation, at low concentrations of thrombin and collagen. Normal binding of P8 to thrombin-stimulated Glanzmann thrombasthenic platelets indicates the presence of a thrombospondin receptor on the platelet surface distinct from the GPIIb-IIIa complex. 相似文献
3.
Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1993,121(2):469-477
The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix. 相似文献
4.
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. 相似文献
5.
Localization of adhesive proteins in two newly subdivided zones in electron-lucent matrix of human platelet alpha-granules 总被引:1,自引:0,他引:1
Platelet alpha-granules have been reported to consist of two zones, nucleoid and electron-lucent matrix, with different densities under electron microscopy. When washed human platelets were prepared by a rapid freeze-substitution method using liquid helium, we found that the electron-lucent matrix could be further subclassified into two zones having different densities: the intermediate and the light zones. The light zone was located at the periphery opposite the most dense nucleoid and contained several tubular structures with diameters of about 20 nm. The intermediate zone often laid between the nucleoid and light zone. By careful inspection, intermediate and light zones could even be identified in the platelets embedded in Lowicryl K4M, which where then used to localize several adhesive proteins in these two zone by immunocytochemical studies using the respective polyclonal antibodies. Fibrinogen, thrombospondin, and fibronectin were detected only in the intermediate zone. In contrast, von Willebrand factor (vWF) was localized only in the light zone, suggesting an association between vWF and the tubular structures in the light zone. In the nucleoid, none of these adhesive proteins were detected. Glycoprotein IIb/IIIa, a receptor for these adhesive proteins on the platelet surface, was detected not only on the outer surface of the cell membranes but also on the inner surface of the alpha-granule membrane. These data indicate that two zones with different densities in electron-lucent matrix and functions exist in the platelet alpha-granules. 相似文献
6.
Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation 总被引:3,自引:0,他引:3
The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist. 相似文献
7.
P J Sims E M Faioni T Wiedmer S J Shattil 《The Journal of biological chemistry》1988,263(34):18205-18212
We have investigated the composition and function of membrane microparticles released from platelets exposed to the C5b-9 proteins of the complement system. Gel-filtered human platelets were incubated with sub-lytic amounts of the purified C5b-9 proteins and the distribution of surface antigens was analyzed using monoclonal antibodies and flow cytometry. C5b-9 assembly caused secretory fusion of the alpha-granule membrane with the plasma membrane and the release of membrane vesicles (approximately 0.1-micron diameter) that contained the plasma membrane glycoproteins (GP) GP Ib and GP IIb-IIIa as well as the alpha-granule membrane protein GMP-140. These microparticles were highly enriched in the C9 neoantigen of the C5b-9 complex. The apparent surface density of C5b-9 on the microparticles was approximately 10(3)-fold higher than on the platelet itself, suggesting that the vesicles were selectively shed from the plasma membrane at the site of C5b-9 insertion. C5b-9 induced the expression of an activation-dependent epitope (recognized by monoclonal antibody, PAC1) in GP IIb-IIIa on the platelet surface but not in GP IIb-IIIa on the microparticles. The surface of the microparticles was also highly enriched in alpha-granule-derived coagulation factor V (or Va), accounting for nearly half of all the membrane-bound factor V detected. The number of potential membrane binding sites for factor Va was probed by adding saturating concentrations of factor Va light chain. Under these conditions, the density of factor Va binding sites on the microparticle surface exceeded that on the C5b-9-treated platelet by three to four orders of magnitude. Moreover, the microparticles provided most of the membrane surface for conversion of prothrombin to thrombin by VaXa. These studies demonstrate that the microparticles shed by C5b-9-treated platelets (and not the platelets themselves) provide the principal binding sites for coagulation factor Va and the principal catalytic surface for the prothrombinase complex. Platelet-derived microparticles formed during complement activation in vivo could provide a membrane surface that facilitates the assembly and dissemination of procoagulant enzyme complexes. 相似文献
8.
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. 相似文献
9.
Synthesis and secretion of thrombospondin by cultured human endothelial cells 总被引:39,自引:14,他引:25
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《The Journal of cell biology》1982,93(2):343-348
Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine- labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin. 相似文献
10.
