Migration of monocytes across endothelium and passage through extracellular matrix involve separate molecular domains of PECAM-1.

During the inflammatory response, the adhesion molecule PECAM plays a crucial role in transendothelial migration, the passage of leukocytes across endothelium. We report here an additional role for PECAM in the subsequent migration of monocytes through the subendothelial extracellular matrix. PECAM has six immunoglobulin (Ig) superfamily domains. Monoclonal antibodies whose epitopes map to domains 1 and/or 2 selectively block monocyte migration through the endothelial junction, whereas those that map to domain 6 block only the migration through the extracellular matrix, trapping the monocyte between the endothelium and its basal lamina. Therefore, transendothelial migration (diapedesis) and passage through extracellular matrix (interstitial migration) are distinct and separable phases of monocyte emigration. Furthermore, distinct and separate Ig domains of PECAM are involved in mediating these two steps.

Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase. Thromboregulation by endothelial cells.

We previously reported that platelets become unresponsive to agonists when stimulated in combined suspension with aspirin-treated human umbilical vein endothelial cells. Inhibition occurred concomitant with metabolism of platelet-derived endoperoxides to prostacyclin by endothelial cells. We now demonstrate that if aspirin-treated platelets which fully respond to appropriate doses of agonists are exposed to aspirin-treated endothelial cells, they remain unresponsive despite absence of prostacyclin. Platelet inhibition is due in large part to ecto-ADPase activity on the endothelial cells. This was established by incubating aspirin-treated endothelial cells with 14C-ADP. Radio-thin layer chromatography and aggregometry demonstrated that 14C-ADP and induction of platelet activation decreased rapidly and concurrently. AMP accumulated transiently, was further metabolized to adenosine, and deaminated to inosine. The apparent Km of the endothelial cell ADPase was 33-42 microM and the Vmax 17-43 nmol/min per 10(6) cells, values in the range of antithrombotic potential. Thus, at least three complementary systems in human endothelial cells control platelet responsiveness: a cell-associated, aspirin-insensitive ADPase which functions in parallel with fluid phase autacoids such as the aspirin-inhibitable eicosanoids, and the aspirin-insensitive endothelium-derived relaxing factor.

Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment.

Erythrocytes are known to influence hemostasis. Bleeding times are prolonged in anemia and corrected by normalizing the hematocrit. We now demonstrate that intact erythrocytes modulate biochemical and functional responsiveness of activated platelets. A two-stage procedure, permitting studies of cell-cell interactions and independently evaluating platelet activation and recruitment within 1 min of stimulation, was developed. Erythrocytes increased platelet serotonin release despite aspirin treatment, enzymatic adenosine diphosphate removal, protease inhibition, or combinations thereof. The data suggested that erythrocyte enhancement of platelet reactivity can reduce the therapeutic effectiveness of aspirin. Erythrocytes metabolically modified platelet arachidonate or eicosapentaenoate release and eicosanoid formation. They promoted significant increases in cyclooxygenase and lipoxygenase metabolites upon platelet stimulation with collagen or thrombin. However, with ionophore, erythrocytes strongly reduced platelet lipoxygenation. These erythrocyte modulatory effects were stimulus-specific. Activated platelet-erythrocyte mixtures, with or without aspirin, promoted 3-10-fold increases in extracellular free fatty acid, which would be available for transcellular metabolism. Erythrocyte-induced increases in free eicosapentaenoate may contribute to antithrombotic and anti-inflammatory effects of this fish oil derivative. These results provide biochemical insight into erythrocyte contributions to thrombosis and hemostasis, and support the concept of thrombus formation as a multicellular event.

Error propagation as a factor in selection of measurement intervals for the determination of polycyclic aromatic hydrocarbons by second-derivative spectrofluorimetry.

The most suitable wavelength intervals were selected for the determination of 4 polycyclic aromatic hydrocarbons (PAHs; benzo[g,h,i]perylene, dibenzo[a,h]anthracene, pyrene, and triphenylene) in very complex mixtures of 11 PAHs: anthracene, benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene, benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene, phenanthrene, pyrene, and triphenylene. The multiple linear regression algorithm was applied to measurements made in several wavelength intervals previously selected on the basis of sensitivity and minimum number of interfering compounds. Of the different models obtained, those displaying minimum error propagation in the analytical result were selected. By applying the models proposed in this study, we precisely and accurately determined benzo[g,h,i]perylene, dibenz[a,h]anthracene, pyrene, and triphenylene in complex mixtures--a feat that could not be achieved by the use of constant-wavelength spectrofluorimetry in combination with second-derivative techniques.

Inhibition of human platelet reactivity by endothelium-derived relaxing factor from human umbilical vein endothelial cells in suspension: blockade of aggregation and secretion by an aspirin-insensitive mechanism.

