Vol. 20 • Issue 7 • Page 42
Thrombosis kills more than 800 people every day1 and is the leading cause of preventable hospital death in U.S. hospitals.2 Yet, there still is no test that: 1) measures thrombotic risk, 2) monitors anti-thrombotic treatment or 3) aids in the development of new anti-thrombotic drugs. Here, new global methods to assaying platelet function in comparison to traditional reference methods such as platelet aggregometry are explored.
Historically, platelet function has been assessed by assays that measure the formation of a platelet aggregate after stimulation with an agonist.3 Platelet aggregometry is based on a turbidimetric technique, i.e., absorbance of a platelet suspension is dependent on the number of particles rather than their size. In a given platelet suspension, as platelet aggregation occurs, the number of particles decreases and light transmission increases.
Aggregometry is not a very reliable method as its accuracy may be influenced by a number of different factors, including platelet count. Coefficients of variation can range from 3.6 to 7.7%, with sources of variability being sample preparation, dietary factors including high levels of lipids in the sample, and aging of the sample. Aggregometry also has poor sensitivity in that only aggregates composed of a minimum of eight platelets are detected.
Click to view Figure 1.
Assays Based on Thrombin Generation
Platelets are known to play an important role in thrombin generation based on several observations:
• fibrin is deposited at sites of vascular injury after platelet adhesion and aggregation have already occurred,
• formation of fibrin occurs in close proximity to the deposited platelets at the site of injury and
• thrombocytopenia and other coagulation disorders affect thrombin generation.
Assays that measure thrombin generation are more reliable than platelet aggregometry but still are not a global function test. There are fluorescence-based methods (e.g., Calibrated Automated Thrombogram® [CAT], Technothrombin® TGA), methods based on optical density such as ETP, and the fibrin/resistance method such as thromboelastrography or thromboelastometry.
In calibrated fluorescent-based methods such as the CAT assay, the plasma specimen is separated into two fractions: the first is activated for thrombin generation, the second is where calibration takes place. The calibrator corrects for color in the sample.4 Color such as that cirrhotic (yellow) plasma can interfere with florescence measurements. The time to reach peak florescence can be plotted against thrombin concentration to create a thrombogram. The peak height, area under the curve (AUC or ETP), lag time and rate of thrombin formation can be measured for each sample,5 with very little within- and between variability observed (Fig. 1).
The CAT assay, which is a research use only assay in the U.S. and Canada, and is not intended for use in diagnostic procedures, allows for continuous real-time measurement of thrombin generation using platelet -poor or platelet-rich plasma samples. The steps in a CAT assay include filling wells with reagents (trigger reagents, plasma, etc.), setting up plates and measurement settings in the program, followed by the measurement of thrombin generation.
Examples of CAT Usage
In one study, a group of researchers have used thrombogram method to study the role of vWF in thrombin generation.6 Since vWF is a plasma substituent required for platelet-dependent thrombin generation, due to the fact that it acts as an adhesive ligand so that platelets can aggregate, the results reconfirmed the importance of the interaction between coagulation and platelets in normal hemostasis.
Click to view Figure 2.
In a second study, thrombogram analysis was used to demonstrate inhibition of thrombin generation of platelet-mediated, tissue factor-induced thrombin generation by an antagonist of platelet glycoproteins.7
Using the thrombogram analysis, researchers found that recombinant coagulation factor VIIa reverses the effect of aspirin or aspirin plus clopidogrel on in vitro thrombin generation.8
Flow Cytometry/VASP Assay
Besides the CAT assay, a number of assays are designed to assess platelet function by flow cytometric methods. One example is a kit that measures platelet responsiveness to clopidogrel (Plavix), the vasodilator-stimulated phosphoprotein (VASP) flow cytometry assay (developed by BioCytex /Diagnostica Stago). As is the case for the CAT assay, VASP is a research use only assay in the U.S. and Canada and is not intended for use in diagnostic procedures.
