Platelet Thrombospondin Research Articles

Endothelial cell mitogenesis induced by LPA: Inhibition by thrombospondin-1 and thrombospondin-2

We examined the effects of thrombospondin-1 (TSP1) and thrombospondin-2 (TSP2) on the uptake of tritiated thymidine by bovine aortic endothelial (BAE) cells in response to two growth factors, basic fibroblast growth factor (bFGF) and lysophosphatidic acid (LPA). bFGF and LPA stimulate cell proliferation through distinct receptors that have convergent signaling pathways. The doses of LPA that trigger proliferation of BAE cells, which have not been reported previously, were 1 to 30 μmol/L, as opposed to the 5 to 100 μmol/L concentrations required to stimulate proliferation of human foreskin fibroblasts. Baseline mitogenic activity and activity stimulated by either bFGF or LPA on BAE cells was inhibited by human TSP1 purified from platelets or a recombinant source with a similar dose response. These results demonstrate that the anti-proliferative effect of platelet TSP1 is not caused by contaminants from the stimulated platelet. Recombinant mouse TSP2 inhibited BAE cell proliferation in response to LPA in a dose range similar to that of TSP1. Inasmuch as TSP2 does not activate latent TGFβ1 (Schultz-Cherry et al., J Biol Chem 1995; 270: 7304), these results show that inhibition of angiogenesis by TSPs is not related to control of activation of TGFβ. Together, these studies suggest that structural motifs common to TSP1 and TSP2 inhibit endothelial cell proliferation. Furthermore, TSPs inhibit cell proliferation stimulated by two growth factor receptors that act through distinct signaling pathways.

Metabolism of Thrombospondin 2: BINDING AND DEGRADATION BY 3T3 CELLS AND GLYCOSAMINOGLYCAN-VARIANT CHINESE HAMSTER OVARY CELLS

Thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2) are members of the thrombospondin family that have a similar structural organization but somewhat different functional activities. Iodinated recombinant mouse TSP2 bound to NIH 3T3 cells and was internalized and degraded through a chloroquine-inhibitable pathway. TSP2 degradation was saturable, specific, and similar to the kinetics of degradation of TSP1. Human platelet TSP1, recombinant mouse TSP1, and recombinant mouse TSP2 cross-competed with one another for degradation by 3T3 cells. Degradation of TSP2 was less sensitive to inhibition by heparin than degradation of TSP1. This is in agreement with differences in heparin-binding affinity of the two TSPs. Degradation of TSP2 was slower in cultures of Chinese hamster ovary (CHO) cells lacking heparan sulfate proteoglycans than in wild type CHO cells or in cultures of 3T3 cells treated with heparitinase than in untreated 3T3 cells. Degradation of TSP2 was inhibited by antibodies against the low density lipoprotein receptor-related protein (LRP) or by the 39-kDa receptor-associated protein, a known antagonist of LRP. This study indicates that TSP2 and TSP1 are metabolized by a common internalization and degradation pathway involving heparan sulfate proteoglycan and LRP. Competition for this pathway is a possible mechanism whereby cells can control the levels and ratio of TSP1 and TSP2 in the extracellular milieu.

An Autosomal Dominant, Qualitative Platelet Disorder Associated With Multimerin Deficiency, Abnormalities in Platelet Factor V, Thrombospondin, von Willebrand Factor, and Fibrinogen and an Epinephrine Aggregation Defect

Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet β-thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 × 109/L; range, 104 to 198 × 109/L). In addition, their platelets failed to aggregate in response to 6 to 10 µmol/L epinephrine despite normal numbers of platelet α2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.

Disturbance of plasma and platelet thrombospondin levels in sickle cell disease.

