Complement activation from protamine sulfate administration after coronary angiography

The cause of hypotension after reversal of heparin by protamine has not been well defined. In this study we evaluated complement activation (C3a and C4a) by the heparin-protamine complex in 46 consecutive patients (40 received protamine sulfate to reverse heparin, and six did not) during and after coronary angiography. In patients receiving protamine sulfate, there was a significant increase in C3a over the value before protamine sulfate administration (P less than .001) or in patients who did not receive protamine sulfate (P less than .05): 807 +/- 100 ng/ml vs. 274 +/- 75 ng/ml. There were no significant changes in C4a after protamine sulfate administration. These results indicate that the alternate complement pathway is activated when protamine sulfate is administered after coronary angiography. This may induce hypotension as well as platelet aggregation and thrombus formation and may contribute to coronary instability. Therefore, in unstable patients, heparin reversal by protamine should not be done routinely.     

In vitro comparison of the complement-scavenging capacity of different intravenous immunoglobulin preparations

Background and Objectives  Complement inhibition is considered important in the mechanism of action of intravenous immunoglobulin (IVIG) in a number of inflammatory and autoimmune disorders. The capacity of different IVIG preparations to 'scavenge' activated C3 and thereby inhibit complement activation was assessed by a new in vitro assay.
Materials and Methods  Diluted human serum as a complement source, with or without addition of different concentrations of IVIG, was incubated in microtitre plates coated with heat-aggregated human IgG. Complement scavenging was measured by detecting reduced C3 binding and determining fluid phase C3b–IgG complex formation. Complement activation induced by the IVIG preparations was measured as C5a formation.
Results  All IVIG preparations exhibited a dose-dependent inhibition of C3b deposition, correlating strongly with binding of C3b to fluid-phase IgG, but the extent of complement scavenging varied considerably between different IVIG preparations. At an IVIG concentration of 0·9 mg/ml, the inhibition of C3b deposition ranged from 72 ± 16% to 22 ± 4·1%. The reduction of C3b deposition on the complement-activating surface was not due to IVIG-induced complement activation in the fluid phase, as shown by the low C5a formation in the presence of serum.
Conclusion  In vitro analysis allows comparison of the complement-inhibitory properties of IVIG preparations. The extent of complement scavenging varies between the products.     

Effect of heparin on complement activation and lysis of paroxysmal nocturnal hemoglobinuria (PNH) red cells.

The effect of heparin upon the binding of the third component of complement (C3) to PNH red cells in vitro and their subsequent hemolysis is described. Heparin, in increasing concentrations, progressively inhibits membrane C3 fixation and hemolysis when the classic complement pathway is activated by anti-red cell antibodies. Heparin has a biphasic effect upon membrane C3 fixation and hemolysis when complement is activated in serum at decreased ionic strength (sucrose lysis) or in serum at decreased pH (Ham test). Heparin in concentrations above 2 U/ml inhibits C3 binding and hemolysis while lower concentrations of heparin enhance the consequences of complement activation by these two procedures. This enhanced complement activation may explain the increased hemolysis sometimes reported in PNH patients treated with heparin, and suggests that heparin may aggravate the consequences of pathologic alternative pathway complement activation in other diseases.     

Intermediate and long-acting insulin preparations without protamine sulphate are complement activators in vitro.

Recently, we have documented an abnormal in vivo complement metabolism in Type 1 diabetic children treated with monocomponent porcine insulin Monotard MC and its correction after switch-over to human insulin Protaphane HM. This prompted us to investigate the ability of different kinds of insulin preparations to induce complement activation in vitro. Freshly collected serum samples from healthy blood donors were incubated with commercial rapid and intermediate or long-acting (by protamine sulphate (PS) or zinc) insulin preparations for 2 hours at 37 degrees C. The C3d content of the supernatants was measured by turbidimetry as a marker of C3 complement fraction consumption. Only long-acting preparations of insulins without protamine sulphate were associated with highly significant increased levels of C3d, whatever the source of insulin, animal or human. Moreover, addition of exogenous protamine sulphate was able to inhibit the C3 conversion. This effect was dose-dependent and peaked at the concentration of commercial NPH insulin preparations. The mechanism by which protamine sulphate inhibits complement activation in vitro could be related to its ability to interfere with the physical nature of the solid surfaces presented by the insulin crystals. Indeed, insulin crystals were rapidly cleared (< 5 min) in the incubated serum when small doses of protamine sulphate were added. The complement activating capacity of long-acting insulin without protamine was dose dependent, equivalent to the known complement activator Zymosan, and abolished in the presence of EDTA. In conclusion, the present study has documented the ability of some protracted insulin preparations to activate the complement system in vitro if they are devoided of protamine sulphate. On the other hand, short-acting and NPH insulins are not complement activators.     

