Original article
A novel modification of the Thrombelastograph assay, isolating platelet function, correlates with optical platelet aggregation

https://doi.org/10.1016/j.lab.2004.01.011Get rights and content

Abstract

Flow cytometry, singlet platelet counting, and optical aggregation have been used to monitor clopidogrel and glycoprotein IIb/IIIa (GPIIb/IIIa) platelet antagonists. Optical aggregation is considered the gold standard, but neither it nor flow cytometry is convenient in larger-scale clinical studies or point-of-care systems. Singlet platelet counting, a point-of-care assay correlated with optical platelet aggregation, only provides a measurement of platelet function at a single point in time. The Thrombelastograph is used to assay whole blood for thrombin-generated maximal clot-shear elasticity, referred to as the maximal amplitude (MA). Although platelet dysfunction, thrombocytopenia, and the in vitro effect of strong inhibitors such as IIb/IIIa antagonists can be observed, with thrombin generation milder platelet inhibitors cannot be assessed. We modified the Thromboelastograph assay, using reptilase and factor XIIIa, to form a clot, without thrombin generation, in heparinized whole blood. The resulting clot MA is dependent on added platelet agonists such as ADP or arachidonic acid, is sensitive to platelet antagonists, and provides a continuous measure of platelet function more analogous and better correlated with optical aggregation. This novel modification of the Thromboelastograph assay should prove to be a useful point-of-care whole-blood assay with which to monitor the effects of GPIIb/IIIa, ADP, and thromboxane A2-receptor–inhibiting drugs in patients.

Section snippets

Materials

Reptilase (batroxobin maranhao; CenterChem, Norwalk, Ct) and factor XIIIa (Nu-Millennium Research, Manhasset, NY) were obtained separately or as a mixture through Hameoscope Corp (Niles, Ill). Monoject sodium heparin and sodium citrate tubes were obtained from Sherwood Medical (St Louis, Mo). We used the GP IIb/IIIa inhibitors eptifibatide (Integrilin injection; Millennium Pharmaceuticals, Cambridge, MA) and abciximab (ReoPro; Eli Lilly, Indianapolis, Ind). PPACK anticoagulant was purchased

Development of the modified Thromboelastograph assay

Several properties can be measured with the standard Thromboelastograph assay, including time to clot formation, rate of clot formation, and development of MA. The in vitro effect of strong inhibitors of platelet function such as GPIIb/IIIa antagonists can be observed with this standard assay in terms of all the parameters measured, including a reduction in MA.22 To measure platelet activation by other agonists and make the assay sensitive to weaker platelet inhibitors such as NSAIDS or

Discussion

Reptilase and thromboelastography have been used previously to study the interaction of platelets with the fibrin network.29 The development of MA has been shown to depend on platelet activation and to be correlated with clot retraction. However, the MA obtained with reptilase and ADP in the absence of factor XIIIa crosslinking was relatively small and slow to develop compared with a thrombin clot.

In this study, the addition of purified factor XIIIa to a reptilase-generated fibrin network

Acknowledgements

We acknowledge the technical assistance of Carolyn C. Snider, Melinda Coker, and Natalie Hammer, as well as the help of the Knoxville Cardiology Group and catheterization-lab staff in collecting blood samples from patients.

References (31)

  • M.J. Alberts et al.

    Clopidogrel plus aspirin for stroke prevention

    Stroke

    (2002)
  • C.M. Samama et al.

    Antiplatelet agents in the perioperative periodexpert recommendations of the French Society of Anesthesiology and Intensive Care (SFAR) 2001—summary statement

    Can J Anaesth

    (2002)
  • J. Smout et al.

    Current practice in the use of antiplatelet agents in the peri-operative period by UK vascular surgeons

    Ann R Coll Surg Engl

    (2003)
  • R. Izaguirre-Avila et al.

    Effect of clopidogrel on platelet aggregation and plasma concentration of fibrinogen in subjects with cerebral or coronary atherosclerotic disease

    Clin Appl Thromb Hemost

    (2002)
  • T. Mueller et al.

    Monitoring aspirin 100 mg and clopidogrel 75 mg therapy with the PFA-100 device in patients with peripheral arterial disease

    Vasc Endovasc Surg

    (2003)
  • Cited by (179)

    View all citing articles on Scopus

    Supported by the University of Tennessee Anesthesiology Research Fund and the Physicians' Medical Education and Research Foundation, as well as Haemoscope Corp, of which Dr Cohen is chief executive officer. Haemoscope Corp may benefit from these studies.

    View full text