Neutrophil P-selectin-glycoprotein-ligand-1 binding to platelet P-selectin enhances metalloproteinase 2 secretion and platelet-neutrophil aggregation

2005 ◽  
Vol 94 (12) ◽  
pp. 1230-1235 ◽  
Author(s):  
Haissam Abou-Saleh ◽  
Jean-François Théorêt ◽  
Daniel Yacoub ◽  
Yahye Merhi
Keyword(s):  
Platelet Activation ◽  
Human Platelets ◽  
Vascular Remodelling ◽  
Adhesive Interaction ◽  
Platelet Binding ◽  
Enhanced Secretion ◽  
Thrombin Activation ◽  
Neutrophil Aggregation ◽  
And Function ◽  
The Impact

SummaryPlatelets and neutrophils constitute a high source of metalloproteinases (MMPs), and their interactions via P-selectin and Pselectin- glycoprotein-ligand-1 (PSGL-1) are involved in thrombosis, vascular remodelling, and restenosis. We investigated the impact of these interactions on platelet MMP-2 secretion and function in platelet and neutrophil aggregation. The secretion of MMP-2 from human platelets was significantly increased threefold after thrombin activation, and enhanced two-fold in the presence of neutrophils. Neutrophil supernatant had no effect on platelet MMP-2 secretion. While no MMP-2 was detected in the supernatant of neutrophils, a high amount of MMP-9 was released by neutrophils, and remained unchanged upon thrombin activation or in the presence of platelets. Platelet P-selectin, which increased significantly after activation, triggered platelet binding to neutrophils that was completely inhibited by P-selectin or PSGL-1 antagonists, and was reduced by 50% with a GPIIb/ IIIa antagonist. P-selectin or PSGL-1 antagonism abolished the enhanced secretion of platelet MMP-2 in the presence of neutrophils and reduced platelet-neutrophil aggregation. Platelet activation and binding to neutrophils enhance the secretion of platelet MMP-2 via an adhesive interaction between P-selectin and PSGL-1, which contribute to increase platelet-neutrophil aggregation.

Talin distribution and phosphorylation in thrombin-activated platelets

1993 ◽  
Vol 106 (4) ◽  
pp. 1189-1199 ◽  
Author(s):  
M.E. Bertagnolli ◽  
S.J. Locke ◽  
M.E. Hensler ◽  
P.F. Bray ◽  
M.C. Beckerle
Keyword(s):  
Platelet Activation ◽  
Subcellular Distribution ◽  
Proteolytic Cleavage ◽  
Human Platelets ◽  
Fourfold Increase ◽  
Activated Platelets ◽  
Thrombin Activation ◽  
And Function ◽  
Adhesion Plaque

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333–3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

scholarly journals Effect of ultrapure lipopolysaccharides derived from diverse bacterial species on the modulation of platelet activation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Thomas M. Vallance ◽  
Divyashree Ravishankar ◽  
Dina A. I. Albadawi ◽  
Harry Layfield ◽  
Jonathan Sheard ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Bacterial Species ◽  
Human Platelets ◽  
Innate Immune Responses ◽  
Experimental Conditions ◽  
Activation Markers ◽  
The Impact ◽  
Inside Out

AbstractPlatelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbβ3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.

scholarly journals Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1475-1482 ◽  
Author(s):  
Wei Zhang ◽  
Robert W. Colman
Keyword(s):  
Cyclic Amp ◽  
Platelet Activation ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Intracellular Camp ◽  
Akt Inhibitor ◽  
Camp Content ◽  
Fibrinogen Receptor ◽  
Thrombin Activation ◽  
Camp Hydrolysis

Abstract Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.

scholarly journals Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hui-Ju Tsai ◽  
Ju-Chien Cheng ◽  
Man-Leng Kao ◽  
Hung-Pin Chiu ◽  
Yi-Hsuan Chiang ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Combined Treatment ◽  
Phosphorylation Site ◽  
Adaptor Protein ◽  
Human Platelets ◽  
Integrin Αiibβ3 ◽  
Activated Platelets ◽  
Phospholipase D1 ◽  
And Function ◽  
Platelet Spreading

