Hemostatic properties and protein expression profile of therapeutic apheresis plasma treated with amotosalen and ultraviolet A for pathogen inactivation.

BACKGROUND: The INTERCEPT Blood System (IBS) using amotosalen-HCl and ultraviolet (UV)A inactivates a large spectrum of microbial pathogens and white blood cells in therapeutic plasma. Our aim was to evaluate to what extent IBS modifies the capacity of plasma to generate thrombin and induces qualitative or quantitative modifications of plasma proteins. STUDY DESIGN AND METHODS: Plasma units from four donors were collected by apheresis. Samples were taken before (control [CTRL]) and after IBS treatment and stored at -80°C until use. The activities of plasma coagulation factors and inhibitors and the thrombin generation potential were determined using assays measuring clotting times and the calibrated automated thrombogram (CAT), respectively. The proteomic profile of plasma proteins was examined using a two-dimensional differential in-gel electrophoresis (2D-DIGE) method. RESULTS: Nearly all of the procoagulant and antithrombotic factors tested retained at least 78% of their initial pre-IBS activity. Only FVII and FVIII displayed a lower level of conservation (67%), which nevertheless remained within the reference range for conventional plasma coagulation factors. The thrombin generation profile of plasma was conserved after IBS treatment. Among the 1331 protein spots revealed by 2D-DIGE analysis, only four were differentially expressed in IBS plasma compared to CTRL plasma and two were identified by mass spectrometric analysis as transthyretin and apolipoprotein A1. CONCLUSION: The IBS technique for plasma moderately decreases the activities of plasma coagulation factors and antithrombotic proteins, with no impact on the thrombin generation potential of plasma and very limited modifications of the proteomic profile.

Cardiovascular mortality in chronic kidney disease patients undergoing percutaneous coronary intervention is mainly related to impaired P2Y12 inhibition by clopidogrel.

OBJECTIVES: We sought to determine whether low platelet response to the P2Y(12) receptor antagonist clopidogrel as assessed by vasodilator-stimulated phosphoprotein flow cytometry test (VASP-FCT) differentially affects outcomes in patients with or without chronic kidney disease (CKD) undergoing percutaneous coronary intervention (PCI). BACKGROUND: Although both CKD and impaired platelet responsiveness to clopidogrel are strong predictors of unfavorable outcome after PCI, the impact of their association is unknown. The platelet VASP-FCT assay is specific for the P2Y(12) ADP receptor pathway. In this test, platelet activation is expressed as the platelet reactivity index (PRI). METHODS: Four-hundred forty unselected patients (CKD: 126, estimated glomerular filtration rate [eGFR] <60 ml/min/1.73 m(2)), no-CKD: 314 eGFR >60 ml/min/1.73 m(2)) undergoing urgent (n = 336) or planned (n = 104) PCI were prospectively enrolled. In each subgroup, patients were classified as low-responders (LR: PRI ≥ 61%) or responders (R: PRI <61%) to clopidogrel. RESULTS: At a mean follow-up of 9 ± 2 months, all-cause mortality, cardiac death, and possible stent thrombosis were higher in CKD than in no-CKD patients. Within the CKD group, the LR status was associated with higher rates of all-cause mortality (25.5% vs. 2.8%, p < 0.001), cardiac death (23.5% vs. 2.8%, p < 0.001), all stent thrombosis (19.6% vs. 2.7%, p = 0.003), and MACE (33.3% vs. 12.3%, p = 0.007). Conversely, in no-CKD patients, the LR status did not affect outcomes. Multivariate analysis identified Killip class ≥ 3, drug-eluting stent implantation, and the interaction between LR and CKD (hazard ratio: 11.96, 95% confidence interval: 1.22 to 116.82; p = 0.033) as independent predictors of cardiac death. CONCLUSIONS: In CKD patients, the presence of low platelet response to clopidogrel is associated with worse outcomes after PCI.

Use of additive solutions and pathogen inactivation treatment of platelet components in a regional blood center: impact on patient outcomes and component utilization during a 3-year period.

BACKGROUND: The Etablissement Français du Sang Alsace (EFS Alsace) successively implemented universal use of platelet additive solutions (PASs) and pathogen inactivation (PI) for platelet components (PCs). To assess the impact of these changes, EFS Alsace evaluated PC use, red blood cell (RBC) component use, and transfusion-related adverse events after implementation of these new technologies. STUDY DESIGN AND METHODS: EFS Alsace prospectively collects data on production, distribution, and response to transfusion of all blood components with greater than 99.5% data acquisition. Adverse events attributed to platelet (PLT) transfusions were collected through a mandatory, active hemovigilance program. A retrospective review of prospectively collected data was conducted covering three periods: 1) apheresis and whole blood-derived PCs in plasma, 2) apheresis and whole blood-derived PCs with PAS, and 3) PCs prepared with PI and PAS. Data on component utilization were analyzed for all patients receiving PCs in each period and for the subset of hematology-oncology patients to evaluate PC use in an intensely transfused population. Values for all continuous variables were summarized as mean and standard deviation, median, and range. RESULTS: Approximately 2000 patients received PCs in each period. PLT and RBC use per patient was not increased after PI (analysis of variance, F = 1.9 and 2.9, respectively) and the incidence of acute transfusion reactions was significantly reduced (p < 0.001). CONCLUSIONS: Universal use of PI was implemented without impacting component use, as indicated by total dose of PLTs per patient, and outcomes to transfusion were improved.

