The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies

1996 ◽  
Vol 76 (05) ◽  
pp. 774-779 ◽  
Author(s):  
John T Brandt ◽  
Carmen J Julius ◽  
Jeanne M Osborne ◽  
Clark L Anderson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Complement Activation ◽  
Thromboembolic Disease ◽  
Clinical Samples ◽  
Hla Antigen ◽  
Aggregation Response ◽  
Immune Mediated ◽  
Dependent Pathway

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

The Effect of Heparin vs. Citrate on the Interaction of Platelets with Vascular Graft Materials

1985 ◽  
Vol 54 (04) ◽  
pp. 842-848 ◽  
Author(s):  
Kandice Kottke-Marchant ◽  
James M Anderson ◽  
Albert Rabinovitch ◽  
Richard A Huskey ◽  
Roger Herzig
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Vascular Graft ◽  
Time Course ◽  
Platelet Reactivity ◽  
Polymer Surfaces ◽  
In Vitro Perfusion ◽  
Citrate Anticoagulation ◽  
Platelet Release

SummaryHeparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

PLATELET ACTIVATION BY HUMAN CANCER CELLS GROWN “IN VITRO” OR DISSOCIATED FROM TUMOUR TISSUES

1987 ◽  
Author(s):  
G Grignani ◽  
L Pacchiarini ◽  
M Zucchella ◽  
L Dezza ◽  
S C Rizzo
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Cancer ◽  
Iodoacetic Acid ◽  
Tumour Cells ◽  
Human Cancer Cells ◽  
Human Tumour Cells ◽  
Dissociated Cells ◽  
Lymphoblastic Cells

The mechanisms of platelet activation by human tumour cells grown “in vitro” or freshly dissociated from tumour tissues have been investigated.MoCCL human T-lymphoblastic cells cultured “in vitro” induced platelet aggregation through the production of ADP, as evidenced by inhibition of the effect by apyrase. The maximum of ADP production by tumour cells was reached after 1 hour and was 225 p moles/106 cells.On the contrary, platelet aggregation induced by 5637 human bladder carcinoma cells was not inhibited by apyrase, but was abolished by hirudin, indicating the important role of thrombin in this effect.Tumour cells dissociated from 3 breast carcinomas showed a very high platelet aggregating activity, which was not inhibited by hirudin or apyrase, but was abolished by iodoacetic acid, suggesting a role for a cystein-protease in platelet activation.These results confirm that platelets can be activated by tumour cells through different mechanisms; they also suggest that the methods employed to obtain the tumour cells can influence the results, probably because of the different cell populations which are present in the dissociated tumour tissues.Informations obtained with freshly dissociated cells are interesting, because this method has been used seldom so far and because it provides a more physiological approach to the study of the interactions of tumours and platelets.

scholarly journals ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway

2018 ◽  
Vol 50 (5) ◽  
pp. 1779-1793 ◽  
Author(s):  
Xiang Wang ◽  
Yun-Feng Fu ◽  
Xiao Liu ◽  
Guo Feng ◽  
Dan Xiong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Mtor Pathway ◽  
Enhanced Chemiluminescence ◽  
Transmission Electron ◽  
Related Proteins

Background/Aims: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. Methods: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. Results: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. Conclusion: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

scholarly journals Platelet Activation and Biofilm Formation by Aerococcus urinae, an Endocarditis-Causing Pathogen

2010 ◽  
Vol 78 (10) ◽  
pp. 4268-4275 ◽  
Author(s):  
Oonagh Shannon ◽  
Matthias Mörgelin ◽  
Magnus Rasmussen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Biofilm Formation ◽  
Platelet Rich Plasma ◽  
Protein Structures ◽  
Prostaglandin E ◽  
Bacterial Surface ◽  
Fibrinogen Receptor ◽  
Aerococcus Urinae

ABSTRACT The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E1. Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.

Intracellular matrix metalloproteinase-2 (MMP-2) regulates human platelet activation via hydrolysis of talin

2014 ◽  
Vol 111 (01) ◽  
pp. 140-153 ◽  
Author(s):  
Christopher Mason ◽  
Stephen Lynch ◽  
James Benjamin ◽  
Dani Ashak ◽  
Jamunabai M. Prakash ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Matrix Metalloproteinase ◽  
Platelet Activation ◽  
Matrix Metalloproteinase 2 ◽  
Activation Pathway ◽  
Extracellular Signals ◽  
Mmp Inhibitor ◽  
Activation Mechanisms ◽  
Vitro Analysis