Thrombospondin (TSP), a large protein found in platelet alpha-granules (as TSP-1), mediates adhesion of sickle reticulocytes to cultured vascular endothelium. To further explore the physiologic relevance of this observation, we have measured plasma TSP levels and platelet TSP-1 content in subjects with sickle cell disease. Plasma TSP levels were similar for normal controls (mean 491 ng/ml, range 331-723) and steady-state HbSS patients (mean 536, range 333-1107) and were significantly (P = 0.012) but variably elevated for HbSS patients presenting with acute painful crisis (mean 868, range 442-2780). Some of these elevated plasma TSP levels reached those previously observed to support maximal red cell adhesion to endothelium in vitro. Compared to normals, both steady-state and in-crisis HbSS patients had significantly (P < 0.001) depressed platelet TSP-1 content (82.6 +/- 11.9, 47.1 +/- 16.0 and 45.9 +/- 20.7 ng/l0(6) platelets, respectively, mean +/- SD). HbSC disease patients, all examined during steady state, had low-normal plasma levels of TSP and either normal or depressed platelet TSP-1 content. Serial observations on three sickle cell anemia subjects indicated a probable relationship between platelet TSP-1 release, elevated plasma TSP levels, and acute vaso-occlusive episodes. These results suggest a state of ongoing release and depletion of TSP-1 from activated platelets in patients with sickle cell disease.

Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase.

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, protein disulfide isomerase, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Protein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isomerase-mediated rearrangement of disulfide bonds in thrombospondin 1. To quantitate disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with protein disulfide isomerase and reduced glutathione. The rate of protein disulfide isomerase-catalyzed disulfide interchange in thrombospondin 1 increased linearly with protein disulfide isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with protein disulfide isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.

Electron Spin Resonance and Fluorescence Studies of the Conformational Environment of the Thiol Groups of Thrombospondin: Interactions with Thrombin

The free thiols of platelet thrombospondin (TSP) were modified with thiol-specific spin labels and fluorescence probes. The conformational effects of thrombin complexation with TSP were monitored by thiol-specific spin labels covalently attached to TSP and active site specific spin labels on thrombin. The results provide evidence supporting speculations that the thiols of the three polypeptide chains in TSP are not conformationally identical. Studies on the effects of Ca2+ and temperature confirm that TSP exists in multiple conformations which are under dynamic equilibrium. The ESR spectra of spin-labeled TSP are sensitive to the proteolytic effects of thrombin in the presence and absence of calcium. Phenylsulfonyl fluoride spin labels specific for the active site of thrombin are excellent indicators of thrombin: TSP complex formation in the absence of calcium. The anticoagulant thrombin inhibitor hirudin competes with TSP for the same binding locus on thrombin (which includes the requirement of an intact anion exosite). The results suggest that the species observed here is the noncovalent complex formed during the first step of the TSP--thrombin interaction, showing also that thrombin activity is not essential for complex formation. ESR and fluorescence studies of thiol-labeled TSP indicate that the sulfhydryls are not affected in the noncovalent thrombin: TSP complex, although they must be playing a major role in the second step, i.e., formation of the covalent complex, through intermolecular thiol exchange.

Regulation of Transforming Growth Factor-β Activation by Discrete Sequences of Thrombospondin 1

Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein secreted by virtually all cells in a latent form. A major mechanism of regulating TGF-beta activity occurs through factors that control the processing of the latent to the biologically active form of the molecule. We have shown previously that thrombospondin 1 (TSP1), a platelet alpha-granule and extracellular matrix protein, activates latent TGF-beta via a protease- and cell-independent mechanism and have localized the TGF-beta binding/activation region to the type 1 repeats of platelet TSP1. We now report that recombinant human TSP1, but not recombinant mouse TSP2, activates latent TGF-beta. Activation was further localized to the unique sequence RFK found between the first and the second type 1 repeats of TSP1 (amino acids 412-415) by the use of synthetic peptides. A peptide with the corresponding sequence in TSP2, RIR, was inactive. In addition, a hexapeptide GGWSHW, based on a sequence present in the type 1 repeats of both TSP1 and TSP2, inhibited the activation of latent TGF-beta by TSP1. This peptide bound to 125I-active TGF-beta and inhibited interactions of TSP1 with latent TGF-beta. TSP2 also inhibited activation of latent TGF-beta by TSP1, presumably by competitively binding to TGF-beta through the WSHW sequence. These studies show that activation of latent TGF-beta is mediated by two sequences present in the type 1 repeats of TSP1, a sequence (GGWSHW) that binds active TGF-beta and potentially orients the TSP molecule and a second sequence (RFK) that activates latent TGF-beta. Peptides based on these sites have potential therapeutic applications for modulation of TGF-beta activation.

