Additive solutions differentially affect metabolic and functional parameters of platelet concentrates.

BACKGROUND: Pathogen inactivation (PI) of platelet concentrates with extension of shelf life to 7 days requires the use of platelet additive solutions (PAS). We examined the quality of platelets resuspended in three different PAS stored for up to 7 days. MATERIALS AND METHODS: Twelve triple adult dose platelet concentrates (PC) were collected using the TrimaAccel® collection system. Each highly concentrated product was divided into three equal parts, and the additive solutions (Composol® or SSP+® or Intersol™) were added to a final concentration of 56% PAS and 44% plasma. Samples were drawn on days 1, 5 and 7 to measure pH, glucose, lactate dehydrogenase (LDH), lactate, mean platelet volume (MPV) and the aggregation response to collagen and the thrombin receptor agonist peptide-6. Further, p-selectin expression on platelets was assessed. RESULTS: No statistically significant changes were observed for pH and MPV during 7 days of storage in all PAS containing PCs, whereas glucose decreased and LDH and lactate increased over time (P < 0·05). These changes were particularly evident in Intersol PCs on days 5 and 7 compared with Composol® PCs or SSP+® PCs (P < 0·05). Platelets from Intersol PCs exhibited the highest baseline activation of p-selectin and showed reduced collagen- and TRAP-6-induced aggregation. CONCLUSION: Resuspension of platelets in Intersol for 7 days results in increased platelet activation and platelet metabolism compared with SSP+® or Composol®. Further clinical studies are needed to evaluate whether the observed differences in PAS-PCs affect the recovery rate or the life span of transfused platelets.

Comparison between the impact of morning and evening doses of rivaroxaban on the circadian endogenous coagulation rhythm in healthy subjects.

UNLABELLED: ESSENTIALS: It is unknown whether single rivaroxaban doses should best be administered in the morning or evening. Circadian rhythm of coagulation/fibrinolysis was measured after morning or evening intake of rivaroxaban. Evening intake of rivaroxaban leads to prolonged exposure to rivaroxaban concentrations. Evening intake of rivaroxaban better matches the morning hypofibrinolysis. BACKGROUND: A circadian variation of the endogenous coagulation system exists with hypercoagulability and hypofibrinolysis and a corresponding peak of cardiovascular thromboembolic events in the morning. So far, no information is given as to whether single daily doses of the new oral anticoagulant drug rivaroxaban should best be administered in the morning or the evening. MATERIALS AND METHODS: Sixteen healthy male or female volunteers with a mean age of 26 ± 7 years were included in this randomized, controlled, analyst-blinded cross-over clinical trial. All subjects were given three morning and three evening single doses of 10 mg rivaroxaban. Circadian rhythms of prothrombin fragment 1 + 2, plasminogen activator inhibitor, and plasmin-antiplasmin complex were measured before any medication intake, as well as after morning or evening medication intake. Rivaroxaban concentrations were determined by an anti-activated factor X assay and liquid chromatography-mass spectrometry. MAIN RESULTS: Concentrations of rivaroxaban were higher 12 h after evening intake of rivaroxaban than 12 h after morning intake (53.3 ng mL(-1) [95% confidence interval 46.0-67.8] vs. 23.3 ng mL(-1) [19.4-29.1, respectively]). Rivaroxaban intake in the evening reduced morning F1+2 concentrations better at 8:00 AM than did administration on awakening (85 ± 25 nmol L(-1) vs. 106 ± 34 nmol L(-1) , CI: 9.4-32.1). In addition, this suppression effect was longer lasting after evening intake. CONCLUSIONS: Evening intake of rivaroxaban leads to prolonged exposure to rivaroxaban concentrations and better matches the morning hypofibrinolysis. These results might help to further improve the efficacy and safety of rivaroxaban treatment.

The association of the Thr715Pro P-selectin genotype with levels of P-selectin in platelet concentrates.

