Skeletal muscle deiodinase type 2 regulation during illness in mice.

We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is upregulated in an animal model of acute illness. However, human studies on the expression of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of skeletal muscle D2 and D2-regulating factors in two mouse models of illness that differ in timing and severity of illness: 1) turpentine-induced inflammation, and 2) Streptococcus pneumoniae infection. During turpentine-induced inflammation, D2 mRNA and activity increased compared to pair-fed controls, most prominently at day 1 and 2, whereas after S. pneumoniae infection D2 mRNA decreased. We evaluated the association of D2 expression with serum thyroid hormones, (de-)ubiquitinating enzymes ubiquitin-specific peptidase 33 and WD repeat and SOCS box-containing 1 (Wsb1), cytokine expression and activation of inflammatory pathways and cAMP pathway. During chronic inflammation the increased muscle D2 expression is associated with the activation of the cAMP pathway. The normalization of D2 5 days after turpentine injection coincides with increased Wsb1 and tumor necrosis factor alpha expression. Muscle interleukin-1beta (Il1b) expression correlated with decreased D2 mRNA expression after S. pneumoniae infection. In conclusion, muscle D2 expression is differentially regulated during illness, probably related to differences in the inflammatory response and type of pathology. D2 mRNA and activity increases in skeletal muscle during the acute phase of chronic inflammation compared to pair-fed controls probably due to activation of the cAMP pathway. In contrast, muscle D2 mRNA decreases 48 h after a severe bacterial infection, which is associated with local Il1b mRNA expression and might also be due to diminished food-intake.

PGC-1alpha regulates the isoform mRNA ratio of the alternatively spliced thyroid hormone receptor alpha transcript.

Transcripts derived from the thyroid hormone receptor alpha (TRalpha) gene are alternatively spliced resulting in a functional receptor TRalpha1 and a non-T3-binding variant TRalpha2 that can exert a dominant negative effect on the transactivation functions of other TRs. There is evidence that the ratio of TRalpha isoform transcripts can be modulated and here, we investigate whether the PPARgamma co-activator alpha (PGC-1alpha) has an effect on this splicing process. PGC-1alpha was discovered not only as a transcriptional co-activator, but also has certain motifs characteristic of splicing factors. We demonstrate that PGC-1alpha alters the ratio of endogenously expressed TRalpha isoform transcripts in HepG2 cells, by decreasing TRalpha1 mRNA levels twofold. This change in isoform ratio is accompanied by a decrease in 5'-deiodinase expression, whereas no differences were found in TRbeta1 expression. Deletion of the RNA-processing domain of PGC-1alpha abrogated the effect on the TRalpha splicing, whereas expression of only the RNA-processing domain favored TRalpha1 expression. PGC-1alpha showed a similar effect on the splicing of a TRalpha minigene containing only the last four exons and introns of the TRalpha gene. These data suggest that PGC-1alpha is involved in the RNA processing of TRalpha transcripts.

Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution.

We investigated the effect of hypothyroidism or hyperthyroidism on mRNA and protein expression, diurnal variation and zonal distribution of thyroid hormone receptor (TR) isoforms TRalpha1, TRalpha2 and TRbeta1 in rat liver. Hypothyroidism results in increased isoform mRNA and protein expression whereas hyperthyroidism shows a decreased TRalpha1 and TRalpha2 mRNA and protein expression. During hyperthyroidism no change is seen in TRbeta1 mRNA, but TRbeta1 protein is upregulated in the light period and downregulated in the dark period. Diurnal changes (measured at 13:30 and 19:30 h) in the TR isoform proteins are abolished in hypothyroidism and hyperthyroidism, with the exception of a reversal in diurnal changes of TRbeta1 in hyperthyroidism. Zonal distribution of the isoforms is not affected by hypo- or hyperthyroidism. We therefore conclude that thyroid hormone influences both the levels and the diurnal expression of its receptor isoforms but not the zonal distribution.

Contribution of interleukin-12 to the pathogenesis of non-thyroidal illness.

