Cardiovascular disease is the major cause of morbidity and mortality globally. Antiplatelet drugs reduce cardiovascular events by decreasing the risk of thrombosis but they also increase the risk of bleeding. There is a compelling need to develop diagnostic assays of platelet function that could be used at the point-of-care and influence clinical management. Interfacial platelet cytometry is a new platform technology with the capability of assessing multiple parameters of platelet function in physiological conditions in a short time using small volumes of whole blood.
by Dr A Lopez-Alonso, Prof. Dermot Kenny and Dr L. Basabe-Desmonts
Platelets play a key role in ensuring primary hemostasis. In the event of vascular injury platelets adhere to the exposed sub-endothelial matrix, become activated and subsequently aggregate with other platelets to form a platelet clot. This ultimately leads to the initiation of thrombus formation, which stops the process of bleeding [1]. Platelet adhesive properties and their ability to respond to thrombotic stimuli vary between individuals. The normal function of platelets can exceed their physiological state and become pathological, making them pivotal not only in wound healing, but also in diseased states such as in inflammation [2], cancer metastasis [3] and atherosclerotic lesion thrombotic events, which can lead to ischemic heart disease and stroke [4]. There is a strong relation between platelet function and acute myocardial infarction; rupture of atherosclerotic plaque triggers platelet function leading to clot formation, vascular occlusion and infarction [4]. This relation has been demonstrated via a decrease in mortality and morbidity in patients on antiplatelet therapies, which interfere with the aggregation mechanism of platelets thereby preventing thrombus formation [5].
However, with the current antiplatelet therapies, cardiovascular disease (CVD) is still the most common cause of death in the developed countries [6] and the number of CVD patients is increasing. Thus further prevention of platelet disorders and CVD is a high priority for the pharmaceutical market.
Although current antiplatelet therapies have been shown to reduce mortality and morbidity in CVD patients, they have limitations: primarily there is a high inter-individual variability in response to these agents. There is also a substantial incidence of arterial thrombosis and bleeding events with patients on antiplatelet agents, due to a lack of or an excess of therapy efficiency [5].
Platelet function assays have shown that increased platelet reactivity can predict major adverse cardiovascular events in patients on antiplatelet medication [7]. Therefore an accurate measure of platelet function during antiplatelet therapy administration and in perioperative settings can influence drug dose management and medical decisions, in order to avoid adverse events. Unfortunately there is not yet clear evidence or consensus on which tests can reliably evaluate platelet function and the drug effect in individual patients [8].
Assessment of platelet function
There are a number of platelet function tests available for clinical use. However there is a lack of correlation between different tests and none of them are routinely used to assess anti-platelet therapy effects. These tests measure different parameters, bleeding or clotting time, platelet aggregation, platelet adhesion, expression of activation markers or the viscoelastic properties of the formed platelet clot. The gold standard technique for platelet function is light transmission platelet aggregometry (LTA) which measures a decrease in optical density or platelet count as platelets aggregate in solution when induced by the addition of platelet activating substances (agonists). There are also point-of-care (POC) aggregometry-based tests which have the advantage of using whole blood as the sample: ICHOR Plateletworks, Multiplate and VerifyNow. The general disadvantages of these tests are that they require the addition of platelet agonists. Aggregation stimulation by agonist results in an all or none answer. Another test measures platelet adhesion and aggregation under physiological shear: the PFA-100. This test shows good sensitivity for von Willenbrand’s disease but does not specifically address efficacy of anti-platelet drug therapy. These techniques have been recently reviewed by Franchini et al [8].
Surprisingly there are only few reports on platelet adhesion assays [9, 10]. Although adhesion is the primary function of platelets and one of the initial events in hemostasis, it is difficult to measure and it has not been possible to develop assays based on this important function of platelets. It was recently reported that a new platelet adhesion assay, performed using whole blood, had the ability to monitor GPIIb/IIIa receptor antagonists and also detected antagonist overdosing that could potentially lead to bleeding complications in patients [10]. The assay is based on the design of specialised polymer particles that are added to a citrated whole blood sample. Fibrinogen in the sample will immediately attach to the special polymer surface and through this, activated platelets by allowing platelets to bind to the fibrinogen-coated particle surface. Quantification of platelets in suspension before and after the assay is used to calculate platelet adhesiveness. The drawback of this method is that platelet adhesion is dependent on the content of fibrinogen in the blood sample and can not measure platelet adhesion to other important vascular protein matrices such as collagen and von Willenbrand factor.
None of the above techniques have the ability to enable the characterisation of individual platelets. Yet each person has a heterogeneous platelet population, with the platelets differing in size, age, and function; an individual’s platelet distribution may have diagnostic or predictive values. For example, only a small fraction of the platelet population, known as COAT platelets, express phosphatidylserine upon coactivation with collagen and thrombin. The size of this COAT platelet subpopulation has been suggested to be clinically informative [11]. Furthermore, a high mean platelet volume has been proposed as a marker for cardiovascular risk [12].
