Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3–92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC₅₀) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.     

Human neutrophils express messenger RNA of vitamin D receptor and respond to 1alpha,25-dihydroxyvitamin D3

1Alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to modulate the production of various cytokines or the expression of certain differentiation markers in human T cells or monocytes. Its effects on neutrophils, however, are poorly understood. In this paper, we show several lines of evidence indicating that neutrophils express functional vitamin D receptors (VDR). Sort-purified neutrophils from human peripheral blood expressed VDR mRNA at a level comparable to that of monocytes. As reported to occur in monocytes, protein expression of CD14 on the cell surface of neutrophils was augmented when the cells were incubated with 1,25(OH)2D3. To investigate the physiological roles for VDR in neutrophils, we investigated possible modulating effects of 1,25(OH)2D3 on the expression of several genes in lipopolysaccharide-stimulated neutrophils by using differential display analysis. Of the genes we identified, trappin-2/elafin/SKALP, which was originally reported to be an inhibitor of elastase, was induced in neutrophils by lipopolysaccharide, but was suppressed significantly in the presence of 1,25(OH)2D3. Under the same conditions, interleukin-1beta expression was also inhibited. These findings suggest that 1,25(OH)2D3 has a potential to affect the inflammatory process by modulating the expression of neutrophil genes.     

Characterization of the hepatic disposition of lanoteplase, a rationally designed variant of tissue plasminogen activator in rodents.

Lanoteplase is a recombinant mutant of tissue-type plasminogen activator (t-PA) that was developed with an aim to overcome the drawback of rapid systemic elimination of t-PA. In this study, we examined the disposition profile of lanoteplase in vivo and the kinetics of receptor-mediated endocytosis (RME) of this recombinant t-PA in vitro to kinetically characterize the mechanism(s) underlying its tissue distribution and elimination. Integration plot analysis of the initial-phase tissue distribution in rats revealed a much lower uptake clearance (CL(uptake)) of lanoteplase in the liver than that of t-PA. Rate constants for cell surface binding, internalization, and degradation of lanoteplase were also lower than those for t-PA in primary cultured rat hepatocytes. These results suggest that the improved stability of lanoteplase in vivo could be accounted for by the delay in the RME of this recombinant protein. The CL(uptake) in the liver decreased with coadministration of lactoferrin, a ligand for the low-density lipoprotein receptor-related protein (LRP) and the asialoglycoprotein (ASGP) receptors in normal mice, and in lrpap1((-/-)) mice, which have a hereditary deficiency of LRP; In contrast, CL(uptake) was not affected by mannose, whereas that of t-PA decreased with both ligands and in the lrpap1((-/-)) mice. Thus, the hepatic disposition of lanoteplase seems to be mediated by common specific receptors for t-PA, including LRP and the ASGP receptors, whereas the mannose receptor seems to be only minimally involved in the disposition of lanoteplase.     

Anti-inflammatory activity of benzo(c) phenanthridine derivatives and possible mechanisms of action (author's transl)]

Of five newly synthesized benzo[c]phenanthridine derivatives tested, the two compounds, BPD-I and BPD-II were found to have potent anti-edematous activity with intraperitoneal administration to S.D. rats. BPD-I showed a marked inhibitory effect against acute inflammation such as induced rat paw edema and leucocyte emigration and protein exudation by means of CMC pouch method and capillary permeability enhancement induced by various phlogists. This compound also inhibited subacute and chronic inflammatory responses such as granuloma formation induced by croton oil or cotton pellet. The anti-inflammatory activities of this compound resembled those of hydrocortisone. The inhibitory effects of carragenan edema and capillary permeability enhancement by ATP were strikingly reduced in adrenalectomized rats suggesting involvement of the hypophysis-adrenal systems. Rat serum corticosterone level and hepatic tyrosine aminotransferase activity (TAT) were then measured after BPD-I injection. The serum corticosterone level was increased and shortly after the elevation of corticosterone, hepatic TAT levels also increased. Thus it is concluded that the corticosterone release from adrenal gland plays a role in the anti-inflammatory action of BPD-I.     

VLA-4 mediates CD3-dependent CD4+ T cell activation via the CS1 alternatively spliced domain of fibronectin

We previously showed that fibronectin (FN) synergized with anti-CD3 in induction of CD4+ T cell proliferation, and that VLA-5 acted as a functional FN receptor in a serum-free culture system. In the present study, we showed that VLA-4 is also involved in this CD3-dependent CD4 cell activation through its interaction with the alternatively spliced CS1 domain of FN. When highly purified CD4 cells were cultured on plates coated with anti-CD3 plus synthetic CS1 peptide-IgG conjugate, significant proliferation could be observed. Neither CS1 alone nor anti-CD3 alone induced this activation. This proliferation was completely blocked by anti-VLA beta 1 (4B4) and anti-VLA-4 (8F2), while anti-VLA-5 (monoclonal antibody [mAb] 16 and 2H6) had no effect. These data indicate that VLA-4 mediates CD3-dependent CD4 cell proliferation via the CS1 domain of FN. Anti-VLA-4 also partially (10-40%) inhibited CD4 cell proliferation induced by native FN plus anti-CD3, implying that the CS1 domain is active in the native plasma FN. However, this native FN-dependent proliferation was entirely abolished by addition of anti-VLA-5 alone. Moreover, when native FN-coated plates were pretreated with anti-FN (mAb 333), which blocks RGDS sites but not CS1 sites, no CD4 cell activation could be observed. These results strongly suggest that CD4 cell activation induced by plasma FN/anti-CD3 may be dependent on both VLA4/CS1 and VLA5/RGDS interactions, although the latter interaction may be required for function of the former.     