Changes in both central and peripheral thyroid hormone (TH) metabolism occur during illness. These changes, known collectively as non-thyroidal illness, are apparently mediated by the proinflammatory cytokines IL-6, TNFalpha and IFNgamma. IL-12 is involved in regulation of IFNgamma and TNFalpha. The aim of this study was to evaluate the role of IL-12 in TH metabolism during illness. We studied TH metabolism both centrally (in the pituitary) and peripherally (in the liver) in IL-12 knock-out (IL-12 (-/-)) and wild type (WT) mice during illness induced by administration of bacterial endotoxin (LPS). LPS induced a similar decrease in serum T (3), T (4) and liver 5'-DI mRNA expression in IL-12 (-/-) and WT mice with the exception of a smaller reduction of serum T (4) in IL-12 (-/-) mice. In the pituitary, the LPS-induced decline in 5'-DI activity in WT mice was not observed in IL-12 (-/-) mice (p < 0.001), whereas the decrease in DII activity tended to be smaller in IL-12 (-/-) mice (p = 0.066). The lower decrease in pituitary activity of both DI and DII in IL-12 (-/-) mice is possibly related to the lower LPS-induced T (4) decrease. In conclusion, IL-12 is involved in the central regulation of the HPT axis during illness but not in the peripheral regulation.

Diurnal variation in rat liver thyroid hormone receptor (TR)-alpha messenger ribonucleic acid (mRNA) is dependent on the biological clock in the suprachiasmatic nucleus, whereas diurnal variation of TR beta 1 mRNA is modified by food intake.

Previous studies have shown a diurnal variation of certain isoforms of thyroid hormone receptors (TR) in rat liver. The genesis of these diurnal changes is still unknown. To clarify whether the biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), is involved, we made selective SCN lesions. Rats with an SCN lesion lost their circadian rhythm of plasma corticosterone and TSH when compared with intact animals. TR alpha 1 and TR alpha 2 mRNA expression of control rats was higher in the light period than in the dark period; changes that were abolished in the rats with SCN lesions. In contrast, liver TR beta 1 mRNA of intact rats showed a diurnal variation that failed to reach statistical significance. To evaluate whether these effects could be explained indirectly by the disappearance of rhythmic feeding behavior in rats with SCN lesions, we performed a second experiment in which otherwise intact animals were subjected to a regular feeding (RF) schedule, with one meal every 4 h. When compared with rats with free access to food, RF only affected TR beta 1 mRNA expression and had no effect on the diurnal changes in TR alpha 1 and TR alpha 2. We conclude that liver TR beta 1 expression is most clearly affected by food intake. Diurnal changes in liver TR alpha 1 and TR alpha 2 are controlled by the biological clock in the SCN but not via changes in the daily rhythm of food intake. The findings may have physiological relevance for diurnal variation of T(3)-dependent gene expression, which is supported by a diurnal variation in the expression of the 5'-deiodinase gene.

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver.

Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein. From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.

A survey of iodine intake and thyroid volume in Dutch schoolchildren: reference values in an iodine-sufficient area and the effect of puberty.

BACKGROUND: Iodine deficiency and endemic goiter have been reported in the past in The Netherlands, especially in the southeast. OBJECTIVE: To evaluate iodine intake and thyroid size in Dutch schoolchildren, contrasting those living in a formerly iodine-deficient region in the east (Doetinchem) with those living in an iodine-sufficient region in the west (Amsterdam area). DESIGN: Cross-sectional survey of 937 Dutch schoolchildren aged 6--18 years, of whom 390 lived in the eastern and 547 in the western part of the country. METHODS: Thyroid size was assessed by inspection and palpation as well as by ultrasound. Iodine intake was evaluated by questionnaires on dietary habits and by measurement of urinary iodine concentration. RESULTS: Eastern and western regions were similar with respect to median urinary iodine concentration (15.7 and 15.3 microg/dl, NS, Mann-Whitney U test), goiter prevalence by inspection and palpation (0.8 and 2.6%, P=0.08, chi-squared test), and thyroid volumes. The P97.5 values of thyroid volumes per age and body surface area group were all lower than the corresponding sex-specific normative WHO reference values. Iodized salt was not used by 45.7% of households. Daily bread consumption was five slices by boys and four slices by girls. Weekly milk consumption was 3 liters by boys and 2 liters by girls. Seafish was consumed once monthly. From these figures we calculated a mean daily iodine intake of 171 microg in boys and 143 microg in girls, in good agreement with the measured median urinary concentration of 16.7 microg/dl in boys and 14.5 microg/dl in girls. The sex difference in iodine excretion is fully accounted for by an extra daily consumption of one slice of bread (20 microg I) and one-seventh of a liter of milk (8.3 microg I) by boys. Thyroid volume increases with age, but a steep increase by 41% was observed in girls between 11 and 12 years, and by 55% in boys between 13 and 14 years, coinciding with peak height velocity. Girls have a larger thyroid volume at the ages of 12 and 13 years, but thyroid volume is larger in boys as of the age of 14 years. CONCLUSIONS: (1) Iodine deficiency disorders no longer exist in The Netherlands. (2) Bread consumption remains the main source of dietary iodine in The Netherlands; the contribution of iodized table salt and seafish is limited. (3) The earlier onset of puberty in girls renders their thyroid volume larger than in boys at the age of 12--13 years, but boys have a larger thyroid volume as of the age of 14 years.

