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Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-3184-0427
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
1997 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 200, no 1-2, 135-143 p.Article in journal (Refereed) Published
Abstract [en]

Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method, flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4°C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 × 107/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1–1.0 U/ml), ADP (10 μM) and epinephrine (100 μM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 μM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.

Place, publisher, year, edition, pages
1997. Vol. 200, no 1-2, 135-143 p.
Keyword [en]
Platelet, Adhesion molecule, P-selectin, CD62P, ELISA, Nitric oxide
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-79738DOI: 10.1016/S0022-1759(96)00198-6OAI: diva2:544101
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2013-09-03Bibliographically approved
In thesis
1. Platelet adhesion and P-selectin surface expression
Open this publication in new window or tab >>Platelet adhesion and P-selectin surface expression
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Upon injury in a blood vessel, platelets become activated and adbere to prevent blood loss. Activated platelets express adhesion molecules on their surface that also make leukocytes adhere to the injury. One such molecule, which also is expressed on endothelium, is the glycoprotein P-selectin. The present studies were focused on how soluble factors and surfaces can affect P-selectin surface expression on human platelets.

This thesis presents an enzyme-linked immunosorbent assay (ELISA) application to study platelet expression and release of P-selectin. The well-known platelet activators collagen, epinephrine, ADP, ristocetin, PMA, and thrombin induced surface expression of P-selectin with different potency and time-dependency in vitro. One of the most powerful platelet activators, thrombin, rapidly mediated both surface expression and release. The surface expression decreased after a short peak upon maximal thrombin-activation. The protein kinase C activator PMA induced surface expression equivalently to thrombin but the release of Pselectin was less as compared to thrombin. Nitric oxide (NO) donors, which mimic a vessel wall mechanism to inhibit platelet activation, decreased expression and release of P-selectin upon activation. Adenosine, another agent that is produced in vivo, acted in concert with NO and totally inhibited P-selectin expression induced by thrombin. NO and adenosine act by increasing the second messengers cGMP and cAMP, respectively.

The effects of an infusion of nicotine on platelet P-selectin expression were studied in nicotine users with normal and impaired renal function. After a peak directly after infusion, the plasma concentration of nicotine declined towards the basal level and the level of NO-products was lower as compared to baseline 2 h after infusion. At the same time, P-selectin expression induced by weak activators, but not by thrombin, increased in both groups. A transient and weak inhibition of collagen-induced platelet aggregation was observed directly after infusion. Studies in vitro showed that nicotine per se inhibits P-selectin expression. Thus, nicotine appears to initially inhibit and then increase platelet activation in vivo.

The adhesion of platelets to surfaces that can be exposed upon vessel wall injury and the subsequent expression of P-selectin were found to be dependent on divalent cations. Mg2+ dose-dependently increased platelet adhesion to both collagen and fibrinogen in the physiological range, but supraphysiological concentrations decreased the adhesion to fibrinogen. Ca2+ did only increase platelet adhesion to fibrinogen. Platelet adhesion to collagen caused more expression of P-selectin than adhesion to fibrinogen. Thus, the surface and the access of divalent cations regulate platelet adhesion and P-selectin surface expression.

In summary, multiple factors rep1late and affect platelet adhesion and P-selectin surface expression. The methods presented in this thesis are suitable for studies to further understand and make pharmacological modulation possible of these complex mechanisms and consequences.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 86 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 641
divalent cations, ELISA, nicotine, nitric oxide, platelet adhesion, P-selectin
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-27555 (URN)12216 (Local ID)91-7219-742-0 (ISBN)12216 (Archive number)12216 (OAI)
Public defence
2000-10-06, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2013-09-03Bibliographically approved

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