Key Points• Pediatric chronic ITP patients with a severe bleeding phenotype exhibit functional platelet defects.• The platelet microaggregation test and the platelet reactivity assay are able to assess platelet function at extremely low platelet count.Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 mL of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts ( <10 9 ). These tests show that platelet function is related to bleeding phenotype in chronic ITP. (Blood. 2014;123(10):1556-1563

BackgroundIn patients with chronic kidney disease studies focusing on platelet function and properties often are non-conclusive whereas only few studies use functional platelet tests. In this study we evaluated a recently developed functional flow cytometry based assay for the analysis of platelet function in chronic kidney disease.MethodsPlatelet reactivity was measured using flow cytometric analysis. Platelets in whole blood were triggered with different concentrations of agonists (TRAP, ADP, CRP). Platelet activation was quantified with staining for P-selectin, measuring the mean fluorescence intensity. Area under the curve and the concentration of half-maximal response were determined.ResultsWe studied 23 patients with chronic kidney disease (9 patients with cardiorenal failure and 14 patients with end stage renal disease) and 19 healthy controls. Expression of P-selectin on the platelet surface measured as mean fluorescence intensity was significantly less in chronic kidney disease patients compared to controls after maximal stimulation with TRAP (9.7 (7.9-10.8) vs. 11.4 (9.2-12.2), P = 0.032), ADP (1.6 (1.2-2.1) vs. 2.6 (1.9-3.5), P = 0.002) and CRP (9.2 (8.5-10.8) vs. 11.5 (9.5-12.9), P = 0.004). Also the area under the curve was significantly different. There was no significant difference in half-maximal response between both groups.ConclusionIn this study we found that patients with chronic kidney disease show reduced platelet reactivity in response of ADP, TRAP and CRP compared to controls. These results contribute to our understanding of the aberrant platelet function observed in patients with chronic kidney disease and emphasize the significance of using functional whole blood platelet activation assays.

BackgroundPlatelets are an underappreciated factor in the classification of the bleeding tendency of patients with hemophilia. In this cross-sectional study, we investigated platelet activation status and responsiveness in relation to residual factor VIII activity and, within the group with severe hemophilia (<1% residual factor VIII activity), to annual factor VIII consumption. Design and MethodsTwenty-one patients with mild-moderate hemophilia A, 13 with severe hemophilia A and 21 healthy controls were studied. The basal level of platelet activation and platelet responsiveness to activation and inhibition were determined by the measurement of platelet P-selectin expression and soluble platelet activation markers. ResultsPatients with severe hemophilia A had a higher percentage of activated platelets at baseline (15.9%) when compared to patients with mild-moderate hemophilia A (8.2%, P=0.014) and controls (6.4%, P<0.001). Both patients with mild-moderate hemophilia A and those with severe hemophilia A had higher levels of the soluble platelet activation markers platelet factor 4 (1.4 and 1.8 pg/10 6 platelets), CXCL7 (65.8 and 48.2 pg/10 6 platelets) and RANTES (12.8 and 9.5 pg/10 6 platelets), compared to controls (platelet factor 4: 0.3 pg/10 6 platelets, P<0.001 and <0.001; CXCL7 20.0 pg/10 6 platelets, P<0.001 and <0.001; RANTES 4.5 pg/10 6 platelets, P<0.001 and =0.003, respectively). In support of these observations, we found clinical evidence that higher platelet P-selectin expression correlates with lower factor VIII consumption in patients with severe hemophilia (Spearman's r -0.65, P=0.043). ConclusionsThis study indicates that platelets from patients with severe hemophilia A are in a pre-activated state and that this pre-activated state is associated with factor VIII consumption.Key words: hemophilia A, platelets, activation, up-regulation, FVIII consumption.Citation: van Bladel ER, Roest M, de Groot PG, and Schutgens REG. Up-regulation of platelet activation in hemophilia A. Haematologica 2011;96(6):888-895. doi:10.3324/haematol.2011 This is an open-access paper. Up-regulation of platelet activation in hemophilia A

Agonist-induced platelet reactivity was significantly correlated to bleeding. Platelet function testing could provide a basis for a personalized transfusion regimen, in which platelet transfusions are limited to those at risk of bleeding.

The effects of coagulation factor concentrate infusion on restoring secondary haemostasis in patients with haemophilia are obvious. It is not known whether coagulation factor concentrate infusion affects primary haemostasis or induces an acute inflammatory response. In this study, the influence of a factor VIII (FVIII) concentrate bolus infusion on platelet activation and responsiveness, endothelial activation, and inflammation in adult patients with severe haemophilia A was assessed. VWF showed a mild, but significant decrease 15 min after FVIII infusion (85.02 IU dL(-1)) vs. before infusion (92.04 IU dL(-1) ; P = 0.017), while ADAMTS-13 levels also show a mild but significant decrease from 66.1 ng mL(-1) before infusion, to 53.9 ng mL(-1) (P = 0.012) 15 min after and 50.8 ng mL(-1) (P = 0.050) 60 min after infusion. Platelet P-selectin expression decreased 15 min (33.3 AU) and 60 min (38.7 AU) after infusion compared to before infusion (41.3 AU; P = 0.018 and 0.036). In conclusion, a single infusion of a high dose FVIII concentrate in haemophilia A patients may influence primary haemostasis by decreasing VWF, ADAMTS-13 and the number of circulating activated platelets. These effects possibly occur as a consequence of binding of the infused FVIII to VWF, influencing its processing. When treating severe haemophilia A patients with coagulation concentrate infusion, one should realize this does not merely correct FVIII levels but also may influence primary haemostasis.