Abstract Background Bidirectional integrin αIIbβ3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function. Results An antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin αIIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin αIIbβ3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling. Conclusions These findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbβ3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

scholarly journals Cyclin-dependent kinase 2 (Cdk2) controls phosphatase-regulated signaling and function in platelets

2020 ◽  
Author(s):  
Paul R. Woods ◽  
Brian L. Hood ◽  
Sruti Shiva ◽  
Thomas P. Conrads ◽  
Sarah Suchko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Platelet Activation ◽  
Human Platelets ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Erk Activation ◽  
Src Signaling ◽  
Energetic State ◽  
And Function

AbstractCell cycle regulatory molecules including cyclin-dependent kinases can be recruited into non-nuclear pathways to coordinate cell cycling with the energetic state of the cell or with functions such as motility. Little is known about the role of cell cycle regulators in anucleate cells such as platelets. We report that cyclin-dependent kinase (cdk2) is robustly expressed in human platelets, is activated by thrombin and is required for platelet activation. Cdk2 activation required Src signaling downstream of the platelet thrombin receptor PAR1. Kinase-active cdk2 promoted the activation of downstream platelet kinases by phosphorylating and inactivating the catalytic subunit of protein phosphatase 1 (PP1). Erk was bound to PP1 in a complex with the PP1 regulator PPP1R12a (MYPT1) in platelets, and cdk2 inhibited the phosphatase activity of PP1 and PPP1R12a bound complexes. The requirement for cdk2 in Erk activation could be replaced by the phosphatase inhibitor calyculin if cdk2 was inhibited. Blockade of cdk2 kinase with chemical and peptide cdk2 inhibitors resulted in suppression of thrombin-induced platelet aggregation, and partially inhibited GPIIb/IIIa integrin activation as well as platelet secretion of P-Selectin and ATP. Together, these data indicate a requirement for cdk2 in platelet activation.

Protease-Activated Receptors and Platelet Function

1999 ◽  
Vol 82 (08) ◽  
pp. 353-356 ◽  
Author(s):  
Shaun Coughlin
Keyword(s):  
Platelet Activation ◽  
Human Platelets ◽  
The Body ◽  
Amino Terminus ◽  
Protease Activated Receptors ◽  
Amino Terminal ◽  
Thrombin Activation ◽  
High Concentrations ◽  
Blocking Antibodies

IntroductionPlatelet activation is critical for normal hemostasis, and platelet-dependent arterial thrombosis underlies most myocardial infarctions. Thrombin is the most potent activator of platelets.1,2 For this reason, understanding the process by which thrombin activates platelets is necessary for understanding hemostasis and thrombosis and may yield novel anti-platelet therapies. This chapter focuses on our recent studies of the receptors that mediate activation of human platelets by thrombin.3,4 Thrombin signaling is mediated, at least in part, by a family of G protein-coupled protease-activated receptors (PARs), for which PAR1 is the prototype.5,6 PAR1 is activated when thrombin binds to and cleaves its amino terminal exodomain to unmask a new receptor amino terminus.5 This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling.5,7,8 The synthetic peptide SFLLRN, which mimics the first six amino acids of the new amino terminus unmasked by receptor cleavage, functions as a PAR1 agonist and activates the receptor independent of thrombin and proteolysis.5,9,10 Such peptides have been used as pharmacological probes of PAR function in various cell types.Our understanding of the role of PARs in platelet activation is evolving rapidly. PAR1 mRNA and protein were detected in human platelets,5,11-13 SFLLRN-activated human platelets,5,9,10 and PAR1-blocking antibodies inhibited human platelet activation by low, but not high, concentrations of thrombin.11,12 These data suggested a role for PAR1 in activation of human platelets by thrombin but left open the possibility that other receptors contribute.Curiously, PAR1 appeared to play no role in mouse platelets.14-16 PAR1-activating peptides did not activate rodent platelets, and platelets from PAR1-deficient mice responded like wild-type platelets to thrombin.16 The latter observation prompted a search for additional thrombin receptors and led to the identification of PAR3.17 PAR3 is activated by thrombin and is expressed in mouse platelets. PAR3 blocking antibodies inhibited mouse platelet activation by low, but not high, concentrations of thrombin,18 and knockout of PAR3 abolished mouse platelet responses to low, but not high, concentrations of thrombin.3 These results established that PAR3 is necessary for normal thrombin signaling in mouse platelets but also pointed to the existence of another mouse platelet thrombin receptor. Such a receptor, PAR4, was recently identified.3,19 PAR4 appears to function in both mouse and human platelets.3 The role of PAR3 in human platelets, if any, remains to be determined, and whether still unidentified receptors contribute to thrombin activation of platelets is unknown. Nonetheless, available data suggest a testable, working model in which PAR3 and PAR4 mediate thrombin activation of mouse platelets and PAR1 and PAR4 mediate activation of human platelets.To determine the roles of PAR1, PAR3, and PAR4 in activation of human platelets by thrombin, we examined PAR mRNA and protein expression in platelets and probed PAR function using specific peptide agonists. We also examined the effect of receptor desensitization, receptor blocking antibodies, and a PAR1 antagonist, used alone and in combination, on platelet activation.4