Complement activation induced by purified Neisseria meningitidis lipopolysaccharide (LPS), outer membrane vesicles, whole bacteria, and an LPS-free mutant.

Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement activation induced by purified Neisseria meningitidis lipopolysaccharide (Nm-LPS), native outer membrane vesicles (nOMVs), LPS-depleted outer membrane vesicles (dOMVs), wild-type meningococci, and an LPS-free mutant (lpxA(-)) from the same strain (44/76) in whole blood anticoagulated with the recombinant hirudin analogue. Complement activation products (C1rs-C1 inhibitor complexes, C4d, C3bBbP, and terminal SC5b-9 complex) were measured by double-antibody EIAs. Nm-LPS was a weak complement activator. Complement activation increased with preparations containing nOMVs, dOMVs, and wild-type bacteria at constant LPS concentrations. With the same protein concentration, complement activation induced by nOMVs, dOMVs, and the LPS-free mutant was equal. The massive complement activation observed in patients with fulminant meningococcal septicemia is, presumably, an indirect effect of the massive endotoxemia. Outer membrane proteins may be more potent complement activators than meningococcal LPSs.     

Cellular mechanisms of pulmonary vasoconstriction in an experimental model of protamine reversal of heparin

The neutralisation of heparin by protamine can cause life-threatening pulmonary hypertension. We studied this reaction in animal experimental models (sheep and rat) to determine the cellular mechanisms of the pulmonary vasoconstriction. The heparin-protamine reaction (H-P) with pulmonary hypertension (peak of mean pulmonary artery pressure = 57.3 +/- 2.2 mmHg), decreased cardiac output (-20%), leukopenia (-30%) and plasma release of high concentrations of thromboxane B2 (6.03 +/- 0.03 ng/ml) was constantly observed in sheep. The reaction was identical in sheep with induced thrombocytopenia by administration of antiplatelet antibodies. On the other hand, the neutralisation of heparin by protamine in rats did not cause thromboxane release or pulmonary vasoconstriction although the leukopenia was identical to that observed in sheep. Therefore, the platelets and white blood cells did not seem to cause the pulmonary vasoconstriction induced by the H-P complexes. The inter-species difference observed suggests that pulmonary intravascular macrophages may be responsible for the liberation of eicosanoids and acute pulmonary vasoconstriction occurring during the neutralisation of heparin by protamine.     

Complement alternative pathway acts as a positive feedback amplification of neutrophil activation

Complement alternative pathway plays an important, but not clearly understood, role in neutrophil-mediated diseases. We here show that neutrophils themselves activate complement when stimulated by cytokines or coagulation-derived factors. In whole blood, tumor necrosis factor/formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate resulted in C3 fragments binding on neutrophils and monocytes, but not on T cells. Neutrophils, stimulated by tumor necrosis factor, triggered the alternative pathway on their surface in normal and C2-depleted, but not in factor B-depleted serum and on incubation with purified C3, factors B and D. This occurred independently of neutrophil proteases, oxidants, or apoptosis. Neutrophil-secreted properdin was detected on the cell surface and could focus "in situ" the alternative pathway activation. Importantly, complement, in turn, led to further activation of neutrophils, with enhanced CD11b expression and oxidative burst. Complement-induced neutrophil activation involved mostly C5a and possibly C5b-9 complexes, detected on tumor necrosis factor- and serum-activated neutrophils. In conclusion, neutrophil stimulation by cytokines results in an unusual activation of autologous complement by healthy cells. This triggers a new amplification loop in physiologic innate immunity: Neutrophils activate the alternative complement pathway and release C5 fragments, which further amplify neutrophil proinflammatory responses. This mechanism, possibly required for effective host defense, may be relevant to complement involvement in neutrophil-mediated diseases.     

Binding of mannose‐binding lectin to fructosamines: a potential link between hyperglycaemia and complement activation in diabetes

Background

Complement activation via the MBL pathway has been proposed to play a role in the pathogenesis of diabetic complications. As protein glycation is increased in diabetes, we tested the possibility that the glycation product fructoselysine is a ligand for MBL and that its interaction with this protein may initiate complement activation.

