scholarly journals SARS-CoV-2 spike protein-induced platelet activation: Mechanism for virus and vaccine-induced thrombotic thrombocytopenia

2021 ◽  
Author(s):  
Huong Nguyen ◽  
Li-Yu Chen ◽  
Nida Khan ◽  
Annerose Lindenbauer ◽  
Van-Chien Bui ◽  
...  
Keyword(s):  
High Rate ◽  
Activation Mechanism ◽  
Functional Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Binding Reaction ◽  
Transcription Process ◽  
Fast Development ◽  
Current Guidelines

Abstract Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies found platelet-activating antibodies against platelet factor 4 (PF4) in both clinically ill Covid-19 patients and vaccine-induced thrombotic thrombocytopenia (VITT) patients. Here, we use various tools to identify the binding reaction of the SARS-CoV-2 spike glycoprotein (SP) with PF4 that results in immunogenic platelet-activating PF4/SP complexes. This binding is evidenced by an increase in mass, optical intensity, and stable binding force observed by quartz crystal microbalance, enzyme immune assay, and force spectroscopy, respectively. The SP induced an increase in the size of PF4 and switched the surface zeta potential of the PF4 from positive to negative values as evaluated by dynamic light scattering. The SP-induced platelet aggregation was identified by functional assay and flow cytometry but in a concentration-dependent manner. Our results indicated that the formed PF4/SP complexes can, on one hand, trigger the formation of PF4-antibodies and on the other hand mediate/activate platelets followed by inducing thrombotic events, which is the mechanism for excessive procoagulant activity observed in Covid-19 patients. With vector-based vaccines, we suggest that soluble SP are produced during the transcription process, forming antigenic PF4/SP complexes that result in a high rate of clotting effects in vaccinated individuals with Ad26.COV2.S and ChAdOx1nCoV-19 vaccines. An additional consideration of PF4/SP complexes in the current guidelines for the diagnosis of VITT will improve the treatment in patients. Our results serve a high demand to develop an effective method to treat Covid-19 patients and improve the safety for Covid-19 vaccination.

scholarly journals Platelet Factor 4 Inhibits and Enhances HIV-1 Infection in a Concentration-Dependent Manner by Modulating Viral Attachment

2016 ◽  
Vol 32 (7) ◽  
pp. 705-717 ◽  
Author(s):  
Zahra F. Parker ◽  
Ann H. Rux ◽  
Amber M. Riblett ◽  
Fang-Hua Lee ◽  
Lubica Rauova ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Viral Attachment ◽  
Hiv 1

A New Mechanism of Platelet Activation and Oxidative Death Induced by ADAMTS-18 and Regulating Bleeding Time.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 133-133
Author(s):  
Zongdong Li ◽  
Michael Nardi ◽  
Ruimin Pan ◽  
Herman Yee ◽  
Simon Karpatkin
Keyword(s):  
Amino Acid ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Activation Mechanism ◽  
Dependent Manner ◽  
Cell Secretion ◽  
Concentration Dependent Manner ◽  
Buffer Control

