Reliability of Platelet Function Tests and Drug Monitoring

A symposium, “The Significance of Drug Monitoring and the Reliability of Platelet Function Tests,” held during the Annual Meeting of the Society of Thrombosis and Haemostasis Research (GTH) in February 2003 in Innsbruck, Austria, summarized the current status of postmarketing surveillance in antithrombotic therapy and elaborated on the progress made in the assessment of reliable and simple platelet function tests for monitoring therapy with antiplatelet drugs.

Until now, platelet function analyses were difficult to perform because they are complex and time-consuming laboratory investigations with a high coefficient of variation. This often led to imprecise interpretations under routine clinical laboratory conditions. Furthermore, platelet function tests were mostly focused on bleeding tendencies and only few evaluated the state of hyperaggregability of platelets.

Recent developments in the understanding of platelet function have led to the development of new and more potent “antiplatelet drugs” such as clopidogrel or abciximab and have also prompted better and more accurate and less time-consuming platelet function tests.[1] [2]

Unfortunately, there seems to be a trend in clinical trials not to perform laboratory monitoring when evaluating new anticoagulant and antiplatelet drugs. This might be due to economic considerations, but it might also be associated with the previous lack of easily performed and reliable laboratory methods for (antiplatelet) therapy monitoring. Many studies correlate the effect of antiplatelet drugs with the opposite clinical endpoints “bleeding” or “thromboembolic events.” Fewer studies correlate the effects with several modifications of the not very reliable bleeding time and there are too few correlations using objective laboratory platelet function tests. This might be the reason that some old questions, such as “perioperative management of platelet inhibition for surgery,” are still controversially discussed and no consensus-formed guidelines have yet been published on this topic.[3] On the other hand, it has been shown in previous studies that involved platelet reactivity or aggregation tests that up to 45% of patients with ischemic strokes or cardiovascular diseases are aspirin “resistant.”[4] [5] [6] However, therapeutic monitoring or testing of aspirin responses in patients who are placed on long-term aspirin is not yet customary. To illustrate this point we searched the literature from 1953 to 2004 (National Library of Medicine-PubMed/NCBI) and found 7,135 entries for the term “drug monitoring,” 14,882 entries for the term “platelet function,” only 68 papers with the combined keywords “drug monitoring and platelet function,” and 30 papers with the combination “drug monitoring and platelet function test.”

According to Tiaden and colleagues, adverse drug reactions (ADRs), caused by anticoagulants and antiplatelet drugs, still belong to the most reported adverse events recorded by the German Spontaneous Reporting System. Although the most frequently reported suspected drug is heparin, followed by phenprocoumon, antiplatelet drugs comprise together 2.4% (thienopyridines 1.6%, acetylsalicylic acid 0.8%) of ADRs. The most-often described ADR symptoms-especially with phenprocoumon and with acetylsalicylic acid (ASA)-are bleeding complications. Other serious ADRs are heparin-induced thrombocytopenia (HIT) and changes in complete blood count (CBC) caused by thienopyridines. During the last few years, a reduction of severe reactions such as fatal cerebral hemorrhage could be achieved by improved clinical management of oral anticoagulant therapy and of adverse events of heparins. Additional measures like postmarketing studies with correct drug monitoring are believed to enhance the drug safety and are expected to reduce the risk and rate of adverse events further.

Introducing the topics of this issue, Jurk and Kehrel review the role of blood platelets in different physiologic and pathologic processes in humans. In addition to the basic processes in primary hemostasis, platelet adhesion, platelet secretion, platelet aggregation, and clot retraction, the new model of thrombin formation on the platelet surface is presented. The different signal transduction pathways in platelets are a main focus of this article.

Matzdorff reviews several platelet function tests and particularly the advantages and disadvantages of flow cytometry to monitor antiplatelet therapy. He concludes that flow cytometry is able to detect and quantify different aspects of platelet function, but the procedure is time-consuming and labor-intensive and preanalytical handling is prone to result in artifacts. Thus it is an extremely valuable tool for research and small clinical studies, but its relevance for routine monitoring purposes remains limited.

