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Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an αIIbβ3- or CD36-independent mechanism
Abstract
Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation. and was inhibited by cytochalasin D. an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors. as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes. as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1. in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.
In this report we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSC), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSC. We report successful modification of a surface chemistry method for use with exosomes, and identify twenty-one surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSC. These glycoprotein identities and functionalities are compared with ninty-three N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The “don’t eat me” molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein alpha (SIRPα) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRPα facilitate exosome-mediated MDSC chemotaxis and migration.
Hantavirus infection is associated with two frequently fatal diseases in humans: Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The pathogenesis of hantavirus infection is complex and not fully understood; however, it is believed to involve virus-induced hyperinflammatory immune responses. Thrombospondin-1 (THBS1) is a large homotrimeric protein that plays a putative role in regulating blood homeostasis. Hyperresponsiveness to inflammatory stimuli has also been associated with defects in the THBS1 gene. Our data suggest that hantavirus infection of human umbilical cord vein endothelial cells (HUVEC) suppress the accumulation of THBS1 in the extracellular matrix. Additionally, this suppression is dependent on virus replication, implying a direct mechanism of action. Our data also imply that the pathogenic Andes and Hantaan strains inhibit THBS1 expression while the non-pathogenic Prospect Hill strain showed little inhibition. These observations suggest that a dysregulation of THBS1 may contribute to the pathogenesis of hantavirus infection.
Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbβ3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbβ3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn2+), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.
Thrombospondin-1 (TSP1) is a multi-domain, multi-functional glycoprotein synthesized by many cells. Matricellular TSP1 modulates cell adhesion and proliferation. TSP1 is involved in angiogenesis, inflammation, wound healing and cancer. As a major platelet protein, for a long time it was postulated to control hemostasis via platelet aggregate stabilization. However, these in vitro findings have been questioned in the absence of corroborating clinical data and of obvious hemostatic defects in TSP1 gene-deficient mice.Yet, the past few years have provided indices to implicate TSP1 in hemostasis. In clinical studies, a correlation exists between a welldefined TSP1 polymorphism and a significant risk of myocardial infarction.At the same time, recent in vivo animal model data imply TSP1 in the multimer size control of von Willebrand factor, in smooth muscle cell regulation and in vascular perfusion. These findings shed new light on the role of TSP1 in hemostasis and prothrombotic vascular pathologies. (Part of a Multi-author Review)
Thrombospondin-1, a multi-modular matrix protein is able to interact with a variety of matrix proteins and cell-surface receptors. Thus it is multifunctional. In this work, we examined the role of thrombospondin-1 in ceramide-induced thyroid apoptosis. We focused on the VVM containing sequence localized in the C-terminal domain of the molecule. Primary cultured thyroid cells synthesize thrombospondin-1 depending on their morphological organization. As it leads thyrocytes to organize into monolayers before inducing apoptosis ceramide can modulate this organization. Here, we established that C2-ceramide treatment decreased thrombospondin-1 expression by interfering with the adenylyl cyclase pathway, thus leading to apoptosis. Furthermore, we demonstrated that the thrombospondin-1-derived peptide 4N1 (RFYVVMWK) abolished ceramide-induced thyroid cell death by preventing intracellular cAMP levels from dropping. Finally, we reported that 4N1-mediated inhibition of ceramide-induced apoptosis was consistently associated with a down-regulation of the caspase-3 processing. Integrin-associated protein receptor (IAP or CD47) was identified as a molecular relay mediating the observed 4N1 effects. Taken together, our results shed light for the first time on anti-apoptotic activities of the thrombospondin-1-derived peptide 4N1 and provide new information on how thrombospondin-1 may control apoptosis of non-tumoral cells.
Platelets abundantly express glycoprotein CD36 with thrombospondin-1 (TSP1) and oxidized low-density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood.
Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca(2+) rises, and resulted in exposure of P-selectin and integrin activation, all in a CD36-dependent manner with additional contributions of α(IIb) β(3) and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co-coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high-shear flow conditions, with increased expression on platelets of activated α(IIb) β(3), P-selectin and phosphatidylserine, again in a CD36-dependent way.
Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase-dependent Ca(2+) signaling mechanism, which enhances collagen-dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.
The glycoprotein CD36, also known as glycoprotein IIIb/IV or FAT, is expressed on the surface of platelets, monocytes, microvascular endothelial cell, smooth muscle cells, cardiomyocytes and other cells of the cardiovascular system. In spite of its abundant presence, CD36 has remained for long a mysterious protein with a poorly understood role. In this paper, we review how CD36 can affect cellular responses by interaction with a variety of ligands, in particular thrombospondin-1, oxidized lipoproteins and fatty acids. Furthermore, given the structure of CD36 with two transmembrane domains and short cytoplasmic tails, we consider how this receptor can induce intracellular signaling, likely in junction with other cellular receptors or associated proteins in the membrane. Current literature points to activation of Src-family and mitogen-activated protein kinases, as well as to activation of the NFκB and Rho pathways. The new insights make CD36 attractive as a therapeutic target to suppress platelet and monocyte/macrophage function and thereby atherothrombosis.
Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known.
