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Background: The use of citrate anticoagulant limits the clinical significance of platelet function tests. Thrombin inhibitors cannot prevent thrombin-induced platelet activation completely. We examined the influence of benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide (BAPA), a dual inhibitor of Factor Xa (FXa) and thrombin, on platelet responsiveness to agonists when measured between 2 and 24 h after venipuncture., Methods: Blood samples from 36 individuals were anticoagulated with citrate and BAPA, respectively. Turbidimetric platelet aggregometry (TPA) and impedance platelet aggregometry (IPA), a whole blood platelet counting assay for measuring platelet aggregation (PCA), and Platelet Function Anlayzer-100 (PFA-100 closure times (CTs) were determined after whole blood storage between 2 and 24 h after venipuncture. Native whole blood was studied over 48 h to determine the inhibition of thrombin generation by BAPA, hirudin and melagatran., Results: BAPA inhibited thrombin generation completely for 48 h, while hirudin and melagatran did not. The use of citrate resulted in significantly reduced TPA induced by arachidonic acid (AA) or adenosine 5'-diphosphate (ADP), and significantly reduced IPA regardless of agonist when measured 10 and 24 h after blood collection. PCA ratios in citrated blood also dropped significantly 10 and 24 h after venipuncture. The length of storage of BAPA-anticoagulated blood samples over 24 h had no significant influence on any platelet response. The reproducibility of platelet function assay results obtained from BAPA-anticoagulated samples was significantly better than corresponding data from citrated blood., Conclusion: TPA, IPA, PCA or PFA-100 CTs remain stable for 24 h when whole blood is anticoagulated with a dual inhibitor of FXa and thrombin. This would greatly simplify the shipment of samples for platelet function testing.

We investigated the relationship between platelet function analyzer (PFA-100) closure times (CT) and bleeding time (BT), platelet aggregation (PA) induced by ADP, arachidonic acid, and collagen, blood cell counts, and von Willebrand factor (VWF) in 120 well-characterised healthy individuals. Pre-analytical and analytical conditions were standardised comprehensively. In a substantial number of cases the differences between duplicate measurements exceeded 15%. The reference range (5th and 95th percentiles) for CT with the collagen/epinephrine (CEPI) and the collagen/ADP (CADP) cartridge was 93-223 s and 64-117 s respectively. Re-examination of 11 individuals with CEPI-CT above the 95th percentile revealed considerable batch-to-batch variation of CEPI-CT. Males had significantly longer CADP than females (P = 0.002). CEPI and CADP-CT measured pm were significantly longer than corresponding values determined am (P = 0.003 and P < 0.0001 respectively). Blood group O was associated with greater CEPI and CADP-CT and lower VWF levels compared with non-O blood groups (P = 0.008, P = 0.0003 and P < 0.0001 respectively). Linear regression analysis revealed association between CEPI-CT, CADP-CT and VWF (P < 0.0001), but no relationship was found between CT and BT or between CT and PA. We conclude that VWF plasma levels modulate PFA-100 CT to a greater extent than platelet function. Establishment of reliable reference ranges and careful standardisation of pre-analytical and analytical conditions is a prerequisite for obtaining reliable PFA-100 results. Duplicate measurements are necessary.

"Aspirin resistance" and "aspirin nonresponsiveness" are terms used both to describe both the failure of aspirin to protect subgroups of individuals from severe vascular events and to evoke an appropriate inhibition of platelet function. Several studies utilizing a broad range of platelet function tests have shown that some subgroups of individuals exhibit a reduced or completely missing antiplatelet response to aspirin. The clinical significance of aspirin nonresponsiveness for the prediction of clinical endpoints remains, however, to be determined. Thus far, only three prospective clinical trials have demonstrated a possible relationship between aspirin nonresponsiveness and subsequent vascular events. Most platelet function tests used in respective clinical studies cannot be reliably performed in clinical routine and are not interchangeable for monitoring antiplatelet treatment. There is a need for a simple and reliable assay for predicting the clinical efficacy of antiplatelet therapy. Recent data demonstrate that none of the currently developed assays, including the PFA-100 system, are presently able to accomplish these objectives.

Several studies have demonstrated with the use platelet function tests (PFT) that subgroups of patients under acetylsalicylic acid (ASA) fail to produce the anticipated antiplatelet effect. This phenomenon as well as the clinical failure of ASA to protect patients from thromboembolic complications has been termed ASA resistance (AR) or ASA nonresponsiveness. Several subtypes of AR can be distinguished by PFT. The following PFT were used to characterize AR: optical aggregometry, platelet aggregation in whole blood, platelet function analyzer (PFA-100), platelet reactivity test or platelet aggregate ratio, flow cytometry and thromboxane B(2) generation. All PFT have in common that their widespread clinical use is substantially limited due to complex preanalytic factors, reduced specificity and poor reproducibility. PFT are not interchangeable for monitoring antiplatelet treatment. Three prospective clinical trials revealed a possible relationship between AR and subsequent cardiovascular events. There is a need for a simple and reliable assay for predicting the clinical efficacy of antiplatelet therapy. Recent data demonstrate that none of the currently available assays including the PFA-100 system are capable to accomplish these objectives.