Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)γ1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC’s activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.

The origin of platelet a-granule fibrinogen (Fg), whether from endogeneous synthesis or exogeneous derivation, remains unknown. Although Fg biosynthesis by megakaryocytes (MK) has been suggested, recent studies have demonstrated that certain a-granular proteins originate primarily from plasma. To study the origin of a-granule Fg, platelet-associated Fg was measured by ELISA and Western blotting, and localized by immunofluorescence and immunoelectron microscopy in a patient with symptomatic congenital afibrinogenemia before and after replacement therapy with cryoprecipitate. a-Granule Fg was detected in the majority of platelets as early as 24 h postinfusion, suggesting that direct platelet uptake was occurring. Platelet Fg reached a maximum value of 42.5% of normal values at 3 d postinfusion and was localized in the a-granules, while plasma levels followed a typical half-life profile. Significant a-granule Fg was still detectable at 13 d postinfusion, with plasma Fg virtually absent. Studies on cultured CFU-MKs from the patient also confirmed that MKs can incorporate exogeneous Fg into a-granules. These results indicate that platelet a-granule Fg can be derived from the circulating plasma pool and that Fg uptake can occur in both platelets and MKs.

Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.

Qualitative/quantitative analysis of von Willebrand factor antigen (vWf:Ag) in either heat or solvent/detergent treated factor VIII concentrates, used for haemophilia replacement therapy, was undertaken to assess their suitability for the treatment of vWD. For the first time immunoaffinity purified vWf:Ag (Monoclate by-product) was also evaluated by in vitro assessment. Potencies of vWf:Ag varied considerably but were consistently higher (28.9-420.5 iu/ml) than factor VIII:C (one-stage) activity (8.13-42.44 iu/ml). The functional activity of vWf was assessed by either Ristocetin Cofactor (vWf:RCo) or collagen binding methods (vWf:CBA) with typical vWf:RCo/vWf:Ag ratios ranging from 0.08 to 0.94. Multimeric analysis confirmed that in vitro biological activity was dependent on the presence of the high molecular weight forms of vWf:Ag. A significant correlation (r = 0.95) between vWf:RCo activity and collagen binding was observed in all of the concentrates with the exception of the immunopurified product. The data suggest that either NHS 8Y (mean vWfRCo/vWf:Ag = 0.94), Haemate P (mean vWf:RCo/vWf:Ag = 0.69) and high purity Octapharma V.I (vWf:RCo/vWf:Ag = 0.82) which contain medium/high MW vWf:Ag multimers are likely to be most cost-effective in the treatment of symptomatic severe vWD patients than other currently available concentrates.

We have previously shown that the engagement of ICAM-1 on brain endothelial cells (EC) results in the propagation of EC signaling pathways that are necessary for efficient lymphocyte migration across the tight vascular barriers of the brain. Signaling via this receptor alone, however, is unlikely to explain the differential recruitment of leukocytes at different vascular beds. In this study, we investigated the role of EC heterotrimeric G-protein-mediated signaling in supporting transendothelial migration of T lymphocytes. Treatment of brain EC monolayers with pertussis toxin (PTX) resulted in ADP-ribosylation of G-protein alpha subunits and inhibition (>80%) of lymphocyte migration without affecting lymphocyte adhesion. Aortic and high endothelial venule EC treated identically resulted in only partial inhibition of lymphocyte migration (<40%). Expression of ribosylation-resistant (PTX-insensitive) G-protein alpha subunits in brain EC restored their ability to support lymphocyte migration after pretreatment with PTX. Treatment of brain EC with PTX did not inhibit ICAM-1-stimulated tyrosine phosphorylation of focal adhesion kinase, suggesting the effects of PTX in inhibiting EC facilitation of lymphocyte migration are distinct from activation of EC through ICAM-1. We conclude that a heterotrimeric G-protein-mediated signaling pathway in brain EC is essential for efficient transendothelial migration of T lymphocytes into the brain.

Factor III (thromboplastin) activity is inhibited by apoB-100, but the mechanism of inhibition is unknown. By examining the effect of purified apoB-100 on factor III activity, we showed that apoB-100 can inhibit factor III via a different mechanism from that caused by the issue-factor pathway-inhibitor, which is mainly carried on the surface of lipoproteins. Although the presence of calcium ions and factors X and VII may enhance the rate of inhibition, they are not a prerequisite for the inhibition of factor III by apoB-100. In addition, by investigating the changes in the UV spectra of apoB-100 on interaction with factor III and factors X and VII and by assigning the shifts in absorption spectra to particular amino acids, we showed that these interactions involve negative and positive residues within these proteins. By following the rates of interactions between apoB-100 and either factors III, X, VII, a two-step mechanism for the inhibition process involving factors X and VII was postulated. In this mechanism, the primary interaction of apoB-100 with factor III is followed by a rate-limiting step that can be accelerated by the presence of either factor X or VII and leads to the inhibition of factor III. Furthermore, a computer-based analysis of the sequences of factor III revealed a possible binding site for apoB-100.

Abstract-The procoagulant activity of tissue factor is regulated by circulating inhibitors such as tissue factor pathway inhibitor (TFPI) and LDL. These 2 inhibitors also readily associate making the distinction between their activities difficult. We have examined the relative contributions of intact and C-terminal truncated TFPI and ApoB100. By following the inhibitory potential of the preparations, over a period of 120 minutes, it was demonstrated that TFPI and LDL-resembling particles inhibited tissue factor at different rates. TFPI was found to be a short, fast-acting inhibitor, whereas the action of LDL-resembling particles was more prolonged but slower. The oxidation of LDL has been closely associated with the development of cardiovascular disease, including atherosclerosis and thrombosis. Positively charged amino acids, particularly lysine residues, are prone to alterations via the formation of adducts by lipid peroxidation products. These residues are important in the inhibition of tissue factor activity by ApoB100. They also play an important role in the inhibitory Kunitz domains of TFPI. We have shown that the decline in the ability of LDL to inhibit tissue factor was as a result of modifications in LDL arising from oxidation. By examining the effects of oxidation on full-length and C-terminal truncated TFPI bound to LDL-resembling particles, we found that TFPI is only affected when in close association with ApoB100. C-terminal truncated TFPI was not affected significantly by oxidation. Finally, chemical modification of lysine and arginine residues reduced the overall inhibition of tissue factor by TFPI. We propose that TFPI and LDL act separately to inhibit tissue factor in vivo. However, the oxidation of LDL can alter both the endogenous activity of ApoB100 and reduce that of closely associated TFPI, compromising normal hemostasis.

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