Impact of P2Y12 inhibition by clopidogrel on cardiovascular mortality in unselected patients treated by percutaneous coronary angioplasty: a prospective registry.

OBJECTIVES: The aim of this study was to determine whether low platelet response to the P2Y(12) receptor antagonist clopidogrel as assessed by Vasodilator-stimulated phosphoprotein flow cytometry test (VASP- FCT) predicts cardiovascular events in a high-risk population undergoing percutaneous coronary intervention (PCI). BACKGROUND: Impaired platelet responsiveness to clopidogrel is thought to be a determinant of cardiovascular events after PCI. The platelet VASP-FCT is a new assay specific to the P2Y(12) adenosine diphosphate receptor-pathway. In this test, platelet activation is expressed as platelet reactivity index (PRI). METHODS: Four-hundred sixty-one unselected patients undergoing urgent (n = 346) or planned (n = 115) PCI were prospectively enrolled. Patients were classified as low-response (LR) and response (R) to clopidogrel, depending on their PRI. Optimal PRI cutoff was determined by receiver-operator characteristic curve analysis to 61% (LR: PRI > or =61% and R: PRI <61%). Follow-up was obtained at a mean of 9 +/- 2 months in 453 patients (98.3%). RESULTS: At follow-up, total cardiac mortality rates and possible and total stent thrombosis were higher in LR patients. Multivariate analysis identified creatinine clearance (hazard ratio [HR]: 0.95; 95% confidence interval [CI]: 0.93 to 0.98, p < 0.001), drug-eluting stent (HR: 5.73; 95% CI: 1.40 to 23.43, p = 0.015), C-reactive protein (HR: 1.01; 95% CI: 1.001 to 1.019, p = 0.024), and LR to clopidogrel (HR: 4.00; 95% CI: 1.08 to 14.80, p = 0.037) as independent predictors of cardiac death. The deleterious impact of LR to clopidogrel on cardiovascular death was significantly higher in patients implanted with drug-eluting stent. CONCLUSIONS: In patients undergoing PCI, LR to clopidogrel assessed by VASP-FCT is an independent predictor of cardiovascular death at the PRI cutoff value of > or =61%. The LR clinical impact seems to be dependent on the type of stent implanted.

Between light and dark, the chimera comes out.

BACKGROUND: Chimerism, especially in the absence of sexual ambiguity, is extremely rare in humans. We report the case of a 6-year-old boy whose skin pigmentary abnormalities revealed chimerism. OBSERVATIONS: The boy had no remarkable previous medical history, and he had normal intelligence and development. On examination, we found a disorder of the skin pigmentation that was difficult to categorize; there was a lighter-appearing skin patch in the median frontal area and also on one-half of the abdominal area, with a sharp midline demarcation. He also had 2 lighter Blaschko-linear bands on the lower extremities and an indefinable mixture of lighter and darker skin on the back and the lateral part of the trunk. It was not possible to ascertain by means of clinical examination of the patient, his parents, and his brother which of the 2 shades was his normal skin color. Because this pattern of pigmentation might be related to mosaicism, we determined his karyotype. We found that his lymphocytes had a normal number of chromosomes, half of them being either 46,XX or 46,XY. In contrast, his fibroblasts were exclusively XY. The chimerism was confirmed by the analysis of the red blood cell antigens, which revealed the presence of 2 different populations. The characterization of the HLA haplotypes of the lymphocytes showed that the boy inherited 2 HLA haplotypes from his mother but only 1 from his father. Interestingly, the ratio of XX to XY cells was expanded in the T-cell subset compared with other peripheral blood mononuclear cell populations. CONCLUSIONS: This is an exceptional case of human chimerism revealed by abnormal skin pigmentation. This boy displayed 2 normal shades of skin color, which we suggest be termed cutis bicolor, as a result of 2 different genetic backgrounds. He also had immune chimerism, which challenges our current comprehension of antigen presentation and tolerance.

Photochemical treatment of plasma with amotosalen and UVA light: process validation in three European blood centers.