Klinkhardt and Harder investigated the effect of antiplatelet agents in a controlled study design as well as under clinical conditions by flow cytometric measurements of platelet-leukocyte aggregate (PLA) formation. They were able to demonstrate considerable reduction of PLA formation during intake of clopidogrel alone or in combination with aspirin. In healthy volunteers, monocyte-PLA formation decreased significantly and dose dependently under clopidogrel. The authors conclude that flow cytometric measurement of PLA formation is well suited for dose response studies of antiplatelet agents in healthy volunteers and may also be used to monitor platelet function in patients under long-term treatment with antiplatelet agents that interfere with the degranulation process.

Haubelt and coworkers review several platelet function tests (PFT) that were used in clinical studies to measure the antiplatelet effect after administration of ASA. All of these tests have drawbacks. Therefore the authors call for a simple and reliable new platelet function assay to monitor antiplatelet therapy. Several subtypes of ASA nonresponsiveness (ASAN) are described; there are only few clinical findings suggesting an association between ASAN determined by PFT and clinical outcome.

Feuring and associates report on the inhibition of platelet function after intake of ASA detected by the PFA-100 system, a platelet function analyzer measuring collagen plus epinephrine- and collagen plus adenosine diphosphate (ADP)- induced platelet plug formation. They found that only 31% of patients with coronary heart disease under low-dose (100 mg q.d.) aspirin therapy showed prolonged closure times, suggesting that only in one third of patients under low-dose aspirin could a sufficient platelet inhibition effect be demonstrated.

Ziegler and coworkers observed no major influence of systemic inflammation on the performance of the PFA-100 system and on the response to therapy with ASA in patients with peripheral vascular occlusive disease.

Crowe and associates studied the phenomenon of aspirin resistance with the PFA-100. Evaluating 31 patients with unstable angina and 105 controls, they found aspirin resistance in 42% of patients, most of whom were shown to be compliant utilizing concomitant salicylate level determinations.

Hertfelder et al evaluated the PFA-100 performance in perioperative monitoring during cardiac surgery in patients undergoing elective coronary artery bypass grafting (CABG). The study encompassed and assessed a large number of current laboratory methods that are used to characterize the alterations of hemostasis during CABG. The data indicate that in standard CABG procedures, heparin- and fibrinolysis-sensitive assays are helpful for the identification of hemostatic defects during bleeding complications and for monitoring therapy inter- and postsurgically.

Wieding and coworkers report on the good performance of the Retention Test Homburg (RTH) in clinical routine and demonstrate its high sensitivity for platelet functions mediated by the adhesive proteins von Willebrand factor (vWF) and fibrinogen via their platelet receptors. They conclude that the RTH is well suited for monitoring therapies with desmopressin and vWF concentrates.

Krischek et al describe the validation of the RTH. In the RTH platelets are exposed to a standardized exogenic surface (Sysmex retention tubes) under defined flow conditions. Platelet counts are performed before and after the blood passes through the Sysmex retention tube. The difference between these values indicates the percentage of retained platelets (retention index, RI). For further evaluation of the retention phenomenon the filters were fixed and examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM and TEM pictures show activated platelets spreading and adhering to the filter surface, similar to their behavior seen at the subendothelium. Also, the influence of different g-forces and centrifugation times were examined on the retention behavior of the platelets in citrated platelet rich plasma (PRP) and whole blood (WB).

In contrast to other materials (for example, collagen, glass pearls, etc.) the filter surface in the retention tubes is nonthrombogenic. Therefore, the RTH seems to be appropriate for measuring a pre-existing (in vivo) over-reactivity of platelets. In a pilot study using the RTH, Krischek et al evaluated the postoperative over-reactivity of platelets in 14 patients and observed a significant heterogeneity in the platelet population concerning size and stickiness. In an in vitro study, the authors evaluated how after administration of platelet aggregation inhibiting drugs in various concentrations (such as ASA, prostaglandin, and abciximab), the ADP-, collagen-, ristocetin-, or suprarenin-induced retention can be reduced. The reaction of the platelets in PRP of different healthy persons is variable to the addition of ADP. The platelet function inhibitor effect is dose dependent. In a clinical pilot study, significant (in vivo) inhibiting effects of clopidogrel were clearly shown with the RTH.