To determine a role for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) in regulation of human platelet reactivity by human endothelial cells (EC), we studied combined suspensions of human umbilical vein endothelial cells (HU-VEC, passage 2 through 3) and washed human platelets. Confluent HUVEC monolayers were treated with aspirin (1 mmol/L) to prevent prostacyclin (PGI2) formation, washed, and harvested. Aspirin-treated platelets alone (58 x 10(6)) were fully aggregated by thrombin at 0.05 U/mL or more. In the presence of 10(6) HUVEC, however, platelet serotonin release and aggregation in response to thrombin at doses as high as 0.5 U/mL were blocked. We demonstrated for the first time that inhibition of aggregation and serotonin release, due to EDRF/NO, occurred in parallel. HUVEC-dependent inhibition of platelet responsiveness was enhanced by superoxide dismutase (SOD) and reversed by hemoglobin. The inhibitory effect was also reversed by preincubation of HUVEC with NG-monomethyl-L-arginine (NMA) or NG-nitro-L-arginine (NNA) through competitive blockade of arginine metabolism. Pretreatment of platelets with methylene blue indicated that EC-dependent inhibition of platelet reactivity occurred through activation of platelet soluble guanylate cyclase. When platelets and HUVEC were separated by a permeable membrane and both cells were stimulated by thrombin, platelets remained unresponsive. This indicated that inhibition was induced by a fluid-phase mediator, independent of direct cell-cell contact. These data demonstrate that EDRF/NO formation from L-arginine by human EC plays an important role as an aspirin-insensitive fluid-phase inhibitor of human platelet reactivity.

Albumin redirects platelet eicosanoid metabolism toward 12(S)-hydroxyeicosatetraenoic acid.

Albumin is a major determinant of eicosanoid formation, affecting autacoids important in cell-cell interactions. We delineated three mechanisms by which albumin controlled platelet eicosanoid formation: 1) Albumin diverted free arachidonate toward 12-lipoxygenation. 2) Albumin enhanced release of arachidonate from phospholipids. 3) Albumin inhibited incorporation of arachidonate from the medium into platelet phospholipids. 12(S)-Hydroxyheptadecatrienoic acid (12-HHTrE) formation was reduced 70% by albumin as compared to that formed in albumin-free medium. In sharp contrast, formation of 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the platelet lipoxygenase product, was much less influenced by albumin. Moreover, 12-HETE production in the presence of albumin was markedly increased and prolonged after aspirin treatment. These data suggested that albumin redirected released endogenous arachidonate from cyclooxygenase to lipoxygenase. Therefore, the metabolic fate of arachidonate present in the medium of stimulated platelets was studied by adding tracer [3H]arachidonate 30 sec before thrombin. Albumin increased arachidonate metabolism by lipoxygenase 7-fold as compared to albumin-free controls, while cyclooxygenation increased 2.7-fold. Redirection of eicosanoid metabolism by albumin toward lipoxygenase products constitutes a heretofore undescribed and potentially important physiological role for albumin. In vitro utilization of albumin may reflect in vivo events in thrombosis and hemostasis more accurately than previous studies without albumin could appreciate.

Resolution of benzo[a]pyrene in complex mixtures of other polycyclic aromatic hydrocarbons. Comparison of two spectrofluorimetric methods applied to water samples.

Two spectrofluorimetric methods, second-derivative constant-energy synchronous luminescence (SDCESL) and constant-wavelength synchronous luminescence (CWSL) in combination with multiple linear regression (MLR), for the quantification of benzo[a]pyrene (BaP) at sub-ng mL-1 levels, in the presence of benzo[b]fluoranthene (BbFt), benzo[k]fluoranthene (BkFt), benzo[ghi]perylene (BghiP) and indeno[1,2,3-cd]pyrene (IP), were developed and compared in detail. SDCESL presents lower limits of detection and quantification than CWSL/MLR and also gives more exact and precise results for levels close to the quantification limit. For BaP, SDCESL achieved quantification limits of 0.019 ng mL-1 in river waters and 0.007 ng mL-1 in drinking waters. This work offers a sensitive, precise, accurate, rapid, simple and economic methodology for monitoring BaP in waters for public consumption, meeting all the requirements of the EC Directive 98/83/CE that fixes the maximum admissible limit for this polycyclic aromatic hydrocarbon in drinking waters at of 0.010 ng mL-1.

Determination of polycyclic aromatic hydrocarbons (PAHs) in a complex mixture by second-derivative constant-energy synchronous spectrofluorimetry.

In this work, the combination of the excellent band narrowing features of derivative spectrometry with constant-energy synchronous fluorescence spectrometry for the simultaneous determination of different polycyclic aromatic hydrocarbons (PAHs) in a mixture of 18 is assessed. This mode of scanning allowed for the identification and quantification of 10 PAHs and the grouping of some of these.

Fast sequential injection determination of benzo[A]pyrene using variable angle fluorescence with on-line solid-phase extraction.

A methodology for the analysis of drinking water for one of the most potent carcinogenic agents known; benzo[a]pyrene (BaP), in the presence of other interfering PAHs is presented. The methodology described is based on the sequential injection analysis of the sample on to a microcolumn (containing 5 mg of C18) where extraction and preconcentration of BaP takes place, followed by elution of BaP with 1 ml of 1,4-dioxane and subsequent detection by using variable angle fluorescence. The advantages of the method include the small amount of stationary phase employed together with the possibility of re-using the phase in order to carry out a large number of injections without the need for column re-packing. Also noteworthy is the small volume of 1,4-dioxane used to elute the BaP retained on the column and the small sample volumes required (9-10 ml) for achieving detection limits at the ng l-1 level. Thus, a methodology for BaP determination is obtained which complies with the requirements of the 98/83/EC Directive which fixes a maximum admissible concentration for this pollutant in waters for public consumption of 10 ng l-1. The variable angle spectra obtained are further processed by means of the multiple linear regression technique. The detection limit for BaP is 2.5 ng l-1, and the linear range is between 7.5 and 280 ng l-1.