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

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 1995-2003 ◽  
Author(s):  
C Legrand ◽  
V Thibert ◽  
V Dubernard ◽  
B Begault ◽  
J Lawler
Keyword(s):  
Platelet Aggregation ◽  
Binding Domain ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Platelet Surface ◽  
Amino Terminal ◽  
Activated Platelets ◽  
Heparin Binding Domain ◽  
Induced Aggregation

Abstract 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.

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

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 1995-2003
Author(s):  
C Legrand ◽  
V Thibert ◽  
V Dubernard ◽  
B Begault ◽  
J Lawler
Keyword(s):  
Platelet Aggregation ◽  
Binding Domain ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Platelet Surface ◽  
Amino Terminal ◽  
Activated Platelets ◽  
Heparin Binding Domain ◽  
Induced 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.

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

2004 ◽  
Vol 91 (04) ◽  
pp. 779-789 ◽  
Author(s):  
Oonagh Shannon ◽  
Jan-Ingmar Flock
Keyword(s):  
Platelet Aggregation ◽  
Potent Inhibitor ◽  
Specific Binding ◽  
Binding Protein ◽  
Dependent Manner ◽  
Platelet Surface ◽  
Putative Receptor ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent

Summary S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

scholarly journals Cellular Internalization and Degradation of Thrombospondin-1 Is Mediated by the Amino-terminal Heparin Binding Domain (HBD)

1997 ◽  
Vol 272 (10) ◽  
pp. 6784-6791 ◽  
Author(s):  
Irina Mikhailenko ◽  
Dmitry Krylov ◽  
Kelley McTigue Argraves ◽  
David D. Roberts ◽  
Gene Liau ◽  
...  
Keyword(s):  
Binding Domain ◽  
Cellular Internalization ◽  
Heparin Binding ◽  
Amino Terminal ◽  
Thrombospondin 1 ◽  
Heparin Binding Domain

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713 ◽  
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

Down Regulation of Thrombin Procoagulant Activity by Prothrombin Activation Fragment 1.2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1946-1946
Author(s):  
Swapan K. Dasgupta ◽  
Perumal Thiagarajan
Keyword(s):  
Active Site ◽  
Procoagulant Activity ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Anionic Phospholipid ◽  
Platelet Surface ◽  
Amino Terminal ◽  
Induced Aggregation ◽  
Kringle 2 ◽  
Prothrombin Activation

Abstract Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Under physiological condition, prothrombin activation catalyzed by the fully assembled prothrombinase complex via an ordered, sequential reaction with primary cleavage at Arg320-lle321 followed by the second cleavage at Arg271-Thr272, forming fragment 1.2 and thrombin. The fragment 1.2 contains the amino-terminal gla domain and the two kringle domains. It has been shown that the kringle 2 domain in fragment 2 can bind to the exosite II of thrombin and inhibit allosterically its activity by inducing conformational changes at the active site. Nascent thrombin that is generated on platelet surface will remains non-covalenty bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate thrombin activity, we tested the effect of anionic phospholipid-bound fragment 1.2 (0–10 μM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on esterase activity towards S2238 (D-Phe-Pip-Arg-pNA.2HCI) suggesting a competitive inhibition of fibrinogen binding. We also labeled thrombin at the active site with fluorescein-FPRCK (5-fluorescein-D-Phe-Pro-Arg chloromethyl-ketone) and monitored fluorescence changes following fragment 1.2 binding in a spectrofluorometer. Anionic phospholipid-bound fragment 1.2 induced changes in the active site similar to fragment 2 with half maximal effect at ~8 μM. We tested the effect of fragment 1.2 on fluorescein-FPR thrombin binding to platelets. Fragment 1.2 inhibited thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that membrane-bound prothrombin fragment 1.2 may play an important role in hemostasis by down regulating thrombin procoagulant activity because of its interaction with exosite 2.

Phosphatidylserine (PS)-Positive Erythrocytes Bind to Both Immobilized and Soluble Thrombospondin-1 (TSP-1) Via Its Heparin Binding Domain.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1568-1568
Author(s):  
Yamaja B.N. Setty ◽  
Suhita Gayen Betal ◽  
Surekha Kulkarni ◽  
Marie J. Stuart
Keyword(s):  
Cell Adhesion ◽  
Binding Domain ◽  
Red Cells ◽  
Binding Motif ◽  
Dependent Manner ◽  
Red Cell ◽  
Heparin Binding ◽  
Cell Binding ◽  
Erythrocyte Adhesion ◽  
Heparin Binding Domain

