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.

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.

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.

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.

Effects of epinephrine, glucagon and insulin on the activity and degree of phosphorylation of fructose-1,6-bisphosphatase in cultured hepatocytes.

The effects of epinephrine, glucagon and insulin on the activity and degree of phosphorylation of fructose-1,6-bisphosphatase in isolated hepatocytes maintained in cell culture for 24 h were investigated. Epinephrine caused a rapid decrease in the apparent Km monitored as the activity ratio between the activity at 12.5 and 83 microM fructose-1,6-bisphosphate, reaching a maximum after 5 min. Glucagon caused a slower and less pronounced activation, and insulin caused an equally slow increase in Km. The effect of epinephrine and glucagon was completely reciprocated by insulin and the action of insulin was totally erased by the other two. Glucagon stimulated the incorporation of [32P]phosphate into fructose-1,6-bisphosphatase from about 2.5 to 4.2 mol/mol enzyme and epinephrine to 3.5 mol/mol. The effect of the two hormones acting together was cumulative. Insulin brought about a decrease in the degree of phosphorylation to 2.0 mol/mol. The effect of epinephrine was shown to be caused by the beta-receptors, since it was completely blocked by propanolol (a beta-antagonist) and remained unaffected by the presence of phentolamine (an alpha-antagonist).

Distinctive expression of neoantigenic C3(D) epitopes on bound C3 following activation and binding to different target surfaces in normal and pathological human sera.

Binding of C3 to sheep erythrocytes in a serum-free milieu (EAC14oxy2, EAC142) has previously been shown to mimic the antigenic change that occurs upon denaturation of C3 in sodium dodecyl sulphate (SDS), whereby neoantigenic C3(D) epitopes are exposed. The present paper deals with C3 bound to various target surfaces which are known to modulate the functional properties of C3 in different ways. Bound C3 fragments on serum-treated human aggregated gammaglobulin, zymosan, rabbit and sheep erythrocytes, and on circulating immune complexes isolated from sera of patients with rheumatoid arthritis and systemic lupus erythematosus, were shown to be mainly in the iC3b form. By RIAs, employing polyclonal antibodies, the range of C3(D) antigenic epitopes of 125I-labelled SDS denatured C3 expressed by the particle-bound iC3b was monitored. The physiologically bound iC3b on all tested particles expressed wide range of C3(D) epitopes and each type of particle-bound C3 exposed its individual range. By competition ELISA specific C3(D) alpha epitopes were monitored, employing monoclonal antibodies. A distinct difference in the expression of these epitopes was observed in iC3b bound to various test particles in the presence of normal serum and in iC3b present on circulating immune complexes from pathological sera. Considering that the neoantigenic C3(D) epitopes have been shown to be associated with different functions of C3, the distinctive antigenic expression of each type of serum-treated particle might reflect different functional forms of the protein.

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver.

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.

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.

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.

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.

Hepatic L-type pyruvate kinase: separation of unphosphorylated, phosphorylated and proteolytically modified in vivo forms.

After partial chromatographic purification of rat liver cell sap on DEAE-cellulose, including the removal of type M2 pyruvate kinase, different forms of type L pyruvate kinase were separated by chromatofocusing. Three fractions of pyruvate kinase activity were found, eluting at pH 5.0, 5.2, and 5.3, respectively. The first one was identified as phosphorylated and the second one as unphosphorylated pyruvate kinase. There were strong indications that the third fraction represented a proteolytically modified form of the enzyme, since it co-migrated with a form modified in vitro and had a similarly increased apparent Km for phosphoenolpyruvate. To rule out the possibility of this being a phosphorylated form of pyruvate kinase, the enzyme was incubated with a phosphoprotein phosphatase and then phosphorylated with cAMP-dependent protein kinase. The enzyme was not phosphorylated, like pyruvate kinase modified with subtilisin or calcium-activated protease. There is some evidence that a proteolytically modified pyruvate kinase exists in vivo. This enzyme form has not previously been demonstrated in cell sap, prior to exposure to proteolytic enzymes. The relative amounts of the three forms were determined in livers from starved rats and rats fed on a normal or a carbohydrate-rich diet.

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.

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.

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.

In vitro phosphorylation of fructose-1,6-bisphosphatase from rabbit and pig liver with cyclic AMP-dependent protein kinase.

Homogeneous preparations of fructose-1,6-bisphosphatase from mouse, man, rabbit, pig, and rat were tested as substrates for cyclic AMP-dependent protein kinase. Up to 1 mol of [32P]phosphate per mole enzyme subunit was incorporated into fructose-1,6-bisphosphatase from pig and rabbit liver, which should be compared with 2.6 mol of phosphate per mole enzyme subunit in the case of the rat liver enzyme. The phosphorylation of fructose-1,6-bisphosphatase from the livers of man and mouse was negligible. Phosphorylation of pig and rabbit fructose-1,6-bisphosphatase decreased the apparent Km for fructose-1,6-bisphosphate, but in contrast to the case of the rat liver enzyme it did not change the inhibition constants for AMP and fructose-2,6-bisphosphate. The phosphorylation sites in rabbit and pig liver fructose-1,6-bisphosphatase were located close to the carboxyterminal of the polypeptide chains, since trypsin treatment of the phosphorylated enzyme quantitatively removed all of the protein-bound radioactivity without significantly altering the subunit molecular weight and with a maintained neutral pH optimum.

Rat liver fructose-1,6-bisphosphatase. Identification of serine 338 as a third major phosphorylation site for cyclic AMP-dependent protein kinase and activity changes associated with multisite phosphorylation in vitro.

Rat liver fructose-1,6-bisphosphatase was phosphorylated with [32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. After digestion with trypsin, two peptides were isolated containing 68 and 32% of the total radioactivity, respectively. The former was found to contain the sequence Ala-Lys-Ser(P)-Arg-Pro-Ser(P)-Leu-Pro. In this fragment, Ser-341, but not Ser-338, had earlier been reported to be a phosphorylation site. The other peptide contained phosphorylated Ser-356. It was demonstrated that all the protein-bound [32P]phosphate was distributed evenly between these three serines in the native enzyme regardless of the degree of phosphorylation. Preservation of the three-dimensional structure, however, was needed to obtain phosphorylation of Ser-356. Peptides containing each phosphorylatable serine residue were sequentially removed by digesting the enzyme with chymotrypsin which cleaved off Ser-356, denaturing it with urea, digesting it further with chymotrypsin, thus removing Ser-341, and finally treating it with trypsin which eliminated the rest of the radioactivity which was bound to Ser-338. Kinetic studies of fructose-1,6-bisphosphatase digested in this manner revealed that phosphorylation of Ser-338 decreased the apparent Km for fructose 1,6-bisphosphatase, whereas phosphorylation of Ser-341 decreased the inhibitory effect of AMP and fructose 2,6-bisphosphatase, Phosphorylation of Ser-356 did not affect these parameters.

Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase.

A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.

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.