The lectin complement pathway serine proteases (MASPs) represent a possible crossroad between the coagulation and complement systems in thromboinflammation.

UNLABELLED: ESSENTIALS: The lectin pathway's MASP-1/2 activates coagulation factors but the trigger of the activation is unknown. MASP-1/2 activation was assessed by quantifying complexes between MASPs and antithrombin/C1-inhibitor. Activated platelets and fibrin were demonstrated to activate MASP-1 and MASP-2 both in vitro and in vivo. These findings may represent a crossroad between the complement and the coagulation systems. BACKGROUND: The activated forms of the complement lectin pathway (LP) proteases MASP-1 and MASP-2 are able to cleave the coagulation factors prothrombin, fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor in vitro. In vivo studies also show that MASP-1 is involved in thrombogenesis. OBJECTIVES: To clarify the not yet identified mechanisms involved in triggering activation of the LP during thrombotic reactions. METHODS: Novel sandwich-ELISAs for detection of complexes between MASP-1 or MASP-2 and the serpins C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2. RESULTS: Activated platelets were shown by flow cytometry to bind Ficolin-1, -2 and -3 but not MBL, which was associated with activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma, bind and activate MASP-1 and MASP-2. As demonstrated by the ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation. CONCLUSIONS: MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation.

MBL1 genotypes in wild boar populations from Sweden, Austria, the Czech Republic, and Japan.

The single nucleotide polymorphism (SNP) G949T in the mannose-binding lectin ( MBL ) 1 gene has been associated with low MBL-A concentration in serum and detected at different frequencies in various European pig populations. However, the origin of this SNP is not known. Part of the MBL1 gene was sequenced in 12 wild boar/Large White crossbred pigs from the second backcross (BC 2 ) generation in a family material originating from two wild boar x Large White intercrosses. Also, MBL-A serum concentration was measured in the entire BC 2 generation (n = 45). Furthermore, the genotypes of 68 wild boars from Sweden, Austria, the Czech Republic, and Japan were determined in regard to five previously described SNPs in MBL1 . The T allele of G949T was present among the BC 2 animals. MBL-A serum concentration in the BC 2 animals showed a bimodal distribution, with one-third of the animals at levels between 0.7 and 1.6 µg mL(-1) and the remaining pigs at levels around 13 µg mL(-1) . There was a co-variation between the presence of the T allele and low MBL-A concentration in serum. The genotyping of the wild boars revealed differences between populations. The T allele of G949T was not detected in the Austrian and Japanese samples and is thus unlikely to be an original feature of wild boars. In contrast, it was present at high frequency (0.35) among the Swedish wild boars, probably representing a founder effect. Five MBL1 haplotypes were resolved. Only two of these were present among the Japanese wild boars compared to four in each of the European populations. This difference may reflect differences in selection pressure and population history.

Extensive polymorphism in the porcine Toll-like receptor 10 gene.

The great importance of the Toll-like receptors (TLRs) in innate immunity is well established, but one family member--TLR10--remains elusive. TLR10 is expressed in various tissues in several species, but its ligand is not known and its function is still poorly understood. The open reading frame of TLR10 was sequenced in 15 wild boars, representing three populations, and in 15 unrelated domestic pigs of Hampshire, Landrace and Large White origin. Amino acid positions corresponding to detected nonsynonymous single nucleotide polymorphisms (SNPs) were analysed in the crystal structures determined for the human TLR1-TLR2-lipopeptide complex and the human TLR10 Toll/Interleukin 1 receptor (TIR) dimer. SNP occurrence in wild boars and domestic pigs was compared, and haplotypes for the TLR10 gene and the TLR6-1-10 gene cluster were reconstructed. Despite the limited number of animals sequenced in the present study (N = 30), a larger number of SNPs were found in TLR10 than recently reported for TLR1, TLR6 and TLR2. Thirty-three SNPs were detected, of which 20 were nonsynonymous. The relative frequency of nonsynonymous (d(N) ) and synonymous (d(S) ) SNPs between wild boars and domestic pigs was higher in TLR10 than recently reported for TLR1, TLR6 and TLR2. However, the polymorphism reported in the present study seems to leave the function of the TLR10 molecule unaffected. Furthermore, no nonsynonymous SNPs were detected in the part of the gene corresponding to the hinge region of the receptor, probably reflecting rigorously acting functional constraint. The total number of SNPs and the number of nonsynonymous SNPs were significantly lower (P < 0.05) in the wild boars than in the domestic pigs, and fewer TLR10 haplotypes were present in the wild boars. The majority of the TLR6-1-10 haplotypes were specific for either wild boars or domestic pigs, probably reflecting differences in microbial environment and population history.

