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Platelet adhesion and P-selectin surface expression
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-3184-0427
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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 641
Keyword [en]
divalent cations, ELISA, nicotine, nitric oxide, platelet adhesion, P-selectin
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27555Local ID: 12216ISBN: 91-7219-742-0 (print)OAI: oai:DiVA.org:liu-27555DiVA: diva2:248107
Public defence
2000-10-06, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2013-09-03Bibliographically approved
List of papers
1. Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application
Open this publication in new window or tab >>Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application
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.

Keyword
Platelet, Adhesion molecule, P-selectin, CD62P, ELISA, Nitric oxide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79738 (URN)10.1016/S0022-1759(96)00198-6 (DOI)
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2017-12-07Bibliographically approved
2. Kinetics of platelet P-selectin mobilization: Concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO Donor SNAP
Open this publication in new window or tab >>Kinetics of platelet P-selectin mobilization: Concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO Donor SNAP
1998 (English)In: Cell Communication & Adhesion, ISSN 1541-9061, E-ISSN 1543-5180, Vol. 6, no 4, 289-300 p.Article in journal (Refereed) Published
Abstract [en]

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1–1.01 µM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.

Keyword
P-selectin mobilization, kinetics, human platelets, thrombin, phorbol ester, nitric oxide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79742 (URN)10.3109/15419069809010788 (DOI)
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2017-12-07Bibliographically approved
3. Acute Effects of Nicotine Infusion on Platelets in Nicotine Users with Normal and Impaired Renal Function
Open this publication in new window or tab >>Acute Effects of Nicotine Infusion on Platelets in Nicotine Users with Normal and Impaired Renal Function
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2000 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 163, no 2, 95-104 p.Article in journal (Refereed) Published
Abstract [en]

The role of platelets in cardiovascular disease associated with smoking is becoming more established, but the effects of nicotine on platelets are unclear. Nicotine therapy is used for smoking cessation in both health and disease. Consequently, the effects of nicotine on platelets are of particular significance in disorders such as renal disease, which is associated with defective platelet function, increased cardiovascular morbidity, and altered nicotine metabolism. Thus, the aim of the present study was to investigate the acute effects of nicotine infusion (NI) on platelets in seven healthy subjects (HS) and seven patients with renal failure (RF). All subjects were nicotine users and had refrained from using nicotine for 36 h before NI. Blood was collected before, immediately after, and 2 h after NI. The plasma concentrations of nicotine and its main metabolite cotinine were determined by gas chromatography. Platelet responsiveness was assessed by aggregometry and flow cytometry in whole blood (P-selectin surface expression, fibrinogen- and von Willebrand factor-binding), P-selectin expression in isolated platelets, and immunoassays of platelet release (β-thromboglobulin, platelet factor 4, and soluble P-selectin) and nitric oxide (NO) products. The plasma levels of cotinine, but not nicotine, were significantly higher in RF compared to HS at all time points. In both groups, collagen-induced platelet aggregation was restrained immediately after NI, when the plasma concentration of nicotine was maximal, and was restored after 2 h. Two hours after NI, activation-dependent P-selectin surface expression in isolated platelets increased in both groups. This increased platelet responsiveness occurred simultaneously with a significant increase of plasma cotinine and a decrease of NO products. Thus, the present study suggests that nicotine, directly or through some secondary mechanism or metabolite, only slightly potentiates some of the platelet responses. Renal failure appears not to influence the effects of nicotine on platelets.

Keyword
Nicotine, cotinine, platelets, P-selectin, nitric oxide, healthy subjects, renal failure
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25232 (URN)10.1006/taap.1999.8853 (DOI)9671 (Local ID)9671 (Archive number)9671 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression
Open this publication in new window or tab >>Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression
2002 (English)In: Blood Coagulation and Fibrinolysis, ISSN 0957-5235, E-ISSN 1473-5733, Vol. 13, no 5, 407-416 p.Article in journal (Refereed) Published
Abstract [en]

At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg2+ and Ca2+ are essential for platelet adhesion and activation, but Mg2+ can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg2+ increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca2+ induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn2+ elicited dose-dependent adhesion only to collagen and fibrinogen. Zn2+, Ni2+ and Cu2+ increased the adhesion of platelets independently of the surface. Ca2+ dose-dependently inhibited adhesion elicited by Mg2+ to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg2+-dependent platelet adhesion to collagen and Ca2+-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26773 (URN)10.1097/00001721-200207000-00005 (DOI)11377 (Local ID)11377 (Archive number)11377 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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