To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology.
Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics.
The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal alpha-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change.
We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.
AIM: Regulatory T cells (Tregs) represent an important immune escape mechanism in advanced melanoma. Essential factors required for Treg induction by melanoma remain unidentified. The matricellular protein thrombospondin-1 (TSP-1) has recently been discovered to generate Tregs through its receptor, CD47. We speculated that melanoma might induce Tregs by secreting TSP-1. METHODS: We examined circulating levels of TSP-1 and Tregs in melanoma patients and normal subjects. We then examined TSP-1 secretion by metastatic melanoma cell lines in melanoma-conditioned media (MCM). The ability of MCM to induce functional Treg from normal human peripheral blood mononuclear cells (PBMCs), with or without TSP-1 or CD47 blockade was determined. Finally, we investigated the ability of recombinant TSP-1 or the CD47-binding peptide 4N1K alone to induce Tregs.RESULTS: Advanced melanoma patients had elevated levels of circulating TSP-1 which correlated with the proportion of peripheral Tregs. Furthermore, MCM contained high levels of TSP-1 and were able to induce Tregs from normal PBMCs. These Tregs were Foxp3+ and suppressed the proliferation of stimulated T responder cells. The amount of TSP-1 in the conditioned media was proportional to the degree of Treg induction and blockade of CD47 or TSP-1 abrogated this induction. However, recombinant TSP-1 or the CD47-binding peptide 4N1K were unable alone to induce Tregs. CONCLUSION: TSP-1 appears to be necessary but not sufficient alone for Treg induction by melanoma. This mechanism provides further understanding of the subversion of immune surveillance by melanoma and may provide a therapeutic target for immune therapies in this and in other malignancies.
Integrins have been reported to mediate cell survival, proliferation, differentiation, and migration programs. For this reason, the past few years have seen an increased interest in the implications of integrin receptors in atherosclerosis. This review considers the potential role of integrins in atherosclerosis and also addresses why integrins present attractive targets for drug design. It discusses the properties of the integrin-based chemotherapeutic agents currently under consideration clinically and endeavours to provide insights into development of cardiovascular drugs using integrins as targets.
By means of its antiangiogenic activity, thrombospondin-1 (TSP-1) exerts indirect antitumoral action on solid tumors. Here, we investigated potential antitumor action in an in vitro cell model for promyelocytic leukemia (NB4-LR1), resistant to retinoid maturation. Purified soluble TSP-1 added to cultures induced a strong dose-dependent growth inhibition and a slowly developing maturation-independent cell death. Recombinant fragments of TSP-1 allowed mapping of these activities to its type 3 repeat/C-terminal domain, features that are distinct from those of TSP-1 action on solid tumors, previously ascribed to the type 1 repeat domain. Cell death in leukemia was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization followed by membrane permeabilization. Mitochondria membrane depolarization was inherent to TSP-1 action but did not produce release of death-promoting proteins (eg, noncaspase apoptosis regulators, apoptosis-induced factor [AIF], endonuclease G, or Omi/HtrA2 or the caspase regulators, cytochrome c or second mitochondrial activator of caspase/direct inhibitor of apoptosis protein-binding protein with low isoelectric point [Smac/DIABLO]). Although detected, reactive oxygen species (ROS) production was likely not involved in the death process. Finally, receptor agonist RFYVVM and RGD peptides indicated that TSP-1 death effects are mediated by membrane receptors CD47 and alphavbeta3. These results demonstrated a new domain-specific antitumoral activity of TSP-1 on a leukemia cell line, which extends TSP-1 therapeutic potential outside the area of vascularized solid tumors.
Integrin-associated protein (IAP or CD47) is a receptor for the cell/platelet-binding domain (CBD) of thrombospondin-1 (TS1),
the most abundant protein of platelet α granules. Although it associates with αIIbβ3, IAP has no known function in platelets.
TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin αIIbβ3, resulting in platelet
spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion
kinase. Furthermore, 4N1K peptide selectively stimulates the phosphorylation of LYN and SYK and their association with FAK.
The phosphorylation of SYK is blocked by pertussis toxin, implicating a Gi-like heterotrimeric G protein. IAP solublized from membranes of unstimulated platelets binds specifically to an affinity
column of 4N1K peptide. Both αIIb and β3 integrin subunits and c-Src bind along with IAP. This complex of proteins is also
detected with immunoprecipitation. Activation of platelets with the agonist peptide 4N1K results in the association of FAK
with the IAP-αIIbβ3 complex. Thus an important function of TS1 in platelets is that of a secreted costimulator of αIIbβ3 whose
unique properties result in its localization to the platelet surface and the fibrin clot.
We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.