Abnormality of the delayed type hypersensitivity (DTH) response to Bordetella pertussis in spontaneously hypertensive rats (SHR)

The delayed type hypersensitivity (DTH) response of spontaneously hypertensive rats (SHR) was compared with that of Wistar Kyoto rats (WKY), a normotensive strain. SHR showed a lower DTH response to Bordetella pertussis than WKY, especially 48 to 72 hr after antigenic challenge. These results were observed before appearance of abnormality of antibody formation or blood pressure. The reduced DTH responses of SHR were partially restored by either transfer of WKY thymocytes or treatment with levamisole. Conversely, the transfer of SHR thymocytes into WKY rats tended to diminish the DTH response. These findings suggest that SHR have a disfunction of T lymphocytes involved in DTH response (e.g., increase of suppressor cells and/or decrease of helper cells).     

Effects of corticosteroid and 1,24R-dihydroxy-vitamin D3 administration on lymphoproliferation and autoimmune disease in MRL/MP-lpr/lpr mice

The pharmacological effects of prolonged administration of a corticosteroid, betamethasone, and active vitamin D3 [1,24R-(OH)2D3] on lymphoproliferation and autoimmune disease of MRL/MP-lpr/lpr (MRL/1) mice were examined. Relatively high doses of betamethasone (0.25 mg/kg/day) prevented lymphoproliferation, reduced serum levels of anti-dsDNA, anti-ssDNA, and anti-poly (ADP-ribose) antibodies, and brought about clinical improvement, such as reduced proteinuria and diminution of skin lesions. It is noteworthy that not only did prevention of lymphoproliferation occur, but also recovery of the Lyt-2+ T cell subset in the thymus and the spleen was observed. The administration of 1,24R-(OH)2D3 (0.1 microgram/kg/day) similarly prevented proteinuria, and produced recovery of a Lyt-2+ subset in the thymus.     

Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats.

Chemokines have been shown to play an important role in leukocyte infiltration into ischemic lesions. Recently, the increased expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) was observed in experimental stroke models where infiltrated leukocytes were supposed to induce tissue injury, however, the protein level and time course of these chemokines have not been fully elucidated. Therefore, we analyzed the time-dependent production of MCP-1 and CINC in the rat brain after transient middle cerebral artery occlusion (MCAO) by means of specific enzyme-linked immunosorbent assay systems. The MCP-1 levels in the ipsilateral hemispheres increased from 6 h, peaked at 2 days, and thereafter gradually decreased. The peak MCP-1 concentration was 89.2+/-28.2 ng/g tissue wet weight (mean +/- SEM, n = 5, 49.3-fold greater than the contralateral value at the same time, P < 0.05), which is supposed to be high enough to exert its biological effects. In contrast, the maximum CINC concentration that corresponded to 2.9+/-0.7 ng/g tissue wet weight (mean +/- SEM, n = 5, 55.0-fold greater than the contralateral value at the same time, P < 0.05), was observed at 6 h. In addition, we confirmed the temporal profile of leukocyte subtypes that infiltrated into the ischemic brain, thus, neutrophil infiltration occurred at early stages (1-3 days), followed by massive infiltration of macrophages at later stages (2-7 days). These studies suggest that MCP-1 in cerebral ischemia actually plays a significant role in the migration of macrophages into the lesion and that the differential temporal production of these chemokines contributes to the regulation of infiltrated leukocyte subtypes.     

Effect of activated human protein C on experimental venous thrombosis induced by stasis with operative invasion in mice

Protein C (PC) is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating blood coagulation and fibrinolysis by inhibiting not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). In this study, it was reported that the antithrombotic effect of a human APC product (designated as CTC-111) compared with that of heparin and human PC on the deep venous thrombosis (DVT) model induced in mice by stasis caused by inferior vena cava ligation and operative invasion. Drugs were injected into a tail vein at -2, 30, 60, and 120 min after the inferior vena cava ligation. One-fifth amount of the total dosage of a given drug was injected at each time point. The wet weight of thrombus formed was reduced by APC or heparin administration, however, PC, which was equal to APC in protein amount, did not show any antithrombotic effect. To confirm whether human PC could be activated by mouse thrombin, PC was treated with mouse or human thrombin to measure the amount of APC formed. Mouse thrombin could activate human PC at a similar activation rate as human thrombin. These results suggest that externally administrated PC cannot exhibit antithrombotic effect in this DVT model due to slow activation rate to APC and that APC is a better antithrombic agent than PC for treating thrombotic diseases.