scholarly journals Role of Streptococcus gordonii Surface Proteins SspA/SspB and Hsa in Platelet Function

2007 ◽  
Vol 75 (12) ◽  
pp. 5740-5747 ◽  
Author(s):  
Steven W. Kerrigan ◽  
Nicholas S. Jakubovics ◽  
Ciara Keane ◽  
Patricia Maguire ◽  
Kieran Wynne ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Infective Endocarditis ◽  
Protein Expression ◽  
Platelet Adhesion ◽  
Surface Proteins ◽  
Dependent Manner ◽  
Streptococcus Gordonii ◽  
Induce Platelet Aggregation ◽  
Bacterial Surface ◽  
Proteomic Approach

ABSTRACT Streptococcus gordonii colonization of damaged heart surfaces in infective endocarditis is dependent upon the recognition of host receptors by specific bacterial surface proteins. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial proteins required remains poorly understood. This study provides clear evidence that several S. gordonii surface proteins participate in the interaction with platelets to support platelet adhesion and induce platelet aggregation. S. gordonii strains were found to support strong (DL1-Challis, SK12, SK184, and Blackburn) or moderate (UB1545 Δhsa and CH1-Challis) adhesion or failed to support platelet adhesion (M5, M99, and Channon). In addition, under flow conditions, platelets rolled and subsequently adhered to immobilized S. gordonii at low shear (50 s−1) in an Hsa-dependent manner but did not interact with S. gordonii DL1 at any shear rate of >50 s−1. S. gordonii strains either induced (DL1-Challis, SK12, SK184, UB1545 Δhsa, and M99) or failed to induce (M5, CH1-Challis, Channon, and Blackburn) platelet aggregation. Using a proteomic approach to identify differential cell wall protein expression between aggregating (DL1) and nonaggregating (Blackburn) strains, we identified antigen I/antigen II family proteins SspA and SspB. The overexpression of SspA or SspB in platelet-nonreactive Lactococcus lactis induced GPIIb/GPIIIa-dependent platelet aggregation similar to that seen with S. gordonii DL1. However, they failed to support platelet adhesion. Thus, S. gordonii has distinct mechanisms for supporting platelet adhesion and inducing platelet aggregation. Differential protein expression between strains may be important for the pathogenesis of invasive diseases such as infective endocarditis.

scholarly journals Human Platelets Recognize a Novel Surface Protein, PadA, on Streptococcus gordonii through a Unique Interaction Involving Fibrinogen Receptor GPIIbIIIa

2009 ◽  
Vol 78 (1) ◽  
pp. 413-422 ◽  
Author(s):  
Helen J. Petersen ◽  
Ciara Keane ◽  
Howard F. Jenkinson ◽  
M. Margaret Vickerman ◽  
Amy Jesionowski ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Protein ◽  
Dependent Manner ◽  
Streptococcus Gordonii ◽  
Wild Type ◽  
Fibrinogen Receptor ◽  
Platelet Receptor

ABSTRACT The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis.

Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs

2004 ◽  
Vol 16 (2) ◽  
pp. 69 ◽  
Author(s):  
S. A. Coonrod ◽  
M. E. Calvert ◽  
P. P. Reddi ◽  
E. N. Kasper ◽  
L. C. Digilio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Indirect Immunofluorescence ◽  
Zona Pellucida ◽  
Surface Expression ◽  
Surface Proteins ◽  
2D Electrophoresis ◽  
Dependent Manner ◽  
Two Dimensional ◽  
Phase Partitioning ◽  
Proteomic Approach

In order to gain a deeper understanding of the molecular underpinnings of sperm–egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode’s solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning. Immunoprecipitation assays also demonstrated that surface re-mZP3 was released from transfected CV-1 in a time-dependent manner. These results demonstrate that ZP3 is specifically associated with the surface of mature eggs and its subsequent release from the cell surface may represent one mechanism by which ZP3 is secreted. Furthermore, the increase in ZP3 surface expression following fertilisation suggests that ZP3 may have a functional role during sperm–oolemma binding and fusion. These results also validate the usefulness of using the 2D proteomic approach to identify and characterise egg-surface proteins.