Flow cytometry is a powerful technique, able to analyse individual platelets and therefore allowing the characterisation of platelet subpopulations. It detects platelet activation markers and platelet interactions with other cells and it can assess receptor expression and inhibition with high sensitivity and specificity [13]. However this is far from physiological conditions, because the technique only measures platelet activation in fluid suspension, a non-physiological situation in the context of key platelet-protein interactions: in vivo, platelet interactions with vessel walls
induce activation.
An ideal tool for platelet function studies would use whole blood samples to avoid sample manipulation, and provide a detailed characterisation of platelet subpopulations, without the need for expensive equipment, while enabling the statistical measurement of platelet adhesion, platelet activation, morphological changes, as well as platelet receptor levels and distributions. The correlation of the quantification of these parameters with physiological events promises to provide a powerful screening, research and diagnostic tool.
Interfacial platelet cytometry
During the last decades, a number of microfabrication techniques such a photolithography have been adopted from the microelectronics industry to develop new tools for biological research. This enabled, for the first time, the analysis of cellular adhesion at the single cell level [14]. Using microcontact printing [15] and surface engineering [16] our group at the Biomedical Diagnostics Institute (BDI) in Ireland has addressed the requirements explained above and developed “interfacial platelet cytometry” (iPC), an innovative platform technology for studying platelet function and subpopulations, which in addition also enables accurate quantification of platelet adhesion [17].
iPC is comprised of micropatterns of platelet-specific proteins like fibrinogen, von Willebrand factor (vWF), collagen or specific antibodies on a flat glass surface. When whole blood is incubated over the patterned surface using gentle rocking (a very low shear system that does not cause platelet activation), platelets selectively adhere to the surface only in the area coated by platelet-specific protein within less than 30 minutes. iPC enables easy and efficient platelet separation from other blood components without need of sample preparation. The size of the pattern motifs controls platelet-platelet interactions in contrast with unpatterned protein surfaces, which offer no means to control the distances and interactions between adhering platelets. iPC allows the creation of single-platelet arrays with no cross-talk between platelets, or the capture of multiple platelets in a single protein “dot”.
The protein pattern in the iPC contains protein dots with sizes ranging from 2-20 µm in diameter. This micrometre scale patterning is achieved using microcontact printing, a soft lithography technique that uses a rubber stamp structured with high density pillar arrays. The density of pillars can exceed 600.000/cm2. The stamp is inked with the protein solution to be patterned, then dried and printed on a flat glass surface, creating protein patterns mirroring the stamp layout. In this way single cell arrays containing more than 600.000 cells/cm2 can be created, providing a high-throughput screening platform [Figure 1].
iPC is based on a flat transparent substrate (glass), making optical analysis of the adhering platelets simple, and allowing statistical studies of multiple parameters simultaneously, including platelet-protein (surface) adhesion, morphology changes, and platelet receptor expression and spatial distribution, the first and last which cannot be measured by flow cytometry.
The capture of platelets on the protein matrix is very specific; different platelet receptors can be targeted to enable the capture. Additionally, the number of platelets that can be captured in a single protein dot depends on the area of the protein dot, the protein matrix (if it induces platelet spreading) and the platelet volume. This facilitates the detection of specific platelet disorders such as Glanzman thrombostenia [18] and May Hegglin disease [19] using iPC. Due to a lack of the GPIIb/IIIa receptor on platelets in Glazman thrombostenia patients, low platelet adhesion is detected on a fibrinogen iPC surface. In the case of May-Hegglin-anomaly patients, single giant platelets that occupy a full fibrinogen protein dot of 12 µm diameter can be rapidly visualised [Figure 2].
On the other hand, patterned surfaces can be classified according to platelet affinity and platelet-induced activation. For example at low shear rates, an anti-CD42b antibody surface has a high-affinity, low-spreading state, therefore platelets are rapidly captured but do not spread, while fibrinogen and VWF surfaces both have moderate-affinity and high-activating platelet characteristics, showing delayed platelet adhesion, but platelet activation signals and spreading is induced. Thus, post-adhesion perturbations of platelets can be measured by choosing the properties of the patterned matrix: non-activating versus activating protein surfaces. For example, the capture of platelets by low-activating surfaces provides a platform for the inspection of single platelets, enabling the exposure of the captured platelets to various conditions and agonists in order to study their effect. On the other hand, high-activating surfaces can allow the degree to which specific platelet pathways are inhibited in patients on antiplatelet therapy.