Glanzmann thrombasthenia is a rare, inherited disorder of the platelet glycoprotein IIb/IIIa (GP IIb/IIIa) complex. We previously identified two distinct populations with this disorder in Israel, Iraqi-Jews and Arabs. The groups are indistinguishable in hemorrhagic symptoms and platelet GP IIB/IIIa receptor deficiency, but they differ in their platelet immunodetectable GP IIIa (beta 3), with the Iraqi-Jewish population expressing no detectable GP IIIa and the Arab population expressing small amounts. We have now examined the platelets of these two populations as well as normal platelets for the alpha v beta 3 vitronectin receptor. Normal platelets contained between approximately 50 to 100 alpha v beta 3 vitronectin receptors as judged by the binding of antibodies to both alpha v (LM142) and the intact alpha v beta 3 vitronectin receptor complex (LM609). In addition, normal platelets bound to immobilized vitronectin in the presence of 1 mmol/LMnCl2; the adhesion was mediated predominantly through GP IIb/IIIa, but with a distinct contribution by the alpha v beta 3 vitronectin receptor, as determined by monoclonal antibody inhibition studies. Iraqi-Jewish patients' platelets had a profound decrease in immunodetectable alpha v beta 3 vitronectin receptors, and their platelets did not adhere well to vitronectin. In contrast, Arab patients' platelets had normal or increased numbers of platelet alpha v beta 3 vitronectin receptors, and these receptors functioned well in the vitronectin adhesion assay, taking over much of the adhesion mediated by GP IIb/IIIa in normal platelets. These studies define further the heterogeneity of the molecular basis of Glanzmann thrombasthenia; they also have more widespread implications for understanding the synthesis and function of the beta 3 family of integrin receptors.
Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.
Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands.
The members of the integrin family of membrane glycoprotein heterodimer complexes function as cell surface receptors for adhesive proteins. We report here on the identification of two integrins on the surface of human platelets that bind to thrombospondin. When platelet membrane proteins are radiolabeled with 125I-lactoperoxidase, solubilized in n-octylglucoside, (Boehringer Mannheim Biochemicals, Indianapolis, IN), and applied to a column of thrombospondin-Sepharose, both complexes are bound to the column and specifically eluted with the peptide GRGDSP. One of these integrins, glycoprotein (GP) IIb-IIIa, appears to bind relatively weakly. The second integrin shares the same beta subunit (beta 3 or GPIIIa), but has a distinct alpha subunit that comigrates with the alpha subunit (alpha v) of the vitronectin receptor (VnR) on endothelial cells and reacts with a monoclonal antibody, LM142, which was raised against an integrin from M21 melanoma cells. The alpha v beta 3 integrin is present on a variety of cell types and appears to act as a receptor for thrombospondin on endothelial and smooth muscle cells. On endothelial and M21 melanoma cells this receptor is also involved in adhesion to fibrinogen, vitronectin, and von Willebrand factor (vWF). The alpha v beta 3 integrin is present at approximately equal levels on normal and thrombasthenic platelets, whereas levels of GPIIb-IIIa are greatly reduced on thrombasthenic platelets. The alpha v beta 3 integrin on thrombasthenic platelets also binds to thrombospondin-Sepharose and can be eluted with the peptide GRGDSP. These data indicate that the alpha v beta 3 integrin on platelets, endothelial cells, and smooth muscle cells functions as an Arg-Gly-Asp (RGD)-dependent receptor for thrombospondin.
Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha-thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400-411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha-thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.
Two distinct murine monoclonal antibodies, designated MA-I and MA-II, and limited proteolysis with thrombin and trypsin have been used to probe the structure of human platelet thrombospondin. The results indicate that each of the constituent chains of thrombospondin comprise four distinct polypeptide segments. The production of these segments is influenced by the presence of calcium, the enzyme employed, the temperature of digestion, and the enzyme-to-substrate ratio. Thrombin digestion in the presence of calcium results in the release of a 30,000-dalton fragment, designated segment I, which contains the epitope for MA-II and the heparin-binding site. Prior EDTA treatment results in the concomitant cleavage of a 25,000-dalton fragment, designated segment IV, from the other terminus. Limited tryptic digestion in the absence of calcium produces a 47,000-dalton fragment (segment III) which is adjacent to segment IV. Segment III contains the epitope for MA-I. Segment II is an 85,000-dalton fragment which contains the interchain disulfide bonds. Calcium inhibits proteolysis at cleavage sites between segments II and III and between segments III and IV. In the presence of calcium, an 85,000-dalton fragment is produced, which is derived from portions of segments II, III, and possibly IV. Electron microscopy of platinum replicas produced by low angle rotary shadowing reveals that thrombospondin is composed of four well-defined globular regions connected by thin flexible regions. Three of the globular regions, designated globular region C, appear to be at the ends of the three thin connecting regions. The fourth globular region, designated globular region N, appears to be close to the site where the chains are cross-linked. Globular region N can be resolved into three separate smaller globular structures which are 70 +/- 7.1 A in diameter. This region is selectively removed by thrombin digestion in the presence of calcium and binds a monoclonal antibody directed against the heparin-binding peptides. These data indicate that globular region N comprises the three NH2-terminal portions (segment I) from each of the three chains of thrombospondin. Globular region C is located at the ends of each of the three thin connecting regions which are each approximately 291 +/- 46 A long. The removal of calcium results in a decrease in the size of globular region C from 118 +/- 18.6 A to 80 +/- 7.4 A and an increase in the length of the adjacent thin connecting region to 383 +/- 30 A.(ABSTRACT TRUNCATED AT 400 WORDS)
Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.