Methods

We investigated the binding of MBL to fructoselysine by chromatography of human serum on fructoselysine‐Sepharose, followed by Western blot and mass spectrometry analysis. We also performed enzyme‐linked immunosorbent assays using purified MBL and fructoselysine‐derivatized (binding assay) or mannan‐coated plates (inhibition assay). Complement activation was determined by the fixation of C3d following incubation of fructoselysine‐derivatized plates with serum from subjects with different levels of MBL.

Results

MBL and its associated proteases were selectively purified from serum by chromatography on fructoselysine‐Sepharose. Competition experiments indicated that MBL had a similar affinity for mannose, fructose and fructoselysine. MBL bound, in a highly cooperative manner, to fructoselysine‐derivatized plates. This binding was associated with complement activation and was much lower with serum from subjects with low‐MBL genotypes.

Conclusions

MBL binding to fructoselysine and the ensuing complement activation may provide a physiopathological link between enhanced glycation and complement activation in diabetes. The cooperative character of this binding may explain the high sensitivity of diabetic complications to hyperglycaemia. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of the half-life of C3 in patients and its relation to the presence of C3-breakdown products and/or circulating immune complexes

Summary Measurement of complement components in serum may not accurately assess the degree of activation of the complement system. An alternative approach is the measurement of conversion products of the complement components. The relation between the presence of an increased concentration of C3-conversion products and the metabolism of C3 was investigated. In a group of patients, circulating immune complexes were also measured (Clq-binding test) to see whether the combination of those markers yielded information on the C3 metabolism. In this study it is shown that static measurements of serum C3 levels is of no value for the degree of complement activation. Measurement of C3-conversion products may indicate C3 hypercatabolism (in 8 of the 11 patients with C3-conversion products), but it does not imply depressed C3 synthesis. Detection of circulating immune complexes by the Clq-binding assay did not always indicate a C3 hypercatabolism. Of 12 SLE patients studied, in 9 of them, a C3 hypercatabolism was detected, and 5 of these patients were clinically characterized by the presence of minor disease symptoms. Overall, the results indicated that detection of circulating immune complexes and/or C3-conversion products could not be used as an absolute measure for insight into the C3 metabolism.     

Thromboxane receptor blockade prevents pulmonary hypertension induced by heparin-protamine reactions in awake sheep

We used competitive thromboxane A2-prostaglandin endoperoxide receptor blockade (SQ 30,741) as a probe to evaluate the role of thromboxane in ovine pulmonary vasoconstriction associated with protamine reversal of heparin anticoagulation. Control heparin-protamine reactions induced rapid release of thromboxane into arterial plasma (more than 1 ng/ml plasma), a 2.5-fold increase of pulmonary artery pressure, a 20% decrease of PaO2, and a 30% reduction in arterial white blood cell concentration. After giving SQ 30,741 despite similar thromboxane release into arterial plasma after heparin-protamine challenge, acute pulmonary hypertension was significantly reduced when 94% of pulmonary vascular smooth muscle thromboxane receptors were occupied with SQ 30,741 (p less than 0.01 at 1 minute after protamine versus control heparin-protamine reaction) and was completely abolished by a 10 mg/kg i.v. bolus (p less than 0.0001 at 1 minute after protamine versus control). Peripheral leukopenia was not affected by SQ 30,741 prophylaxis, but hypoxemia was prevented. We conclude that thromboxane causes pulmonary vasoconstriction in ovine heparin-protamine-induced pulmonary hypertension. Pulmonary vasoconstriction and hypoxemia can be completely prevented by thromboxane receptor blockade.     

Effect of C3b inactivator on monocyte-bound C3-coated human erythrocytes

As a model of IgM-induced hemolytic anemia in man, human erythrocytes were sensitized with IgM antibody and coated with complement components, including C3 and C4, using human serum as a source of complement. These coated red cells were then interacted with monolayers of human mononuclear phagocytic cells (monocytes). Complement-coated red cells so bound could be displaced from their monocyte attachment site in a dose- and time-dependent manner by serum factors, including C3b inactivator (C3bINA). These factors were more efficient in inactivating red cell-bound complement components prior to interaction of the coated cells with monocytes. With large amounts of complement per erythrocyte, measured as membrane-bound C3, the ability of the serum inactivating factor(s) to remove the complement-coated red cells from the monocyte surface was compromised and persistently bound red cells were progressively phagocytosed. These studies implicate C3bINA in the displacement of complement-coated erythrocytes, formed from the interaction of IgM antibody and serum complement, from the hepatic macrophage in IgM-induced immune hemolysis. They suggest that both the concentration of complement components, especially on the erythrocyte surface, and the level of C3bINA and perhaps other inactivators may be important features regulating hemolysis in this disorder.     

