Levels of procoagulant microparticles expressing phosphatidylserine contribute to bleeding phenotype in patients with inherited thrombocytopenia.

Inherited thrombocytopenia is a heterogeneous group of hereditary disorders with varying bleeding tendencies, not simply related to platelet count. Platelets transform into different subpopulations upon stimulation, including procoagulant platelets and platelet microparticles (PMPs), which are considered critical for haemostasis. We aimed to investigate whether abnormalities in PMP and procoagulant platelet function were associated with the bleeding phenotype of inherited thrombocytopenia patients. We enrolled 53 inherited thrombocytopenia patients. High-throughput sequencing of 36 inherited thrombocytopenia related genes was performed in all patients and enabled a molecular diagnosis in 57%. Bleeding phenotype was evaluated using the ISTH bleeding assessment tool, dividing patients into bleeding (n = 27) vs. nonbleeding (n = 26). Unstimulated and ADP, TRAP or collagen-stimulated PMP and procoagulant platelet functions were analysed by flow cytometry using antibodies against granulophysin (CD63), P-selectin (CD62P), activated GPIIb/IIIa (PAC-1) and a marker for phosphatidylserine expression (lactadherin). Procoagulant platelets were measured in response to collagen stimulation. An in-house healthy reference level was available. Overall, higher levels of activated platelets, PMPs and procoagulant platelets were found in nonbleeding patients compared with the reference level. Nonbleeding patients had higher proportions of phosphatidylserine and PMPs compared with bleeding patients and the reference level, in response to different stimulations. Interestingly, this finding of high proportions of phosphatidylserine and PMPs was limited to PMPs, and not present in procoagulant platelets or platelets. Our findings indicate that nonbleeding inherited thrombocytopenia patients have compensatory mechanisms for improved platelet subpopulation activation and function, and that generation of phosphatidylserine expressing PMPs could be a factor determining bleeding phenotype in inherited thrombocytopenia.

Evaluation of the quality of blood components obtained after automated separation of whole blood by a new multiunit processor.

BACKGROUND: The Reveos system (Terumo BCT) is a fully automated device able to process four whole blood (WB) units simultaneously into a plasma unit, a red blood cell (RBC) unit, and an interim platelet (PLT) unit (IPU). Multiple IPUs can be pooled to form a transfusable PLT product. The aim of our study was to evaluate the quality of components made with the Reveos system from either fresh (2-8 hr) or overnight-held WB. STUDY DESIGN AND METHODS: A prototype of the Reveos system was used to process WB. RBCs were resuspended in SAGM, leukoreduced, and assayed for in vitro quality variables during a 42-day storage period at 2 to 6 °C. Twenty-four-hour in vivo recovery was determined on Day 42. Plasma was assayed for cellular contamination and activation variables. IPUs were pooled with SSP+ additive solution for in vitro quality assessments during a 7-day storage period at room temperature. RESULTS: Reveos-produced RBCs and plasma units met the predefined requirements. RBC recovery was superior to control units. On Day 42, hemolysis was below 0.8% and in vivo recovery was above 75% for all RBCs. Cellular contamination was lower for Reveos-produced plasma. PLT yield was higher with overnight-stored WB. PLT quality was well maintained during storage with no significant differences between the two groups. CONCLUSION: Blood components prepared with the Reveos from fresh or overnight-held WB meet quality criteria without any relevant difference between the two groups. The Reveos system has the potential to increase efficacy and standardization of blood component preparation.

Hemostatic function of buffy coat platelets in additive solution treated with pathogen reduction technology.

BACKGROUND: Pathogen reduction technologies (PRTs) may influence the hemostatic potential of stored platelet (PLT) concentrates. To investigate this, buffy coat PLTs (BCPs) stored in PLT additive solution (SSP+) with or without Mirasol PRT treatment (CaridianBCT Biotechnologies) were compared by functional hemostatic assays. STUDY DESIGN AND METHODS: We performed in vitro comparison of PRT (PRT-BCP) and control pooled-and-split BCPs (CON-BCP) after 2, 3, 6, 7, and 8 days' storage. Hemostatic function was evaluated with thrombelastography (TEG) and impedance aggregometry (Multiplate), the latter also in a sample matrix (Day 2) with or without addition of red blood cells (RBCs), control plasma, and/or PRT-treated plasma. RESULTS: PRT treatment of 8-day-stored BCPs influenced clot formation (TEG) minimally, with reductions in maximum clot strength (maximum amplitude, p = 0.014) but unchanged initial fibrin formation (R), clot growth rate (α), and fibrinolysis resistance. In the absence of RBCs and plasma, PRT impaired aggregation (Multiplate) in stored BCPs, with reduced aggregation against thrombin receptor activating peptide-6 (p < 0.001), collagen (p = 0.014), adenosine 5'-diphosphate (p = 0.007), and arachidonic acid (p = 0.070). Addition of RBCs and PRT-treated or untreated plasma to PRT-BCP and CON-BCP, respectively, enhanced aggregation in both groups. CONCLUSIONS: Mirasol PRT treatment of BCPs had a minimal influence on clot formation, whereas aggregation in the absence of RBCs and plasma was significantly reduced. Addition of RBCs and plasma increased agonist-induced responses resulting in comparable aggregation between PRT-BCP and CON-BCP. The clinical relevance for PLT function in vivo of these findings will be investigated in a clinical trial.