Präparation von Thrombospondin aus Thrombozytenkonzentraten

Thrombospondin (TSP), a 420-kDa glycoprotein found in human platelet alpha gra-nula, is thought to play an important role in platelet haemostatic function. The aim of this study was to develop a rapid method to isolate TSP for functional and structural studies. For preparation we used two PRPs, each unit containing 2.6 ×1010 platelets. Washed platelets were stimulated with thrombin (1.5 U/ml), protease activity was inhibited by hirudin (5 U/ml) and DIFP (1 mM). Cellular fragments and micro-somes were separated by centrifugation (700g, 5min; 48,000g, 30min). The clear protein solution was further purified by HiTrap Heparin affinity chromatography and a following MonoQ ion exchange chromatography using a NaCl gradient (0-2 M) on an FPLC System. 800 µg/U TSP were isolated within a total separation time of 60min. Purity of the isolated fraction was proved by SDS PAGE showing no additional proteins. TSP-binding studies are now made feasible without long-term planning and set-up. This method may be a helpful tool for further characterisation of platelet TSP receptor.

Calcium Ion Binding to Thrombospondin 1

We have quantified the binding of Ca2+ to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with 45CaCl2. Ca2+ binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 microM CaCl2, 90% occupancy at 100 microM CaCl2, and a Hill coefficient of 2.4 +/- 0.2 The average apparent Kd was 52 +/- 5 microM. Maximum binding, assuming Mr = 450,000 and epsilon = 0.918 (A280/mg/ml), was 35 +/- 3 Ca2+/TSP1. This value is close to the 33 sites (11 per subunit) predicted based on homology of the epidermal growth factor (1 site) and aspartate-rich (10 sites) regions to known Ca2+ binding sequences. Ca2+ protected the aspartate-rich region from trypsin proteolysis, but not until nearly all of the Ca2+ binding sites were filled. At lower occupancy of Ca2+ binding sites, several limited tryptic digest products were obtained. This finding and the previous demonstration of extensive thiol-disulfide isomerization within the aspartate-rich regions suggest that subregions of the aspartate-rich region are stabilized in different conformers. Zn2+, Cu2+, Mn2+, Mg2+, Co2+, Cd2+, and Ba2+ were tested for their ability to modulate Ca2+ binding and protease sensitivity of TSP1. Zn2+ inhibited 40% of the Ca2+ binding but neither protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward trypsin proteolysis. These results provide direct evidence for high capacity, cooperative and specific binding of Ca2+ to conformationally labile aspartate-rich repeats of TSP1.

Purification of thrombospondin from human platelets

Thrombospondins are a rapidly growing family of adhesive proteins that have diverse activities to modulate cellular growth, motility, and gene expression. Thrombospondin-1 (TSP) was the first identified member of this family and is the major form of thrombospondin in human platelets. A method is described to prepare TSP from human platelets in biologically active form with minimal degradation or contamination with other platelet proteins.

Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family.

Platelet Thrombospondin Interactions with Human High and Low Molecular Weight Kininogens

Multifunctional proteins, e.g. high molecular weight kininogen (HK, 120 kDa) and the homotrimer, thrombospondin (TSP, 540 kDa), which have more than one domain on a single polypeptide chain, are particularly well-suited to be structural elements of extracellular matrices because of their ability to bind to several macromolecules. We now demonstrate that 125I-high molecular weight kininogen (HKa) cleaved by purified kallikrein forms a complex with purified intact platelet TSP (540 kDa). HK also complexed with a proteolytic fragment (450 kDa) of TSP, lacking its three identical heparin-binding domains (HBD, 30 kDa), but failed to bind to a more extensively proteolysed molecule (210 kDa) lacking the C-terminal globular domain indicating that the binding on TSP-450 kDa is confined to the C-terminus. The binding of HK to intact TSP and to its 450 kDa fragment was of high affinity (Kd = 17-52 nM), specific, concentration dependent and saturable. Furthermore, we found both forms of the light chain (LC) of HK (56 and 46 kDa) resulting from cleavage by plasma kallikrein bound to both intact TSP and HBD independent of the presence of calcium ions. However, neither the epitope recognized by monoclonal antibody (MAb) C11C1 on domain 5 nor the prekallikrein binding site on domain 6 are involved, suggesting that the intervening proline-rich region may be the site of interaction. The heavy chain (HC) of HK required ionized calcium to bind to intact TSP or its 450 kDa homotrimer fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Diverse mechanisms for cell attachment to platelet thrombospondin