Abstract Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P<0.001). Other anionic polysaccharides including high molecular weight dextran sulfate and chondroitin sulfate A also inhibited PS+ve erythrocyte adhesion to immobilized TSP with the magnitude of the inhibitory effects comparable to heparin. These results suggested that the heparin-binding domain on TSP may be involved in PS-mediated red cell adhesion to immobilized TSP. We have extended these studies to characterize the PS-binding site on TSP using monoclonal antibodies directed against specific cell-binding domains on the molecule and also using specific TSP peptides. We demonstrate that pre-incubation of immobilized TSP with an antibody directed against the heparin-binding domain on TSP (TSP-Ab9, Lab Vision) blocked PS-mediated red cell adhesion to immobilized TSP (80% inhibition compared to an isotype-matched negative control antibody, n=7, P<0.001), whereas an antibody that recognizes the collagen-binding domain on TSP (TSP-Ab4) did not affect this process. In addition, incubation of PS+ve erythrocytes with a TSP peptide containing the specific heparin-binding motif, KKTRG, inhibited PS-mediated red cell adhesion by 55% (P<0.001, n=6), whereas a peptide lacking the binding motif had no effect. Since protein confirmation of immobilized TSP appears to be different from that of soluble TSP, we next investigated whether soluble TSP, like immobilized TSP, also interacted with PS+ve erythrocytes. Erythrocytes containing 8 to 10% PS+ve cells were incubated in the absence or the presence of increasing concentrations of soluble TSP (0.1 to 10 μg/ml), and then analyzed by flow cytometry for surface bound TSP using both adhesion blocking (TSP-Ab9) and non-blocking (TSP-Ab4) anti-TSP antibodies. We demonstrate that soluble TSP binds to PS+ve erythrocytes in a concentration-dependent manner with 3 to 11% TSP-positive (TSP+ve) red cells measured at soluble TSP concentrations between 1 to 10 μg/ml (n=4). In addition, TSP binding could be detected only with the non-adhesion blocking antibody TSP-Ab4, which recognizes the collagen-binding domain on TSP. The adhesion blocking antibody TSP-Ab9 that interacts with the heparin binding domain, failed to detect any TSP+ve red cells. No TSP+ve erythrocytes were detected when PS-negative control red cells were evaluated in binding assays. In parallel adhesion experiments, soluble TSP inhibited PS+ve erythrocyte adhesion to immobilized TSP at concentrations at which significant TSP binding to erythrocytes occurred (43% and 44% inhibition at 5 and 10 μg of soluble TSP per ml, n=4). These results conclusively demonstrate that PS-positive erythrocytes interact with both immobilized and fluid phase TSP through the heparin-binding domain, and that heparin blocks this interaction.

Bevacizumab Immune Complexes Induce Thrombocytopenia and Thrombosis in Mice Transgenic for Human IgG Receptor FcYIIa

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 114-114
Author(s):  
Ali Amirkhosravi ◽  
Todd V Meyer ◽  
Liza Robles-Carillo ◽  
Florian Langer ◽  
Theresa Robson ◽  
...  
Keyword(s):  
Immune Complexes ◽  
Binding Domain ◽  
Vascular Endothelial ◽  
Heparin Binding ◽  
Surface Binding ◽  
Platelet Surface ◽  
Heparin Binding Domain ◽  
Surface Localization

Abstract Background: The anti-VEGF drug, bevacizumab (Bev), has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. Prevailing opinion on the molecular mechanism behind Bev-associated bleeding and thrombosis is that tissue factor driven coagulation, secondary to vascular endothelial cell dysfunction, may cause thrombosis due to VEGF suppression by Bev. Bev forms immune complexes (IC) with VEGF (vascular endothelial growth factor), a heparin-binding protein. In our previous in vitro studies we showed that, in the presence of heparin, Bev+VEGF immune complexes activate platelets via the IgG receptor FcγRIIa —a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia (HIT). Objectives: First, we investigated whether Bev-associated thrombosis might be replicated in mice. Because mouse platelets do not carry FcγRIIa, we used mice transgenic for this human IgG receptor (hFcR mice) in order to enable the signaling pathway identified above. Second, using human platelets in vitro, we studied the functional roles of heparin and platelet surface localization of IC in Bev-induced FcγRIIa activation. Methods: Bev+VEGF IC were preformed using VEGF165 or VEGF121 (similar to VEGF165 but lacking the heparin-binding domain). Platelet dense granule release and aggregation were measured by the serotonin release assay (SRA) and Chrono- Log aggregometers, respectively. Platelet surface localization was assessed by flow cytometry (50,000 events/test condition) and fluorescence microscopy using Alexa488- labeled Bev (Bev488). For in vivo studies, Bev+VEGF+Heparin IC (60–500 nM) or control reagents were injected intravenously into wild-type (WT) or hFcR mice. Platelet counts were measured 10–60 minutes following IC injection after obtaining blood (0.45 ml) by cardiac puncture. Immediately afterward, lungs were processed for hematoxylin and eosin staining and analyzed microscopically for evidence of thrombosis. Results: IC consisting of Bev+VEGF165+Heparin (0.2U/ml) caused thrombotic thrombocytopenia in hFcR but not WT mice, showing a requirement for FcγRIIa. Injection of Bev+VEGF121+Heparin (0.2U/ml) into hFcR mice did not cause thrombocytopenia, suggesting a requirement for the VEGF165 heparin binding domain. Bev+VEGF165 was without effect in the absence of heparin or in the presence of excess (200 U/ml) heparin demonstrating that a limited range of heparin concentrations enable Bev-induced thrombocytopenia and thrombosis. This mechanism is similar to that observed in HIT and our in vivo results were consistent with SRA and aggregation in vitro studies. By flow cytometry, maximal Fab-dependent Bev488 platelet surface binding occured only with VEGF165+0.2U/ml heparin. Saturating IV.3 (anti-FcγRIIa antibody) concentrations, present in all samples, excluded Bev-Fc binding to FcγRIIa. Furthermore, binding of Bev488+VEGF121+0.2 U/ml heparin was not detected, suggesting the VEGF heparin binding domain is required for heparin-enhanced surface binding. Conclusions: In the presence of heparin, Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of FcγRIIa receptor. This mechanism may be relevant to the thromboembolic complications observed in patients receiving Bev therapy.

scholarly journals Platelet thrombospondin mediates attachment and spreading of human melanoma cells.

1987 ◽  
Vol 104 (1) ◽  
pp. 131-139 ◽  
Author(s):  
D D Roberts ◽  
J A Sherwood ◽  
V Ginsburg
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Attachment ◽  
Melanoma Cells ◽  
Binding Domain ◽  
Heparin Binding ◽  
Amino Terminal ◽  
Sulfated Fucan ◽  
Heparin Binding Domain ◽  
Carboxyl Terminal ◽  
Inhibition Studies

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.

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