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Background: Light transmission aggregometry (LTA) is still the most used test for the identification and diagnosis of platelet function defects (PFD). Disadvantages of LTA include its laborious nature, the large volumes of blood required and the relative insensitivity to small changes in platelet function. We investigated if the flow cytometry-based whole blood platelet activation test (WB-PACT) correlates with LTA or could be used as a complimentary test. Methods: WB-PACT quantifies αIIbβ3 activation and P-selectin expression in response to 3 agonists and VWF binding to platelets in response to ristocetin. In total, 212 patients (51 on dual antiplatelet therapy (acetylsalicylic acid and ADP receptor antagonist), 161 suspected for bleeding diathesis) were tested. For WB-PACT parameters, the 2.5th, 10th and 25th percentiles were determined in 56 healthy donors to score the patient samples. For LTA, maximal aggregation, disaggregation and prolongation of the lagtime in response to 4 agonists and ristocetin was scored. Patients with at least one parameter lower than the 10th percentile measured with WB-PACT in healthy donors or at least one value deviating from the normal range measured with LTA were diagnosed with PFD. A bleeding score (BS) was calculated with the ISTH/SSC bleeding assessment tool (Rodeghiero et al., JTH, 2010). Results: A moderate correlation between LTA versus WB-PACT was found with a R of 0.60 (Figure 1). In total, 95 patients were diagnosed with PFD by both tests (κ=0.40, Table 1) and BS were recorded for 28/95 patients. Of these 28 patients, 25% had an elevated BS (adapted according to gender and age: >5 in women, >3 in men and >2 in children) and 36% had a BS>3. BS were recorded for 30/38 and 22/24 patients who were diagnosed with PFD according to WB-PACT only and LTA only, respectively. Interestingly, 27% of patients with PFD according to WB-PACT had an elevated BS and 40% had a BS>3. For LTA, only 13% of patients with PFD had an elevated BS and 13% had a BS>3. Conclusions: Flow cytometric analysis of platelet function by WB-PACT gives additional value to LTA to determine PFD. Disclosures No relevant conflicts of interest to declare.

Introduction In hemophilia, unravelling the mechanisms behind interindividual variability in phenotype remains a big challenge. Objectives To study platelet activation and platelet responsiveness in relation to hemostatic phenotypes in hemophilia B by 1) comparing hemophilia B patients to healthy controls and 2) relating these to the clinical phenotype of the individual patients with severe hemophilia B. Methods Platelet reactivity was determined in whole blood measuring expression of P-selectin and binding of fibrinogen with FACS analysis after stimulating or inhibiting platelets with concentration series of several (ant-)agonists, including adenosine diphosphate (ADP), convulxin (CVX) and thrombin receptor associated protein (TRAP)and iloprost. Concentration of soluble platelet activation markers (soluble P-selectin, β-Thromboglobulin and RANTES) were measured in platelet poor plasma by ELISA. Markers of platelet activation and responsiveness were compared between healthy controls (n=12), patients with mild/moderate (n=12) or severe hemophilia B (n=19). In severe hemophilia patients, annual FIX consumption/kg was calculated over the last 6 years. Data were analysed with linear regression using SPSS 20. Results Levels of basal platelet activation (both P-selectin and fibrinogen) were equal between controls, mild/moderate and severe hemophilia B (Figure 1). For basal P-selectin expression, the mean area under the curves for were 28.5 (95% CI 23.3-33.6), 33.0(95% CI 24.4-41.6) and 28.4(95% CI 23.9-32.9) respectively (p=0.4). After stimulation with ADP, CVX and TRAP (with and without iloprost), platelet responsiveness was similar in all groups. There was no relation between annual FIX consumption and any marker of platelet activity. For basal P-selectin expression, the beta coefficient was -0.212 (p=0.38)(Figure 2). Conclusion In hemophilia B, platelet activity and responsiveness was similar to healthy controls. In severe hemophilia patients, there was no relation between FIX consumption and platelet activity. Figure 1. Basal P-selectin activity between healthy controls and hemophilia patients Figure 1. Basal P-selectin activity between healthy controls and hemophilia patients Figure 2. Basal P-selectin activity according to FIX consumption in severe hemophilia B Figure 2. Basal P-selectin activity according to FIX consumption in severe hemophilia B Disclosures Schutgens: Pfizer: Research Funding.

Platelet thrombus formation on collagen is initiated by platelet GPIb interaction with von Willebrand factor (vWF) bound to collagen, followed by firm attachment of the platelet to collagen by the integrin alpha(2)beta(1). Platelet and plasma vWF levels and alpha(2)beta(1) density on the platelet surface are highly variable among normal subjects; however, little is known about the consequences of this variability on platelet adhesion to collagen. A population of 32 normal subjects was studied to evaluate the relation between genetic and phenotypic variations of alpha(2)beta(1) density on the platelet surface, plasma vWF levels, platelet vWF levels, and adenosine diphosphate and adenosine triphosphate concentrations on the one hand and platelet adhesion to collagen under flow on the other hand. Platelet adhesion to collagen types I and III under flow was correlated with plasma levels of vWF (r(2) = 0.45 and 0.42, respectively) and alpha(2)beta(1) density on the platelet surface (r(2) = 0.35 and 0.17, not significant). Platelet adhesion to collagen type IV under flow was significantly correlated with platelet vWF levels (r(2) = 0.34) and alpha(2)beta(1) density on the platelet surface (r(2) = 0.42). Platelet adhesion to collagen types I and III depends on both plasma levels of vWF and alpha(2)beta(1) density on the platelet surface, whereas platelet adhesion to collagen type IV is mediated by both platelet vWF levels and alpha(2)beta(1) density on the platelet surface. (Blood. 2000;96:1433-1437)