Circulating immune complexes in patients with progressive systemic sclerosis

Forty-one patients with progressive systemic sclerosis were studied for the presence of immune complexes by the fluid- and solid-phase C1q binding, C1 activation, and the fluid-phase conglutinin assays. Complement activation and autoantibodies were also studied. Immune complexes were detected in only 6 patients (15%); activation of complement was found in 5 others. The clinical and serologic features of patients with complexes were compared with those in whom complexes were not identified. No significant difference was found with respect to serology. Organ involvement was generally more frequent in the group with immune complexes, but the difference was statistically significant only with respect to lung involvement. The present data suggest that, although complement-fixing immune complexes are infrequently detected in progressive systemic sclerosis, they may play a role in the pathogenesis of lung lesions associated with the disease.     

The detection of complement binding immune complexes by means of 51Cr-labelled indicator cells

A radioimmunoassay is described for the detection of circulating complement binding immune complexes. The method was found to be highly sensitive but of limited specificity. It is based on the ability of the complexes to bind added complement (C1q-component) so that they are not available for the lysis of 51Cr-labelled chicken red blood cells in a haemolytic system. Sera from 241 patients and 87 healthy persons were studied for the presence of circulating immune complexes. The complement binding capacity is dependent on the disease groups. Patients with immune complex diseases showed a high significant difference to the healthy persons. The experimental conditions and the quality control of the method were described. There was found a very good correlation between complement binding capacity and concentration of heat-aggregated immunoglobulin.     

Complement activation in sickle cell disease: Dependence on cell density,hemolysis and modulation by hydroxyurea therapy

The complement system is an innate immune defense cascade that can cause tissue damage when inappropriately activated. Evidence for complement over activation has been reported in small cohorts of patients with sickle cell disease (SCD). However, the mechanism governing complement activation in SCD has not been elucidated. Here, we observe that the plasma concentration of sC5b-9, a reliable marker for terminal complement activation, is increased at steady state in 61% of untreated SCD patients. We show that greater complement activation in vitro is promoted by SCD erythrocytes compared to normal ones, although no significant differences were observed in the regulatory proteins CD35, CD55, and CD59 in whole blood. Complement activation is positively correlated with the percentage of dense sickle cells (DRBCs). The expression levels of CD35, CD55, and CD59 are reduced in DRBCs, suggesting inefficient regulation when cell density increases. Moreover, the surface expression of the complement regulator CD46 on granulocytes was inversely correlated with the plasma sC5b-9. We also show increased complement deposition in cultured human endothelial cells incubated with SCD serum, which is diminished by the addition of the heme scavenger hemopexin. Treatment of SCD patients with hydroxyurea produces substantial reductions in complement activation, measured by sC5b-9 concentration and upregulation of CD46, as well as decreased complement activation on RBCs in vitro. In conclusion, complement over activation is a common pathogenic event in SCD that is associated with formation of DRBCs and hemolysis. And, it affects red cells, leukocytes and endothelial cells. This complement over activation is partly alleviated by hydroxyurea therapy.     

C3 activation products, C3 containing immune complexes, the terminal complement complex and native C9 in patients with rheumatoid arthritis

Complement activation products, C9 and C3-containing circulating immune complexes (CIC), were evaluated in plasma and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and osteoarthritis. C3 activation products and the fluid phase terminal complement complex were considerably elevated in SF from RA patients reaching levels five- to eighttimes that in plasma, consistant with a local activation of the whole cascade in the joints. The results emphazise the importance of detecting C3 activation by neoepitope expression instead of single fragment determinations. The concentration of native C9 was lower in synovial fluid compared with plasma, consistant with the excessive local complement activation. Increased CIC levels which correlated with the degree of complement activation were also found in the SF from the RA patients.     

Evidence for enhanced rates of complement activation in serum from patients with newly diagnosed insulin-dependent diabetes mellitus exposed to rat islet cells and complement-dependent induction of islet cell apoptosis.

