Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant- induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine- xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.     

Graded responses of human neutrophils induced by serum-treated zymosan

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3'- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.     

Management of heparin resistance during cardiopulmonary bypass: the effect of five different anticoagulation strategies on hemostatic activation

OBJECTIVE: Attenuation of hemostatic activation is a central goal during CPB. However, this poses a problem in patients insensitive to heparin. The present investigation was performed to assess different strategies of managing patients with heparin resistance during CPB. DESIGN: A randomized, prospective clinical investigation. SETTING: A major European heart center. PARTICIPANTS: Five groups with 20 patients each were investigated. INTERVENTIONS: The groups were handled as follows: (1). maintenance of a target ACT, (2). maintenance of the target unfractionated heparin (UFH) level and supplementation of a UFH level-based strategy with (3). AT III, (4). the direct thrombin inhibitor r-hirudin, or (5). the short-acting platelet glycoprotein (GP) IIb/IIIa antagonist tirofiban. Platelet count and generation of contact factor XIIa, thrombin, and soluble fibrin were assessed. Samples were obtained before CPB and after CPB before protamine infusion. MEASUREMENTS AND MAIN RESULTS: There were no differences observed in the generation of factor XIIa. The UFH-based strategy and supplementation with AT III, r-hirudin, and tirofiban resulted in significantly reduced (p < 0.05) thrombin generation compared with ACT management. A significant reduction of fibrin formation was seen only in patients who received AT III, r-hirudin, or tirofiban supplementation to the UFH. The administration of tirofiban resulted in a significant preservation of the platelet count compared with the other groups. There were no significant differences in the postoperative blood loss. CONCLUSIONS: Activation of hemostasis during CPB in heparin-resistant patients most likely has to be attributed to stimulation of the tissue factor pathway. Even the sole use of high concentrations of UFH does not effectively inhibit this activation. Therefore, in these patients anticoagulation during CPB with UFH should be supplemented with either AT III, a short-acting direct thrombin inhibitor, or a short-acting platelet glycoprotein IIb/IIIa antagonist.     

Hemostatic activation and inflammatory response during cardiopulmonary bypass: impact of heparin management

BACKGROUND: Cardiac surgery involving cardiopulmonary bypass (CPB) leads to fulminant activation of the hemostatic-inflammatory system. The authors hypothesized that heparin concentration-based anticoagulation management compared with activated clotting time-based heparin management during CPB leads to more effective attenuation of hemostatic activation and inflammatory response. In a randomized prospective study, the authors compared the influence of anticoagulation with a heparin concentration-based system (Hepcon HMS; Medtronic, Minneapolis, MN) to that of activated clotting time-based management on the activation of the hemostatic-inflammatory system during CPB. METHODS: Two hundred elective patients (100 in each group) undergoing standard cardiac surgery in normothermia were enrolled. No antifibrinolytic agents or aprotinin and no heparin-coated CPB systems were used. Samples were collected after administration of the heparin bolus before initiation of CPB and after conclusion of CPB before protamine infusion. RESULTS: There were no differences in the pre-CPB values between both groups. After CPB there were significantly higher concentrations ( < 0.05) for heparin and a significant reduction in thrombin generation (25.2 +/- 21.0 SD vs. 34.6 +/- 25.1), d-dimers (1.94 +/- 1.74 SD vs. 2.58 +/- 2.1 SD), and neutrophil elastase (715.5 +/- 412 SD vs. 856.8 +/- 428 SD), and a trend toward lower beta-thromboglobulin, C5b-9, and soluble P-selectin in the Hepcon HMS group. There were no differences in the post-CPB values for platelet count, adenosine diphosphate-stimulated platelet aggregation, antithrombin III, soluble fibrin, Factor XIIa, or postoperative blood loss. CONCLUSION: Compared with heparin management with the activated clotting time, heparin concentration-based anticoagulation management during CPB leads to a significant reduction of thrombin generation, fibrinolysis, and neutrophil activation, whereas there is no difference in the effect on platelet activation. The generation of fibrin even in the presence of high heparin concentrations most likely has to be attributed to the reduced antithrombin III concentrations or reduced inhibition of clot-bound thrombin. Therefore, in addition to maintenance of higher heparin concentrations, monitoring and substitution of antithrombin III should be considered to ensure more efficient antithrombin activity during CPB.     

The activation of the contact phase of coagulation by physiologic surfaces in plasma: the effect of large negatively charged liposomal vesicles

The endogenous, negatively charged surface that induces activation of the contact coagulation factors was investigated in plasmas taken from women in late pregnancy and control subjects of child-bearing age. The plasmas from the two groups of subjects were incubated at 4 degrees C for 24 hours either in plastic or in glass tubes and the factor VII coagulant activity (VIIc) was assayed in the treated plasmas. The activation of factor VII under these conditions involves the generation of enzymes derived from factor XII (XIIa). The contact surface is rate- limiting for the activation of factor VII in the plasmas in both groups of subjects and can be supplemented by large multilamellar liposomal vesicles carrying the appropriate density of negative charge. The size of these vesicles is within the range of sizes of the large lipoprotein particles (chylomicrons, very low and intermediate-density lipoproteins). The relationship between the density of negative charge on the liposomal vesicles and VIIc was similar in the late pregnancy and the control plasmas incubated in plastic tubes. At a saturating density of negative charge the observed relative VIIc was similar in both sets of plasmas. The incubation of late pregnancy or control plasma in plastic tubes in the presence of sodium stearate caused VIIc to increase with increasing concentration of the added fatty acid. These results suggest that large lipoprotein particles carrying the appropriate free fatty acid at a sufficient density of negative charge could provide the contact surface that induces the generation of factor XIIa and the subsequent activation of factor VII. Moreover, plasmas from women in late pregnancy have a higher concentration of potential surface and a higher density of negative charge than the plasmas from nonpregnant women.     

冠状循环药理学

直到最近为止,冠心病的内科治疗原则仍是降低心肌需氧量。由于粥样硬化病变一直认为是固定不变的狭窄,不可能增加冠脉血流量,因此认为用药物扩张冠状动脉是徒劳的。在近几年内这些概念有了基本转变。然而仅能引起远端心肌内小动脉扩张的药物却起着相反的作用。当心外膜冠状动脉有粥样硬化时,冠状小动脉扩张可加重血流分布异常,导致心肌缺血。冠状循环由近端大冠状动脉及其分枝小动脉组成的心肌内动脉网构成。近端大冠状