BACKGROUND AND OBJECTIVES: P-selectin is stored in the alpha granules of platelets and in the Weibel Palade bodies of endothelial cells; upon activation, it is translocated to the cell surface and released into the plasma in soluble form. One variant of the P-selectin gene, the Thr715Pro polymorphism, is strongly associated with the plasma levels of soluble P-selectin. In platelet concentrates soluble P-selectin can be regarded mainly platelet derived. MATERIALS AND METHODS: The relation of the genotype with soluble P-selectin, platelet expressed P-selectin and the sum of all forms of P-selectin - comprising soluble P-selectin, platelet surface P-selectin and P-selectin from the alpha granules - was assessed in fresh whole blood and in apheresis platelets suspended in 35% plasma/65% SSP+ obtained from 89 platelet donors. RESULTS: Levels of total P-selectin were genotype associated (P = 0·025); likewise, in fresh whole blood there was an association of soluble P-selectin with genotype (P = 0·02). In platelets suspended in additive solution, however, levels of the storage-associated or TRAP-6 agonist induced increase of platelets' P-selectin were not associated with the genotype. A correlation between levels of soluble P-selectin and surface expression of P-selectin was observed on day 3 of storage in Thr715Thr individuals (P < 0·0001), but not in heterozygotes (Thr715Pro, P = 0·2). CONCLUSION: The donors' genotype has only little influence on levels of soluble P-selectin in apheresis platelets suspended in 35% plasma/65% SSP+.

The effect of plasma removal from apheresis platelet concentrates on platelet function.

The effect of plasma removal on platelet function has scarcely been investigated. Plasma removal from apheresis platelet concentrates was achieved by centrifugation at 5000 g for 6 min or 2000 g for 10 min. After resting for 1 h, platelet concentrates were resuspended in 0·9% NaCl. Platelet function was tested before centrifugation and after resuspension by multiple electrode impedance aggregometry (MEA) and light transmission aggregometry (LTA). Plasma removal resulted in 10-14% lower response to TRAP-6 by MEA using both washing procedures, whereas TRAP-6-inducible aggregation by LTA increased slightly (2-5%). Neither plasma removal method affected collagen-induced aggregation. Thus, platelet function did not deteriorate significantly by either method.

Racial differences in endotoxin-induced tissue factor-triggered coagulation.

BACKGROUND: Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (=DARC, Duffy antigen receptor of chemokines) and coagulation. OBJECTIVES: Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation. PATIENTS/METHODS: Healthy male volunteers (16 Duffy-negative Africans, 16 Duffy-positive Caucasians) received 2 ng kg(-1) LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). RESULTS: LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin-antithrombin (TAT) complexes (10.4 pg mL(-1) vs. 23.0 pg mL(-1), P<0.0001) and less prothrombin fragments (F1+2) (337 pmol mL(-1) vs. 819 pmol mL(-1), P<0.0001). Consistently, Africans also had decreased fibrin formation (D-dimer: 0.3 pg mL(-1) vs. 0.5 pg mL(-1), P=0.02). CONCLUSION: Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.

Erythropoietin increases platelet reactivity and platelet counts in patients with alcoholic liver cirrhosis: a randomized, double-blind, placebo-controlled study.

BACKGROUND: Patients with liver cirrhosis have a complex haemostasis disturbance including thrombocytopenia and abnormal bleeding time. Erythropoietin is the primary stimulator for erythrocyte production and also induces megakaryocyte formation. In healthy men erythropoietin increased platelet count and platelet reactivity. AIM: As patients with liver cirrhosis often undergo invasive procedures, we were interested to study whether erythropoietin could improve platelet function in addition to thrombocytopenia. METHODS: In total, 22 thrombocytopenic (platelet counts < 120 g/L) patients with alcoholic liver cirrhosis received either 100 IE/kg erythropoietin or placebo on days 1, 3 and 5 in a 2:1 randomized, placebo-controlled double-blind fashion. Platelet counts and platelet reactivity (activator-stimulated expression of P-selectin on platelets measured by flow cytometry) were determined on study days 1, 3, 5 and 9. RESULTS: Median platelet count was 80 g/L which is borderline for major elective surgical interventions. Baseline values were not different between groups (P > 0.05). Treatment with erythropoietin increased platelet count by 25% (P = 0.01) and platelet reactivity twofold (P < 0.01) vs. baseline. The increase in platelet count vs. baseline was more pronounced in patients with platelet counts <80 g/L. No significant effect was observed in the placebo group. CONCLUSIONS: Treatment with erythropoietin significantly increased platelet counts and platelet reactivity in patients with alcoholic liver cirrhosis. Preoperative treatment with erythropoietin is therefore expected to yield higher platelet levels and better platelet function.