Abstract Anti-platelet integrin GPIIIa49-66 Ab obtained from HIV-ITP patients (or raised in rabbits) induces complement-independent platelet oxidative fragmentation and death by activating platelet 12-lipoxygenase (generation of 12(S)-HETE) and NADPH oxidase (JCI, 113:973, 2004). Platelet oxidative fragmentation is measured by flow cytometry of generated microparticles as well as intracellular DCFH oxidation. We now report that oxidative fragmentation in human platelets is preceded by Ca++ flux and P-selectin activation, n=6. However, the activation mechanism is different from classic platelet activation in that it is not inhibited by PGE1 or dibutryl cyclic AMP and is operative with Gαq−/− mouse platelets, whereas under these conditions, thrombin-induced platelet activation is completely inhibited, n=5–6. We chose to identify putative physiologic ligands that behave similarly to the GPIIIa49-66 Ab, and are therefore capable of regulating platelet reactive oxygen species (ROS) as well as arterial thrombus formation. The GPIIIa49-66 platelet peptide was used as bait to screen a 7-mer peptide phage display library. A peptide was found with 70% homology at the C-terminal position of ADAMTS-18, an ‘orphan’ disintegrin and metalloproteinase with thrombospondin (TSR)-like motifs, with unknown substrate. We have found it present in HUVEC as well as human pulmonary artery endothelial cells, on fixed sections of pathology specimens employing immunohistochemistry with a specific rabbit Ab raised against a C-terminal 18 mer peptide ADAMTS-18 (no staining with preimmune Ab). Recombinant ADAMTS-18 was produced in HEK 293 T cells and shown to induce ROS and oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. HUVEC ADAMTS-18 activity could be inhibited by a human scFv Ab raised against its C-terminal 18 mer peptide, as well as the ADAMTS-18 peptide itself, but not by a rabbit Ab against the N-terminal domain or an irrelevant peptide. Endothelial cell secretion and activation of ADAMTS-18 was optimally induced with 0.5 u/ml thrombin at 2 – 4 hrs, n=3–4. The truncated 385 amino acid C-terminal rADAMTS-18 fragment containing the 4 TSR motifs (produced in E.coli) had full activity at (<0.3 uM) whereas the C-terminal 66 amino acid fragment not containing the 18-mer binding site was inactive at 65 fold higher concentration, n=4. The physiologic significance of ADAMTS-18 was supported by demonstrating its secretion into plasma following iv injection of 4–16 u/ml thrombin into mice. Wild type mice have no detectable ADAMTS-18 in their plasma, with a sensitive ELISA assay (1 ng detectability). Thrombin stimulated mice secrete ADAMTS-18 in a concentration dependent manner. Platelet aggregates produced ex vivo with ADP and fibrinogen were destroyed with ADAMTS-18 as documented by LDH release at 1, 2 and 4 hrs of 83, 241 and 260 fold respectively, of PBS buffer control. In vivo tail vein bleeding time was shortened 4.5 fold with 1 hr prior infusion of 25 ug of a polyclonal rabbit IgG against ADAMTS-18, but not with preimmune IgG, n=10. Thus, a new mechanism is proposed for platelet activation, ROS release, death and platelet thrombus regulation, via platelet membrane oxidative fragmentation induced by thrombin-induced secretion and activation of ADAMTS-18.

Inhibition Of Contact Activation By Platelet Factor 4

1981 ◽  
Author(s):  
K Weerasinghe ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Platelet Factor 4 ◽  
Molar Ratio ◽  
Factor Xii ◽  
Dependent Manner ◽  
Dextran Sulphate ◽  
Contact Activation ◽  
Concentration Dependent Manner ◽  
Platelet Factor ◽  
Dependent Inhibition ◽  
Repeated Freezing

The contact activation of FXII by dextran sulphate (DS, MW 500,000) has been shown to be inhibited by platelet factor 4 (PF4). Plasma was prepared from blood taken with EDTA, theophylline and PGE1. Contact activation was initiated by the addition of an equal volume of DS (10μg/ml) and incubating at 0° (8 min) or 37° (2 min). The kallikrein generated was measured using S2302. Addition of purified PF4 to plasma caused a concentration dependent inhibition with complete inhibition at greater than 2.0μg/PF4/ml plasma (molar ratio of 20:1). PF4 was also observed in a concentration dependent manner to inhibit clotting of plasma as initiated by dextran sulphate.Repeated freezing of human PRP (Av. platelet count=3.55 × 108/ml) released PF4 (as measured by radioimmunoassay) and caused inhibition of contact activation. By dilution with PPP, the degree of inhibition was found to be related to the concentration of PF4. The amount needed for 50% inhibition varied from 0.2-1.5μg/ml in different donors. Platelet aggregation with epinephrine (2μg/ml) released 4.8μg/ml (±1.7) of PF4 and showed 55% (±22) inhibition of contact activation (n=ll). Collagen (5.7μg/ml) caused 4.9μg/ml (±1.3) of PF4 to be released, but the degree of inhibition varied (n=13). The discrepancy between this and the freezing experiment may be due to the “contact-product activator” properties of collagen and the intact platelet membrane. Control experiments performed using indomethacin or EDTA-PRP showed no release of PF4 or inhibition of contact activation.The effect observed appears to be related directly to the ability of PF4 to bind to negatively charged polysaccharides since PF4 was not found to inhibit activated factor XII or kallikrein. These results indicate another role for PF4 as an inhibitor of contact activation. Together with its known antiheparin properties it indicates that PF4 may be closely involved in the control of haemostasis.

