PS1475 COATED PLATELET FUNCTION IS RELATED TO BLEEDING PHENOTYPE IN PATIENTS WITH INHERITED THROMBOCYTOPENIA

Abstract

Background:Platelets transform into different subpopulations following stimulation. Coated‐platelets (CPs) are a subpopulation of highly procoagulant platelets with an outer membrane coating of procoagulant proteins. Also, platelet microparticles (PMPs) released from the platelet membrane have procoagulant properties. Though CPs and PMPs are inferred critical for hemostasis, they have mainly been investigated in thrombotic and inflammatory diseases. Only few studies have investigated their role in bleeding disorders and thrombocytopenia. Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders with varying bleeding tendencies, not simply related to platelet count.Aims:To investigate platelet, PMP and CP phenotypes and function in IT patients with different bleeding phenotypes.Methods:We enrolled 34 IT patients. Sequencing of 36 IT related genes enabled a molecular diagnosis in 47%. Bleeding phenotype was evaluated using ISTH bleeding assessment tool (BAT) with significant bleeding defined as BAT score >5 (women) or >3 (men). Hereby patients were divided into bleeding (n = 19) vs. non‐bleeding (n = 15) patients. Platelet, PMP and CP phenotype and function were evaluated by flow cytometry: Activation and granule release were examined by antibodies against granulphysin (CD63), P‐selectin (CD62P), activated GPIIb/IIIa (PAC‐1) and phosphatidylserine (PS) (lactadherin) unstimulated and ADP, TRAP or collagen stimulated. Coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and GPIbα (CD42b). Normal healthy reference levels were available.Results:The platelet count in bleeding (72x109/l) and non‐bleeding (68x109/l) patients was comparable (P = 0,66). Bleeding patients had a higher BAT score compared to non‐bleeding patients (10 vs. 2, P < 0,01). The proportion of CPs was normal in all patients. However, in non‐bleeding patients the proportion of PS+CPs and per cell PS expression (MFI) (86,16% and 4,96MFI) were higher, compared to bleeding patients (64,08% and 2,44MFI, both p < 0,05), and the proportion of PS+CPs correlated negatively with BAT score (r2 = 0,26, p < 0,01) (figure 1). CD63+CP was higher in non‐bleeding (97,25% and 10,54MFI) compared to both bleeding patients (94,88% and 6,89MFI) and significantly higher than the reference level (88,44% and 5,36MFI, both p < 0,05). Finally, the proportion of PS+PMPs was normal in bleeding patients, but their PMPs expressed higher than reference PS per cell, both unstimulated and for all agonist (134,12 MFI unstimulated vs 32,38 MFI reference, P < 0,01).Summary/Conclusion:Patients with IT exhibited different bleeding tendency despite comparable thrombocytopenia. In non‐bleeding patients the proportion and per cell level of PS+ were higher, indicating that generation of CPs with high PS expression is a critical factor determining bleeding phenotype. The finding of high PMP PS per cell level in bleeding patients could represent an inadequate compensation for lack of CP function, indicating that procoagulant PMPs may be less important than CPs for thrombocytopenic bleeding. Quantification and characterization of CPs may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in IT and other conditions with bleeding diathesis and/or thrombocytopenia. More studies investigating this field are warranted.image