Complement activation products in liquid stored plasma and C3a kinetics after transfusion of autologous plasma.

BACKGROUND AND OBJECTIVES: Keeping a small stock of liquid plasma readily available for transfusion is common practise in Sweden. We report data on complement activation markers in plasma components during storage in the liquid state and the kinetics of C3a-(desArg) after transfusion of autologous plasma with high content of C3a-(desArg). MATERIAL AND METHODS: Plasma components were prepared by apheresis or from whole blood. C3 fragments (C3a-(desArg), C3d,g, iC3), and soluble terminal complement complex (sC5b-9) were investigated. C3a-(desArg) kinetics was investigated in regular apheresis donors. RESULTS: Apheresis plasma prepared by membrane centrifugation had significantly higher level of C3a-(desArg), C3d,g and sC5b-9 from day 0 and low iC3, than plasma prepared by other methods. By storage day 7, C3a-(desArg)-levels were above the reference value in 88% of all components. After re-infusion of autologous plasma with high C3a-(desArg) content, there were rapid a(1) and a(2)-distribution followed by a slower b-elimination phase. CONCLUSION: Plasma components prepared by different methods and stored in the liquid phase differ significantly in the amount and timing of complement activation. C3a-(desArg) present in plasma is rapidly eliminated after transfusion. Autologous plasma could be used to study complement kinetics in different clinical situations.

Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets.

BACKGROUND: Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets. OBJECTIVE: Here we test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase. METHODS AND RESULTS: Thrombin receptor-activating peptide (TRAP)-6 was used to activate platelets in platelet-rich plasma and blood, anticoagulated with the thrombin inhibitor lepirudin. TRAP activation induced fluid-phase complement activation, as reflected by the generation of C3a and sC5b-9, which could be attenuated by the C3 inhibitor compstatin. Chondroitinase ABC treatment of supernatants from activated platelets totally inhibited the activation, indicating that platelet-derived CS had initiated the complement activation. Furthermore, addition of purified CS to plasma strongly triggered complement activation. C1q was identified as the recognition molecule, as it bound directly to CS, and CS-triggered complement activation could be restored in C1q-depleted serum by adding purified C1q. TRAP activation of whole blood increased the expression of CD11b on leukocytes and generation of leukocyte-platelet complexes. It was demonstrated that these leukocyte functions were dependent on C3 activation and signaling via C5a, as this expression could be inhibited by compstatin and by a C5aR antagonist. CONCLUSIONS: We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a.

Immune complex-stimulated production of interleukin-12 in peripheral blood mononuclear cells is regulated by the complement system.

Immune complexes (IC) can induce cytokine production in vitro. While immune aggregates (IA) consisting of heat-aggregated gamma globulin (HAGG) as model IC increased interleukin (IL)-10 levels in cell cultures with native human serum, IL-12p40/p70 production was inhibited. Three series of experiments suggested that the effects of IA on IL-12 production depended on a functionally intact complement system: (1) heat-inactivation of serum inverted the inhibitory effect of IA on IL-12p40/p70 production; (2) IA-induced IL-12p40 production in a C4 deficient serum was lowered by addition of C4; and (3) addition of the peptide compstatin, which blocks C3 activation, mimicked the effects of heat inactivation on IL-12p40 levels. Neutralization of IL-12 resulted in modestly increased IL-10 levels, while neutralization of IL-10 had no effects on IL-12p40 production. IA-induced production of IL-10 was partially blocked by anti-Fcgamma RII antibodies, whereas Fcgamma R or CR blockade had no effect on IL-12p40 production. IC and local or systemic complement activation characterize rheumatoid arthritis, systemic lupus erythematosus and many malignancies. Different and complement-dependent effects on the production of IL-10 and IL-12 can be of importance in these diseases, where control of the complement system might be a way to direct IC-induced cytokine production in either a type 1 or type 2 direction.

Binding of a model regulator of complement activation (RCA) to a biomaterial surface: surface-bound factor H inhibits complement activation.