Quality of packed red blood cells and platelet concentrates collected by multicomponent collection using the MCS plus device.

The demand for blood components is constantly increasing, while the exclusion criteria for donors are strengthened in order to reach maximal safety for donors and patients. To counterbalance reduced availability of volunteers, multicomponent collections (MCC) is an attractive approach to produce more than one component during a single apheresis procedure from one donor, such as packed red blood cells (PRBCs) and platelet concentrates (PCs). Further, the exposures of patients to a limited number of donors reduces the possibility of alloimmunization and transfusion-related diseases. We measured the quality of PRBCs and PCs obtained by MCC, using the MCS+ device with the LDPRBC program, Revision B, and compared them with the quality of manually collected PRBCs and PCs collected with the Revision C2 of the MCS+. We found higher pH levels and lower hemolysis assessed by means of fHb and K+ in the supernatant of PRBCs over the whole storage period of 42 days in MCC-derived PRBCs. The functional metabolism assessed by intracellular ATP was higher in PRBCs collected by MCC than in manually collected units. Furthermore, PCs obtained during MCC showed an increase in p-selectin expression on day 5 of storage compared to PCs collected with the Revision C2 of the MCS+. The p-selectin expression on MCC platelets was within the range of p-selectin expression found in PCs obtained by other apheresis devices. These results indicate less storage lesion in MCC-derived PRBCs compared to manually collected units and no compromise in the quality of MCC PCs obtained in the same apheresis procedure.

Impaired platelet function among platelet donors.

BACKGROUND: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. STUDY DESIGN AND METHODS: We determined the inter-donor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in alpha2 integrin polymorphism and density. RESULTS: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the alpha2-integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. CONCLUSION: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation.

Fy phenotype and gender determine plasma levels of monocyte chemotactic protein.

BACKGROUND: In vitro studies indicate that the Fy blood group system antigens serve as receptors for chemokines such as monocyte chemotactic protein-1 (MCP-1) and RANTES. However, it is unclear whether subjects with the Fy(a-b-) phenotype exhibit altered clearance and hence altered plasma levels of chemo-kines, because they still express Fy on endothelial cells. STUDY DESIGN AND METHODS: To clarify a possible in vivo role of Fy on RBCs in the regulation of chemo-kine levels, healthy young volunteers of common Fy phenotypes were compared in a cross-sectional study. RESULTS: More than 90 percent of the 34 subjects of African origin were Fy(a-b-), one black volunteer was Fy(a+b-), and two were Fy(a-b+). As expected, all 65 white volunteers were positive for either Fy(a) and/or Fy(b). Unexpectedly, persons expressing either Fy(a) and/or Fy(b) had significantly higher plasma levels of MCP-1 than Fy(a-b-) volunteers (women: 154 vs. 110 ng/L, p<0.01; men: 179 vs. 169 ng/L, p = 0.03). Surprisingly, plasma levels of MCP-1 were found to be sex-dependent: median MCP-1 levels averaged 180 ng per L in men but only 139 ng per L in women (p<0.001). Further, MCP-1 levels decreased significantly throughout the menstrual cycle of 18 women studied longitudinally. CONCLUSION: MCP-1 levels are about 30 percent higher in men than in premenopausal women, and MCP-1 levels are also higher in persons with RBCs expressing Fy antigens than in Fy(a-b-) persons. These findings have direct implications for the concept and interpretation of clinical studies measuring MCP-1 levels; the role of the observed differences in MCP-1 levels for the pathogenesis of MCP-1-dependent diseases, such as atherosclerosis, merits further investigation.

A new flow cytometric method for simultaneous measurement of residual platelets and RBCs in plasma: validation and application for QC.

