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Platelet Adhesion to Proteins in Microplates: Applications in Experimental and Clinical Research
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1068
Keyword [en]
platelets, platelet adhesion, platelet assay, synergistic activation, thrombosis, anti-platelet treatment
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:liu:diva-11733ISBN: 978-91-7393-863-1 (print)OAI: oai:DiVA.org:liu-11733DiVA: diva2:18140
Public defence
2008-06-03, Aulan, Hälsans Hus 240, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2008-05-21 Created: 2008-05-21 Last updated: 2013-09-03Bibliographically approved
List of papers
1. Measurement of adhesion of human platelets in plasma to protein surfaces in microplates
Open this publication in new window or tab >>Measurement of adhesion of human platelets in plasma to protein surfaces in microplates
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]

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.

Keyword
Antiplatelet agents; Collagen; Fibrinogen; Human; Methods; Platelet activation; Platelet adhesion; Platelet assay
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13246 (URN)10.1016/j.vascn.2005.06.002 (DOI)
Available from: 2008-05-21 Created: 2008-05-21 Last updated: 2013-09-03
2. Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion
Open this publication in new window or tab >>Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion
2006 (English)In: Blood Coagulation and Fibrinolysis, ISSN 0957-5235 (print), 1473-5733 (online), Vol. 17, no 5, 359-368 p.Article in journal (Refereed) Published
Abstract [en]

Lysophosphatidic acid (LPA) and adrenaline are weak platelet activators considered important for thrombus formation, and were previously shown to synergistically increase platelet aggregation. Here we investigate synergistic activation by LPA and adrenaline when measuring platelet adhesion. Platelet-rich plasma from healthy blood donors together with adrenaline and/or LPA were added to protein-coated microplates. Platelets were allowed to adhere and the amount of adhesion detected enzymatically. The LPA and adrenaline combination induced a synergistic increase of platelet adhesion to a normally non-adhesive albumin surface. The degree of synergy varied markedly between individuals; these variations could not be explained by age, gender, blood type or different amounts of platelets, oxidized low-density lipoprotein, insulin or glucose in plasma. There was a trend indicating increased synergistic effect for platelets sensitive to adrenaline stimulation. The synergistic effect was blocked by the α2-adrenoceptor antagonist yohimbine and inhibited by the ADP scavenger system creatine phosphate/creatine phosphokinase and antibodies against αIIbβ3. Furthermore, platelets adhering to albumin after adrenaline and LPA treatment expressed P-selectin. In conclusion, LPA and adrenaline act synergistically to increase αIIbβ3-mediated platelet adhesion to albumin, dependent on α2-adrenoceptor signalling and platelet secretion. We also confirm that synergistic platelet activation achieved with LPA and adrenaline is highly donor dependent.

Keyword
adrenaline, albumin, lysophosphatidic acid, platelet adhesion, synergism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13247 (URN)10.1097/01.mbc.0000233366.18605.b2 (DOI)
Available from: 2008-05-21 Created: 2008-05-21 Last updated: 2013-09-03
3. Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates
Open this publication in new window or tab >>Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates
2009 (English)In: BLOOD COAGULATION and FIBRINOLYSIS, ISSN 0957-5235 , Vol. 20, no 3, 197-206 p.Article in journal (Refereed) Published
Abstract [en]

Platelet adhesion is a complex and important event for prevention of blood loss after vessel injury. This study investigated fundamental adhesive mechanisms occurring in an in-vitro assay developed for the measurement of static adhesion of human platelets in plasma. The aim was to gain methodological knowledge that could be used for interpretations of results from other studies using this specific assay. Involvement of adhesive receptors was investigated by the use of various antibodies as well as therapeutic drugs (abciximab, eptifibatide and tirofiban). Inhibitors of adenosine 5-diphosphate receptors (cangrelor, MRS2179) and of thromboxane A(2) signalling (BM-531) were used to estimate the role of autocrine activation. Adhesion to collagen was found to be mainly mediated by alpha(2)beta(1) and to some extent by alpha(IIb)beta(3) Adhesion to fibrinogen was mediated by alpha IIb beta 3. In addition, adenosine 5-diphosphate-induced adhesion to albumin was dependent on alpha(IIb)beta(3). Furthermore, experiments with cangrelor and BM-531 showed that the majority of the adhesive interactions tested were dependent on adenosine 5-diphosphate or thromboxane A(2). We conclude that the mechanisms of adhesion measured by the static platelet adhesion assay are in accordance with the current knowledge regarding platelet activation and adhesion. Despite its simplicity, we suggest that this adhesion assay could be used as a screening device for the study of the influence of various surfaces and soluble substances on platelet adhesion.