SummaryMatrix metalloproteinase (MMP) activity is generally associated with normal or pathological extracellular processes such as tissue remodeling in growth and development or in tumor metastasis and angiogenesis. Platelets contain at least three MMPs, 1, 2 and 9 that have been reported to stimulate or inhibit agonist-induced platelet aggregation via extracellular signals. The non-selective Zn+2 chelating MMP inhibitor, 1,10-phenanthroline, and the serine protease inhibitor, AEBSF, were found to inhibit all tested agonist-induced platelet aggregation reactions. In vitro analysis demonstrated that 1,10-phenanthroline completely inhibited MMP-1,2,and 9 but had little to no effect on calpain activity while the converse was true with AEBSF. We now demonstrate that MMP-2 functions intracellularly to regulate agonistinduced platelet aggregations via the hydrolytic activation of talin, the presumed final activating factor of glycoprotein (GP)IIb/IIIa integrin (the inside-out signal). Once activated GPIIb/IIIa binds the dimeric fibrinogen molecule required for platelet aggregation. The active intracellular MMP-2 molecule is complexed with JAK 2/STAT 3, as demonstrated by the fact that all three proteins are co-immunoprecipitated with either anti-JAK 2, or anti-STAT 3 antibodies and by immunofluorescence studies. The MMP-2 platelet activation pathway can be synergistically inhibited with the non-selective MMP inhibitor, 1,10-phenanthroline, plus a JAK 2 inhibitor. This activation pathway is distinct from the previously reported calpain-talin activating pathway. The identification of a new central pathway for platelet aggregation presents new potential targets for drug regulation and furthers our understanding of the complexity of platelet activation mechanisms.

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

2005 ◽  
Vol 98 (4) ◽  
pp. 1414-1419 ◽  
Author(s):  
Antonino Coppola ◽  
Ludovico Coppola ◽  
Liliana dalla Mora ◽  
Francesco M. Limongelli ◽  
Antonio Grassia ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
B Lymphocytes ◽  
Platelet Activation ◽  
Critical Role ◽  
Strenuous Exercise ◽  
Platelet Glycoprotein ◽  
Vigorous Exercise ◽  
Platelet Aggregates

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

scholarly journals Novel Antibodies Against Glycoprotein Ibα Inhibit Pulmonary Metastasis By Affecting Vwf-Gpibα Interaction

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1133-1133
Author(s):  
Qi Yingxue ◽  
Wenchun Chen ◽  
Ke Xu ◽  
Fengying Wu ◽  
Xuemei Fan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Binding Sites ◽  
Tumor Metastasis ◽  
Cancer Metastasis ◽  
Platelet Glycoprotein ◽  
Induce Platelet Aggregation ◽  
Metastasis Model

Abstract Background: Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy. Methods and Results: To generate antibodies that bind to mouse platelet GPIbα, washed mouse platelet lysate was used as the antigen for rat immunization. Obtained hybridoma clones were screened in ELISA for binding affinity to the GPIb-IX complex. Positive clones were further screened for their abilities to inhibit platelet-cancer cell adhesion. Finally, at static condition, two antibodies, 2B4 and 1D12, had virtually no effect on the activation of integrin αIIbβ3, which is used to indicate platelet activation. Then, we characterized the binding sites of 2B4 and 1D12 by 20 purified recombinant GPIbα fragments binding. Results showed that 2B4 and 1D12 shared the same binding sites with vWF. To determine whether 2B4 and 1D12 affect vWF binding, we tested the binding by flow cytometry using recombined mouse vWF, and then, we investigated platelet aggregation induced by several agonists, including vWF binding agonist ristocetin. Our data demonstrated clearly that 2B4 and 1D12 could inhibit vWF binding. To investigate whether the inhibition of vWF-GPIbα interaction was associated with tumor metastasis, we examined the effect of 2B4 and 1D12 in each of the following interactions in vitro: between activated platelets and tumor cells, platelets and endothelial cells. Meanwhile, We further investigated the inhibitory effect of these antibodies in vivo using the experimental metastasis model and the spontaneous metastasis model. Results showed that 2B4 and 1D12 could potently inhibit the adhesion of cancer cells in vitro, and metastasisin vivo. We next investigated whether 2B4 and 1D12 could affect platelet activation and/or induce thrombocytopenia in vivo. Results showed that the addition of 2B4 or 1D12 to PRP did not induce platelet aggregation and injection of 2B4 or 1D12 Fab at appropriate dose did not affect tail-bleeding time and platelet count. Based on the above findings, we obtained anti-human platelet GPIbα monoclonal antibody YQ3 using the same approach to explore the role of human GPIbα in cancer metastasis. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia. Conclusion: Our results therefore supported the hypothesis that GPIbα contributes to tumor metastasis, and suggested potential value of using anti-GPIbα mAb to suppress cancer metastasis. Disclosures Li: Neoletix: Consultancy, Equity Ownership.

The Antiplatelet Effects of a New Nitroderivative of Acetylsalicylic Acid - An In Vitro Study of Inhibition on the Early Phase of Platelet Activation and on TXA2 Production

1996 ◽  
Vol 76 (05) ◽  
pp. 791-798 ◽  
Author(s):  
Clara Lechi ◽  
Giuseppe Andrioli ◽  
Stefania Gaino ◽  
Rosamaria Tommasoli ◽  
Valeria Zuliani ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Methylene Blue ◽  
Platelet Aggregation ◽  
Acetylsalicylic Acid ◽  
Platelet Activation ◽  
Cyclic Gmp ◽  
Thromboxane A2 ◽  
Antiplatelet Activity ◽  
Ncx 4016

SummaryWe studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[l-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclooxygenaseThe effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 × 10−5 M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IMIIa (CD41 / αIIbβ3) (IC50 = 3.4 × 10−5 M) and P-selec-tin (CD62 / GMP-140) (IC50 = 4.9 × 10−5 M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 × 10−5 M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylyl-cyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.

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