The complement system is an important inflammatory mediator during procedures such as cardiopulmonary bypass and hemodialysis when blood is exposed to large areas of biomaterial surface. This contact between blood and the biomaterials of implants and extracorporeal circuits leads to an inflammatory response mediated by the complement system. The aim of this study was to assess the ability of a complement regulator (factor H) immobilised on a biomaterial surface to inhibit complement cascade mediated inflammatory responses. The cross-linker N-succinimidyl 3-(2-pyridyldithio) propionate was used to immobilise factor H on a model biomaterial surface without affecting the biological activity of the inhibitor. Binding of factor H was then characterised using quartz crystal microbalance-dissipation (QCM-D) and enzyme immunoassays for products of complement activation: bound C3 fragments and soluble C3a, sC5b-9, and C1s-C1INA. Immobilised factor H reduced the amount C3 fragments deposited on the biomaterial surface after incubation with serum, plasma. or whole blood. In addition, lower levels of soluble C3a and sC5b-9 were generated after incubation with whole blood. In summary, we have demonstrated that complement activation on a highly activating model surface can be inhibited by immobilised factor H and have defined prerequisites for the preparation of future biomaterial surfaces with immobilised regulators of complement activation.

Contact between a polymer and whole blood: sequence of events leading to thrombin generation.

The mechanism by which thrombin is generated on a polymer surface in an extracorporeal circuit is not yet fully understood. To address this question we have developed an in vitro chamber model in which whole blood containing heparin (1 IU/mL) comes in contact with a commonly used biomaterial, polyvinyl chloride (PVC). Incubation of blood in the chamber for 60 minutes at 37 degrees C resulted in the binding of platelets to the material surface and the generation of thrombin-antithrombin complexes. Corn trypsin inhibitor, a specific inhibitor of factor XIIa, inhibited this thrombin-antithrombin complex generation in blood in contact with PVC, which is not considered an efficient activator of factor XII. The addition of the glycoprotein IIb/IIIa inhibitor Ro44-9883 abrogated platelet binding and aggregation and resulted in decreased generation of thrombin-antithrombin complexes. Thrombin-antithrombin generation was also negligible in platelet-rich plasma but could be partially restored in the presence of erythrocytes. Taken together, these data are compatible with a model in which thrombin generation is triggered by factor XII. The response to contact with PVC appears to begin with a low-grade generation of thrombin that involves both erythrocytes and leukocytes and that activates platelets, followed by the activation of a platelet-dependent amplification loop that produces most of the thrombin.

Complement C3b interactions studied with surface plasmon resonance technique.

The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.

Phosphorylation of coagulation factor XI by a casein kinase released by activated human platelets increases its susceptibility to activation by factor XIIa and thrombin.

Previous studies suggest that activated platelets facilitate the cleavage of factor XI by both factor XIIa and thrombin. Extracellular phosphorylation is a mechanism by which the function of plasma proteins can be regulated. Phosphorylation is mediated by a casein kinase which is released by activated platelets concomitant with large amounts of ATP and Ca2+. The purpose of this study was to investigate if factor XI is phosphorylated by a platelet casein kinase and whether phosphorylation may affect its activation properties. It was shown that supernatants from platelets which contain platelet casein kinase phosphorylated factor XI. By Western blot analysis it was shown that phosphorylation of factor XI substantially increased its susceptibility to cleavage by factor XIIa, and, to a lesser extent, by thrombin. The generated factor XIa was functionally active in that it cleaved the chromogenic substrate S2366, and in that factor XIa-antithrombin and thrombin-antithrombin complexes were generated when phosphorylated factor XI was added to blood plasma. The present study indicates that platelet-mediated phosphorylation of factor XI enhances the cleavage of factor XI into XIa and that the generated XIa possesses functional activity. Phosphorylation of factor XI might be an essential regulatory mechanism by which platelets mediate amplification of the coagulation cascade.

Titanium is a highly thrombogenic biomaterial: possible implications for osteogenesis.

Titanium has superior osteointegrating properties compared to other biomaterials. The mechanism for this is unknown. During the initial phase of bone implantation the biomaterial comes into direct contact with whole blood. In this study we use a newly developed in vitro chamber model to compare different commonly used biomaterials in contact with whole blood. These materials were selected with respect to their different osteointegrating properties in order to correlate these properties with the response to whole blood. In the presence of 3 IU/ml of heparin only titanium induced macroscopic clotting. This was reflected by the generation of thrombin-antithrombin which was much increased in blood in contact with titanium compared with steel and PVC. The coagulation activation caused by titanium was triggered by the intrinsic pathway because the generation of FXIIa-AT/C1 esterase inhibitor paralleled that of thrombin-antithrombin, and both thrombin-antithrombin complex and FXIIa-AT/C1 esterase inhibitor generation were abrogated by corn trypsin inhibitor, which is a specific inhibitor of FXIIa. The binding of platelets was increased on the titanium surface compared to the other biomaterial surfaces and the state of platelet activation was much more pronounced as reflected by the levels of beta-thromboglobulin and PDGF. This study indicates that titanium is unsuitable as a biomaterial in devices which are in direct contact with blood for a prolonged period. Furthermore, PDGF and other alpha-granule proteins e.g. TGF-beta, are known to be potent promotors of osteogenesis which suggests that the pronounced thrombogenic properties of titanium might contribute to the good osteointegrating properties.

Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.

Compstatin inhibits complement and cellular activation in whole blood in two models of extracorporeal circulation.

Recently, a C3-binding cyclic synthetic peptide (Compstatin) has been identified that binds to complement component C3 and inhibits complement activation. Here we have examined the influence of Compstatin on complement activation and its indirect effects on cellular responses in whole blood in two models for extracorporeal circulation. Compstatin effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/ C3 fragments to the polymer surface. As a result of the inhibition of complement activation, the activation of polymorphonuclear leukocytes (PMNs; as assessed by the expression of CD11b) and the binding of these cells (CD16(+)) to the polymer surface were almost completely lost. In contrast, blood cell counts were not affected. Using surface plasmon resonance technology, we have confirmed that Compstatin exerts its inhibitory effect on complement activation by binding to native C3. These data show that complement activation, leading to activation and binding of PMNs to the biomaterial surface, can be abolished by the addition of Compstatin. The properties of Compstatin make Compstatin a promising drug for use in extracorporeal circuits to avoid bioincompatibility reactions, eg, during cardiopulmonary bypass, but also a favorable precursor peptide for the development of an anticomplement drug for oral use.

Inhibition of complement activation by soluble recombinant CR1 under conditions resembling those in a cardiopulmonary circuit: reduced up-regulation of CD11b and complete abrogation of binding of PMNs to the biomaterial surface.

The influence of soluble recombinant CR1 (sCR1) on complement activation, and its indirect effects on the coagulation system and cellular responses were assessed in two models for the study of blood/surface and blood/air interactions, as are encountered in e.g. cardiopulmonary bypass circuits. The concentrations of C3a and sC5b-9 and the amount of bound C3/C3 fragments were analyzed as indicators of complement activation. Thrombin-antithrombin complexes, the platelet count, surface-ATP, beta-thromboglobulin, and the expression of CD11b on leukocytes were the parameters analyzed to reflect coagulation and cellular responses. In addition, immunochemical analyses of the phenotypes of surface-bound leukocytes and platelets were performed. Recombinant sCR1, at doses ranging between 0.1-0.25 mg/ml, was found to completely inhibit the generation of sC5b-9, and of C3a by two thirds; the binding of C3 and/or C3 fragments to the surface was almost entirely abolished. As a result of the inhibition of complement activation, the expression of CD11b on PMNs, and the binding of these cells to the biomaterial surface was almost completely lost. In contrast, the thrombin-antithrombin complexes, the platelet count, and the adherence of platelets to the surface, as reflected by the ATP binding and the release of beta-thromboglobulin, were not affected. These data show that complement activation, in association with extra-corporeal treatment, causes activation and binding of PMNs to the biomaterial and that these effects can be completely abolished by the addition of soluble recombinant sCR1.

Increased phosphate content in complement component C3, fibrinogen, vitronectin, and other plasma proteins in systemic lupus erythematosus: covariation with platelet activation and possible association with thrombosis.

OBJECTIVE: To investigate whether extracellular phosphorylation of plasma proteins takes place in vivo in patients with systemic lupus erythematosus (SLE), to determine possible correlations between phosphate levels and clinical and/or laboratory parameters, and to identify individual phosphorylated plasma proteins. METHODS: Sera from SLE patients were analyzed for total amounts of protein-bound phosphate by a colorimetric technique, and for levels of beta-thromboglobulin by radioimmunoassay. In addition, the ability of these sera to activate platelets, resulting in the release of protein kinase, was tested using an assay in which platelet-rich plasma from healthy blood donors was incubated with sera or immune complexes from SLE patients. In this assay, [gamma-32P]ATP was added, and 32P-labeled C3 was quantified. Phosphate in individual proteins was detected by Western blot analysis. RESULTS: 32P-labeled, activated platelets were able to phosphorylate exogenously added proteins, without the addition of ATP or cations. Platelet-rich plasma from healthy blood donors became activated by sera or by polyethylene glycol-precipitated immune complexes from patients with SLE, which led to the extracellular phosphorylation of plasma proteins, exemplified in the C3 assay. The phosphate content in plasma proteins was increased in SLE patients with previous thrombosis. The degree of phosphorylation increased up to 3-fold in serial samples obtained from 2 SLE patients during periods of disease exacerbation. Substantial phosphate increases were seen in C3 and fibrinogen. The changes were linked to platelet activation because of the observed covariation with the levels of beta-thromboglobulin. CONCLUSION: In SLE patients, the phosphate content in plasma proteins (including C3 and fibrinogen) increases due to platelet activation.