Platelet adhesion to a protein matrix in iPC is assessed using single platelet arrays. Ordered protein dot arrays provide a straightforward means to quantify capture and non-capture events, since only one single platelet can be captured in each dot. The percentage of occupied protein dots allows assessment of platelet adhesion
properties [Figure 3].
iPC is well suited for developing low cost devices for platelet function research and clinical diagnostics. Platelets are separated from whole blood without sample preparation, and it is a surface-confined assay where a wealth of information can be obtained with analysing a micrometre scale area. For these reasons, it can be easily incorporated into microfluidic devices, integrating the steps of rinsing and fixing while allowing the testing of very small blood volumes, facilitating, for example, studies of mouse blood or neonatal blood sample assays and multiplexing. The development of a disposable test for POC applications based on this technology platform is straightforward.
Conclusions
A major cause of morbidity and mortality in cardiovascular diseases (the leading cause of death in the western world) is due to platelet-mediated thrombosis. For this reason the importance of anti-platelet therapeutic strategies is increasing, with the global market for anti-platelet drugs currently valued at US$10 billion. Antiplatelet drugs such as aspirin and clopidogrel (Plavix) have reduced mortality in patients with vascular disease. However, despite the widespread use of these drugs, significant numbers of patients either have recurrent thrombosis or suffer serious bleeding events, suggesting that a personal, rapid reliable assay of platelet function could guide therapy. There is clearly a compelling need to both monitor platelet function in individual patients and identify patients at increased risk of cardiovascular events. However there is no consensus on how to measure the effect of anti-platelet drugs rapidly and effectively. Tests of platelet function are not widely available, largely because laboratory assessments of platelet function are time-consuming, expensive, require dedicated, trained personnel, are not physiological, and, frequently, do not give information to the clinician that can rapidly influence clinical management.
Interfacial platelet cytometry has emerged as a new tool-box to measure platelet function close to physiological conditions. The single-step separation of platelets from whole blood is well suited to the development of low-cost devices for clinical diagnostics and antiplatelet therapy monitoring, particularly when eventually coupled with an automated high-throughput version of the digital quantification of platelets by on-chip iPC.
References
1. Michelson AD. Platelets. 2006; Academic Press.
2. Kulkarni S et al. Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading. Blood 2007; 110: 1879-1886.
3. Jurasz P et al. Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: Regulation by nitric oxide. Cancer Research 2001; 61(1): 376-382.
4. Davi G and Patrono C. Mechanisms of disease: Platelet activation and atherothrombosis. New England Journal of Medicine 2007; 357(24): 2482-2494.
5. Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nature Reviews Drug Discovery 2010; 9(2): 154-169.
6. Cardiovascular diseases. 2007. World Health Organization. p. WHO Fact sheet N°317.
7. Snoep JD et al. Association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events - A systematic review and meta-analysis. Archives of Internal Medicine 2007; 167(15): 1593-1599.
8. Lippi G et al. Laboratory assessment and perioperative management of patients on antiplatelet therapy: From the bench to the bedside. Clinica Chimica Acta 2009; 405(1-2): 8-16.
9. Eriksson AC and Whiss P.A. Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates. Blood Coagulation & Fibrinolysis 2009;. 20(3): 197-206.
10. Nowak G et al. Platelet adhesion assay - A new quantitative whole blood test to measure platelet function. Seminars in Thrombosis and Hemostasis, 2005; 31
(4): 470-475.
11. Prodan CI et al. Coated-platelets in ischemic stroke: differences between lacunar and cortical stroke. Journal of Thrombosis and Haemostasis, 2008; 6(4):
609-614.
12. Endler G et al. Mean platelet volume is an independent risk factor for myocardial infarction but not for coronary artery disease. British Journal of Haematology 2002; 117(2): 399-404.
13. Matzdorff A. Platelet function tests and flow cytometry to monitor antiplatelet therapy. Seminars in Thrombosis and Hemostasis 2005; 31(4): 393-399.
14. Chen C.S et al. Geometric control of cell life and death. Science 1997; 276(5317): 1425-1428.
15. Bernard A et al. Microcontact printing of proteins. Advanced Materials 2000; 12(14): 1067-1070.
16. Falconnet D et al. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006; 27(16): 3044-3063.
17. Basabe-Desmonts L et al. Single-Step Separation of Platelets from Whole Blood Coupled with Digital Quantification by Interfacial Platelet Cytometry (iPC). Langmuir 2010; Jan 28
18. Franchini M, Favaloro EJ and Lippi G. Glanzmann thrombasthenia: An update. Clinica Chimica Acta 2010; 411(1-2): 1-6.
19. Saito H and Kunishima S. Historical hematology: May-Hegglin anomaly. American Journal of Hematology 2008;. 83(4): 304-306.
The authors
Dr Ana Lopez-Alonso1, Prof. Dermot Kenny1,2
and Dr Lourdes Basabe-Desmonts*1,2
1 Molecular & Cellular Therapeutics
Royal College of Surgeons
in Ireland (RCSI),
Dublin
IRELAND
2 Biomedical Diagnostics Institute (BDI),
Dublin City University
Dublin
IRELAND
* Author for correspondence