The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells.

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.

Platelet thrombus formation on collagen at high shear rates is mediated by von Willebrand factor-glycoprotein Ib interaction and inhibited by von Willebrand factor-glycoprotein IIb/IIIa interaction.

We studied the role of von Willebrand Factor (vWF) in platelet thrombus formation in flowing blood by using a perfusion system and mutant forms of vWF lacking either interaction with glycoprotein Ib (GpIb) or with glycoprotein IIb/IIIa (alphaIIb-beta3). These mutants were added to the blood of patients with severe von Willebrand's disease (vWD) or to normal blood reconstituted with a human albumin solution instead of plasma. This blood was then perfused over collagen type III spray-coated on a glass surface and preincubated for 2 hours with 20 microg/mL plasma vWF. In this way, the adhesion step was mediated by the preincubated plasma vWF bound to collagen type III, whereas thrombus formation was mediated by mutant vWF added to the perfusate. Thrombus formation was absent at all 3 shear rates studied (300, 800, and 2600 s(-1)) when DeltaA1-vWF, lacking interaction with GpIb, was added to the perfusate, indicating the importance of GpIb-vWF interaction for thrombus formation. The interaction of vWF and GpIb is currently thought to be possible under physiological conditions in which the conformation of vWF has been changed by adsorption to a surface. Our results regarding the role of GpIb-vWF interaction in thrombus formation suggest that a second mechanism may operate by which a change may occur in GpIb on the surface of adhered platelets either by activation of the molecule or as a consequence of shear stress. Increased thrombus formation was observed when the Arg-Gly-Gly-Ser-vWF, which does not interact with alphaIIb-beta3, was added to vWD blood and perfused at 2600 s(-1). This increase was not observed in vWD blood at lower shear rates or after addition of Arg-Gly-Gly-Ser-vWF to reconstituted normal blood. Thrombus formation at a high shear rate was largest when either vWF or fibrinogen was present as a single ligand for alphaIIb-beta3 at a high shear rate. When both were present, thrombus formation was decreased. We postulate that thrombus formation is less efficient because of incomplete bridge formation when vWF and fibrinogen are both present as ligands for alphaIIb-beta3.

Recombinant vWF type 2A mutants R834Q and R834W show a defect in mediating platelet adhesion to collagen, independent of enhanced sensitivity to a plasma protease.