By competing for binding with platelet agonist adenosine diphosphate (ADP), clopidogrel and other anti-P2Y12 drugs inhibit clotting by directly interfering with signaling through ADP/P2Y12, a platelet cell surface receptor. Normally, binding of ADP to ADP/P2Y12 ultimately changes the phosphorylation state of VASP (Fig. 2). By triggering prostaglandin E1 (PGE1) receptor-mediated signaling, binding of (PGE1) alone leads to phosphorylation of VASP and platelet inactivation, while incubating platelets with both ADP and PGE1 has the opposite effect. In contrast, when platelets are first treated with a P2Y12 antagonist (such as clopidogrel) then incubated with PGE1 either alone or in combination with ADP, VASP is constitutively phosphorylated and the platelets do not become activated.
VASP is the most reliable molecular reporter of platelet function in that it is not affected by platelet count and is thus not subjected to significant inter-patient variability compared with platelet aggregometry. The assay workflow takes 30 minutes to complete at room temperature. Uncoagulated whole blood is collected from patients currently taking anti-P2Y12 drug. A sample of specimen is incubated with PGE1 in the presence and absence of ADP, followed by fixation and permeabilization. The cells are then labeled with antibody against the phosphorylated VASP, followed by staining with a fluorophore-conjugated antibody for detection of the cells by flow cytometry.
Dr. Netterwald is a licensed medical technologist and freelance biomedical writer based in New Jersey.
1. Gerotziafas and Samama. CurrOpin PulmMed. 10:356-365.
2. Geerts et al. Chest 126(suppl): 338S- 400S.
3. Platelet Function: Assessment, Diagnosis, and Treatment. Eds: Martin Quinn and Desmond Fitzgerald. Human Press. 2005. Totowa, NJ.
4. de Smedt, et al. Thromb Haemost 2008; 100:343-349.
5. Hemker et al. Thromb Haemost 2006; 96: 553-561.
6. Keularts et al. Thromb Haemost 2000; 84: 638-42.
7. Reverter et al. J. Clin. Invest. 1996;98 (3): 863-874.
8. Altman et al. Journal of Thrombosis and Haemostasis 4:2022-2027.
Role of Platelets in Cardiovascular Disease
By Donna D. Castellone, MS
Platelets play a crucial role in thrombosis and inflammation, immunity and atherogenesis. Clinical evidence has also demonstrated that in patients that present with an elevated platelet count, platelet activation and platelet hyper-reactivity may be associated with adverse cardiovascular events.1
When vascular injury occurs, platelets adhere to exposed von Willebrand factor (vWF) through a membrane receptor glycoprotein (GP) Ib/IX/V; the exposed vWF and collagen bind to circulating platelets and activate them. Extracellular collagen is recruited through other receptors of GP Ia/IIa and GPVI, which contributes to platelet shape change and activation. The contents of the alpha and dense granules are released (degranulation) to recruit more platelets to the site of vessel damage and potentiate platelet activation. The platelets also synthesize and release thromboxane, another mediator of activation. The fibrinogen receptor, GP IIb/IIIa, undergoes a conformation change binding to fibrinogen and fibrin. This leads to platelet aggregation and thrombus formation.1
When atherosclerotic plaque ruptures, the arterial extracellular matrix is exposed and platelet aggregation is initiated, called atherothrombosis. Simultanously, the coagulation cascade is activated via tissue factor pathway. These events are responsible for clot formation and a possible clinical diagnosis of either stroke or myocardial infarction (MI).
Three factors determine the extent of thrombus formation once plaque ruptures-the degree of plaque disruption, the degree of stenosis and the physicochemical properties of the surface exposed to the circulating blood. As stenosis, or narrowing of the vessels, increases, so does the deposition of platelets, resulting in a larger thrombus formation. The site can be influenced by the placement of deposition in relation to blood flow.
Donna D. Castellone is clinical project manager, Siemens Healthcare Diagnostics, Tarrytown, NY, and an ADVANCE editorial advisory board member.
Marchant K. Importance of platelets and platelet response in acute coronary syndromes. Cleveland Clinic Journal of Medicine 2009;76:1.