Effect of a collagen-derived octapeptide on phosphoinositide turnover and 43K protein phosphorylation in collagen-activated platelets

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.

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Changes in Phosphoinositide Turnover, Ca2+ Mobilization, and Protein Phosphorylation in Platelets From NIDDM Patients

Diabetes ◽

10.2337/diab.39.12.1561 ◽

1990 ◽

Vol 39 (12) ◽

pp. 1561-1568 ◽

Cited By ~ 15

Author(s):

H. Ishii ◽

F. Umeda ◽

T. Ashimoto ◽

H. Nawata

Keyword(s):

Protein Phosphorylation ◽

Phosphoinositide Turnover ◽

Niddm Patients

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Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Physiologia Plantarum ◽

10.1034/j.1399-3054.1994.920123.x ◽

1994 ◽

Vol 92 (1) ◽

pp. 181-187

Author(s):

Maria T. Giardi ◽

Josef Komenda ◽

Jiri Masojidek

Keyword(s):

Photosystem Ii ◽

Protein Phosphorylation

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Anti-β2 Glycoprotein I Antibodies Cause Inflammation and Recruit Dendritic Cells in Platelet Clearance

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616059 ◽

2001 ◽

Vol 86 (11) ◽

pp. 1257-1263 ◽

Cited By ~ 6

Author(s):

Attilio Bondanza ◽

Angelo Manfredi ◽

Valérie Zimmermann ◽

Matteo Iannacone ◽

Angela Tincani ◽

...

Keyword(s):

Dendritic Cells ◽

Inflammatory Responses ◽

Glycoprotein I ◽

Activated Platelets ◽

Tnf Α ◽

Per Se ◽

Antigen Presenting ◽

Antiinflammatory Cytokine

SummaryScavenger phagocytes are mostly responsible for the in vivo clearance of activated or senescent platelets. In contrast to other particulate substrates, the phagocytosis of platelets does not incite pro-inflammatory responses in vivo. This study assessed the contribution of macrophages and dendritic cells (DCs) to the clearance of activated platelets. Furthermore, we verified whether antibodies against the β2 Glycoprotein I (β2GPI), which bind to activated platelets, influence the phenomenon. DCs did not per se internalise activated platelets. In contrast, macrophages efficiently phagocytosed platelets. In agreement with the uneventful nature of the clearance of platelets in vivo, phagocytosing macrophages did not release IL-1β, TNF-α or IL-10. β2GPI bound to activated platelets and was required for their recognition by anti-ββ2GPI antibodies. DCs internalised platelets opsonised by anti-ββ2GPI antibodies. The phagocytosis of opsonised platelets determined the release of TNF-α and IL-1β by DCs and macrophages. Phagocytosing macrophages, but not DCs, secreted the antiinflammatory cytokine IL-1β0. We conclude that anti-ββ2GPI antibodies cause inflammation during platelet clearance and shuttle platelet antigens to antigen presenting DCs.

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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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Scientific and Standardization Committee Communications Comparison of Test Methods for the Lupus Anticoagulant: International Survey on Lupus Anticoagulants-I (ISLA-1)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647340 ◽

1990 ◽

Vol 64 (03) ◽

pp. 478-484 ◽

Cited By ~ 28

Author(s):

Thomas Exner ◽

Douglas A Triplett ◽

David A Taberner ◽

Margaret A Howard ◽

E Nigel Harris

Keyword(s):

Lupus Anticoagulant ◽

Test Methods ◽

Positive Sample ◽

Direct Proportion ◽

Clotting Time ◽

Preliminary Screening ◽

Activated Platelets ◽

Tissue Thromboplastin ◽

Kaolin Clotting Time ◽

Induced Defects

SummarySix lyophilized plasma samples were sent to 20 “expert” laboratories for assessment of lupus anticoagulant (LA). Four samples contained pooled LA of graded potency mixed with aged normal plasma. One contained LA plus cephalin phospholipid and one contained a nonspecific venom anticoagulant. Sixteen methods were used overall with some participants using up to 8 methods. Results were scored in regard to the known potencies of LA in the samples and other known induced defects.Activated partial thromboplastin time (APTT) tests used by most participants for preliminary screening were relatively sensitive, but non-specific. Platelet or phospholipid neutralization procedures (PNP) appeared to be sensitive and specific but showed a non-linear response to increased LA content. Kaolin clotting time (KCT) tests showed the most sensitive response to increased LA content but the weaker LA were not scored as abnormal by most laboratories as the samples may have contained platelet fragments. Other commonly used tests such as the tissue thromboplastin inhibition (TTI) test and the dilute Russell’s viper venom test (DRVVT) were carried out somewhat inconsistently. The variability in performance of tests in different laboratories indicates that standardization of methodology is urgently required.Generally it seemed that most clotting tests were “bypassed” by the addition of phospholipid to a known LA-positive sample in apparently direct proportion to their sensitivity. Sample preparation, especially prevention of contamination with activated platelets is a vital preliminary part in the assay of LA.

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A Monoclonal Antibody to the Human Platelet Glycoprotein II b/III a Complex Induces Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647637 ◽

1988 ◽

Vol 60 (01) ◽

pp. 068-074 ◽

Cited By ~ 33

Author(s):

Piet W Modderman ◽

Han G Huisman ◽

Jan A van Mourik ◽

Albert E G Kr von dem Borne

Keyword(s):

Human Platelet ◽

Adenosine Diphosphate ◽

Dense Granule ◽

Platelet Glycoprotein ◽

Activated Platelets ◽

Complex Functions ◽

Slow Aggregation ◽

Irreversible Aggregation ◽

Platelet Release ◽

Aggregation Of Platelets

SummaryThe platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex. Whereas C17 inhibited the binding of fibrinogen to platelets and platelet aggregation induced by adenosine diphosphate (ADP) or collagen, 6C9 caused irreversible aggregation of platelets, both in the presence and absence of extracellular fibrinogen. When incubated with unstirred (nonaggregating) platelets, 6C9 induced release of alpha and dense granule-constituents as well as binding of 125I-fibrinogen to platelets. The latter was evidently mediated in part by platelet-derived ADP, since it was inhibited to a large extent by apyrase, the ADP-hydrolyzing enzyme. F(ab’)2 fragments of 6C9 did not induce platelet-release reactions but caused (slow) aggregation of platelets in the presence of extracellular fibrinogen.These results indicate that binding of an antibody to a specific site on the platelet GPIIb/IIIa complex may cause fibrinogen-mediated aggregation. The Fc part of the platelet-bound antibody appears to be involved in the induction of platelet release.

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