J Niewiarowska C S Cierniewski G P Tuszynski 《The Journal of biological chemistry》1984,259(10):6181-6187
Triton-insoluble cytoskeletons prepared from either normal or thrombasthenic platelets were found to contain approximately 1.3 micrograms of fibronectin/10(9) platelets as measured by a radioimmunoassay. Total endogenous platelet fibronectin was quantitatively retained on the platelet cytoskeleton, whereas 70% of exogenously added fibronectin that bound the surface of thrombin-activated platelets was recovered with the Triton-insoluble cytoskeleton. The exogenously added fibronectin specifically bound platelets and cytoskeletons with the same affinity giving an apparent binding constant of 1.47 X 10(-7) M. The possibility that fibrin associated with the platelet cytoskeleton could serve as the fibronectin receptor was investigated by measuring the binding constant of fibronectin for polymerizing fibrin and by measuring the amount of fibronectin associated with cytoskeletons of thrombasthenic platelets which contain 4-fold less fibrin than controls. The binding constant of fibronectin for polymerizing fibrin was 14-fold lower than that for cytoskeletons and cytoskeletons prepared from thrombasthenic platelets contained approximately the same amount of fibronectin as controls. Therefore, it is unlikely that fibrin is the platelet fibronectin receptor. These results support the hypothesis that platelet fibronectin is released from platelet alpha granules upon thrombin stimulation and becomes bound to the platelet surface and cytoskeleton either directly or through some intermediate protein that spans the membrane and interacts both with fibronectin and the internal cell cytoskeleton. 相似文献
11.
A polyclonal antisera specific for human platelet thrombospondin (TSP) has been utilized to study the biosynthesis and secretion of TSP in the M21 human melanoma cell line. Pulse-chase indirect immunoprecipitation analysis reveal that human melanoma cells rapidly synthesize and secrete this platelet alpha-granule associated glycoprotein. Topographical analysis of the melanoma cell surface by indirect immunofluorescence indicate that the TSP molecules have no obvious extracellular organization. The implications of thrombospondin synthesis in the metastatic process of melanoma are discussed. 相似文献
12.
Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry 总被引:7,自引:0,他引:7
Tomer A 《Journal of biological regulators and homeostatic agents》2004,18(2):172-177
Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity. 相似文献
13.
K R McCrae S J Shattil D B Cines 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(10):3920-3927
Platelet contain Fc gamma RII. However, little is known about how the expression of these receptors is regulated. Inasmuch as platelet activation by a variety of agonists increases the expression of several proteins on the platelet surface, we used flow cytometry to study the effect of platelet activation on the expression of platelet Fc gamma R by measuring the binding of fluorescein-labeled oligomeric IgG (FITC-IgG oligomer) and fluorescein-labeled mAb IV.3 (FITC-IV.3), a mAb that recognizes Fc gamma RII, to platelets. The number of Fc gamma R per platelet was determined by relating the binding to platelets of FITC-IV.3, measured by flow cytometry, to the binding of 125I-labeled IV.3, measured using a standard filtration assay. Nonactivated, gel-filtered platelets from nine healthy donors expressed a mean of 891 Fc gamma R per platelet, whereas platelets activated at 25 degrees C by thrombin or PMA expressed a mean of 1382 Fc gamma R an average increase of 55% (p less than 0.001). Binding of FITC-IgG oligomer increased to a similar extent when platelets were stimulated by these agonists. A smaller increase in the number of Fc gamma R expressed on the platelet surface was measured when platelets were stimulated with ADP, though no increase was observed with epinephrine. The agonist-dependent increase in Fc gamma R expression did not occur when platelets were studied at 4 degrees C or in the presence of agents that elevate intracellular levels of cAMP, suggesting that platelet activation was required for this process. Agonist-stimulated Fc gamma R expression did not depend on dense-granule secretion, because it was observed at low agonist concentrations in the absence of 14C-serotonin release. These studies demonstrate that the number of Fc gamma R expressed on the platelet surface increases when platelets are activated by several agonists, perhaps as a result of the exposure of Fc gamma R located along the surface-connected open canalicular system, or the fusion of platelet alpha-granule and plasma membranes during the activation process. Increased Fc gamma R expression may promote the clearance of IgG-containing immune complexes from the circulation, and contribute to the development of immune complex-mediated thrombocytopenia. 相似文献
14.