Structure and Function of Murine αIIbβ3 (GPIIb/IIIa): Studies Using Monoclonal Antibodies and β3-null Mice

2000 ◽  
Vol 84 (12) ◽  
pp. 1103-1108 ◽  
Author(s):  
Lesley Scudder ◽  
Susan Smyth ◽  
Dimitrios Tsakiris ◽  
Barry Coller
Keyword(s):  
Platelet Activation ◽  
Wild Type Mouse ◽  
Immunoblot Analysis ◽  
Human Platelets ◽  
Structure And Function ◽  
Binding Studies ◽  
Integrin Receptor ◽  
Β3 Integrin ◽  
And Function ◽  
Induced Aggregation

SummaryThe αIIbβ3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet αIIbβ3, utilizing both β3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine αIIbβ3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of αIIbβ3 (60-80,000 copies/platelet). Like their human counterparts, murine αIIb and β3 exhibit different electrophoretic motilities under nonreducing (αIIb 135k Da; β3 92k Da) and reducing (αIIb 120k Da; β3 108k Da) conditions, and the αIIbβ3 complex is dissociated by EDTA at pH 8 and 37 ºC. Murine β3 is less susceptible to proteolysis by plasmin than is human β3. In addition to defective platelet aggregation, mouse platelets lacking αIIbβ3 and αVβ3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, β3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, β3-null platelets have altered tyrosine phosphorylation patterns following thrombinand collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Abstract Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

scholarly journals Spirochete-platelet attachment and thrombocytopenia in murine relapsing fever borreliosis

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 2843-2850 ◽  
Author(s):  
Kishore R. Alugupalli ◽  
Alan D. Michelson ◽  
Isabelle Joris ◽  
Tom G. Schwan ◽  
Kairbaan Hodivala-Dilke ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Human Platelets ◽  
Bacterial Attachment ◽  
Severe Thrombocytopenia ◽  
Relapsing Fever ◽  
Integrin Αiibβ3 ◽  
Platelet Binding ◽  
Functional Defect ◽  
High Level

Abstract Thrombocytopenia is common in persons infected with relapsing fever Borreliae. We previously showed that the relapsing fever spirochete Borrelia hermsii binds to and activates human platelets in vitro and that, after platelet activation, high-level spirochete-platelet attachment is mediated by integrin αIIbβ3, a receptor that requires platelet activation for full function. Here we established that B hermsii infection of the mouse results in severe thrombocytopenia and a functional defect in hemostasis caused by accelerated platelet loss. Disseminated intravascular coagulation, immune thrombocytopenic purpura, or splenic sequestration did not play a discernible role in this model. Instead, spirochete-platelet complexes were detected in the blood of infected mice, suggesting that platelet attachment by bacteria might result in platelet clearance. Consistent with this, splenomegaly and thrombocytopenia temporally correlated with spirochetemia, and the severity of thrombocytopenia directly correlated with the degree of spirochetemia. Activation of platelets and integrin αIIbβ3 were apparently not required for bacterium-platelet binding or platelet clearance because the bacterium-bound platelets in the circulation were not activated, and platelet binding and thrombocytopenia during infection of β3-deficient and wild-type mice were indistinguishable. These findings suggest that thrombocytopenia of relapsing fever is the result of platelet clearance after β3-independent bacterial attachment to circulating platelets.

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