We have previously postulated that surface membrane proteins become specifically associated with the internal platelet cytoskeleton upon platelet activation. Four lines of evidence are in support of this general hypothesis since we now show that platelet surface receptors for fibrin become specifically associated with the platelet Triton-insoluble cytoskeleton. 1) Fibrin was detected immunologically in the washed Triton-insoluble cytoskeletons of thrombin-activated platelets under conditions where fibrin polymerization and resultant precipitation was blocked with Gly-Pro-Arg-Pro, a synthetic peptide that inhibits polymerization of fibrin monomer. 2) Radiolabeled fibrin bound to thrombin-activated platelets and became associated with the cytoskeleton. 3) The amount of radiolabeled fibrin bound to thrombin-activated thrombasthenic platelets and their cytoskeletons amounted to about 20% of the fibrin bound to thrombin-activated control platelets and their cytoskeletons. 4) The association of fibrin with cytoskeletons and with the platelet surface was nearly quantitatively blocked by an antibody prepared against cytoskeletons (anti-C), an antibody against isolated membranes of Pronase-treated platelets (anti-M1), and a monoclonal antibody to the platelet surface glycoprotein complex, GPII(b)-GPIII (anti-GPIII). These antibodies blocked ADP and thrombin-induced platelet aggregation as well as thrombin-induced clot retraction. Analysis of the immunoprecipitates obtained with anti-C, anti-M1, and anti-GPIII from detergent extracts of 125I-surface labeled platelets revealed that these antibodies recognized GPII(b)-GPIII. These data suggest that thrombin activation of platelets results in the specific association of fibrin with the platelet cytoskeleton, that this association may be mediated by the GPII(b)-GPIII complex, and that these mechanisms may play an important role in platelet aggregation and clot retraction induced by thrombin.
Thrombospondin (TSP), the major alpha-granule protein of human platelets, binds to the activated platelet surface upon platelet stimulation. TSP has hemagglutinating (lectin-like) activity and forms a specific complex with fibrinogen. Based on these observations, it was postulated that the interaction of TSP and fibrinogen on the activated platelet surface may be an important step in the platelet aggregation process. To test this hypothesis, monospecific, affinity-purified anti-TSP Fab fragments were prepared and their effects on platelet aggregation and platelet fibrinogen binding were studied. Anti-TSP Fab caused significant interference with thrombin- and collagen-induced platelet aggregation, as monitored by both turbidometric aggregometry and particle counting measuring the disappearance of single platelets. Phase-contrast microscopy revealed that anti-TSP Fab caused a marked decrease in platelet macroaggregates and an increase in microaggregates and nonaggregated single platelets. Anti-TSP Fab did not affect the initial phase of ADP-induced platelet aggregation but caused rapid platelet disaggregation with the abolition of the secondary phase of aggregation. The effect of anti-TSP Fab was not mediated by a direct inhibition of platelet secretion. The effect of anti-TSP Fab on specific binding of labeled fibrinogen to thrombin-stimulated platelets was also studied. Anti-TSP Fab caused a marked decrease in the affinity of fibrinogen binding to the receptors on the activated platelet surface. Kinetic analyses revealed significant displacement of labeled fibrinogen by unlabeled fibrinogen in the presence of anti-TSP Fab, suggesting that TSP serves to stabilize fibrinogen binding to the activated platelet surface and reinforces the strength of interplatelet interactions. It is proposed that platelet aggregation is a dynamic, multistep process, governed initially by the platelet membrane glycoprotein IIb/IIIa-fibrinogen interaction, with the TSP-fibrinogen interaction playing an important role in determining the size and reversibility of platelet aggregates.
The platelet plasma membrane is lined with a membrane skeleton composed of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of the cell through association with the cytoplasmic domains of transmembrane receptors. In detergent-lysed platelets, cytoplasmic actin filaments are sedimented by centrifugation at 15,600 x g, but the sedimentation of membrane skeleton fragments requires higher g-forces (100,000 x g). In the present study, we show that the major platelet integrin, glycoprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 100,000 x g together with fragments of the membrane skeleton that contain the cytoskeletal proteins spectrin, vinculin, and talin. In addition, this cell fraction contained the tyrosine kinases pp60c-src and pp62c-yes and the p21ras GTPase-activating protein (GAP). After thrombin-induced platelet aggregation mediated by fibrinogen binding to GP IIb-IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sediments at 15,600 x g. The redistribution of these proteins from the high-speed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-defective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activated platelets were present in detergent-insoluble fractions. These results are consistent with the possibilities that 1) GP IIb-IIIa, pp60c-src, pp62c-yes, and GAP associate with a membrane skeleton fraction that contains spectrin, vinculin, and talin, 2) the association of GP IIb-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplasmic actin filaments, and 3) many of the proteins phosphorylated on tyrosine residues in activated platelets are components of the cytoskeleton. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets. Further, GP IIb-IIIa-induced redistribution of components of the membrane skeleton and associated signaling molecules may represent an important step in regulating integrin-induced motile events in platelets.
Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.