Activation of maxi-anion channel by protein tyrosine dephosphorylation

2009 ◽  
Vol 297 (4) ◽  
pp. C990-C1000 ◽  
Author(s):  
Abduqodir H. Toychiev ◽  
Ravshan Z. Sabirov ◽  
Nobuyaki Takahashi ◽  
Yuhko Ando-Akatsuka ◽  
Hongtao Liu ◽  
...  
Keyword(s):  
Single Channel ◽  
Tyrosine Phosphatase ◽  
Anion Channel ◽  
Activation Mechanism ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Channel Activation ◽  
Phosphatase Inhibitors ◽  
Protein Tyrosine

The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5′-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)ζ, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPζ gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPζ in mouse fibroblasts, but not in C127 cells.

scholarly journals Antiatherosclerotic effect of dehydrocorydaline on ApoE−/− mice: inhibition of macrophage inflammation

2021 ◽  
Author(s):  
Bin Wen ◽  
Yuan-ye Dang ◽  
Su-hua Wu ◽  
Yi-min Huang ◽  
Kong-yang Ma ◽  
...  
Keyword(s):  
Vascular Inflammation ◽  
Chinese Herb ◽  
Gene Clusters ◽  
High Rate ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protein Levels ◽  
Atherosclerosis Development

AbstractDespite improvements in cardiovascular disease (CVD) outcomes by cholesterol-lowering statin therapy, the high rate of CVD is still a great concern worldwide. Dehydrocorydaline (DHC) is an alkaloidal compound isolated from the traditional Chinese herb Corydalis yanhusuo. Emerging evidence shows that DHC has anti-inflammatory and antithrombotic benefits, but whether DHC exerts any antiatherosclerotic effects remains unclear. Our study revealed that intraperitoneal (i.p.) injection of DHC in apolipoprotein E-deficient (ApoE−/−) mice not only inhibited atherosclerosis development but also improved aortic compliance and increased plaque stability. In addition, DHC attenuated systemic and vascular inflammation in ApoE−/− mice. As macrophage inflammation plays an essential role in the pathogenesis of atherosclerosis, we next examined the direct effects of DHC on bone marrow-derived macrophages (BMDMs) in vitro. Our RNA-seq data revealed that DHC dramatically decreased the levels of proinflammatory gene clusters. We verified that DHC significantly downregulated proinflammatory interleukin (IL)-1β and IL-18 mRNA levels in a time- and concentration-dependent manner. Furthermore, DHC decreased lipopolysaccharide (LPS)-induced inflammation in BMDMs, as evidenced by the reduced protein levels of CD80, iNOS, NLRP3, IL-1β, and IL-18. Importantly, DHC attenuated LPS-induced activation of p65 and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Thus, we conclude that DHC ameliorates atherosclerosis in ApoE−/− mice by inhibiting inflammation, likely by targeting macrophage p65- and ERK1/2-mediated pathways.

scholarly journals Disruption of ADAMTS13-Autoantibody Complexes with an ADAMTS13 Spacer Domain As a Potential Therapeutic Strategy for Immune Thrombotic Thrombocytopenic Purpura