BACKGROUND: A photochemical treatment (PCT) process has been developed to inactivate pathogens and white blood cells (WBCs) in therapeutic plasma. Process validation studies were performed in three European blood centers under routine operating conditions. STUDY DESIGN AND METHODS: Each center prepared 30 apheresis and 30 to 36 whole blood-derived plasma units for PCT. Each whole blood-derived plasma unit contained a mixture of two to three matched donations. After removal of pretreatment control samples (control fresh-frozen plasma [C-FFP]), 546 to 635 mL of plasma was treated with 15 mL of 6 mmol per L amotosalen, 3 J per cm(2) UVA treatment, and removal of residual amotosalen with a compound adsorption device. After processing, plasma samples (PCT-FFP) were withdrawn, frozen at -60 degrees C within 8 hours of collection, and assayed for coagulation factors and residual amotosalen. RESULTS: A total of 186 units of plasma were processed. The mean prothrombin time (12.2 +/- 0.6 sec) and activated partial thromboplastin time (32.1 +/- 3.2 sec) of PCT-FFP were slightly prolonged compared to C-FFP. Fibrinogen and Factor (F)VIII were most sensitive to PCT (26% mean reduction). PCT-FFP, however, retained sufficient levels of fibrinogen (217 +/- 43 mg/dL) and FVIII (97 +/- 29 IU/dL) for therapeutic plasma. Mean levels of FII, FV, FVII, F IX, FX, FXI, and FXIII in PCT-FFP were comparable to C-FFP (81%-97% retention of activity). Antithrombotic proteins were not significantly affected by PCT with retention ranging between 83 and 97 percent. Mean residual amotosalen levels were 0.6 +/- 0.1 micromol per L. CONCLUSION: Process validation studies in three European centers demonstrated retention of coagulation factors in PCT-FFP within the required European and respective national standards for therapeutic plasma.

Plasma glycoprotein V levels in the general population: normal distribution, associated parameters and implications for clinical studies.

Soluble glycoprotein V (sGPV) is a new plasma marker of thrombosis released from the platelet surface by thrombin. sGPV levels are increased in patients with atherothrombotic diseases, but the determinants of sGPV levels are unknown in the general population. Identification of these potential confounding factors is needed for correct design and analysis of clinical studies on cardiovascular diseases. The aim of this study was to determine the normal range of plasma values and the factors controlling sGPV levels in a population of normal individuals. Three hundred blood donors were recruited at the Etablissement Français du Sang-Alsace for the measurement of plasma levels of sGPV, platelet factor 4 (PF4), thrombin-antithrombin complexes (TAT) and D-dimers. The plasma level of sGPV was (median [interquartile range]) 27.5 [23.5-34.4] microg/l and displayed a Gaussian distribution. sGPV had a lower interindividual coefficient of variation (33%) than PF4 (176%), TAT (87%) or D-dimers (82%). sGPV levels were independent of age and sex but sensitive to red cell (r = 0.412; p < 0.0001) and platelet counts (r = 0.267; p = 0.001), total cholesterol (r = -0.313; p < 0.0001), food intake (r = 0.184; p = 0.0014) and smoking (r = -0.154; p = 0.039). Contrary to PF4 and TAT, sGPV did not differ between venous and arterial blood samples of 12 healthy individuals. Red cell and platelet counts, total cholesterol, current smoking and recent food intake are important determinants of sGPV levels and must be taken into account in clinical studies using sGPV as a thrombosis marker. Normal distribution of sGPV levels in the general population supports its use in clinical applications.

Soluble platelet glycoprotein V is a marker of thrombosis in patients with ischemic stroke.

BACKGROUND AND PURPOSE: The diagnosis and management of patients with acute ischemic stroke still lack an informative biochemical test. Soluble glycoprotein V (sGPV) is a new plasmatic marker of thrombosis released from the platelet surface by thrombin. The objective of this prospective study was to compare the levels of sGPV in stroke and control patients. METHODS: Consecutive patients with acute ischemic stroke (n=159) and controls (n=70) were recruited for sGPV measurement. RESULTS: Plasmatic levels of sGPV were significantly increased in ischemic stroke compared with control patients (median [interquartile range] 39.4 [31.8 to 52.9] versus 28.1 [24.0 to 37.9] ng/mL; P<0.0001). In multivariate analysis, ischemic stroke was the major determinant of the sGPV increase (odds ratio [95% CI], 5.87 [2.62 to 13.12]; P<0.0001). CONCLUSIONS: sGPV is a new marker of arterial thrombosis and represents a tool to study the mechanisms involved in ischemic stroke. The increase in plasmatic sGPV observed in stroke patients supports a role of platelets and thrombin in the events leading to ischemic stroke.

[Platelets and arterial thrombosis]. / Plaquettes et thrombose artérielle.

The pathological mechanisms involved in arterial thrombus formation are similar to the mechanisms involved in physiological hemostasis. Arterial thrombosis is initiated following lesion of the vessel wall, either through rupture of an atherosclerotic plaque containing lipids, adhesive proteins and tissue factor or after angioplasty exposing the thrombogenic subendothelial matrix. Platelets play a major role in arterial thrombus formation through ADP secretion and thrombin generation on their activated surface. Arterial thrombosis is a frequent complication of atherosclerotic lesions and leads to acute ischemic events. These events are therapeutic emergencies which require administration of antithrombotic drugs inhibiting platelet functions and thrombin.