The interaction of human thrombospondin (TSP) with GPIa-IIa and GPIIb-IIIa was studied. The binding for both proteins became time-independent after 60 min. A 7-fold excess concentration of unlabelled GPIa-IIa added either initially, or after time-dependent binding, resulted in a 50% inhibition of GPIa-IIa bound to TSP. GPIa-IIa and GPIIb-IIIa specifically bound TSP since: (a) the binding of GPIIb-IIIa to TSP was dependent on the presence of 1 mM MgCl2 and 1 mM CaCl2, whereas binding of GPIa-IIa was ion-independent. (b) The binding was saturable, with dissociation constants of 0.69 +/- 0.17 microM and 3.77 +/- 1.02 microM for GPIa-IIa and GPIIb-IIIa respectively. (c) GPIIb-IIIa and GPIa-IIa did not significantly bind to BSA. (d) GPIIb-IIIa bound fibrinogen ion-specifically, whereas little or no binding of GPIa-IIa was detectable. (e) Both GPIIb-IIIa and GPIa-IIa bound collagen in an ion-independent manner. (f) GPIIb-IIIa did not compete with GPIa-IIa for binding to TSP. (g) Binding of GPIa-IIa to TSP was inhibited with anti-(GPIa-IIa) (6F1), whereas mouse IgG and anti-(GPIIb-IIIa) (AP-2) had no effect. (h) The interaction of GPIa-IIa with TSP is 5.5-fold more favourable than that of GPIIb-IIIa suggesting that GPIa-IIa may be a preferred binding protein for TSP-mediated platelet adhesion.
Thrombospondin, a major secretory product of the alpha-granules of activated platelets, is a large trimeric glycoprotein that plays an important role in platelet aggregation. On resting platelets, thrombospondin binds to a single receptor in a cation-independent manner, but upon platelet activation it binds at least two further, distinct receptors that are both dependent upon divalent cations. Each of these receptors on the platelet surface binds to different regions of the thrombospondin molecule, and such binding may be responsible for the multifunctional role of thrombospondin in aggregation. We show here that a peptide from the carboxyl terminus of thrombospondin, RFYVVMWK, directly and specifically induces the activation and aggregation of washed human platelets from different donors at concentrations of 5-25 microM. At lower concentrations the peptide synergizes with suboptimal concentrations of ADP to induce aggregation. Peptide affinity chromatography and immunoprecipitation with a monoclonal antibody were used to identify the receptor for the carboxyl-terminal peptide as the integrin-associated protein. The integrin-associated protein remained bound to the RFYVVMWK-containing peptide column when washed with a scrambled peptide in the presence of 5 mM EDTA, indicating a divalent cation-independent association. It is suggested that integrin-associated protein is the primary receptor for thrombospondin on the surface of resting platelets and is implicated in potentiating the platelet aggregation response.
The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.
Integrin-associated protein (IAP or CD47) is a receptor for the cell/platelet-binding domain (CBD) of thrombospondin-1 (TS1), the most abundant protein of platelet alpha granules. Although it associates with alphaIIbbeta3, IAP has no known function in platelets. TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin alphaIIbbeta3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase. Furthermore, 4N1K peptide selectively stimulates the phosphorylation of LYN and SYK and their association with FAK. The phosphorylation of SYK is blocked by pertussis toxin, implicating a Gi-like heterotrimeric G protein. IAP solublized from membranes of unstimulated platelets binds specifically to an affinity column of 4N1K peptide. Both alphaIIb and beta3 integrin subunits and c-Src bind along with IAP. This complex of proteins is also detected with immunoprecipitation. Activation of platelets with the agonist peptide 4N1K results in the association of FAK with the IAP-alphaIIbbeta3 complex. Thus an important function of TS1 in platelets is that of a secreted costimulator of alphaIIbbeta3 whose unique properties result in its localization to the platelet surface and the fibrin clot.
Integrin-associated protein (IAP; CD47) is a thrombospondin receptor that forms a signaling complex with beta3 integrins resulting in enhanced alphavbeta3-dependent cell spreading and chemotaxis and, in platelets, alphaIIbbeta3-dependent spreading and aggregation. These actions of CD47 are all specifically abrogated by pertussis toxin treatment of cells. Here we report that CD47, its beta3 integrin partner, and Gi proteins form a stable, detergent-soluble complex that can be recovered by immunoprecipitation and affinity chromatography. Gialpha is released from this complex by treatment with GTP or AlF4. GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid decrease in intraplatelet cyclic AMP levels, a Gi-dependent event necessary for aggregation. Finally, 4N1K stimulates the binding of GTPgamma35S to membranes from cells expressing IAP and alphavbeta3. This functional coupling of CD47 to heterotrimeric G proteins provides a mechanistic explanation for the biological effects of CD47 in a wide variety of systems.
Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of alphaIIbbeta3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin alpha2beta1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP-/- mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of alpha2beta1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and alpha2beta1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.
Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of IIbβ3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin 2β1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP−/− mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of 2β1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and 2β1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.
We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.
Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands.
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Integrins are remarkably multifunctional: they mediate cell adhesion and migration, orchestrate organization of the actin-based cytoskeleton, and activate signal transduction pathways. Recent studies have identified a variety of steps and hierarchies in these intracellular cytoskeletal and signaling responses, laying the groundwork for future studies on specificity and coordination with responses to growth factors.
