Activation of protease-activated receptors by gingipains fromPorphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity

The bacterium Porphyromonas gingivalis is a major etiologic agent in the pathogenesis of adult periodontitis in humans. Cysteine proteinases produced by this pathogen, termed gingipains, are considered to be important virulence factors. Among many other potentially deleterious activities, arginine-specific gingipains-R (RgpB and HRgpA) efficiently activate coagulation factors. To further expand knowledge of the interaction between gingipains and the clotting cascade, this study examined their effects on cellular components of the coagulation system. The enzymes induced an increase in intracellular calcium in human platelets at nanomolar concentrations and caused platelet aggregation with efficiency comparable to thrombin. Both effects were dependent on the proteolytic activity of the enzymes. Based on desensitization studies carried out with thrombin and peptide receptor agonists, and immunoinhibition experiments, gingipains-R appeared to be activating the protease-activated receptors, (PAR)-1 and -4, expressed on the surface of platelets. This was confirmed by the finding that HRgpA and RgpB potently activated PAR-1 and PAR-4 in transfected cells stably expressing these receptors. Cumulatively, the results indicate the existence of a novel pathway of host cell activation by bacterial proteinases through PAR cleavage. This mechanism not only represents a new trait in bacterial pathogenicity, but may also explain an emerging link between periodontitis and cardiovascular disease.

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Cancer-Associated Thrombosis: A Two-Way Street

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0039-1693472 ◽

2019 ◽

Vol 45 (06) ◽

pp. 559-568 ◽

Cited By ~ 8

Author(s):

Bal Krishan Sharma ◽

Matthew J. Flick ◽

Joseph S. Palumbo

Keyword(s):

Cancer Progression ◽

Molecular Mechanisms ◽

Coagulation Factor ◽

Coagulation Factors ◽

Coagulation System ◽

Cancer Pathogenesis ◽

Bidirectional Relationship ◽

Clotting Cascade ◽

Tumor Growth And Metastasis ◽

Cancer Associated Thrombosis

AbstractPathological activation of the coagulation system occurs with virtually all forms of cancer, particularly epithelial malignancies. Accordingly, thrombosis is one of the most common comorbidities associated with cancer. Indeed, cancer-associated thromboembolism is the second leading cause of death for cancer patients, second only to the cancer itself. The identification of specific molecular mechanisms whereby tumor cells activate the coagulation system and drive thrombosis has been an active area of investigation for several decades. Studies in animal models and human trials have revealed that there is a bidirectional relationship between coagulation factor activity and cancer, whereby the pathological hemostatic system activation associated with cancer not only promotes thromboembolism but also drives progression of the malignancy. Numerous studies indicate that factors up and down the clotting cascade can contribute to various stages of cancer, including tumorigenesis, primary tumor growth, and metastasis. Although there are some mechanistic points of commonality, there are also clearly context-dependent contributions of coagulation components to cancer progression dependent on the type of cancer and stage of disease. It is also notable that in some instances, coagulation factors appear to contribute to cancer progression independently of their traditional roles in hemostasis and thrombosis. Here, the authors review the current state of the field with regard to hemostatic factor-driven cancer pathogenesis.

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cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets

Cell Communication and Signaling ◽

10.1186/s12964-019-0428-1 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 3

Author(s):

Stephanie Makhoul ◽

Katharina Trabold ◽

Stepan Gambaryan ◽

Stefan Tenzer ◽

Daniele Pillitteri ◽

...

Keyword(s):

Platelet Aggregation ◽

Tyrosine Phosphorylation ◽

Intracellular Signaling ◽

Light Transmission ◽

Thrombus Formation ◽

Coagulation Factors ◽

Human Platelets ◽

Induce Platelet Aggregation ◽

Akt Phosphorylation ◽

Camp And Cgmp

Abstract Background The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. Methods Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. Results EB-induced platelet aggregation was dependent on integrin αIIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin−/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. Conclusion EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα−/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα. Graphical abstract

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FbsA, a fibrinogen-binding protein from Streptococcus agalactiae, mediates platelet aggregation

Blood ◽

10.1182/blood-2004-06-2149 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1052-1059 ◽

Cited By ~ 44

Author(s):

Giampiero Pietrocola ◽

Axel Schubert ◽

Livia Visai ◽

Mauro Torti ◽

J. Ross Fitzgerald ◽

...