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 88-88
Author(s):  
Konstantine Halkidis ◽  
X. Long Zheng
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Variable Region ◽  
Functional Assay ◽  
Dependent Manner ◽  
Adamts13 Activity ◽  
Single Chain ◽  
Concentration Dependent Manner ◽  
Thrombocytopenic Purpura ◽  
Rich Domain ◽  
Normal Hemostasis

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is primarily caused by acquired immunoglobulin (Ig) G against ADAMTS13, a member of the A Disintegrin and Metalloprotease with Thrombospondin Type 1 repeats family, which specifically cleaves von Willebrand factor (VWF). Such proteolytic cleavage is essential for normal hemostasis. An inability to cleave VWF due to IgG autoantibody-mediated inhibition of plasma ADAMTS13 activity results in an accumulation of ultra large VWF multimers and disseminated microvascular thromboses, a pathognomonic feature of TTP. Previous studies have demonstrated that the spacer domain of ADAMTS13 is the primary target of IgG autoantibody; also, spacer domain is important for recognition of VWF under various conditions. Objective: In the present study, we explored if an isolated spacer domain can compete or reverse IgG autoantibody-mediated inhibition of ADAMTS13 activity, an important question to be addressed for developing a new strategy to treat iTTP. Methods:A recombinant ADAMTS13-spacer domain was expressed and purified to homogeneity; additionally, a single-chain fragment of the variable region of anti-ADAMTS13 IgG (i.e. scFv4-20) was isolated via phage display from a patient with iTTP, which was shown to specifically target the spacer domain and potently inhibit plasma ADAMTS13 activity with sub-nanomolar affinity. A functional assay with a fluorescein-labeled VWF73 substrate was used to determine the ADAMTS13 activity. Results: First, when a purified recombinant spacer domain (0-1.0 µM) was pre-incubated with scFv4-20 at various concentrations, followed by an addition of normal human plasma (NHP) that provides wild-type ADAMTS13 and a fluorescein-labeled recombinant VWF73 (F-rVWF73) substrate, we demonstrated that the inhibitory activity of scFv4-20 towards plasma ADAMTS13 decreased in a concentration-dependent manner. As a control, recombinant Cys-rich domain had no effect on blocking the scFv4-20-mediated inhibition of plasma ADAMTS13 activity under the same conditions. When scFv4-20 was pre-incubated with plasma ADAMTS13, recombinant spacer domain could also reverse the scFv4-20-mediated inhibition of ADAMTS13 activity but to a lesser degree. At the final concentration of 1.0 µM, recombinant spacer domain was able to increase ADAMTS13 activity by 2.1-fold when the recombinant spacer domain was added prior to the formation of ADAMTS13-antibody complexes but only by 1.5-fold if the recombinant spacer domain was added after the ADAMTS13 and antibody complexes were formed. Conclusion: Our results demonstrate that autoantibody-mediated inhibition of plasma ADAMTS13 is reversible and can be blocked by a molecular mimic that is the primary antibody-binding region of the full-length ADAMTS13 protein. The data suggest that it may be feasible to explore the development of agents capable of displacing inhibitory antibodies against ADAMTS13 in patients with iTTP. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx/Sanofi: Consultancy, Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.

scholarly journals Platelet Factor 4 Induces Leukocyte Responses through Integrin Mac-1 (CD11b/CD18)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2529-2529
Author(s):  
Nataly P. Podolnikova ◽  
Valentin P. Yakubenko ◽  
Tatiana P. Ugarova
Keyword(s):  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Structural Similarity ◽  
Peptide Library ◽  
Platelet Factor 4 ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
Platelet Factor