BACKGROUND: Guidelines for the preparation of FFP in Austria and Germany require the quantification of residual RBCs in plasma. However, currently there is no validated method for enumeration of RBC counts as low as 1 x 10(9) per L. STUDY DESIGN AND METHODS: A new flow cytometric method was developed for QC, to simultaneously determine if fresh unfrozen plasma contains residual platelets and RBCs. This method uses test tubes (TruCount, Becton Dickinson Immunocytometry Systems) that contain a known number of brightly fluorescent polystyrene beads. Plasma is pipetted into these tubes and mixed with FITC-labeled anti-CD41 and PE-conjugated anti-glycophorin A MoAbs. Acquisition can be performed on a flow cytometer after an incubation period of 15 minutes. RESULTS: Individual standard curves for platelet counts revealed excellent correlation coefficients (r>0.998). Platelet counts were validated against simultaneous measurements by using a cell counter and a hemocytometer. While the flow cytometric method slightly overestimated platelet counts of >40 x 10(9) per L by 10 percent, its precision in the critical range was very good-that is, a deviation of platelets < or = 1 x 10(9) per L from target values, which was even better than microscopic enumeration. The limit of sensitivity was <0.5 x 10(9) per L of platelets. RBC counts were also validated against simultaneous measurements made with a second cell counter. Standard curves for RBC counts also revealed excellent correlation coefficients (r>0.997). The limit of sensitivity was <0.25 x 10(9) RBCs per L. Platelet counts in plasma obtained by plateletpheresis or plasmapheresis ranged from 3 to 60 x 10(9) per L. About 25 percent of all plasma samples had platelet counts greater than the allowed upper limit of 25 x 10(9) per L, while all plasma samples contained fewer RBCs than the upper limit of 6 x 10(9) per L. CONCLUSION: This newly developed method provides a simple, quick, precise, and easily reproducible tool for simultaneous measurement of residual platelets and RBCs in fresh plasma.

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis.

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.

Effects of anticoagulation on thrombopoietin release during endotoxemia.

Several lines of evidence suggest that coagulation may induce the release of thrombopoietin (TPO) into plasma and that TPO levels are higher in disseminated intravascular coagulation. Therefore we set out to illuminate the mechanism of TPO release in the setting of experimental endotoxemia, which induces activation of coagulation and platelets. Endotoxin (lipopolysachharide [LPS], 2 ng/kg) was infused into a total of 54 healthy men in two subsequent studies. Volunteers received infusions of unfractionated heparin, low-molecular-weight heparin, lepirudin, or placebo in a randomized, placebo-controlled fashion after bolus injection of LPS. TPO levels increased on average by 27% to 38% in all groups at 6 hours (P <.05 vs baseline), although all active drugs effectively blocked coagulation. Platelet counts dropped by about 15% at 1 hour after LPS infusion, recovered after 2 days, and exceeded baseline values by 8% to 18% after 7 days (P <.001 vs baseline for all groups). Yet lepirudin blunted the LPS-induced increase in circulating P-selectin by one half (P <.005 vs placebo), whereas both heparins did not diminish the increase in this platelet or endothelial activation marker as compared with placebo. Endotoxemia enhances TPO plasma levels independent of the degree of coagulation induction, which eventually results in increased platelet numbers. Of potential clinical interest is the observation that the direct thrombin inhibitor lepirudin, in contrast to heparins, mitigated LPS-induced platelet activation.

Granulocyte colony-stimulating factor (G-CSF) downregulates its receptor (CD114) on neutrophils and induces gelatinase B release in humans.

Despite the increasing use of granulocyte colony-stimulating factor (G-CSF) for the mobilization of stem cells and neutrophils, its pharmacodynamic actions are not fully understood. Because of the roles of G-CSF and gelatinase B in leucokinetics, we set out to characterize the interaction of G-CSF with its receptor in humans and its effects on gelatinase B release. G-CSF was infused at bolus doses of 1 microg/kg and 5 microg/kg, and compared to placebo and dexamethasone (1 mg/kg b.i.d), which enhances the plasma levels of endogenous G-CSF. The study was randomized, double-blind, four-way crossover, in eight healthy male volunteers. G-CSF dose-independently induced profound neutropenia (> 95%) within minutes and downregulated its own receptor (CD114) on neutrophils by 75%. The G-CSF/CD114 interaction dose-independently induced degranulation of neutrophils as evidenced by a 300-400% increase in CD11b expression. Degranulation induced up to a 10-fold increase in plasma levels of gelatinase B, an enzyme known to precipitate neutropenia and subsequent neutrophilia in animals. In this study, it was shown that G-CSF downmodulates CD114 expression on the surface of neutrophils in humans and the consequent degranulation enhances gelatinase B release into plasma, which may contribute to mobilization of neutrophils or stem cells.