• Extracellular Hb alters the GPIba-VWF interaction.Intravascular hemolysis occurs in patients on extracorporeal membrane oxygenation. High levels of free acellular adult hemoglobin (free HbA) are associated with clotting in this mechanical device that can result in thrombotic complications. Adsorption of fibrinogen onto the surface of biomaterial correlates with platelet adhesion, which is mediated by von Willebrand factor (VWF). Because free Hb interacts with VWF, we studied the effect of hemoglobin (Hb) on platelet adhesion to fibrin(ogen) under conditions of different hydrodynamic forces. This effect was investigated using purified human HbA and fibrinogen, extracellular matrix, collagen, or purified plasma VWF as surface-coated substrates to examine flow-dependent platelet adhesion. Antibodies and VWF-deficient plasma were also used. Free Hb ( ‡50 mg/dL) effectively augmented platelet adhesion, and microthrombi formation on fibrin(ogen), extracellular matrix, and collagen at high shear stress. The effect of free Hb was effectively blocked by anti-glycoprotein Iba (GPIba) antibodies or depletion of VWF. Unexpectedly, free Hb also promoted firm platelet adhesion and stable microthrombi on VWF. Lastly, we determined that Hb interacts directly with the A1 domain. This study is the first to demonstrate that extracellular Hb directly affects the GPIba-VWF interaction in thrombosis, and describes another mechanism by which hemolysis is connected to thrombotic events. (Blood. 2015; 126(20):2338-2341 IntroductionThe excessive release of hemoglobin (Hb) from erythrocytes into the circulation of patients on mechanical circulatory support devices is a well-recognized major clinical complication.1 Increasing incidence of hemolysis and thrombosis is associated with morbidity and mortality in patients on extracorporeal membrane oxygenation (ECMO).2 Prevention of circuit clotting in ECMO can improve clinical outcome.von Willebrand factor (VWF) is a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation under high flow conditions. 3-7 Plasma VWF mediates platelet adhesion to surfaces coated with fibrin(ogen), 8,9 which is adsorbed onto surfaces of many materials used in biomedical instruments, including ECMO.10,11 Previously, we reported that free Hb interacts with the A2 domain of VWF 12 and, moreover, we and many others have described that the A2 domain regulates the binding of its neighboring A1 domain in VWF to platelet receptor glycoprotein Iba (GPIba).13-15 Thus, in this study, we examined the effect of the free Hb-VWF interaction on mediating platelet adhesion to immobilized fibrin(ogen) at high shear stress; a mechanism not previously investigated. Study design ReagentsPurified Hb and plasma VWF were obtained using established methods.13,16 Human collagen type III was purchased from Advanced BioMatrix, human fibrinogen from Calbiochem, and extracellular matrix (ECM) from Sigma-Aldrich. Anti-GPIba antibody 6D1 was a gift from Dr Barry Coller (The Rockefeller University, New Yor...
An association between alcoholism and abnormal red blood cell (RBC) size and shape has long been recognized (1). However, the underlying pathophysiologic mechanisms responsible for the morphologic alterations are incompletely understood (2-6). Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been shown to be incorporated into phospholipid bilayers up to 30 mol % of total membrane fatty acids (7). FAEEs have been implicated as mediators of ethanolinduced organ damage (8-13) and have clinical utility as markers for ethanol intake (14, 15). Serum and plasma FAEE levels closely correlate with blood ethanol levels, but these circulating FAEEs in blood persist for at least 24 h after ethanol ingestion, long after ethanol is no longer detectable (15, 16).Although the largest reservoir of FAEE synthetic capability appears to reside in the pancreas and the liver (17, 18), enzyme activity that catalyzes FAEE synthesis has been detected in RBCs, white blood cells (WBCs), and platelets. Given the much higher concentration of RBCs in the blood, it has been suggested that RBCs may provide a significant portion of the total FAEE synthase activity in whole blood (19). With this information, the possibility that RBC membranes might synthesize and sequester FAEEs in the blood has been raised.We conducted a controlled clinical trial to determine if FAEEs accumulate and persist within RBCs following ethanol ingestion. The objectives of this study were to determine: 1 ) whether FAEEs appear in RBCs following ethanol ingestion and whether RBC-associated FAEEs correlate with blood ethanol concentration; 2 ) how long FAEEs persist in RBCs after ethanol intake is discontinued; 3 ) whether the fatty acid composition of RBC FAEEs is distinct from the fatty acid composition of FAEEs in plasma; and 4 ) whether RBC and plasma FAEE species undergo a remodeling process with changes in fatty acid composition following discontinuation of ethanol ingestion. METHODSThe study was approved by the institutional review board of the Massachusetts General Hospital. A written informed consent was obtained from each volunteer prior to subject enrollment.Abbreviations: AIC, akaike information criteria; E16:0, ethyl palmitate; E18:0, ethyl stearate; E18:1, ethyl oleate; E18:2, ethyl linoleate; FAEE, fatty acid ethyl ester; GC-MS, gas liquid chromatography-mass spectrometry; RBC, red blood cell; SPE, solid-phase extraction; WBC, white blood cell.
Predicting the risk of bleeding or thrombosis in cirrhotic patients is difficult due to reduced levels and dysregulation of both procoagulant and anticoagulant factors. We utilized thrombin generation and microvesicle analysis to better understand the regulation of haemostasis in cirrhotic patients. We studied 24 patients with cirrhosis vs. 21 healthy controls. Cirrhotic patients had reduced prothrombin activity (40 ± 9 vs. 112 ± 15), protein C activity (36 ± 10 vs. 114 ± 19) and antithrombin activity (43 ± 14 vs. 109 ± 10). Peak thrombin generation was reduced in cirrhotic patients (165 ± 47 vs. 232 ± 101), but the ratio of peak thrombin generation to prothrombin activity was increased in cirrhotic patients (4.2 ± 1.0 vs. 2.1 ± 0.9) indicating a relative increase in thrombin generation in cirrhosis. The termination time ratio was increased in cirrhotic patients (7.2 ± 1.9 vs. 3.1 ± 0.7) and correlated with reduced antithrombin levels, indicating that cirrhotic patients took longer to stop thrombin generation than controls. Cirrhotic patients showed reduced procoagulant microvesicles from platelets (39 500 ± 24 800 vs. 107 700 ± 74 200) and other cells, but levels overlapped with controls. Cirrhotic patients showed a wide range of procoagulant and anticoagulant levels leading to variability in the regulation of thrombin generation. In conclusion, compared with healthy controls, patients with cirrhosis have lower antithrombin levels that lead to slower downregulation of thrombin generation and more overall thrombin being produced for a given procoagulant level in blood, but also low normal levels of procoagulant microvesicles that would slow initiation of thrombin generation. Whether an individual cirrhosis patient is at a greater risk of bleeding vs. thrombosis may depend on their specific imbalance in procoagulants vs. anticoagulants.
PIVKA-II seems to be a sensitive indicator of mild VK deficiency. Further studies are needed to investigate the lack of relationship between PIVKA-II and functional protein C or S levels.
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