Marie Lordkipanidzé

Comparison of four tests to assess inhibition of platelet function by clopidogrel in stable coronary artery disease patients

AIMS We investigated the comparability of platelet function tests in quantifying platelet inhibition achieved by clopidogrel. METHODS AND RESULTS This pre-specified substudy of a randomized, double-blind trial included 116 patients with stable coronary artery disease requiring diagnostic angiography. Patients received clopidogrel for 1 (300 or 600 mg) or 7 days (300 + 75 or 150 mg daily) before the procedure. Blood samples obtained before clopidogrel initiation and before diagnostic coronary angiography were assayed using light transmission aggregometry [adenosine diphosphate (ADP) 5 and 20 microM as the agonist], whole-blood aggregometry (ADP 5 and 20 microM), PFA-100 (Collagen-ADP cartridge), and VerifyNow P2Y12. Although all assays studied were found sensitive to clopidogrel ingestion, none could distinguish categorically between patients who had, or not, ingested clopidogrel. Agreement between assays to identify patients with insufficient inhibition of platelet aggregation by clopidogrel was low. CONCLUSION The assessment of platelet function inhibition by clopidogrel is highly test-specific. Decision to increase clopidogrel dosage may vary on the basis of the assay used, thus highlighting the need for unambiguous guidelines with respect to assay selection, as platelet function assays are not interchangeable. At present, platelet function testing evaluating clopidogrel efficacy cannot be recommended in routine clinical practice.

Possibility of a rebound phenomenon following antiplatelet therapy withdrawal: A look at the clinical and pharmacological evidence

The importance of regular administration of antiplatelet drugs in patients suffering from coronary artery disease stands on firm grounds, as large meta-analyses have shown these therapies to drastically reduce the risk of death. Although the current guidelines published jointly by the American Heart Association, the American College of Cardiology, the Society for Cardiovascular Angiography and Interventions, the American College of Surgeons and the American Dental Association stress the hazards of premature discontinuation of antiplatelet drugs, abrupt withdrawal remains widespread, with potentially catastrophic consequences. In the limited state of knowledge on antiplatelet drug withdrawal, an early sound of alarm has risen from early thromboembolic complications reported after the interruption of treatment in patients who require antiplatelet therapy for prevention of ischemic vascular disease. Acute thrombotic complications are not immediate and usually follow interruption of aspirin or clopidogrel therapy after a mean delay of 8-25 days, a time lapse consistent with normal platelet turnover required to replace the platelet pool in circulation and suggestive of a rebound phenomenon. This review article describes the thrombotic risks associated with discontinuing antiplatelet therapy and the bleeding risks associated with continuing these drugs. By integrating the current understanding of the pharmacology of antiplatelet agents and the kinetics of platelet function recovery, this article unveils the possibility of a pharmacological rebound phenomenon which could lead to adverse ischemic events, and supports the warning against premature discontinuation of antiplatelet drugs issued in current guidelines.

Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders.

The ISTH bleeding assessment tool (ISTH‐BAT) was developed to record bleeding symptoms and to aid diagnosis in patients with a possible bleeding disorder.

Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel

Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects

We analyzed candidate platelet function disorder genes in 13 index cases with a history of excessive bleeding in association with a significant reduction in dense granule secretion and impaired aggregation to a panel of platelet agonists. Five of the index cases also had mild thrombocytopenia. Heterozygous alterations in FLI1 and RUNX1, encoding Friend leukemia integration 1 and RUNT-related transcription factor 1, respectively, which have a fundamental role in megakaryocytopoeisis, were identified in 6 patients, 4 of whom had mild thrombocytopenia. Two FLI1 alterations predicting p.Arg337Trp and p.Tyr343Cys substitutions in the FLI1 DNA-binding domain abolished transcriptional activity of FLI1. A 4-bp deletion in FLI1, and 2 splicing alterations and a nonsense variation in RUNX1, which were predicted to cause haploinsufficiency of either FLI1 or RUNX1, were also identified. Our findings suggest that alterations in FLI1 and RUNX1 may be common in patients with platelet dense granule secretion defects and mild thrombocytopenia.