ROLE OF PLATELET GLYCOPROTEINS lb AND llb/llla IN TUMOR CELL INDUCED PLATELET AGGREGATION AND TUMOR CELL ADHESION TO EXTRACELLULAR MATRIX. I. Grossi

1987 ◽  
Author(s):  
L Grossi ◽  
K V Honn ◽  
B F Sloane ◽  
J Thomopson ◽  
D Ohannesian ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Platelet Aggregation ◽  
Tumor Cell ◽  
Platelet Adhesion ◽  
Eicosatetraenoic Acid ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Tumor Cell Adhesion ◽  
Dose Dependent

Platelet glycoproteins are known to play a role in platelet platelet interactions, platelet activation, and platelet adhesion to extracellular matrix (ECM). Monoclonal antibody to human platelet glycoprotein lb (mAblb) and polyclonal antibodies to the llb/llla complex (pAbllb/llla) were used to evaluate the involvement of these glycoproteins in tumor cellinduced platelet aggregation (TCIPA and tumor cell adhesion to the ECM. We have demonstrated that human cervical carcinoma (MS5I7), human colon carcinoma (Clone A), and rat Walker 256 carcinosarcoma (W256) cells induce aggregation of homologous platelets via thrombin generation. MAblb and pAbllb/llla were shown to inhibit TCIPA by MS517, Clone A, and W256 in a dose dependent manner. MAblb was also shown to inhibit platelet thromboxane B2 production in response to tumor cells in a dose dependent manner. Neither mAblb nor pAbllb/llla had any effect on ADP stimulated platelet aggregation. Concentrations of mAblb and pAbllb/llla which produced half maximal inhibition alone were combined resulting in complete inhibition of TCIPA. Preincubation of MS5I7 and W256 with mAblb also resulted in inhibition of TCIPA, while preincubation of Clone A with mAblb did not, suggesting the presence of this glycoprotein on the cell membranes of MS5I7 and W256, but not on Clone A. Immunofluorescence studies confirmed the presence of this glycoprotein on the cell plasma membrane of the MS5I7 and W256, but not on Clone A. Preincubation of MS5I7 and W256 with both mAblb and pAbllb/llla alone or in combination, also resulted in decreased (12S)-12 -hydroxy -5, 8,10, 14 -eicosatetraenoic acid (12-HETE) production, while platelets preincubated with these antibodies had no effect on the concentration of 12-HETE produced. Isolation of platelet membranes and released platelet contentswere tested separately and in combination on platelet adhesion to ECM. Platelet release factors were ineffective, while isolated platelet membrane ghosts enhanced adhesion. Disruption of the platelet cytoskeleton andinhibition of the formation of the llb/llla complex decreased platelet enhanced tumor cell adhesion. These findings suggest a role for these platelet glycoproteins in TCIPA, platelet enhanced tumor cell adhesion to ECM and subsequent tumor metastasis.

Interaction of Red Cell Microparticles (RMP) with Platelets: Potential Role of RMP in Hemostasis and Thrombosis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3263-3263
Author(s):  
Wenche Jy ◽  
Max E Johansen ◽  
Carlos Bidot ◽  
Powei Chen ◽  
Lawrence L Horstman ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Low Dose ◽  
Potential Role ◽  
Image Analyzer ◽  
Venous Blood Flow ◽  
Color Flow ◽  
Induce Platelet Aggregation ◽  
Blood Calcium ◽  
Platelet Aggregates

