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Measurement of adhesion of human platelets in plasma to protein surfaces in microplates
Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-3184-0427
2005 (English)In: Journal of Pharmacological and Toxicological Methods, ISSN 1056-8719, Vol. 52, no 3, 356-365 p.Article in journal (Refereed) Published
Abstract [en]


Platelet adhesion is an initial, crucial and complex event for inhibiting blood loss upon vascular injury. Activation and adhesion of platelets also play a fundamental role in the development of thrombosis. A combination of exposed extracellular matrix proteins in the vascular wall and release of activating compounds from the participating cells activate the platelets. New potent anti-platelet agents are in progress but there is a shortage of methods that measure the concerted action of adhesive surfaces and soluble compounds upon platelet adhesion in vitro. The aim of this work was to develop a method to measure adhesion of platelets in plasma with standard laboratory equipment.


Platelet-rich plasma from healthy humans was used in studies to optimise the conditions of the present assay. Different proteins were coated in microplate wells and various soluble platelet activators and inhibitors were added to establish the ability of the current method to detect increased as well as decreased platelet adhesion. The amount of platelet adhesion was measured by the reaction between p-nitrophenyl phosphate and the intracellular enzyme acid phosphatase.


Adhesion of platelets in plasma to microplate wells coated with albumin, collagen, fibrinogen and activated plasma showed significant surface dependency. The known soluble platelet activators adenosine diphosphate, adrenaline and ristocetin enhanced the levels of adhesion. Available anti-platelet agents such as prostacyclin, forskolin, acetylsalicylic acid and RGD containing peptides caused dose-dependent inhibition of platelet adhesion.


This report describes a further development of a previously described method and offers the advantage to use platelets in plasma to measure platelet adhesion to protein surfaces. The assay is simple and flexible and is suitable in basic research for screening and characterisation of platelet adhesion responsiveness.

Place, publisher, year, edition, pages
2005. Vol. 52, no 3, 356-365 p.
Keyword [en]
Antiplatelet agents; Collagen; Fibrinogen; Human; Methods; Platelet activation; Platelet adhesion; Platelet assay
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-13246DOI: 10.1016/j.vascn.2005.06.002OAI: diva2:18135
Available from: 2008-05-21 Created: 2008-05-21 Last updated: 2013-09-03
In thesis
1. Platelet Adhesion to Proteins in Microplates: Applications in Experimental and Clinical Research
Open this publication in new window or tab >>Platelet Adhesion to Proteins in Microplates: Applications in Experimental and Clinical Research
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelets are crucial for prevention of blood loss after vessel injury. Platelet adhesion to disrupted vessel walls is mediated by receptors such as the GPIb-IX-V complex that binds von Willebrand factor and the collagen-binding integrin α2β1. Also cross-linking of platelets, mediated by αIIbβ3 that binds to fibrinogen, results in platelet aggregation that further contributes to hemostasis. Platelets are also important pathophysiologically because of their role in thrombus formation following atherosclerotic plaque rupture. Pharmacological treatments aimed to prevent such events include use of platelet inhibitors such as acetylsalicylic acid (ASA) and clopidogrel. Despite the presence of several different platelet function assays, no one has so far been considered useful for clinical evaluation of the effect of anti-platelet treatment. The aim of this thesis was to evaluate possible applications in experimental as well as in clinical research for a platelet adhesion assay performed during static conditions. In principle, platelets in plasma are allowed to attach to protein coated microplates. Adhered platelets are then detected by induction of an enzymatic reaction followed by spectrophotometric measurements of the developed product. Our results show that the platelet adhesion assay is able to detect experimentally induced activation as well as inhibition of platelets. The assay also seems useful for investigation of synergistically induced platelet activation, especially when the coated surface consists of albumin. This is exemplified by the combination of lysophosphatidic acid and adrenaline, which induced a synergistically increased platelet adhesion to albumin that was dependent on αIIbβ3-receptors and on the secretion of ADP. Furthermore, secretion of ADP as well as TXA2 seems to contribute to several adhesive reactions investigated with this assay. The dependence on secretion, together with results showing that adhesion to collagen and fibrinogen is dependent on α2β1- and αIIbβ3-receptors respectively, indicate that the adhesive interactions occurring in the assay is in accordance with the general knowledge about platelet function. Regarding clinical applications, we found that platelet adhesion was increased for patients with essential thrombocythemia (ET) compared to controls. This is in line with the in vivo function of ET-platelets since a common complication for ET-patients is thrombosis. Furthermore, the assay was able to detect effects of treatment with clopidogrel in patients with unstable angina. To some extent it also measured the effects of ASA-treatment. In conclusion, our results suggest that the assay is suitable for experimental research and that further studies should be performed aimed at developing the assay into a clinically useful device.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 67 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1068
platelets, platelet adhesion, platelet assay, synergistic activation, thrombosis, anti-platelet treatment
National Category
Pharmacology and Toxicology
urn:nbn:se:liu:diva-11733 (URN)978-91-7393-863-1 (ISBN)
Public defence
2008-06-03, Aulan, Hälsans Hus 240, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Available from: 2008-05-21 Created: 2008-05-21 Last updated: 2013-09-03Bibliographically approved

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