Original article
Measurement of adhesion of human platelets in plasma to protein surfaces in microplates

https://doi.org/10.1016/j.vascn.2005.06.002Get rights and content

Abstract

Introduction

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.

Methods

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.

Results

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.

Discussion

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.

Introduction

Platelet adhesion is an initial, crucial and complex event for inhibiting blood loss after vessel wall injury. Upon injury, platelets become activated by a combination of exposed extracellular matrix proteins such as collagen, release of activating agents such as adenosine diphosphate (ADP) from platelets and other damaged cells, and local thrombin generation (Gawaz, 2004). Platelet adhesion to extracellular matrix is mediated by von Willebrand factor (vWf) and collagen binding to different glycoprotein (GP) receptors expressed by the platelets. Platelet activation also leads to a conformational change of another receptor, GPIIb/IIIa that facilitates fibrinogen binding and platelet aggregation.

In various diseases and conditions, excessive platelet activation can lead to thrombosis, whereas insufficient platelet function can cause bleeding. Thus, evaluation of platelet function is important for estimating the risk of bleeding or thrombosis in numerous conditions (Harrison, 2005). However, laboratory investigation of platelets is complicated because the mechanisms of platelet activation are highly complex. Drawing correct conclusions about platelet function is therefore dependent on the combined use of different platelet assays. Aggregometry, Ca2+-measurements, western blot and flow cytometry are all widely used methods in platelet research. In contrast, considerably less data is published regarding platelet adhesion, despite that this is an initial event in hemostasis and probably the most important physiological function of platelets. In addition platelet adhesion might also influence plaque progression and stability in the development of atherosclerosis (Ruggeri, 2002).

Platelets are of special importance in the development of arterial thrombosis but despite therapy with recommended doses of currently approved anti-platelet agents, many patients suffer thrombotic events (Patrono et al., 2004). In addition to acetylsalicylic acid (ASA), the prototype anti-platelet agent, many new and more potent anti-platelet inhibitors are today in progress or already available for use upon thrombotic risk. ASA, as well as newly developed platelet inhibitors, are all reported to exert inter-individual effects (Van De Graaff & Steinhubl, 2001) making the value of a simple and flexible platelet function assay obvious.

As an alternative and/or complement to new equipment and the oldest but still most common method for evaluating platelet function, aggregometry, we have further developed a previously described method to measure adhesion of isolated platelets with a microplate reader (Bellavite et al., 1994). In the present study we have used this technique to evaluate adhesion of platelets in plasma to different protein surfaces and the influence of various soluble activators. We have also used the method to study pharmacological inhibition of platelet adhesion in vitro. We suggest that this assay is suitable for screening and characterisation of platelet adhesion responsiveness and for evaluating the effects of new potential anti-platelet drugs. A future goal is to use the method to monitor thrombotic risk and the effects of anti-platelet drugs ex vivo.

Section snippets

Preparation of platelet-rich plasma (PRP)

The study conforms with the principles outlined in the Declaration of Helsinki, Finland 1964 and later revisions, and was approved by the local ethical committee (Linköping, Sweden). Venous blood from healthy volunteers was consecutively collected, after informed consent, into sodium heparin tubes (Greiner bio-one, Kremsmünster, Austria) or siliconised 10 mL tubes (Becton Dickinson, Oxford, UK) containing 1 mL 3.8% trisodium citrate. Only blood donors declaring that they had not used any drugs

Optimal conditions of the platelet adhesion assay

After the centrifugation procedure, the mean recovery of platelets in PRP prepared from 21 individuals was 151 and the median was 169 × 109 platelets/L. A set of experiments was initially carried out to establish that the measurement of product development by acid phosphatase correlated to the platelet number under the present conditions. PRP was diluted 1 / 2; 1 / 4; 1 / 8; 1 / 16; 1 / 32 and 1 / 64 with 0.9% NaCl. Undiluted PRP did not result in increased absorbance compared to a dilution 1 / 2 in NaCl. In

Assay conditions

The aim of this report is to present an assay that, with minimal effort, preparation steps and equipment, can be used to study platelet adhesion to various surfaces together with the influence of various soluble agents. We used the method described by Bellavite et al. (1994) as starting point and used different approaches to make this assay more convenient. The main difference is the use of PRP in the present study instead of isolated platelets. Thus, this assay does not require counting of

Acknowledgements

The authors thank Ulrika Engdahl, Department of Natural Science and Biomedicine, School of Health Sciences at Jönköping University (presently at the Laboratory of Clinical Chemistry at Värnamo Hospital) for performing some of the initial experiments. The extraordinary staff at the Blood Donor Centre, University Hospital in Linköping and the County Hospital in Jönköping is acknowledged for skilful help with the blood sampling. This work was supported by grants from the County Council of

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