Abstract Abstract 3263 Introduction: We previously reported data indicating that RMP are well suited for use as hemostatic agent for treating bleeding disorders (Jy et al, Hemophilia 17:4, 2011). Previous studies have shown that RMP can contribute to RBC-related thrombotic complications such as sickle cell disease and PNH. Microparticles (MP) derived from platelets (PMP), endothelia (EMP), and leukocytes (LMP) are believed to play a role in hemostasis and thrombosis. They can adhere to blood cells and endothelia, facilitating prothrombotic and proinflammatory reactions. However, less is known about interaction of RMP with cells and their potential role in hemostasis and thrombosis. Here we report evidence of interaction of RMP with platelets resulting in enhanced platelet aggregation and increased size of adherent platelet aggregates induced by shear stress. Methods: (i) RMP were prepared by high-pressure extrusion of washed RBC. (ii) Platelet aggregation was performed in a Chrono-log aggregometer. PRP (490 μL) was mixed with 10 μL of RMP (1 × 108 /mL final conc.) for 5 min, then low-dose activating agent (ADP 0.2 μM, or arachidonic acid (AA) 0.3 mM) was added. (iii) Shear-induced platelet adhesion was measured in a cone-and-well device (Diamed Impact-R). Whole blood was pre-incubated with RMP as above for 10 min, then subjected to various shear rates (900, 1800, 2700 sec−1) for 1 min. The adherent platelets were then washed, stained, and quantitated by image analyzer. (iv) RMP-platelet interaction employed 2-color flow cytometry. RMP-platelet conjugates were identified by co-expression of α-CD41-FITC and α-glycophrin A-PE, in both the free platelet and micro-aggregated platelet populations. Results: (1) Platelet aggregation: Addition of RMP to PRP did not induce platelet aggregation. However, RMP enhanced platelet aggregation induced by low-dose ADP or AA. Low-dose ADP alone induced a transient increase of aggregation peaking at 25–35% followed by slow disaggregation to 0–5% at 10 min; but in presence of RMP, a similar rate (slope) of aggregation was seen but peaking at 50–60% and disaggregation was abolished. Using AA, the RMP also potentiated aggregation from 20–30% to 50–60%. These results were obtained with heparinized PRP. Interestingly, when citrated PRP was used, the RMP effect was negligible. (2) Shear-induced platelet adhesion: At 1800 sec−1 shear rate, which approximates venous blood flow, addition of RMP increases the adhered mean aggregate size from 47 to 53 μm2 (p<0.03) but decreased the number of adhering objects from 1380 to 1242 (p<0.01). At lower (900 sec−1) or higher (2700 sec−1) rate, the RMP effects disappeared. (3) Two-color flow cytomrtry showed that RMP do not conjugate with single platelets but do with platelet micro-aggregates induced by ADP. When platelet micro-aggregates are diluted with PBS (1:10), they usually disaggregate rapidly (t1/2 = 10–15 min) but in presence of RMP, the rate of dissociation was much slower (t1/2 = 30–40 min). Conclusions: These results reveal that RMP can interact with weakly activated platelets to enhance platelet adhesion and aggregation and stabilize platelet aggregates. Since these effects were seen with heparinized but not with citrated blood, calcium may be a cofactor for this interaction. We suggest that RMP-platelet interaction could play a role in hemostasis, and that therapeutic RMP may improve hemostatic abnormality in thrombocytopenia and platelet dysfunction partly by this mechanism, augmenting the limited platelet function. Disclosures: No relevant conflicts of interest to declare.

Human Anti-Streptokinase Antibodies Induce Platelet Aggregation in an Fc Receptor (CD32) Dependent Manner

1995 ◽  
Vol 74 (03) ◽  
pp. 938-942 ◽  
Author(s):  
Jamal Lebrazi ◽  
Gérard Helft ◽  
Mustapha Abdelouahed ◽  
Ismaïl Elalamy ◽  
Massoud Mirshahi ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Response Curve ◽  
Human Platelets ◽  
Antibody Concentration ◽  
Dependent Manner ◽  
Platelet Response ◽  
Induce Platelet Aggregation ◽  
Fibrinogen Binding ◽  
Two Factors ◽  
Unimodal Response

SummaryExposure to streptokinase (SK) elicits anti-SK antibodies (Abs), which inhibit fibrinolysis and induce platelet aggregation. The mechanism of the latter is not fully understood, although it seems to involve platelet binding by a plasminogen streptokinase and anti-SK ternary complex. Anti-SK Abs were purified by affinity chromatography from serum of patients having received SK for acute myocardial infarction (AMI), and were shown to be of the IgG type. Their effects were studied with (i) human platelets in citrated plasma in the presence of SK or acetylated plasminogen-SK activator complex (APSAC), and (ii) in washed platelets, resuspended in Tyrode buffer after lowering the ionic strength, in the presence of APSAC (which provides both SK and plasminogen). An antibody concentration-response curve was obtained, showing a plateau in the presence of 0.1 mg/ml IgG. By increasing the concentration of APSAC, we obtained a unimodal response curve, the optimal concentration of APSAC being 0.05 U/ml. Aggregation was suppressed by chelating calcium with EDTA, blocking fibrinogen binding by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), and raising intraplatelet cAMP with Iloprost (a prostacyclin analogue). Aggregation required the interaction of the anti-SK Ab Fc domain with the platelet Fc-gamma receptor type II, also known as CD32, since: (i) it was blocked by the monoclonal antibody IV-3 directed against CD32, (ii) it did not occur with F(ab)’2 fragments, which block the response to the intact IgG. The clinical relevance of these platelet-activating anti-SK antibodies remains to be determined. Two factors might influence clinical outcome: (i) the amount and type of pre-existing anti-SK Abs; (ii) the known interindividual variability of the platelet response to binding and activation by IgG involving the CD32 molecule.