In vitro cell quality of buffy coat platelets in additive solution treated with pathogen reduction technology.

BACKGROUND: Pathogen reduction technologies (PRTs) may induce storage lesion in platelet (PLT) concentrates. To investigate this, buffy coat PLTs (BCPs) in PLT additive solution (AS; SSP+) with or without Mirasol PRT (CaridianBCT Biotechnologies) were assessed by quality control tests and four-color flow cytometry. STUDY DESIGN AND METHODS: In vitro comparison of PRT and control pooled-and-split BCPs after 2, 3, 6, 7, and 8 days of storage was made. PLT concentration, count per unit, swirl, metabolism, activation (CD62P, PAC1, CD42b/GPIb, CD63, CD40L/CD154, CD40, annexin V), and microparticle, sCD40L, and sCD62P release were evaluated. RESULTS: PRT induced a minor initial PLT loss (Day 2 [mean±SD], 302×10(9) ±44×10(9) PLTs/unit vs. 325× 10(9) ±46×10(9) PLTs/unit; p<0.001) but the decline was comparable to control BCP. Swirling was comparable and declined with similar rates in PRT-treated and control BCPs during storage. PRT enhanced PLT metabolism and activation, evidenced by lower pH(22) ; increased glucose consumption and lactate production rates (p<0.01); early increases in CD62P-, PAC1-, CD63-, CD40L-, CD40-, and annexin V-positive PLTs; reduced GPIb expression; and enhanced release of PLT-derived MPs and sCD40L (all p<0.05). CD62P and PAC1 expression changed with different kinetics during storage and varying GPIb expression was displayed within the CD62P/PAC1-positive PLT subsets. CONCLUSION: PRT treatment of BCP in AS induced a minor initial PLT loss and enhanced metabolism and PLT activation. The clinical relevance for PLT function in vivo of these findings will be investigated in a clinical trial.

The in vitro effect of recombinant factor VIIa on coated platelet formation and clot dynamics of stored platelet concentrates.

BACKGROUND: Storage can negatively impact the hemostatic potential of platelet concentrates (PCs) used for transfusion. At the site of vascular injury, normal platelets (PLTs) are hypothesized to change into highly procoagulant-coated PLTs upon costimulation with collagen and thrombin. We investigated whether activated recombinant factor VII (rFVIIa, NovoSeven, Novo Nordisk A/S) could improve the ability of stored PLTs to support coagulation under conditions that promote the formation of coated PLTs. STUDY DESIGN AND METHODS: PCs stored for 1, 4, 6, and 8 days were costimulated with thrombin and with convulxin, a collagen glycoprotein VI receptor agonist, to create coated PLTs. The effect of rFVIIa on the ability of stored PCs to form coated PLTs, generate thrombin, and impact clot dynamics was evaluated by flow cytometry, a plasma-based assay, and thrombelastography, respectively. RESULTS: Coated PLT formation decreased significantly with increasing storage time (80% vs. 50%-55%, p < 0.05), and this was not affected by the addition of rFVIIa. rFVIIa accelerated thrombin generation (p < 0.001) and clot formation (p < 0.001) and significantly increased thrombin generation throughout the storage period (p < 0.001). Resistance to fibrinolysis was impaired at the end of storage, and this was not affected by the addition of rFVIIa. CONCLUSION: rFVIIa accelerated thrombin and clot formation throughout storage, with the most pronounced effect observed in the PCs that had been stored for the shortest length of time (Day 1). Resistance to fibrinolysis was gradually impaired throughout the storage period and was not affected by the addition of rFVIIa.

Impairment of the hemostatic potential of platelets during storage as evaluated by flow cytometry, thrombin generation, and thrombelastography under conditions promoting formation of coated platelets.

BACKGROUND: The increasing demand for platelet (PLT) transfusions has focused attention on appropriate use. Coated PLTs are a subpopulation of highly procoagulant PLTs formed by simultaneous stimulation by the agonist's collagen and thrombin hypothesized to drive clot formation at the site of vascular injury. Prolonged storage of PLTs may reduce their ability to support optimal hemostasis upon transfusion. STUDY DESIGN AND METHODS: PLT concentrates (PCs) stored for 1, 4, 6, and 8 days were costimulated with thrombin and the collagen glycoprotein VI (GPVI) receptor agonist convulxin, and their ability to form coated PLTs was determined by flow cytometry. Further, a plasma-based thrombin generation assay and thrombelastography were used to evaluate the aged PCs' capacity to support thrombin generation and clot formation, respectively. The stored PCs were additionally tested by standard quality control methods. RESULTS: PLT quality as measured by standard analyses was acceptable according to current practice. The hemostatic potential, however, was impaired with increasing storage time. The formation of coated PLTs decreased significantly from approximately 85 to 55 percent with increasing storage time (p<0.05). The velocity of clot formation was significantly increased from Day 4 (p<0.05). The velocity of thrombin generation and resistance against fibrinolysis were significantly reduced on Day 8 compared to Day 1 of storage (p<0.05). CONCLUSION: Data in the present study suggest that storage significantly reduced the stored PLTs' ability to respond to conditions expected to exist at the site of vascular injury and that storage-induced reduction in PLT activation sensitivity correlated with a loss of hemostatic potential.