In order to better understand granule release from platelets, we developed an alpha-toxin permeabilized platelet model to study alpha-granule secretion. Secretion of alpha-granules was analyzed by flow cytometry using P-selectin as a marker for alpha-granule release. P-selectin surface expression occurred when platelets were permeabilized in the presence of Ca2+. Responsiveness to Ca2+ was lost 30 min after permeabilization but could be reconstituted with MgATP. Alpha-toxin-permeabilized, MgATP-exposed platelets also degranulated within a pH range of 5.4-5.9 without exposure to and independent of Ca2+. ATP, GTP, CTP, UTP, and ITP supported Ca2+-induced alpha-granule secretion, while H+-induced alpha-granule secretion occurred only with ATP and GTP. Both Ca2+- and H+-induced alpha-granule secretion required ATP hydrolysis. Kinase inhibitors blocked both Ca2+- and H+-induced secretion. These data suggest that alpha-granule secretion in this permeabilized platelet system shares many characteristics with granule secretion studied in other permeabilized cell models. Furthermore, these results show that H+ can trigger alpha-granule release independent of Ca2+. 相似文献
15.
Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid 总被引:11,自引:6,他引:5
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《The Journal of cell biology》1994,127(5):1447-1459
Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet- poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino- terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing. 相似文献
16.
Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding 总被引:15,自引:0,他引:15
Thrombospondin synthesized and secreted by human endothelial cells in culture binds specifically to fibronectin immobilized on Sepharose beads. It can also bind to immobilized platelet-derived thrombospondin but not to immobilized gelatin or albumin. These interactions are not dependent on the presence of divalent cations or of other secreted materials. Purified platelet thrombospondin binds to fibronectin and fibrinogen immobilized on plastic surfaces with dissociation constants of 1.12 +/- 0.37 X 10(-7) M and 1.27 +/- 0.41 X 10(-7) M respectively, and to thrombospondin immobilized on plastic with dissociation constant of 4.82 +/- 1.01 X 10(-7) M. The affinities of interaction are not significantly affected by removal of divalent cations. Soluble fibrinogen inhibits binding of thrombospondin to fibronectin regardless of which of the latter two is surface-bound. Thrombospondin-fibronectin interaction is also inhibited by soluble thrombospondin. The binding of soluble thrombospondin to surface-bound fibrinogen is inhibited both by soluble fibronectin and soluble fibrinogen. These results suggest that thrombospondin plays a role both in platelet-platelet aggregation and in platelet-substratum adhesion, and that it may also take part in the construction of the extracellular matrix. 相似文献
17.
The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins. 相似文献
18.
Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin 总被引:2,自引:0,他引:2
Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 +/- 0.2 x 10(-7) M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 +/- 1.5 x 10(-7) M and the N-terminal domain binds with a Kd of 8.8 +/- 1.8 x 10(-7) M. Binding of the intact molecule is Ca2+-independent. By contrast, following tryptic fragmentation, binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule. 相似文献
19.
PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes 总被引:83,自引:0,他引:83
E Larsen A Celi G E Gilbert B C Furie J K Erban R Bonfanti D D Wagner B Furie 《Cell》1989,59(2):305-312
PADGEM (platelet activation dependent granule-external membrane protein) is an integral membrane protein of the alpha granules of platelets and Weibel-Palade bodies of endothelial cells that is expressed on the plasma membrane upon cell activation and granule secretion. Activated platelets, but not resting platelets, bind to neutrophils, monocytes, HL60 cells, and U937 cells. This interaction is inhibited by anti-PADGEM antibodies, PADGEM, and EDTA; anti-GPIIb-IIIa, anti-thrombospondin, anti-GPIV, and thrombospondin produce no effect. Neutrophils and U937 cells, in contrast to Jurkatt cells, contain PADGEM recognition sites, as shown by binding of PADGEM contained in phospholipid vesicles. These results indicate that PADGEM mediates adhesion of activated platelets to monocytes and neutrophils. Therefore, PADGEM shares not only structural but also functional homology with ELAM-1 and MEL-14, members of a new family of vascular cell adhesion molecules. 相似文献
20.
G Kanigür B Kan D Tiryaki E Bermek 《Biochemical and biophysical research communications》1983,117(1):135-140
Fibronectin is a large dimeric glycoprotein found in plasma, on cell surfaces and as a component of the extracellular matrix which has been implicated in a variety of adhesive processes. The role of fibronectin in platelet function has not been clarified. The present investigation demonstrates that an excess of exogenously added fibronectin inhibits platelet aggregation induced by either thrombin or A23187 at a step subsequent to platelet activation and secretion. Similar concentrations of fibrinogen or von Willebrand factor, both of which also bind to the surface of activated platelets, are not inhibitory. These results are consistent with the concept that fibronectin is one of the important mediators of platelet aggregation. 相似文献