Keyword
adhesion receptor, antiplatelet agents, autocrine signalling, platelet adhesion, platelet assay
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18037 (URN)10.1097/MBC.0b013e328327353d (DOI)
Note
This is a non-final version of an article published in final form: Andreas Eriksson and Per Whiss , Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates, 2009, BLOOD COAGULATION and FIBRINOLYSIS, (20), 3, 197-206. http://dx.doi.org/10.1097/MBC.0b013e328327353d Copyright: Lippincott Williams and Wilkins; 1999 http://www.lww.com/ Available from: 2009-05-14 Created: 2009-05-04 Last updated: 2013-09-03Bibliographically approved
4. Enhanced platelet adhesion in essential thrombocythemia after in vitro activation
Open this publication in new window or tab >>Enhanced platelet adhesion in essential thrombocythemia after in vitro activation
2010 (English)In: TURKISH JOURNAL OF HEMATOLOGY, ISSN 1300-7777, Vol. 27, no 2, 82-90 p.Article in journal (Refereed) Published
Abstract [en]

Objective: Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by elevated platelet counts and increased risk of thrombosis. Ex vivo data suggest increased platelet reactivity in agreement with the increased thrombosis risk, while in vitro tests often detect decreased platelet activity. The present study aimed to investigate adhesion of ET-platelets in vitro, which is an aspect of platelet function that has been addressed in only a few studies on ET patients. Material and Methods: The study included 30 Er patients and 14 healthy controls. Platelet adhesion was measured with a static platelet adhesion assay. Results: The main finding was that ET-platelets were more readily activated by adhesion-inducing stimuli in vitro than control platelets. This was particularly evident in elderly patients and when using multiple stimuli, such as surfaces of collagen or fibrinogen combined with addition of adenosine 5-diphosphate or ristocetin. Such multiple stimuli resulted in adhesion above the control mean +2 standard deviations for approximately 50% of the patients. Conclusion: The results are in accordance with the concept of increased platelet activity in ET, but opposite to most other in vitro studies. We suggest that the conditions in the adhesion assay might mimic the in vivo situation regarding the presence of chronic platelet activation. (Turk J Hematol 2010; 27: 82-90)

Place, publisher, year, edition, pages
Aves Yayincilik, 2010
Keyword
Essential thrombocythemia; platelet activation; adhesion; thrombosis; platelet assay
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13249 (URN)10.5152/tjh.2010.05 (DOI)000278947500005 ()
Available from: 2008-05-21 Created: 2008-05-21 Last updated: 2013-09-03
5. Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study
Open this publication in new window or tab >>Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study
Show others...
2009 (English)In: Journal of Translational Medicine, ISSN 1479-5876, E-ISSN 1479-5876, Vol. 7, no 42Article in journal (Refereed) Published
Abstract [en]

Background: Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment. Methods: With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B-2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis. Results: The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r(2) = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone. Conclusion: The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.

Place, publisher, year, edition, pages
BioMed Central, 2009
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-19665 (URN)10.1186/1479-5876-7-42 (DOI)000267242100001 ()19508722 (PubMedID)
Note

Original Publication: Andreas Eriksson, Lena Jonasson, Tomas Lindahl, Bo Hedbäck and Per Whiss, Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study, 2009, JOURNAL OF TRANSLATIONAL MEDICINE, (7), 42. http://dx.doi.org/10.1186/1479-5876-7-42 Licensee: BioMed Central http://www.biomedcentral.com/

Available from: 2009-08-28 Created: 2009-07-10 Last updated: 2017-12-13Bibliographically approved

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