Human anti-streptokinase antibodies induce platelet aggregation in a Fc/CD32-dependent manner

1994 ◽  
Vol 8 ◽  
pp. 40
Author(s):  
J. Lebrazi ◽  
G. Helft ◽  
A. Vacheron ◽  
M.M. Samama ◽  
M. Mirshahi ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Dependent Manner ◽  
Induce Platelet Aggregation

scholarly journals The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin*

1996 ◽  
Vol 315 (3) ◽  
pp. 939-945 ◽  
Author(s):  
Hana S. SUIDAN ◽  
Kenneth J. CLEMETSON ◽  
Marianne BROWN-LUEDI ◽  
Simone P. NICLOU ◽  
Jeannine M. CLEMETSON ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Serine Protease ◽  
Morphological Changes ◽  
Thrombin Receptor ◽  
Neural Cells ◽  
Dependent Manner ◽  
Induce Platelet Aggregation ◽  
Granzyme A ◽  
Neurite Retraction ◽  
Induced Aggregation

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.

Beta2-Glycoprotein I Interferes with von Willebrand Factor-Dependent Platelet Adhesion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 415-415
Author(s):  
Peter J. Lenting ◽  
Janine J. Hulstein ◽  
Bas de Laat ◽  
Ronald H. Derksen ◽  
Rob Fijnheer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Antiphospholipid Syndrome ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Dependent Manner ◽  
Binding Studies ◽  
Binding Conformation ◽  
Platelet Binding ◽  
Von Willebrand

Abstract Patients with the antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity with the presence of antibodies against phospholipid-binding proteins, such as beta2-Glycoprotein (beta2-GPI) or prothrombin. In particular, antibodies against beta2-GPI strongly correlate with thrombotic complications. One model that explains this correlation involves an antibody-induced gain-of-function of beta2-GPI, which results in prothrombotic properties of this plasma protein. In an alternative model, beta2-GPI may display anti-thrombotic properties that disappear in an antibody-dependent manner. Of course, both possibilities may occur simultaneously. It should be noted, however, that little is known about the physiological function of beta2-GPI. Several in vitro-based studies have pointed to beta2-GPI as a potential inhibitor of fibrinolysis and/or the intrinsic pathway of coagulation. In the present study, we investigated the hypothesis that beta2-GPI affects platelet function. Addition or immune-depletion of beta2-GPI from platelet-rich plasma had little effect on ADP- or collagen-induced platelet aggregation, whereas it did modulate ristocetin-induced platelet aggregation. In a system employing washed platelets, beta2-GPI completely inhibited platelet aggregation induced by VWF/ristocetin or by a recombinant VWF type 2B mutant (VWF/R1306Q). This suggests that beta2-GPI is directed to the von Willebrand (VWF)-glycoprotein Ib axis. Indeed, beta2-GPI interfered with platelet adhesion to VWF-coated coverslips under conditions of flow as well, resulting in a 2-fold reduction of platelet coverage. In direct binding studies, wt-VWF displayed weak binding to immobilized beta2-GPI. However, conversion of latent VWF into a platelet-binding conformation (either via ristocetin or via a type 2B mutation) induced efficient, dose-dependent and saturable binding of VWF to beta2-GPI. By using a number of recombinant deletion-mutants and individual domains, we found that binding was mediated by the VWF A1 domain. Interestingly, anti-beta2-GPI antibodies isolated from patients with the antiphospholipid syndrome not only interfered with the interaction between beta2-GPI and VWF, but also neutralized the inhibitory effect of beta2-GPI towards VWF-dependent platelet aggregation. Finally, we analysed plasma of antiphospholid-syndrome patients for the presence of VWF that is in its platelet-binding conformation by using a specific nanobody (Hulstein et al (2005) Blood106:3035). Levels of active VWF were increased 1.7-fold in patients compared to healthy individuals or patients that lack beta2-GPI-directed antibodies (p&lt;0.0001). This increase in active VWF is similar to that observed in patients with acquired TTP. In conclusion, we demonstrate that beta2-GPI interacts preferentially with the platelet-binding conformation of VWF, thereby inhibiting VWF-dependent platelet adhesion and aggregation. Anti-beta2-GPI antibodies obtained from antiphospholipid-syndrome patients reverse this anti-thrombotic effect of beta2-GPI. We propose that beta2-GPI functions as a natural first-line barrier that prevents small amounts of active VWF to interact with platelets.