Thrombospondin-1 is a component of the extracellular matrix which is thought to play important roles in cell migration and proliferation, during embryogenesis and wound repair. To understand the basis for these activities, we are mapping the regions of the molecule with cell adhesive activity. Here, we use antagonists of specific cell binding sites, adhesion-perturbing thrombospondin monoclonal antibodies and proteolytic fragments of platelet thrombospondin, to investigate the adhesive mechanisms used by G361 melanoma cells, human intestinal smooth muscle cells (HISM), epidermal keratinocytes and MG-63 osteosarcoma cells. When attached to the same preparations of platelet thrombospondin, HISM and MG-63 cells underwent spreading, whereas G361 cells and keratinocytes did not. Attachment of all four cell types involved the carboxyterminal domain. The type 1 repeats and the amino-terminal heparin binding domain were important for stable attachment of G361, HISM and MG-63 cells, but were not involved in keratinocyte attachment. GRGDSP peptide caused near complete inhibition of HISM and MG-63 cell attachment, partially inhibited G361 attachment, but did not inhibit keratinocyte attachment. Attachment of HISM and MG-63 cells involved the alpha v beta 3 integrin. The integrity of the thrombospondin molecule was important for its adhesivity towards G361, HISM, and MG-63 cells, whereas keratinocytes attached to the 140 kDa tryptic fragment as effectively as they did to the intact molecule. These results show that cell attachment to platelet thrombospondin typically involves multiple binding interactions, but the exact profile of interactions is cell type specific. Usage of particular cell-binding sites does not predict whether cells will undergo spreading or not. These data may, in part, explain some of the current controversies surrounding the mechanisms of cell attachment to thrombospondin.

Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis.

We have investigated the cell surface recognition mechanisms used by human monocyte-derived macrophages (M phi) in phagocytosis of intact aging human neutrophils (PMNs) undergoing apoptosis. This study shows that the adhesive protein thrombospondin (TSP) was present in the interaction, both associated with the M phi surface and in solution at a mean concentration of 0.59 micrograms/ml. The interaction was inhibited by treatment of M phi (but not aged PMN) with cycloheximide, but could be "rescued" by replenishment with exogenous TSP. Under control conditions, M phi recognition of aged PMNs was specifically potentiated by purified platelet TSP at 5 micrograms/ml, present either in the interaction or if preincubated with either cell type, suggesting that TSP might act as a "molecular bridge" between the two cell types. In support, both aged PMN and M phi were found to adhere to TSP, and phagocytosis of aged PMN was specifically inhibited by (a) excess soluble TSP; (b) antibodies to TSP that also inhibit TSP-mediated adhesion to aged PMN; and (c) down-regulation of M phi receptors for TSP by plating M phi on TSP-coated surfaces. Furthermore, inhibition with mAbs/Arg-Gly-Asp-Ser peptide of the candidate M phi receptors for TSP, CD36, and alpha v beta 3 exerted synergistic effects on both M phi recognition of aged PMN and M phi adhesion to TSP, indicating that "two point" adhesion of TSP to these M phi structures is involved in phagocytosis of aged PMN. Our findings indicate newly defined roles for TSP and CD36 in phagocytic clearance of senescent neutrophils, which may limit inflammatory tissue injury and promote resolution.

Localization of two binding domains for thrombospondin within fibronectin

Thrombospondin is a major glycoprotein of the platelet α-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 K d of 1.35 × 10 −9 m. The K d for the 31-kDa domain of fibronectin was 2.28 × 10 −8 m. The 40-kDa carboxy-terminal fragment bound with a K d of 1.65 × 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.

Heparin-binding peptides from the type I repeats of thrombospondin. Structural requirements for heparin binding and promotion of melanoma cell adhesion and chemotaxis.