Type 2A von Willebrand Disease (vWD) is characterized by the absence of high molecular weight von Willebrand factor (VWF) multimers in plasma which is caused by enhanced extracellular proteolysis or defective intracellular transport. We identified in vWD type 2A patients two mutations in the A2 domain at position 834 in which arginine (R) was substituted for glutamine (R834Q) or tryptophan (R834W). We reproduced these mutations in vWF cDNA and expressed the recombinant proteins in furin cDNA containing baby hamster kidney (fur-BHK) cells. The subunit composition and the multimeric structure of both mutants was similar to wild-type (WT) vWF. Characterization of mutant R834Q by ristocetin or botrocetin induced platelet binding, and by binding to heparin showed no abnormality. R834W had normal botrocetin induced platelet binding, but ristocetin induced platelet binding and binding to heparin were decreased. Under static conditions R834Q and R834W, at 10 microg/ml, bound equally well to collagen type III as WT-vWF. At high shear rate conditions both mutants supported platelet adhesion normally when coated to a glass surface or preincubated on collagen. When R834Q or R834W was added to the perfusate, adhesion to collagen type III was 50% of the WT-vWF value, which was not due to a decreased collagen binding under flow. A divalent cation dependent protease, purified from plasma, degraded the 2A mutants rapidly while WT-vWF was not affected. In conclusion, the mutations present in the A2 domain of vWF result in an enhanced proteolytic sensitivity to a divalent ion-dependent protease. When present in the perfusate, R834Q and R834W show a decrease in platelet adhesion to collagen type III under flow conditions, which is not caused by decreased binding of the mutant vWF to collagen or enhanced proteolysis.

von Willebrand factor proteolysis is deficient in classic, but not in bone marrow transplantation-associated, thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) after bone marrow transplantation (BMT) differs from classic TTP in its clinical course and therapy. A characteristic of classic TTP is the inhibition of a plasma protease that specifically cleaves von Willebrand factor (vWF), thus reducing its multimeric size. We investigated whether this protease was also inhibited in BMT-associated TTP. Plasma from patients with classic or BMT-associated TTP was incubated with recombinant vWF R834Q, a vWF mutant with enhanced sensitivity to the protease. The proteolysis of vWF multimers was analyzed and quantified on Western blot. Metalloprotease activity was strongly inhibited in the classic TTP patient group. However, metalloprotease activity was normal in the BMT-associated TTP patient group. The difference in activity between the two patient groups was highly significant (P =.0016). The results indicate that the etiologies of classic and BMT-associated TTP are indeed different and provide an explanation for the lack of success of plasma exchange in BMT-associated TTP.

von Willebrand factor without the A2 domain is resistant to proteolysis.

von Willebrand factor (vWF) is a complex multimeric plasma glycoprotein, that plays a critical role in the mediation of platelet adhesion to the damaged vascular wall, and functions as a carrier protein for factor VIII. vWF has a domain structure consisting of repeated A, B, C, and D domains. The A1 domain is involved in binding to the platelet receptor glycoprotein (GP) Ib, and the A3 domain has a binding site for collagen. A function of the A2 domain has not been described, although point mutations identified in von Willebrand disease (vWD) type 2A patients are localized in this domain. To study the role of the A2 domain a deletion mutant was constructed which lacked the A2 domain, delta A2-vWF. Previous studies have shown that this approach is a powerful tool to study the function of a domain in a protein since it does not affect the activity of other domains. After expression in baby hamster kidney (BHK) cells, delta A2-vWF was compared to wild-type (WT) vWF, and to delta A1-vWF (Lankhof et al., Blood 86: 1035, 1995). Ristocetin induced platelet binding was slightly increased but botrocetin induced platelet binding was normal as was binding to heparin and collagen type III. Adhesion studies to surface coated purified delta A2-vWF or to delta A2-vWF preincubated on collagen under flow conditions showed no abnormalities. Incubation with normal human plasma showed that delta A2-vWF like WT-vWF was not sensitive to proteolysis. After addition of urea, WT-vWF becomes sensitive to the protease, indicating that unfolding of the molecule is necessary for exposure of the cleavage site. delta A2-vWF tested under the same conditions was resistant, indicating that the protease sensitive site is located in the A2 domain.

Functional studies on platelet adhesion with recombinant von Willebrand factor type 2B mutants R543Q and R543W under conditions of flow.

