Immune-dependent thrombocytopaenia in mice infected with Schistosoma mansoni.

As has been shown previously, immunologically intact mice with patent Schistosoma mansoni infections had a significantly lower mean platelet number than intact uninfected mice (P<0.0001). However, platelet numbers in T-cell deprived mice with patent infections were not significantly different from those in uninfected T-cell deprived mice. Also, platelet counts in both the infected and uninfected T-cell deprived groups were not significantly different from those in intact uninfected mice. The S. mansoni-induced thrombocytopaenia in mice is thus seemingly immune dependent. Immunologically intact mice with chronic 12-week-old S. mansoni infections had IgG antibodies that were reactive in an ELISA-type assay with whole fixed platelets of both mouse and human origin. In Western immunoblots the IgG antibodies from chronically-infected mice reacted in particular against mouse and human platelet antigens of 90, 37 and 30 kDa. Antisera raised from 2 rabbits, immunized respectively with mouse and human platelet antigens, cross-reacted with antigens of the larval, adult worm and egg stages of S. mansoni. These results support the hypothesis that an anti-platelet antibody response may be the cause of the thrombocytopaenia observed in mice with patent schistosome infections.

Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium.

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.

Schistosoma mansoni egg attachment to cultured human umbilical vein endothelial cells: an in vitro model of an early step of parasite egg excretion.

To model the attachment of Schistosoma mansoni eggs to the endothelium of the mesenteric vasculature, the interaction between S. mansoni eggs and cultured human umbilical vein endothelial cells (HUVEC) in vitro was investigated. S. mansoni eggs rapidly attached to monolayers of both HUVEC and bovine aortic endothelial cells but more slowly to monolayers of cultured fibroblasts and smooth muscle cells. While both native and glutaraldehyde-fixed eggs attached equally well to HUVEC, eggs attached only to live, metabolically active HUVEC. Attachment was enhanced by both serum and plasma factors. In addition, platelet release products increased egg attachment by 75%. Preincubation of S. mansoni eggs with soluble egg antigens promoted attachment; in contrast, preincubation of HUVEC with the antigens inhibited attachment. These results suggest that interaction of S. mansoni eggs with HUVEC is an active process that can be modulated by molecules secreted by the egg and by platelets during egg extravasation.

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.

A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells.

Cleavage by thrombin of the platelet thrombin receptor exposes a new N-terminal segment SFLLRNPNDKYEPF (SFLL) which acts as a tethered ligand. The free peptide activates platelets and induces platelet aggregation. We now show that SFLL can also activate human umbilical vein endothelial cells (HUVEC) and induce rises in both cytosolic free calcium ([Ca2+]i) and prostacyclin (PGI2) production. These responses were time- and concentration-dependent and were similar to those for native thrombin except that they were not blocked by hirudin. Initial activation of HUVEC with thrombin desensitized the subsequent response to SFLL for both rises in [Ca2+]i and PGI2 production. Thus, SFLL alone can activate HUVEC and elevate [Ca2+]i and induce PGI2 production suggesting that the thrombin receptors on platelet and endothelial cells are functionally and structurally similar.

Blood platelets and schistosome egg excretion.

The eggs of helminths of the Schistosoma genus require to be extravasated in order to continue the life cycle of the parasite. The possible mode by which this takes place was investigated in a mouse model. Suppression of platelet activity in Schistosoma mansoni-infected mice by administering rabbit anti-mouse platelet serum or a selection of "antiplatelet drugs" resulted in a significant reduction of parasite egg excretion. This reduction was best achieved when antiplatelet agents were administered just before the onset of parasite egg excretion. The association between parasite eggs and platelets was illustrated in vivo and in vitro where platelet aggregates on egg surfaces were seen in both light and electron microscopy. In addition, eggs that had been isolated from infected mouse tissues induced platelet aggregation in whole mouse blood, and this was inhibited by preincubation with the beta-lactam antibiotic, ticarcillin. Isolated eggs were also capable of inducing ex vivo platelet aggregation in mice, which was dependent on presensitization with eggs. These data suggest a role for platelets in the extravasation and excretion of parasite eggs in schistosomiasis.