Synthetic peptides derived from the type I repeats of human platelet thrombospondin containing a consensus sequence Trp-Ser-Xaa-Trp bind to heparin, promote cell adhesion, and inhibit heparin-dependent interactions of melanoma cells with extracellular matrix components (Guo, N. H., Krutzsch, H. C., Nègre, E., Vogel, T., Blake, D. A., and Roberts, D. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3040-3044). In the present study, we further examined the structural requirements for activity of these peptides. The minimal active sequence for heparin or sulfatide binding based on inhibition studies is Trp-Ser-Pro-Trp, although an octapeptide is required for optimal activity. The 2 Trp residues and the Ser residue are essential. Peptides with more than 2 residues between the Trp residues are inactive. The Pro residue is essential for activity of the pentapeptide Trp-Ser-Pro-Trp-Ser, but some larger peptides with substitutions for the Pro residue are active. For direct high affinity binding to heparin, both the consensus sequence and a flanking sequence of basic amino acids are essential. Peptides containing the consensus sequence promote cell adhesion and act cooperatively with the adjacent basic amino acid sequence to promote cell spreading. Chemical modification of the Trp residues in the peptides with amino-terminal basic amino acids abolished both cell adhesion and heparin-binding. Peptides containing the consensus sequence and basic amino acids are chemotactic for A2058 human melanoma cells. The functional importance of this novel heparin and sulfatide-binding motif is suggested by its conservation in other members of the thrombospondin gene family, complement components, and in many members of the cytokine receptor and transforming growth factor beta superfamilies.

Molecular Requirements for the Interaction of Thrombospondin With Thrombin-Activated Human Platelets: Modulation of Platelet Aggregation

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.

Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation

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.

Expression and mutagenesis of thrombospondin

Thrombospondin is a 420,000-dalton adhesive glycoprotein that is composed of three subunits of equivalent molecular weight. When the cDNA for the complete coding region of the human endothelial cell thrombospondin subunit is expressed in mouse NIH 3T3 cells, a 420,000-dalton protein is synthesized and secreted. The expressed protein comigrates with human platelet thrombospondin both in the presence and in the absence of a reducing agent. The expressed protein binds to a monoclonal anti-thrombospondin antibody, heparin, and calcium. In addition to the 420,000-dalton protein, the transfected cell lines also express a variable amount of a 140,000-dalton polypeptide. When the culture supernatants that are produced by cells that are expressing thrombospondin are applied to heparin-Sepharose, the 420,000-dalton and the 140,000-dalton proteins are bound to the column and are eluted with buffer containing 0.55 and 0.3 M NaCl, respectively. The 140,000-dalton protein only binds to heparin-Sepharose in the presence of calcium. Deletion of the region of homology with procollagen results in defective assembly of the trimer. Deletion of the type 1 or type 2 repeats results in decreased stability of the subunit with the predominant polypeptides that are expressed having molecular weights of 127,000 and 130,000, respectively. These polypeptides retain low-affinity heparin-binding activity. High-affinity heparin binding is markedly diminished by mutations in either of two sequence motifs that include clusters of lysines and arginines.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet thrombospondin and glycoprotein IV abnormalities in patients with essential thrombocythemia: Effect of α‐interferon treatment

Platelet aggregability and some biochemical parameters were evaluated in seven patients with essential thrombocythemia (ET) compared with seven patients with secondary thrombocytosis (ST). Defective platelet aggregation with one or more agonists was seen in five patients with ET whereas aggregation was increased in two other patients. In addition, three patients with ET demonstrated spontaneous platelet aggregation in citrated plasma. This was associated with increased level of thrombospondin (TSP) in the plasma membrane. Interestingly, the presence of a proteolyzed 160 kDa form of TSP was detected in all patients with ET, whereas it was never found in patients with ST. Furthermore, three patients with ET demonstrated increased levels of platelet surface glycoprotein IV (GP IV), the putative receptor for TSP in the plasma membrane. In two of these patients, this correlated with increased surface expression of TSP and spontaneous platelet aggregation. The results suggest a possible link between the increased number of plasma membrane GP IV molecules, the spontaneous expression of TSP on the platelet surface and platelet hyperaggregability in some ET patients. The levels of plasma membrane GP IV and platelet surface-associated TSP tended to be normalized during alpha-interferon treatment, whereas the presence of an altered form of TSP persisted. This last parameter might be of practical usefulness in the characterization of the disease, permitting a clear distinction from ST.