Tubing loops as a model for cardiopulmonary bypass circuits: both the biomaterial and the blood-gas phase interfaces induce complement activation in an in vitro model.

We describe here a model for the study of blood/surface and blood/air interaction as encountered in cardiopulmonary bypass (CPB) circuits. Polyethylene tubing was filled with serum or blood and closed end to end into loops whereby the volume of the remaining air bubble was inversely varied with respect to that of the fluid. The loops were rotated vertically in a water bath at 37 degrees C. The profiles of C3a, iC3, and TCC generation were similar to those observed at surgery, involving CPB. Soluble heparin and heparan sulfate inhibited both C3a and TCC formation, but surface-conjugated heparin had only a minor effect. Binding of C3 and/or C3 fragments to the heparin surface was much reduced compared to the amine matrix to which heparin was linked, but compared with the polyethylene surface the effect was less pronounced. These data suggest that, in addition to the biomaterial surface, the blood-gas interface seems to play an important role in the activation of complement and that this activation is inhibitable by high concentrations of soluble glucose aminoglycans.

Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b.

Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes.

Impairments in complement receptor- and Fc receptor-mediated functions in vivo in patients with psoriasis.

The function of the fixed macrophage system in 18 psoriasis patients was evaluated by measuring the elimination rate of injected autologous erythrocytes coated with iC3b or IgG. The mean half-life of iC3b-coated erythrocytes was significantly prolonged in patients with psoriasis compared with healthy controls (4.7 +/- 0.8 vs 2.7 +/- 0.2 min, P = 0.01). There was also a decrease in the total number of cells eliminated from the circulation (2.5 +/- 0.2 x 10(8) vs 3.3 +/- 0.2 x 10(8), P = 0.01). There was an even more pronounced increase in the half-life of IgG-coated erythrocytes (85 +/- 18 vs 20 +/- 5 min, P < 0.001), with normal values in only 5 of 15 patients, and 4 of these 5 patients were receiving systemic treatment. The slow elimination was interpreted as being caused by primary or secondary defects in receptor function rather than by blocking of the receptors by immune complexes, since patients with psoriasis show normal levels of circulating immune complexes. Further studies are needed to elucidate the nature of these defects.

Detection and characterization of immunoconglutinins in patients with systemic lupus erythematosus (SLE): serial analysis in relation to disease course.

The levels of IgA, IgG and IgM immunoconglutinins (IK) were assessed in sera from 20 patients with SLE which were followed for 8-month periods. At the time of the exacerbation, IgG IKs were significantly increased to 226 +/- 90 arbitrary units (mean +/- s.e.m.) compared with both the minimum value of 75 +/- 28 in the SLE patients and with 31 +/- 2 in healthy controls (P < 0.05). There was no difference between SLE patients and controls in the levels of IgM and IgA IKs. Most of the SLE patients in this material showed maximal IgG IK levels before exacerbation, but there was no correlation between the clinical disease index and the levels of IgG IK. The specificity of IgG IKs showed a broad diversity for microtitre-fixed C3b, iC3b, C3c and C3dg. The antibodies were of IgG1, IgG3 and in two patients, IgG4 subclass. IgG IKs were correlated to the C3d/C3 ratio which suggested that the IK responses were secondary to C3 activation. In summary, unlike other conditions associated with complement activation where elevated IgM IKs are common, an increase in IgG IK levels was observed. It is possible that this diverging IK response contributes to the pathophysiology of the disease.

Further studies on the phosphorylation and kinetics of rat liver fructose-1,6-bisphosphatase: importance of the three-dimensional structure of a substrate to protein kinase A.

Rat liver fructose-1,6-bisphosphatase was phosphorylated by cAMP-dependent protein kinase to 2.6 mol phosphate/mol subunit but not by Ca2+/phospholipid-dependent and Ca2+/calmodulin-dependent protein kinases. It was demonstrated that phosphorylation of Ser-341 and Ser-356, and to a much lower extent, Ser-338, was dependent on the presence of intact arginine residues. This observation implicates that the intact three-dimensional structure of the substrate is necessary for phosphorylation of Ser-356 since the closest arginine is located at a six amino acid residue distance.