Keyword(s):

Platelet Aggregation ◽

Virulence Factor ◽

Streptococcus Agalactiae ◽

Prostaglandin E1 ◽

Binding Protein ◽

Human Platelets ◽

Etiologic Agent ◽

Fibrinogen Binding ◽

Important Virulence Factor ◽

Aggregation Of Platelets

AbstractThe bacterium Streptococcus agalactiae is an etiologic agent in the pathogenesis of endocarditis in humans. FbsA, a fibrinogen-binding protein produced by this pathogen, is considered an important virulence factor. In the present study we provide evidence that S agalactiae clinical isolates bearing FbsA attach to fibrinogen and elicit a fibrinogen-dependent aggregation of platelets. Mutants of S agalactiae lacking the fbsA gene lost the ability to attach to fibrinogen and to aggregate platelets. Plasmid-mediated expression of fbsA restored the capability for fibrinogen binding and platelet aggregation in S agalactiae fbsA mutants, and allowed Lactococcus lactis to interact with fibrinogen and to aggregate human platelets. Moreover, a monoclonal anti-FbsA antibody inhibited bacterial adherence to fibrinogen and S agalactiae–induced platelet aggregation. Platelet aggregation was inhibited by aspirin, prostaglandin E1, the peptide RGDS, and the antibody abciximab, demonstrating the specificity of platelet aggregation by S agalactiae and indicating an involvement of integrin glycoprotein IIb/IIIa in the induction of platelet aggregation. Aggregation was also dependent on anti-FbsA IgG and could be inhibited by an antibody against the platelet FcγRIIA receptor. These findings indicate that FbsA is a crucial factor in S agalactiae–induced platelet aggregation and may therefore play an important role in S agalactiae–induced endocarditis.

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Protease-activated Receptors in Vascular Biology

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616227 ◽

2001 ◽

Vol 86 (07) ◽

pp. 298-307 ◽

Cited By ~ 139

Author(s):

Shaun Coughlin

Keyword(s):

Cell Activation ◽

Tissue Injury ◽

Coagulation Factors ◽

Coagulation Cascade ◽

Endothelial Cell Activation ◽

Protease Activated Receptors ◽

Disease States ◽

Cellular Behaviors ◽

Vessel Development ◽

G Protein Coupled

SummaryG protein-coupled protease-activated receptors (PARs) provide one answer to the question of how coagulation factors and other proteases regulate cellular behaviors. In concert with the coagulation cascade, these receptors provide an elegant mechanism that links mechanical information in the form of tissue injury or vascular leak to cellular responses. Roles for PARs in hemostasis and thrombosis, inflammation, and perhaps even blood vessel development are beginning to emerge. Our current understanding of the role of PARs in platelet and endothelial cell activation and their potential importance in normal and disease states is discussed.

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An Antibody Targeted To The Anionic Region Of Human Protease Activated Receptor 4 Inhibits Thrombosis Without Influencing Bleeding

Blood ◽

10.1182/blood.v122.21.2376.2376 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2376-2376

Author(s):