Genotyping and phenotyping of platelet function disorders

The majority of patients with platelet function disorders (PFDs) have normal platelet counts and mild day‐to‐day bleeding symptoms, but are at risk of major hemorrhage at times of trauma, surgery, or childbirth. This group is challenging to investigate, because the assays are often time‐intensive and labour‐intensive, and interpretation is difficult, especially in patients with mild disorders. In addition, interuser variability in performance of the assays, including the currently accepted gold standard, light transmission aggregometry, makes the results difficult to compare between laboratories. Furthermore, a similar pattern of mucocutaneous bleeding is seen in disorders in other components of the hemostatic pathway, including type 1 von Willebrand disease (VWD). We have undertaken an extensive investigation of patients with clinically diagnosed excessive bleeding, using a genotyping and platelet phenotyping approach based on lumi‐aggregometry, and other specialist tests of platelet function, in combination with Sanger and next‐generation sequencing (NGS). We found a functional defect in ~ 60% of patients, the majority being associated with feedback pathways of platelet activation. Function‐disrupting mutations were identified in known and novel genes, and coinheritance with other genetic disorders of hemostasis, including type 1 VWD, was shown. A significant number of mutations are heterozygous and unlikely to cause extensive bleeding in isolation, consistent with incomplete penetrance of inheritance of bleeding disorders and a multifactorial etiology for excessive bleeding in many patients. Mucocutaneous bleeding is a complex trait, and this has important implications for NGS in the assessment of a PFD.

The role of platelets in the recruitment of leukocytes during vascular disease

Abstract Besides their role in the formation of thrombus during haemostasis, it is becoming clear that platelets contribute to a number of other processes within the vasculature. Indeed, the integrated function of the thrombotic and inflammatory systems, which results in platelet-mediated recruitment of leukocytes, is now considered to be of great importance in the propagation, progression and pathogenesis of atherosclerotic disease of the arteries. There are three scenarios by which platelets can interact with leukocytes: (1) during haemostasis, when platelets adhere to and are activated on sub-endothelial matrix proteins exposed by vascular damage and then recruit leukocytes to a growing thrombus. (2) Platelets adhere to and are activated on stimulated endothelial cells and then bridge blood borne leukocytes to the vessel wall and. (3) Adhesion between platelets and leukocytes occurs in the blood leading to formation of heterotypic aggregates prior to contact with endothelial cells. In the following review we will not discuss leukocyte recruitment during haemostasis, as this represents a physiological response to tissue trauma that can progress, at least in its early stages, in the absence of inflammation. Rather we will deal with scenarios 2 and 3, as these pathways of platelet–leukocyte interactions are important during inflammation and in chronic inflammatory diseases such as atherosclerosis. Indeed, these interactions mean that leukocytes possess means of adhesion to the vessel wall under conditions that may not normally be permissive of leukocyte–endothelial cell adhesion, meaning that the disease process may be able to bypass the regulatory pathways which would ordinarily moderate the inflammatory response.

Use of next‐generation sequencing and candidate gene analysis to identify underlying defects in patients with inherited platelet function disorders

Inherited platelet function disorders (PFDs) are heterogeneous, and identification of the underlying genetic defects is difficult when based solely on phenotypic and clinical features of the patient.

Testing platelet function.

Platelet function tests have been traditionally used to aid in the diagnosis and management of patients with bleeding problems. Given the role of platelets in atherothrombosis, several dedicated platelet function instruments are now available that are simple to use and can be used as point-of-care assays. These can provide rapid assessment of platelet function within whole blood without the requirement of sample processing. Some tests can be used to monitor antiplatelet therapy and assess risk of bleeding and thrombosis, although current guidelines advise against this. This article discusses the potential utility of tests/instruments that are available.

Assessment of VerifyNow P2Y12 assay accuracy in evaluating clopidogrel-induced platelet inhibition.

The emergence of point-of-care assays enabling bedside testing such as the VerifyNow P2Y12 system might prove useful in clinical settings. The aim of this study was to evaluate the ability of the VerifyNow P2Y12 assay to estimate the inhibition of platelet aggregation provided by clopidogrel in the absence of baseline off-drug aggregation data. Sixty-eight patients with coronary artery disease scheduled to initiate clopidogrel therapy underwent platelet aggregation testing by VerifyNow P2Y12 at baseline and after clopidogrel administration. The inhibition reported by the VerifyNow assay (relative to thrombin receptor activating peptide-induced platelet aggregation, serving as baseline) was compared with that calculated with the actual adenosine diphosphate-induced baseline obtained with the same methodology. The postclopidogrel thrombin receptor activating peptide-induced aggregation showed a great discordance with that induced by adenosine diphosphate before clopidogrel with a bias of 24 units (95% limits of agreement from −142 to 190 units). Moreover, the inhibition reported by the assay overestimated the standard before-and-after testing data by an average of 8% (95% limits of agreement from −49% to 65%), making its use without a true baseline comparator unsatisfactory. The VerifyNow P2Y12 assay fails to accurately quantify platelet inhibition achieved by clopidogrel compared with before-and-after testing. Further studies are required to establish the clinical usefulness of the VerifyNow P2Y12 assay to accurately predict the occurrence of major adverse cardiovascular events in patients with reduced clopidogrel efficacy before it can be implemented in clinical practice. At present, the use of this assay in clinical care cannot be recommended for monitoring clopidogrel therapy.