Glycoprotein IIIb (GPIV, CD36) has been proposed as the platelet receptor for thrombospondin (TSP). We found two healthy blood donors, whose platelets were shown to be GPIIIb deficient. These platelets expressed endogeneous TSP as control platelets and their binding capacity for exogeneous TSP was the same. These results indicate that GPIIIb is not the major TSP receptor on platelets. However, it is not yet possible to exclude that in GPIIIb-deficient platelets other proteins may substitute for GPIIIb in TSP binding.
To characterize the interaction between thrombospondin and human platelets, thrombospondin was purified from the supernatant of thrombin-activated human platelets, labeled with iodine 125, and allowed to interact with the washed platelets. With concentrations of 10 to 50 micrograms/ml, only minute amounts of 125I-labeled thrombospondin bound to resting platelets or to platelets activated by adenosine diphosphate. In contrast, when platelets were stimulated with thrombin, binding increased fivefold to sixfold in a time-dependent and 125I-labeled thrombospondin concentration-dependent manner. Binding of 125I-labeled thrombospondin to thrombin-activated platelets required the presence of divalent cations, proceeded concomitantly with platelet release, and at a concentration of 1 nmol/L thrombin, reached a maximum of 2200 +/- 260 molecules of 125I-labeled thrombospondin bound per platelet. After its binding to platelets, 125I-labeled thrombospondin was not internalized, because up to 85% of the 125I-labeled thrombospondin was dissociated from the cell surface by adding ethylenediaminetetraacetic acid. Using various experimental approaches, including studies with severe type I thrombasthenic platelets, we further demonstrated that the interaction of 125I-labeled thrombospondin with thrombin-stimulated platelets occurred as a fibrinogen- and fibrin-independent process, and that the glycoprotein IIb-IIIa complex did not function as a physiologic plasma membrane receptor for 125I-labeled thrombospondin. Last, about 60% of the 125I-labeled thrombospondin molecules bound to the platelet surface were found to be associated with the platelet cytoskeleton recovered from platelets solubilized with Triton X-100. On Western blot analysis, this cytoskeletal fraction lacked detectable glycoprotein IV, the putative platelet receptor for thrombospondin. These results suggest that on the surface of thrombin-activated platelets, a fraction of 125I-labeled thrombospondin does not associate with glycoprotein IV but instead with other plasma membrane components that have yet to be identified.
The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet-rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.
Fibrinogen and thrombospondin are major constituents of human platelet alpha-granules and contribute to cell-cell interactions following their release. Glanzmann's thrombasthenia is characterized by the absence of platelet aggregation and reduced levels of GP IIb-IIIa complexes and platelet fibrinogen. The level of thrombospondin is thought to be normal but has not so far been quantified. Using an electroimmunoassay method adapted from Laurell, we have measured fibrinogen and thrombospondin in platelet extracts of four patients with classical Glanzmann's thrombasthenia and two variants with abnormal platelet aggregation associated with subnormal levels of GP IIb-IIIa complexes. Triton X-100 lysates were prepared in the presence of leupeptin or EDTA to avoid endogenous calcium-dependent protease activation during the solubilization procedure. Platelet fibrinogen was not detected in one patient with type I Glanzmann's thrombasthenia; it was reduced to 5-10% of normal values in two other type I patients and to 65% of normal values in one type II patient. It was normal in patient R.P., a variant of Glanzmann's thrombasthenia with 60% of GP IIb-IIIa complexes but decreased in patient A.P. a newly described variant with 35% of GP IIb-IIIa complexes. These findings support a role for GP IIb-IIIa complexes in the packaging of fibrinogen into alpha-granules. Normal or subnormal amounts of thrombospondin were measured in thrombasthenic platelets. Patient A.P., who was investigated on two different occasions, demonstrated variable levels of thrombospondin. This underlines the need for quantifying this protein when evaluating its expression in this disorder.
Participation of fibrinogen in platelet aggregation is contingent upon the capacity of various stimuli to induce specific receptors for the molecule on the surface of the cell. The interaction of fibrinogen with this receptor results directly in platelet aggregation, and dissociation of fibrinogen is associated with disaggregation. While the role of exogenous fibrinogen in this process has been fully documented, the mechanisms which control the surface exposure of platelet fibrinogen are less understood. In the present study Fab fragments of antibodies monospecific for fibrinogen have been used to examine the surface expression of intracellular fibrinogen and its involvement in platelet aggregation. Radiolabelled Fab fragments did not interact with non-stimulated platelets but significant binding was observed when the cells were stimulated by ADP, thrombin, collagen and Ca ionophore A23187. Binding was specific for fibrinogen, was not observed with thrombasthenic platelets and was dependent upon the presence of extracellular calcium. With all stimuli tested, the binding of the Fab probe to platelets correlated with platelet secretion. At the following concentrations of stimuli: 30 microM ADP, 4 micrograms/ml collagen, 3 microM A23187 and 0.05 U/ml thrombin, the immune Fab fragments inhibited platelet aggregation. A monoclonal antibody to glycoprotein IIb/IIIa complex and a synthetic peptide gamma 400-411, that inhibited the interaction of plasma fibrinogen with platelets, did not inhibit the binding of 125I-FAB fragments. Taken together these results support the hypothesis that endogenous fibrinogen becomes surface-expressed during stimulation of the cell and can support platelet aggregation, particularly that induced by low concentrations of stimuli. The mechanism for the surface expression of platelet fibrinogen may be distinct from that for the binding of plasma fibrinogen.