Detection of Platelet Adhesion/Aggregation to Immobilized Ligands on Microbeads by an Aggregometer

1996 ◽  
Vol 76 (06) ◽  
pp. 1072-1079 ◽  
Author(s):  
Yoko Minamoto ◽  
Takaaki Hato ◽  
Shingo Nakatani ◽  
Shigeru Fujita
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Signaling ◽  
Platelet Adhesion ◽  
Kinase Inhibitor ◽  
Light Transmission ◽  
Microscopic Analysis ◽  
Electron Microscopic ◽  
Induce Platelet Aggregation ◽  
Immobilized Ligands ◽  
Transmission Electron Microscopic Analysis

SummaryPlatelet aggregation is believed to follow platelet adhesion to vascular injury sites. We have developed a turbidimetric assay for platelet aggregation following platelet adhesion to immobilized ligands using an aggregometer. The addition of polystyrene beads coated with von Willebrand factor (vWF) or fibrinogen (Fg) to platelet suspensions caused prompt aggregation of beads and platelets, which was detected as an increase in light transmission. Electron microscopic analysis revealed that platelets adhered to the bead surfaces and that additional platelets adhered to already adhering platelets, leading to the formation of platelet aggregates. vWF-coated beads induced larger aggregates than Fg-coated beads. The interaction of vWF-coated beads with platelets was abolished by both GPIb and GPIIb-IIIa blockers, while that of Fg-coated beads was abolished by GPIIb-IIIa blockers. vWF-coated beads induced modest secretion of granules from platelets but no thromboxane B2 synthesis. Fg-coated beads induced neither reaction. However, pleckstrin phosphorylation and protein tyrosine phosphorylation was induced by both types of bead. Platelet aggregation following platelet adhesion to both types of bead was inhibited by ADP scavengers, a protein kinase C inhibitor and a tyrosine kinase inhibitor, but not by aspirin. These findings suggest that vWF- and Fg-coated beads can induce platelet aggregation following platelet adhesion through specific ligand-receptor interactions and intracellular signaling. Our simple assay using these beads may represent a useful test for immobilized ligand-induced platelet adhesion and aggregation.

scholarly journals Multiple sites on Streptococcus gordonii surface protein PadA bind to platelet GPIIbIIIa

2013 ◽  
Vol 110 (12) ◽  
pp. 1278-1287 ◽  
Author(s):  
Helen J. Petersen ◽  
Dorothea O. Tilley ◽  
Jennifer Haworth ◽  
Dermot Cox ◽  
Howard F. Jenkinson ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Platelet Adhesion ◽  
Protein A ◽  
Surface Protein ◽  
Dense Granule ◽  
Site Directed Mutagenesis ◽  
Streptococcus Gordonii ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Spreading

SummaryInfective endocarditis is a life threatening disease caused by a bacterial infection of the endocardial surfaces of the heart. The oral pathogen, Streptococcus gordonii is amongst the most common pathogens isolated from infective endocarditis patients. Previously we identified a novel cell wall protein expressed on S. gordonii called platelet adherence protein A (PadA) that specifically interacts with platelet GPIIb/IIIa. The interaction between PadA and GPIIb/IIIa resulted in firm platelet adhesion, dense granule secretion and platelet spreading on immobilised S. gordonii. This study set out to identify specific motifs on the PadA protein that interacts with platelet GPIIb/IIIa. Proteomic analysis of the PadA protein identified two short amino acid motifs which have been previously shown to be important for fibrinogen binding to GPIIb/IIIa and contributing to the generation of outside-in signalling. Site directed mutagenesis on the PadA protein in which 454AGD was substituted to AAA, and the 383RGT was substituted to AAA suggests the RGT motif has no role in supporting platelet adhesion however plays a role in dense granule secretion and platelet spreading. In contrast to this the AGD motif has no role to play in supporting firm platelet adhesion or dense granule secretion however plays a role in platelet spreading. These results suggest that multiple sites on S. gordonii PadA interact with GPIIb/IIIa to mediate a number of platelet responses that likely contribute to the thrombotic complications of infective endocarditis.

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