The decreased bioavailability of arginine (Arg) and the resulting lower nitric oxide (NO) production has been shown to be an important factor in the pathology of sickle cell disease (SCD) and thalassemia. Vascular alterations leading to pulmonary hypertension are important factors of heart failure and death in these hemoglobinopathies. Red blood cell (RBC) hemolysis in these patients will release arginase into the circulation and contribute to the reduction of plasma Arg levels, change the Arg-to-ornithine ratio, and increase other downstream amino acid metabolites. Such compounds, including proline and polyamines, may contribute to vascular and airway remodeling. To study the contribution of arginase released from RBC, we measured the arginase activity and arginase protein concentration in the plasma and RBC lysates of normal controls, SCD patients, and thalassemia patients.

BACKGROUND: Although trauma patients often suffer direct lung damage, an equally destructive mechanism of lung injury involves postinjury systemic inflammation. We postulate that secretory phospholipase A, (sPLA,) release induced by trauma relates to systemic inflammation that compromises both lung function and clinical status after injury. The objectives of this study were: to relate Injury Severity Score to postinjury sPLA(2); to determine whether circulating sPLA, relates to pulmonary oxygenation and compliance; and to determine whether early or persistent increases in sPLA, are associated with abnormal chest x-ray at 72 hours after injury.STUDY DESIGN: The prospective cohort study comprised 54 consecutive intensive care admissions in patients with traumatic injury admitted over a 6-month period from November 1, 1996, to May 1, 1997.RESULTS: Postinjury peak sPLA(2) values were associated with increased ISS (r = 0.49, r(2) = 0.24, p < 0.001). Patients with elevated sPLA(2) had poor oxygenation compared with those with normal sPLA(2) levels (Pa0(2)/Fi0(2) ratio 164 +/- 16 versus 260 +/- 26 mmHg [mean +/- SEM], p < 0.01) and also required additional PEEP (5.5 +/- 0.9 versus 2.5 +/- 0.4 cm H2O, P = 0.01) Secretory PLA(2) levels in patients with abnormal chest x-ray 72 hours after injury were higher (1.08 +/- 0.2 versus 0.34 +/- 0.1 activity units, p < 0.001) than levels seen in patients with normal x-rays.CONCLUSIONS: Increasing injury magnitude is associated with elevated sPLA(2) levels, and increased sPLA(2) is related to postinjury hypoxemia and clinical status. (C) 2005 by the American College of Surgeons.

Secretory phospholipase A2 (sPLA2) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA2 will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA2, at levels of sPLA2 observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nm/108 RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 μm LPA, and the combination of PS-exposing red blood cells with PLA2 caused a similar increase in permeability. In the presence of N-palmitoyl l-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 μm LPA in vivo were abolished. The present study demonstrates that increased sPLA2 activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.

Previous studies from our institution have shown that serum secretory phospholipase A2 (sPLA2) levels are markedly increased in sickle cell disease (SCD) patients with acute chest syndrome (ACS)[1][1] and that sequential measurements of sPLA2 are useful in predicting the subsequent development of

Phosphatidylserine (PS) is found exclusively in the inner monolayer of plasma membranes. During programmed cell death this well-preserved asymmetry of phopholipid distribution is lost, and PS is exposed on the outer surface. PS exposure is also found in subpopulations of red blood cells (RBC) in disease states such as sickle cell disease (SCD). PS exposure leads to increased cell-cell interactions as well as recognition and removal by macrophages. PS serves as a binding site for factors involved in blood coagulation and provides a target for secretory phospholipase A2 (sPLA2) mediated membrane degradation. Measurement of PS-exposing cells (PSRBC) relies on the use of proteins that bind to PS and can be fluorescently labeled and visualized by flow-cytometry. The human protein annexin V (AV) has a high affinity for PS and has been shown to interfere with the physiologic processes induced by the exposure of PS. To improve detection of PS exposing cells, and develop a probe that may be used in vivo, we generated plasmids encoding a dimer of human AV (DAV), and expressed the protein in E.coli. We show that DAV has a 5–8 fold higher sensitivity than AV in binding to PSRBC, and effectively identifies PSRBC in SCD blood samples. Moreover, DAV protects PSRBC from sPLA2-induced hemolysis. While normal RBC show only 2% hemolysis after incubation with 100 ng/ml sPLA2 for one hour, sPLA2 induces more than 90% hemolysis in PSRBC. DAV, when added to PSRBC before incubation with sPLA2, protects more than 90% of the PSRBC from hemolysis. Our data show that DAV effectively competes with factor Va for PS binding and inhibits thrombin formation in the presence of the pro-thrombinase complex. At low concentrations, DAV was significantly more efficient in inhibiting thrombin formation than AV (P<0.001). The increased size of DAV gives the molecule a significantly increased survival time in the murine circulation compared to AV, and allows visualization of PS exposing membranes in vivo. Together this data shows that DAV is a superior probe to identify PS-exposing membranes, protects PSRBC from sPLA2-induced hemolysis, and interferes with cellular hemostasis. The use of DAV will allow more precise determination of PS exposure by flow-cytometry as compared to currently available commercial probes. Moreover, it will allow the measurement of PS exposing membranes in vivo, and may ultimately be proven to be useful as a therapeutic agent in SCD pathology by masking PS exposing cell membranes.