Michele M. Mumaw ◽

Maria de la Fuente ◽

Amal Arachiche ◽

Daniel N. Nobel ◽

Marvin T. Nieman

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Preliminary Evidence ◽

Human Platelets ◽

Control Treatment ◽

Dependent Manner ◽

Protease Activated Receptors

Abstract Protease activated receptors (PARs) are G-protein coupled receptors which are activated by cleavage of their N-terminus by thrombin. This generates a tethered ligand which is then able to activate the corresponding receptor. Human platelets express PAR1 and PAR4, which both have crucial roles in mediating the response of platelets to injury. Our hypothesis is that PAR4 is an ideal target for new anti-platelet therapies because it is required for stable clot formation and has limited tissue distribution. We have previously determined a region on PAR4 that is required for efficient activation by thrombin. A polyclonal antibody (CAN12) targeted to this region of the PAR4 exodomain does not cross react to PAR1. Initial studies determined that CAN12 is able to block thrombin-induced human platelet aggregation with an IC50 of 10 ng/ml. Control IgG does not inhibit aggregation at 2 mg/ml. In mouse platelets, CAN12 inhibits P-selectin expression and integrin activation. In the Rose-Bengal mouse model of carotid artery thrombosis, CAN12 (1 mg/kg) administered 10 minutes prior to injury was able to completely inhibit the formation of a thrombus in a dose dependent manner. The antibody delayed thrombosis to greater than 90 min; the experiment was terminated at 90 minutes. In contrast, control treatment (2 mg/kg IgG or saline) resulted in complete occlusion at ∼40 minutes. Further, the minimal dose of CAN12 required for complete inhibition of thrombosis (0.5 mg/kg) administered fifteen minutes after injury also delayed thrombosis from ∼50 minutes to ∼80 minutes. This indicates that CAN12 is able to disrupt a thrombus after it has been initiated. Preliminary evidence indicates that CAN12 is able to delay the cleavage of PAR4. Importantly, CAN12 (2 mg/kg) treatment does not increase bleeding time or blood loss in the tail clip assay compared to control IgG (2 mg/kg) treatment. There was also no significant increase in bleeding in the saphenous vein assay. The mice treated with CAN12 (2 mg/kg) had an average bleeding time of 102 seconds for 12 clot formations in 20 minutes compared to the control mice (IgG 2 mg/kg) which had an average bleeding time of 143 seconds for 11 clot formations. These data demonstrate that we are able to inhibit platelet aggregation in vitro and thrombosis in vivo without influencing bleeding time. Overall, these studies provide insight towards the development of new anti-platelet therapies and, specifically, PAR4 as an antiplatelet therapy target. Disclosures: No relevant conflicts of interest to declare.

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Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614247 ◽

1998 ◽

Vol 79 (01) ◽

pp. 177-185 ◽

Cited By ~ 10

Author(s):

Ashia Siddiqua ◽

Michael Wilkinson ◽

Vijay Kakkar ◽

Yatin Patel ◽

Salman Rahman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Human Platelets ◽

Western Blots ◽

Activated Platelets ◽

Reducing Conditions ◽

Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642389 ◽

1994 ◽

Vol 71 (01) ◽

pp. 091-094 ◽

Cited By ~ 15

Author(s):

M Cattaneo ◽

B Akkawat ◽

R L Kinlough-Rathbone ◽

M A Packham ◽

C Cimminiello ◽

...

Keyword(s):

Cardiovascular Disease ◽

Platelet Aggregation ◽

Prostaglandin E1 ◽

Human Platelet ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Before And After ◽

Normal Human ◽

Platelet Aggregates ◽

Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

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The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645696 ◽

1990 ◽

Vol 63 (01) ◽

pp. 112-121 ◽

Cited By ~ 18

Author(s):

David N Bell ◽

Samira Spain ◽

Harry L Goldsmith

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Red Blood Cells ◽

Blood Cells ◽

Platelet Rich Plasma ◽

Mean Transit Time ◽

White Blood Cells ◽

Aggregate Size ◽

Human Platelets ◽

Induced Aggregation

SummaryThe effect of red blood cells, rbc, and shear rate on the ADPinduced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23°C the rate of single platelet aggregtion was upt to 9× greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates Ḡ = 41.9,335, and 1,920 s−1, and at both 0.2 and 1.0 µM ADP. At 0.2 pM ADP, the rate of aggregation was greatest at Ḡ = 41.9 s−1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At Ḡ ≥335 s−1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 µM ADP, the initial rate of single platelet aggregation was still highest at Ḡ = 41.9 s1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at Ḡ ≥335 s−1 but the rate of single platelet aggregation was markedly greater than at 0.2 pM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

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Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648166 ◽

1991 ◽

Vol 65 (04) ◽

pp. 432-437 ◽

Cited By ~ 9

Author(s):

A W J Stuttle ◽

M J Powling ◽

J M Ritter ◽

R M Hardisty

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Aggregation ◽

Calcium Ions ◽

Human Platelet ◽

Human Platelets ◽

In Vivo Detection ◽

Fibrinogen Binding ◽

Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650238 ◽

1996 ◽

Vol 75 (01) ◽

pp. 168-174 ◽

Cited By ~ 13

Author(s):

Shigeru Tokita ◽

Morio Arai ◽

Naomasa Yamamoto ◽

Yasuhiro Katagiri ◽

Kenjiro Tanoue ◽

...

Keyword(s):

Platelet Aggregation ◽

Heparan Sulfate ◽

Disulfide Bonds ◽

Dextran Sulfate ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent Fashion ◽

Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

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