Type 2B von Willebrand disease (vWD) is characterized by the absence of the very high molecular weight von Willebrand factor (vWF) multimers from plasma, which is caused by spontaneous binding to platelet receptor glycoprotein Ib (GPIb). We studied two mutations in the A1 domain at position 543 in which arginine (R) was replaced by glutamine (Q) or tryptophan (W), respectively. Both mutations were previously identified in vWD type 2B patients. The mutations R543Q and R543W were cloned into a eukaryotic expression vector and subsequently transfected in baby hamster kidney cells overexpressing furin (fur-BHK). Stable cell lines were established by which the mutants were secreted in the cell culture supernatant. The subunit composition and multimeric structure of R543Q and R543W were similar to wild-type (WT) vWF. The mutants showed a spontaneous binding to GPIb. R543Q and R543W showed normal binding to collagen type III or heparin. Both mutants supported platelet adhesion under conditions of flow, usually when preincubated on a collagen type III surface. A low dose (2.5% of the concentration present in normal pooled plasma) of recombinant R543Q or R543W added to normal whole blood inhibited platelet adhesion to collagen type III. No inhibition was found when vWF was used as an adhesive surface. These results indicate that point mutations identified in vWD type 2B cause bleeding symptoms by two mechanisms: (1) the mutants cause platelet aggregation, which in vivo is followed by removal of the aggregates leading to the loss of high molecular weight multimers and thrombocytopenia, (2) on binding to circulating platelets the mutants block platelet adhesion. Relatively few molecules are required for the latter effect.

Immunoneutralization of interleukin-1, tumor necrosis factor, interleukin-6 or interferon does not prevent the LPS-induced sick euthyroid syndrome in mice.

The sick euthyroid syndrome is a state of altered thyroid hormone metabolism which occurs during illness. The pathogenesis is incompletely understood but recent studies indicate a role of cytokines. It is unknown if cytokines released during illness are directly responsible for the changes in thyroid hormone metabolism. Therefore we studied if previous immunoneutralization of cytokines can prevent endotoxin (lipopolysaccharide LPS), induced sick euthyroid syndrome. LPS administration resulted in systemic illness, an increase in serum tumor necrosis factor (TNF alpha) and interleukin (IL)-6 and a decrease in serum triiodothyronine (T3) and thyroxine (T4). Immunoneutralization of the effects of cytokines was accomplished by administration of monoclonal antibodies against mouse IL-1 type-1 receptor (IL-1R), TNF alpha, IL-6 or interferon (IFN gamma) prior to LPS. The LPS-induced release of cytokines was affected by previous immunoneutralization as compared with control experiments with normal immunoglobulin (IgG): anti-IL-1R did not affect serum TNF alpha but decreased serum IL-6, anti-TNF alpha decreased serum TNF alpha but not IL-6, anti-IL-6 did not affect serum TNF alpha but hugely increased IL-6 and anti-IFN gamma decreased both serum TNF alpha and IL-6. Specific immunoneutralization of IL-1, TNF alpha or IFN gamma did not prevent the LPS-induced decrease in serum T3, T4 and liver 5'-deiodinase mRNA. However, immunoneutralization of IL-6, although not preventing the fall in serum T3 and T4, did mitigate the LPS-induced decrease in liver 5'-deiodinase mRNA. In view of possible non-specific effects of the huge dose of immunoglobulins (1 mg), used only in the immunoneutralization of IL-6, we repeated the experiment with F(ab')2 fragments of anti-IL-6 antibodies. Compared with F(ab')2 fragments of control IgG, anti-IL-6 F(ab')2 did not affect the LPS-induced rise in serum TNF alpha or the decrease in serum T3 and T4 and liver 5'-deiodinase mRNA. Serum IL-6 levels induced by LPS were, however, cleared more rapidly from the circulation when anti-IL-6 F(ab')2 fragments rather than intact anti-IL-6 were administered. In conclusion, immunoneutralization of IL-1, TNF alpha or IFN gamma did not prevent the LPS-induced sick euthyroid syndrome in mice; immunoneutralization of IL-6, however, transiently inhibits the LPS-induced decrease of liver 5'-deiodinase mRNA.

Relationship between serum 3,5,3'-triiodothyronine and serum interleukin-8, interleukin-10 or interferon gamma in patients with nonthyroidal illness.