Introduction. Membrane phospholipids are distributed asymmetrically in normal cells, with phosphatidylserine (PS) confined to the inner monolayer. Externalization of PS is a marker for apoptosis and follows ischemia-reperfusion injury. Circulating levels of secretory phospholipase A2 (sPLA2) are elevated in sepsis, the acute respiratory distress syndrome, and following trauma, pointing at an active role of the enzyme in disease-associated tissue destruction. PLA2 hydrolyzes membrane phospholipids and is an early and rate-limiting step in the production of powerful inflammatory mediators. We hypothesized that (1) normal cells are not susceptible to sPLA2, but that (2) erythrocytes with externalized PS are hemolyzed by sPLA2 at clinically relevant concentrations, (3) binding the externalized PS would protect the cells from sPLA2-induced lysis, and also that (4) inhibition of PLA2 would protect cells with externalized PS from hemolysis. Methods. Erythrocytes were experimentally induced to expose PS. Normal erythrocytes and PS-exposing erythrocytes were exposed to sPLA2, and vulnerability was measured by flow cytometry and hemolysis. Annexin V, and its dimmer, DiAnnexin V, were tested as cytoprotective agents given their affinity for binding externalized PS. Finally, PX-18 was tested for its ability to inhibit enzymatic activity of sPLA2 and protect the PS-exposing cells. Results. Normal erythrocytes are not susceptible to sPLA2, however, sPLA2 hemolyzes PS-exposing erythrocytes at clinically relevant concentrations in a dose- and time-dependent fashion. Increase in hemolysis was inversely related to removal of PS-exposing cells, indicating that PLA2 targets externalized PS as a marker for cytotoxicity. Annexin V and Di-Annexin V effectively prevented PLA2-induced hemolysis at 7 nmol per 5 × 107 cells, confirming that PLA2 requires externalization and recognition of PS for cytotoxicity. At 10 μM, PX-18 reduced hemolysis of PS-exposing cells to baseline levels, indicating that sPLA2 inhibition also protects PS-exposing cells. Conclusion. Secretory PLA2 effectively hemolyzes PS-exposing erythrocytes. Cellular protection can be achieved both by binding PS and by inhibiting sPLA2 activity. These inhibitors may have therapeutic efficacy in inflammatory diseases.

Phosphatidylserine (PS), exclusively present in the inner monolayer of the normal red blood cell (RBC) membrane, is exposed in subpopulations of sickle cells. PS-exposing RBCs were found predominantly among the densest and the very light sickle cells. Within the light RBC fraction, PS exposure was found on reticulocytes, transferrin receptor-expressing reticulocytes, and mature RBCs. The last subset contained low-density valinomycin-resistant RBCs, previously shown to have high Na(+) and low K(+) content. This subpopulation contained the highest percentage of PS-exposing cells. The PS-exposing sickle cells did not show the sustained high cytosolic Ca(++) levels that have been shown to activate scramblase activity. Data from this study indicate that PS exposure can occur at different stages in the life of the sickle RBC and that it correlates with the loss of aminophospholipid translocase activity, the only common denominator of the PS-exposing cells. The additional requirement of scramblase activation may occur during transient increases in cytosolic Ca(++). (Blood. 2001;98:860-867)

Phosphatidylserine (PS) asymmetry was determined in red blood cells from patients with hereditary spherocytosis and elliptocytosis. No PS-exposing subpopulations were detected using the very sensitive method with fluorescently labeled annexin V. Treatment with N-ethylmaleimide or adenosine triphosphate (ATP) depletion to inactivate the flipase did not lead to formation of PS-exposing subpopulations in these cells, but elevated intracellular calcium levels did lead to extensive scrambling of the PS asymmetry. Although interactions of the membrane skeleton with the phospholipid bilayer have been suggested to stabilize the asymmetric distribution of PS across the bilayer, our data show that red blood cells with a severely damaged membrane skeleton are able to preserve asymmetry, even under conditions in which restoration of the asymmetric distribution is excluded. Moreover, the loss of membrane asymmetry in these cells requires active scrambling involving high levels of intracellular calcium as in normal cells. Our data show that the severe disorder of the membrane skeleton found in these cells does not affect the activity of flipase or scramblase, indicating that these proteins are not regulated by, nor coupled to the membrane skeleton assembly, and that possible thrombotic events in spherocytosis patients are not likely associated with altered PS topology of the red blood cells.