Thrombospondin with fibrinogen, fibronectin, and von Willebrand factor binds to platelets stimulated with agonists and support platelet adhesive functions. The receptors for the latter three proteins are associated with membrane glycoprotein GPIIb-IIIa. Thrombasthenic platelets deficient in GPIIb-IIIa have been utilized to examine the role of this membrane protein in the interactions of thrombospondin with platelets. Radioiodinated thrombospondin bound to thrombin-stimulated platelets from normal and thrombasthenic donors with a similar affinity and capacity. As monitored with a monoclonal antibody to thrombospondin, the divalent ion-dependent and -independent pathways for the expression of the endogenous pool of thrombospondin on the surface of thrombin-stimulated platelets from normal and thrombasthenic donors were also qualitatively and quantitatively similar. GPIIb-IIIa or ligands associated with GPIIb-IIIa thus are not essential for the binding of thrombospondin to platelets. Therefore, thrombospondin interacts with unique receptors on platelets.
The distribution and transport of thrombospondin (TSP), fibrinogen (Fbg), fibronectin (Fn), and Factor VIII-related antigen (VIII:RAg) in resting and thrombin-stimulated platelets was investigated by immunofluorescence microscopy. In resting intact cells, little surface staining was seen for these proteins. In permeable resting cells, punctate staining similar to that reported for platelet factor 4 was observed. Double-label immunofluorescence staining for Fbg and either beta-thromboglobulin (beta TG), TSP, or Fn demonstrated co-localization, indicating their presence in the same intracellular structures. VIII:RAg showed general co-localization; however, the staining was finer, suggesting a possible differential intragranular localization. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins. In thrombin-stimulated cells, co-localization of all proteins in these masses was observed by double label immunofluorescence. Thus, TSP, Fbg, Fn, and beta TG are localized in the same structure in resting cells. Thrombin stimulates formation of common larger masses of these proteins prior to their release, suggesting that they reach the cell surface through a common intermediate.
Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution
of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src
activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src
from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated
in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which
are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there
was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly
activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal
fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is
dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize
and anchor activated enzymes involved in signal transduction.
Adhesive interactions play critical roles in directing the migration, proliferation, and differentiation of cells; aberrations in such interactions can lead to pathological disorders. These adhesive interactions, mediated by cell surface receptors that bind to ligands on adjacent cells or in the extracellular matrix, also regulate intracellular signal transduction pathways that control adhesion-induced changes in cell physiology. Though the extracellular molecular interactions involving many adhesion receptors have been well characterized, the adhesion-dependent intracellular signaling events that regulate these physiological alterations have only begun to be elucidated. This article will focus on recent advances in our understanding of intracellular signal transduction pathways regulated by the integrin family of adhesion receptors.
The platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments which fill the cytoplasm and mediate contractile events. The other is the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability. In the unstimulated platelet, only 30-40% of the actin is polymerized into filaments; the rest is thought to be prevented from polymerizing by the association of thymosin beta 4 with monomeric actin and by the association of gelsolin with the barbed ends of pre-existing actin filaments. When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, myosin binds to cytoplasmic actin filaments, causing them to move towards the center of the platelet. As platelets aggregate, additional cytoskeletal reorganizations occur: GP IIb-IIIa associates with adhesive ligand in a platelet aggregate; this results in the association of GP IIb-IIIa, membrane skeleton proteins, and signaling molecules with cytoplasmic actin. Future studies should help to elucidate the significance of the cytoskeleton in regulating signal transduction events in platelets.
We have recently shown that several components from the platelet plasma membrane were also present at different rates in the alpha-granule membrane. This is the case for the glycoprotein (GP) IIb-IIIa (CD41), CD36, CD9, PECAM1, and Rap1b, while the GPIB-IX-V complex was considered to escape the rule. In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the resting platelets of normal subjects, patients with Bernard-Soulier syndrome, patients with Gray platelet syndrome, and human cultured megakaryocytes. Ultra-thin sections of the cells were labeled with antibodies directed against glycocalicin, GPIb, GPIX, and GPV. We have shown that a significant and reproducible labeling for the three GPs was associated with the alpha-granule membrane, accounting for approximately 10% of the total labeling. Furthermore, GPIb labeling appears Willebrand factor (vWF). After thrombin activation, vWF remained close to the limiting membrane of the open canalicular system (OCS), suggesting an early association of both receptor and ligand. Plasma membrane and alpha-granule labeling was virtually absent from the Bernard-Soulier platelets (characterized by a GPIb deficiency), thus proving the specificity of the reaction. In Gray platelets (storage granule deficiency syndrome), the small residual alpha-granules were also occasionally labeled for GPIb, GPIX, and GPIX. Cultured megakaryocytes that displayed the classical GPIb distribution, eg, demarcation and plasma membranes, exhibited also a discrete labeling associated to the alpha-granules. In conclusion, this study shows that, evenly for these three GPs, the alpha-granule membrane mirrors the plasma membrane composition. This might occur through an endocytotic process affecting each plasma membrane protein to a different extent and could have a physiologic relevance in further presentation of a receptor bound to its alpha-granule ligand to the platelet surface.