In order to further delineate the role of cytokines in the pathogenesis of the low T3 syndrome, we studied the association between serum T3 and serum interferon gamma (IFN gamma), interleukin-8 (IL-8) and interleukin-10 (IL-10) in 99 consecutive patients hospitalised because of a wide variety of nonthyroidal diseases; none used drugs affecting thyroid hormone metabolism. Patients were divided in group A (normal serum T3 and T4), group B (subnormal serum T3 and normal T4) and group C (subnormal T3 and T4). Serum IFN gamma, IL-8 and IL-10 were not different between group A, B and C (with the exception of a small increase of IFN gamma in group B). Serum T3 was slightly related to serum IL-8 (r = 0.25, p < 0.05), but not to IFN gamma and IL-10. Stepwise multiple regression indicated that these serum cytokines were not independent determinants of the variability in serum T3. The results do not support any role of circulating IFN gamma, IL-8 or IL-10 in the pathogenesis of the low T3 syndrome during illness.

A3 domain is essential for interaction of von Willebrand factor with collagen type III.

von Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To study the functional importance of these binding sites, we have made two deletion mutants that lack the A1 domain (residues 478-716; delta A1-vWF; Sixma et al. Eur. J. Biochem, 196, 369, 1991 [1]) or the A3 domain (residues 910-1113; delta A3-vWF). After transfection in baby hamster kidney cells overexpressing furin, the mutants were processed and secreted efficiently. Ristocetin or botrocetin induced platelet binding was normal for delta A3-vWF as was binding to heparin and factor VIII. As reported by Sixma et al. (1) delta A1-vWF still binds to collagen type III, indicating that the A3 domain is sufficient for the interaction. In the current study, we investigated the binding of delta A3-vWF to collage type III. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able to support platelet adhesion when coated directly to a glass surface. The binding of 125I-delta A3-vWF to collagen was specific but maximal binding was about 40 times less compared to 125I-vWF. When added at 25 times excess, delta A3-vWF did not compete with 125I-vWF for binding to collagen type III, whereas delta A1-vWF did. The binding of 125I-delta A3-vWF could be blocked by excess unlabeled vWF but not by delta A1-vWF. In conclusion, we demonstrate that the A3 domain in vWF contains the major collagen binding site. The major binding site present on the A3 domain and the minor site present on A1 bind to different sites on collagen.

Selective macrophage depletion in the liver does not prevent the development of the sick euthyroid syndrome in the mouse.

A decreased serum triiodothyronine (T3) level is one of the main characteristics of the sick euthyroid syndrome, caused mainly by a decreased 5'-deiodination of thyroxine (T4) in the liver. Cytokines have been implicated in the pathogenesis of the changes in thyroid hormone metabolism during illness. We therefore investigated the role of cytokines produced by the liver macrophages (Kupffer cells) in the development of the sick euthyroid syndrome, which was induced in mice by a single injection of bacterial endotoxin (lipopolysaccharide) or by 24-h starvation. Experiments were carried out with or without previous selective depletion of liver macrophages by intravenous administration of liposome-encapsulated dichloromethylene diphosphonate. Relative to saline-injected pair-fed controls, the administration of lipopolysaccharide caused a decrease of serum T3 and T4 and liver 5'-deiodinase mRNA. Selective depletion of liver macrophages, did not affect these changes. Starvation for 24h decreased serum T3 and T4, associated with a slight decrease of liver 5'-deiodinase mRNA. There were no differences between macrophage-depleted and non-depleted animals in this respect. In summary, selective depletion of liver macrophages did not affect the decrease in serum T3, T4 or liver 5'-deiodinase mRNA induced by lipopolysaccharide or 24-h starvation in mice. We conclude that cytokines produced by Kupffer cells are not involved in the pathogenesis of the sick euthyroid syndrome in this experimental model.

Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C.