Another innovative technique for monitoring antiplatelet therapy is the platelet-reactivity test (Grotemeyer-test, PR-test), a modification of the original method by Wu and Hoak (1974), introduced by Grotemeyer in 1983. The test determines circulating platelet aggregates using different buffer solutions. Platelet aggregates dissolve when blood is sampled in ethylenediaminetetraacetic acid- (EDTA) buffer, but remain fixed when EDTA-formalin-buffer is used. Therefore, the platelet count in the EDTA-formalin-buffer sample is lower proportionally to the number of platelet aggregates. Koscielny and colleagues present a pilot study comparing sensitivity and specificity of the platelet reactivity test (Grotemeyer test) with other newer platelet function tests such as the RTH and PFA-100. They conclude that the PR test could be a valid method for drug monitoring during antiplatelet therapies.

Nowak et al describe a new and innovative platelet adhesion assay (PADA). With only a small investment of time and equipment, the PADA provides quantitative measurements of platelet adhesiveness. Special polymer particles are added to freshly drawn citrated whole blood. A defined shear grade is induced by a short shaking period of the sample. Proteins in the blood specimen, especially fibrinogen, and thereafter also activated platelets, bind to the specific polymer surface. Following platelet counts in both the sample and in a control (blood without particles), the adhesion index (AI) is calculated as a quantitative measure of platelet adhesiveness. AI was shown to be nearly independent of current platelet counts and hematocrit as well as of fibrinogen levels. Age and gender of the volunteers also had almost no influence on the AI.

According to Schumann and coworkers, the PADA is also useful for measuring the influence of drugs on the first phase of platelet activation, adhesion. Adhesion is triggered by glycoproteins (GP) on the platelet surface, mainly by GPIIb/IIIa, and to lesser extent by GPIb/V/IX. Since fibrinogen serves as adhesive protein for GPIIb/IIIa receptors, the PADA is able to monitor GPIIb/IIIa receptor antagonists and to detect overdose that can be accompanied by bleeding complications. Comparing the PADA adhesion index with GPIIb/IIIa receptor occupancy, determined by FACS, a good consistency of the data was shown. Via intracellular signaling, ADP receptor mechanisms are closely involved in the activation of GPIIb/IIIa receptors so that ADP receptor antagonists of the thienopyridine type, especially clopidogrel, can also be quantitatively determined using the PADA. In patients under clopidogrel therapy, the therapeutic effect was monitored and also individual dose adjustments were detected. Furthermore, patients with clopidogrel resistance as well as with a potential overdose with ADP receptor antagonists can be detected with the PADA.

Graff and Harder investigated the PADA for its sensitivity toward characterization of antiplatelet therapy. Healthy volunteers were investigated before and after a single dose of aspirin. Samples were spiked in vitro with increasing doses of abciximab, eptifibatide, and AR-C 69931MX in citrated and hirudinized whole blood. The AI, as the characteristic parameter of the PADA test, decreased in a dose-dependent fashion for abciximab and eptifibatide. Variable effects between the different anticoagulants were shown for eptifibatide. Aspirin yielded no additional effect, while inhibition by AR-C 69931 MX could be demonstrated in hirudinized blood. Different levels of GPIIb/IIIa inhibitors and ADP receptor inhibitors can be assessed with the PADA test, although different anticoagulants lead to different results. For the routine applicability of this test, its suitability has to be elucidated in further patient studies.

With these and other new and carefully standardized test systems, the time has come to look at platelet function tests in a new light. One is encouraged to implement these tests in clinical routines as well as in new trials to answer old questions of hemostasis and to improve significantly the scientific background of postmarketing surveillance.

We express appreciation to our colleagues B. Krischek (Marburg), J. deHaan (Marburg), J. Walenga (Chicago), and B. von Stritzky (Berlin), for several critical readings of the manuscripts at different stages in their evolution. We also wish to express our deep gratitude to Prof. Dr. K. Schrör for a fruitful discussion and for helpful and critical reading of this editorial. Appreciation also goes to W. Heinrich (Homburg/Saar) for careful management of the reference databank and to I. Neubauer (Jena) and T. Zahour (Vienna) for skillful technical assistance. We also thank E. Mammen and Thieme Medical Publishers for their excellent editorial review and cooperation.

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