Abstract The proteins/peptides secreted from α-granules of activated platelets not only aid in thrombus formation and blood coagulation, but also exert various immune-modulating effects. Among many secreted products, Platelet Factor 4 (PF4), a chemokine that belongs to the CXC family, is one of the most abundant platelet proteins. While PF4 assignment to the chemokine family is based on its structural similarity with other CXC chemokines and chemotactic activity, to date no receptor for PF4 on leukocytes has been identified. Our recent elucidation of the recognition specificity of a major leukocyte integrin αMβ2 (Mac-1) allowed the prediction that PF4 contains several putative Mac-1 recognition motifs and thus could potentially interact with this receptor. Using a peptide library spanning the sequence of PF4, we showed that the αMI-domain of Mac-1 bound several overlapping PF4-derived peptides. The biolayer interferometry analyses demonstrated that PF4 bound recombinant active αMI-domain of Mac-1 in a concentration-dependent manner with a KD of 1.3 ± 0.2x10-6 M. No interaction of PF4 with the inactive αMI-domain (α7 helix extended) or the αLI-domain of a homologous integrin αLβ2 was detected. The full-length recombinant PF4 and the αMI-domain-binding peptide (residues 58-70) identified in the peptide library supported strong adhesion and spreading of Mac-1-expressing cells, including neutrophils, U937 monocytic and Mac-1-transfected HEK293 cells. The cell adhesion to PF4 was partially inhibited by anti-Mac-1 mAbs and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface proteoglycans act cooperatively with integrin Mac-1. PF4 induced a potent migratory response of wild-type, but not Mac-1-deficient, macrophages in a Transwell system. PF4 also enhanced phagocytosis: coating of E. coli bacteria or latex beads with PF4 enhanced ~ 4-fold their phagocytosis by macrophages, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by wild-type, but not Mac-1-deficient, macrophages. These results identify PF4 as a ligand for integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated via interaction with Mac-1. Disclosures No relevant conflicts of interest to declare.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Clinical evaluation of a new thyroglobulin immunoradiometric assay in the follow-up of differentiated thyroid carcinoma

2003 ◽  
Vol 42 (02) ◽  
pp. 71-77 ◽  
Author(s):  
I. Schreivogel ◽  
C. Angerstein ◽  
U. Siefker ◽  
K. Lehmann ◽  
G. Altenvoerde ◽  
...  
Keyword(s):  
Thyroid Carcinoma ◽  
Residual Disease ◽  
False Negative ◽  
Differentiated Thyroid Carcinoma ◽  
Threshold Concentration ◽  
High Rate ◽  
Functional Assay ◽  
Hypothyroid Patient ◽  
Assay Sensitivity ◽  
Significant Difference

SummaryAim: Formal and clinical comparison of a new 3rd-gene-ration-Tg-IRMA (3-G-IRMA; Dynotest®Tg-plus) with a conventional Tg-IRMA (3-G-IRMA; SELco®Tg-assay) for patients with differentiated thyroid carcinoma. In addition we evaluated, if thyroglobulin (Tg) levels above a specific threshold concentration indicate the need for further investigations for residual disease. Patients, methods: Tg concentration of 105 sera of 93 consecutive patients with a differentiated thyroid cancer was determined with both assays and compared at different cut-off values (Dynotest®Tg-plus: 0.2, 1, 2 ng/ml; SELco®Tg-assay: 0.5, 1, 2 ng/ml) with the clinical results in respect to the corresponding TSH concentration. Results: Tg concentration did not show any significant difference (SELco®Tg-assay 0.5 ng/ml, Dynotest® Tg-plus 0.2 ng/ml). The Tg-values of both assays correlated with 97%. However, correlation of recovery in both assays was small (40%). The sensitivities and specificities of both assays at different cut-offs and TSH values did not reveal significant differences. In patients with TSH concentration >30 µU/ml the functional assay sensitivity was superior to arbitrary cut-offs in the decision to start further evaluations. Conclusions: In our study neither formal nor clinical significant differences between two Tg-assays were found. In a hypothyroid patient (TSH >30 µU/ml, Tg concentration exceeding the functional assay sensitivity) further investigations for residual disease are warranted. Higher thresholds are of limited value, due to a inacceptable high rate of false negative results.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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