The interaction of factor VIII with von Willebrand factor (vWF) was investigated on a quantitative and qualitative level. Binding characteristics were determined using a solid phase binding assay and protection of factor VIII by vWF from inactivation by activated protein C (aPC) was studied using three different assays. Deletion mutants of vWF, a 31-kD N-terminal monomeric tryptic fragment of vWF that contained the factor VIII binding site (T31) and multimers of vWF of different size were compared with vWF purified from plasma. We found that deletion of the A1, A2, or A3 domain of vWF had neither an effect on the binding characteristics nor on the protective effect of vWF on factor VIII. Furthermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on factor VIII of vWF was not related to the size of the multimers of vWF. A 20-fold lower binding affinity was observed for the interaction of T31 with factor VIII, and T31 did not protect factor VIII from inactivation by aPC in a fluid-phase assay. Comparable results were found for a mutant of vWF that is monomeric at the N-terminus (vWF-dPRO). The lack of multimerization at the N-terminus may explain the decreased affinity of T31 and vWF-dPRO for factor VIII. Because of this decreased affinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO. We hypothesized that this fraction was protected from inactivation by aPC but that this protection was not observed due to the presence of an excess of unbound factor VIII in the fluid phase. Therefore, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VIII from unbound factor VIII in the fluid phase. Subsequently, the protective effect of these forms of vWF on bound factor VIII was studied. In this approach, all forms of vWF were able to protect factor VIII against inactivation by aPC completely. We conclude, in contrast with earlier work, that there is no discrepancy between binding of factor VIII to vWF and protection of factor VIII by vWF from inactivation by aPC. The protective effect of T31 was not recognized in previous studies due to its low affinity for factor VIII. The absence of multimerization observed for T31 and vWF-dPRO may explain the low affinity for factor VIII. No other domains than the binding site located at the D' domain were found to be involved in the protection of factor VIII from inactivation by aPC.

Recombinant leech antiplatelet protein specifically blocks platelet deposition on collagen surfaces under flow conditions.

Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP). This protein was cloned and expressed in yeast and blocks collagen-mediated platelet aggregation and the adhesion of platelets to collagen-coated plates under static conditions. In the current study we investigated the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions. rLAPP completely inhibited platelet adhesion on collagen types I, III, and IV with IC50 values of 70, 600, and 90 nmol/L, respectively (shear rate = 1600 s-1). Approximately 10-fold more rLAPP was required to obtain a similar inhibition at a low shear rate of 375 s-1. rLAPP caused a concentration-dependent inhibition of binding of 125I-von Willebrand factor (vWF) to collagen type III and was able to displace prebound vWF even after 24 hours. Since platelet adhesion at low shear rate is less dependent on vWF than at high shear rate, this property of rLAPP may explain why less rLAPP is needed at high shear rate than at low shear rate to produce the same effect. Platelet adhesion to collagen type VI was only partially inhibited by rLAPP (maximal 44% with 3 mumol/L rLAPP). rLAPP also caused a pronounced inhibition of platelet deposition to cross sections of human atherosclerotic coronary arteries but had no effect on matrices of cultured human umbilical vein endothelial cells. rLAPP is a potent platelet adhesion inhibitor at high shear rate, which binds to collagen and works by inhibiting binding of vWF to collagen.

The role of cytokines in the lipopolysaccharide-induced sick euthyroid syndrome in mice.

To evaluate the role of cytokines in the sick euthyroid syndrome, we tried to establish an animal model of non-thyroidal illness in mice by the administration of a sub-lethal dose of bacterial endotoxin (lipopolysaccharide; LPS) which induces a variety of cytokines, including tumour necrosis factor (TNF alpha), interleukin-1 (IL-1 alpha), interleukin-6 (IL-6) and interferon-gamma (IFN gamma). When compared with pair-fed controls, a single dose of LPS resulted in (a) systemic illness, (b) induction of TNF alpha and IL-6 and (c) a decrease of liver 5'-deiodinase mRNA from 4 h onwards followed by a decrease of serum tri-iodothyronine (T3) and thyroxine (T4) at 8 h and of serum free T3 (fT3) and free T4 (fT4) at 24 h; serum TSH remained unchanged. We then studied whether a single dose or a combination of IL-1 alpha, TNF alpha, IL-6 or IFN gamma could induce the sick euthyroid syndrome in mice, again using pair-fed controls. None of the cytokines except IL-1 alpha caused systemic illness, and IL-1 alpha was the only cytokine that decreased liver 5'-deiodinase mRNA transiently. IL-1 alpha, TNF alpha or IL-6 did not decrease serum T3, T4 and TSH, but administration of IFN gamma decreased serum T4, T3 and fT3 in a dose-dependent manner without changes in serum TSH. Administration of all four cytokines together had no synergistic effects; observed changes were of a smaller magnitude than after LPS. The following conclusions were reached.(ABSTRACT TRUNCATED AT 250 WORDS)