Previously, we showed that a subpopulation of the major platelet integrin, alphaIIbbeta3, co-sediments from detergent lysates with talin and other membrane skeleton proteins. Once alphaIIbbeta3 has bound adhesive ligand in a platelet aggregate, the detergent-insoluble alphaIIbbeta3 redistributes (along with the detergent-insoluble membrane skeleton proteins and a variety of signaling molecules) to a fraction that contains cytoplasmic actin filaments. Concomitantly, certain signaling molecules are activated. The present study shows that, in intact platelets, alphaIIbbeta3 forms clusters when occupied by ligand and is selectively moved into the open canalicular system; alphaIIbbeta3 that has not bound ligand remains diffusely distributed at the periphery of the cell. When cytoplasmic actin filaments are depolymerized by cytochalasins, the ability of alphaIIbbeta3 to bind ligand is decreased, and the movement of ligand-occupied alphaIIbbeta3 is prevented. Together with the previous findings, these results suggest that (i) membrane skeleton-associated alphaIIbbeta3 is selectively induced to bind ligand in activated platelets, (ii) ligand-induced transmembrane signaling causes an altered association of membrane skeleton-associated alphaIIbbeta3 with the cytoplasmic component of the cytoskeleton, (iii) ligand-induced cytoskeletal reorganizations stabilize the interaction between ligand and integrin, and (iv) ligand-occupancy triggers cytoskeletal reorganizations that result in selective movements of occupied ligand.
The sequence β3203-228 is involved, in a yet undetermined manner, in α(IIb)β3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for β3211-221, binds to α(IIb)β3 on adenosine diphosphate (ADP)- activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding-site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the γ-chain carboxy- terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of α(IIb)β3. Its binding to thrombin-stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of α(IIb)β3 colocalizing with P-selectin and suggesting the presence of α(IIb)β3-ligand complexes in the α-granule membrane. There was little binding of AP6 to surface α(IIb)β3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to α(IIb)β3 within the platelet. Thus, our results provide evidence of (1) a pool of α-granule α(IIb)β3 occupied by ligand in nonactivated platelets, (2) thrombin-induced activation of α(IIb)β3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound α(IIb)β3 to the surface.
Thrombospondin (TSP) is a platelet alpha-granule adhesive glycoprotein (M(r) = 450,000) that is released in large amounts from activated platelets and participates in thrombus formation. The aim of the present study was to assess the effect of peptides corresponding to sequences within the NH2-terminal region and type 1 repeats of TSP on platelet aggregation induced by thrombin in washed platelet suspensions. We found that TSP18 (amino acids 1-174), used at micromolar concentrations, inhibited platelet aggregation by 30-50%, reducing the size of the aggregates formed. Similar results were obtained with the hexapeptide Cys-Ser-Val-Thr-Cys-Gly (amino acids 429-434 and 486-491) used at 1.2 mM. The shorter peptide Val-Thr-Cys-Gly was even more inhibitory whereas the peptide Val-Thr-Lys-Gly, which lacks a cysteine, had no effect. Interestingly, we have constantly found that inhibition of platelet aggregation by these peptides was accompanied by an inhibition of alpha and dense granule secretion, suggesting that the binding of secreted TSP to the plasma membrane may participate in the platelet signaling process. We conclude that peptides of TSP may prove useful in the treatment of thrombosis by impairing both the release of proaggregating substances and platelet macroaggregate formation.
Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin alphaIIb beta3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack alphaIIb beta3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% alphaIIb beta3, functional), and variant type (50% alphaIIb beta3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional alphaIIb beta3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by alphaIIb beta3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of alphaIIb beta3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.
In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein alpha-actinin in a solid-phase binding assay. Stored in the alpha-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. alpha-Actinin is an actin-binding and actinin-cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of alpha-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence. alpha-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of alpha-actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as alpha-granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both alpha-actinin and TSP-1 were seen redistributing from the alpha-granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic alpha-actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of 125I-surface-labeled, thrombin-activated platelets further demonstrated that alpha-actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could from molecular complexes with TSP-1 on activated platelets. These results suggest that alpha-actinin found to be present on the platelet surface together with TSP-1 originates in the alpha-granules by fusion of the alpha-granules with the plasma membrane during platelet exocytosis.
Considerable progress has been made towards understanding the function of thrombospondin-1 and-2. The description of the phenotype of mice with thrombospondin-1 and-2 knocked-out supports in vitro biochemical and cell-biological data and has opened new avenues of research. Recently, our understanding of the roles of thrombospondins in the activation of TGFbeta, inhibition of angiogenesis and the initiation of signal transduction has advanced.