In this paper we report the concentration of terminal complement complexes (TCCs, SC5b-9, an index of complement activation) in newly diagnosed insulin-dependent diabetes mellitus (IDDM) patient serum and normal human serum. In the nine patients studied, levels of serum soluble TCCs were approximately 1.6-fold higher than in sera obtained from normal control individuals. On incubation of rat islet cells with diluted serum (10%, v/v, concentration), complement activation was increased at a significantly faster rate and the total TCC concentration was significantly higher in culture medium containing IDDM patient serum than in medium containing control serum. The concentration of anti-(glutamic acid decarboxylase) autoantibodies in newly diagnosed IDDM patient serum was on average 60-fold higher than in normal human control serum. IDDM patient serum (10%, v/v) induced apoptosis in islet cells, as determined by islet cell density changes and DNA fragmentation patterns. However, serum from IDDM patients was not able to induce apoptosis of the cells when complement components (C1q and C3) or antibodies were depleted. In addition, glutamine and the potent antioxidant 1-pyrrolidinecarbodithioic acid partially reversed cell death induced by IDDM patient serum in a concentration-dependent manner. The ATP concentration in islet cells incubated for 24 h in the presence of diluted IDDM patient serum was reduced to 4.4% of that observed in islet cells incubated in fetal calf serum or 7.3% of that observed in islet cells incubated in normal human serum. On the basis of these observations, we suggest that the pathway of IDDM patient serum-induced islet cell apoptosis may involve antibody-dependent complement activation, free radical generation and a precipitous fall in ATP levels.     

Platelet activation and binding of complement components to platelets induced by immune complexes

Clinical disorders such as malignant diseases, infectious diseases or autoimmune diseases are associated with circulating immune complexes. These immune complexes can activate the complement system in the blood or interact with complement or Fc receptors on the surface of cells. Complement activation may cause cytolysis and the immune complex interaction with receptors may cause activation of cells. We have used flow cytometry and labelled chicken antibodies to study the in vitro effects of model immune complexes on platelets and show that such immune complexes activate platelets and deposit Clq, C4 and C5 on them. Either low levels or no C3 could be detected on the platelets by flow cytometry. The immune complexes also induced formation of microparticles from purified platelets. Flow cytometry might become a useful tool in estimation of risk of thrombosis or thrombocytopenia in patients with autoimmune disease. Chicken antibodies are superior to mammalian antibodies for the measurement of platelet bound plasma proteins as they do not induce complement activation or platelet activation.     

Platelet factor 4 efficiently reverses heparin anticoagulation in the rat without adverse effects of heparin-protamine complexes.

BACKGROUND. It has been observed that the reversal of heparin anticoagulation in humans by protamine sulfate (PS) results in various adverse reactions including leukopenia, thrombocytopenia, activation of complement, increased vascular permeability, systemic hypotension, pulmonary vasoconstriction, and pulmonary edema. The purpose of this study was to compare the efficacy and effects of native platelet factor 4 (PF4) and recombinant platelet factor 4 (rPF4) with those of PS in heparin neutralization in vivo, using a rat model. METHODS AND RESULTS. Sprague-Dawley rats were anesthetized with sodium pentobarbital, and the right femoral vein and carotid artery were cannulated. For determination of activated partial thromboplastin time, platelet count, white blood cell count, and complement titer, arterial blood samples were taken before and immediately after heparin (10 units/100 g) infusion and at several time points after the infusion of the neutralizing agent (PS, 0.1 mg/100 g; PF4, 0.5 mg/100 g). In separate groups of animals, mean arterial blood pressure was monitored throughout identical protocols and the lungs were prepared for histological examination. The anticoagulant activity of heparin was effectively reversed by all of the neutralizing agents (PS, PF4, and rPF4). Platelet count (48% of initial), white blood cell count (52% of initial), complement titer (60% of initial), and mean arterial pressure (20% decrease) decreased significantly in heparinized animals receiving PS but not in those receiving PF4 or rPF4. Lung interstitium appeared normal when heparin was followed by PF4; however, interstitial edema and hemorrhage were observed with heparin-PS. CONCLUSIONS. These results suggest that PF4 efficiently reverses heparin anticoagulation in the rat without the adverse effects of heparin-protamine complexes. Therefore, rPF4 may be an appropriate substitute for PS in patients undergoing cardiovascular surgery and other procedures that require heparin anticoagulation.     

Activation of the alternative complement pathway in ascitic fluid during spontaneous bacterial peritonitis

We studied complement and immunoglobulin profiles on the serum and ascitic fluid of a patient before and during gram-negative spontaneous bacterial peritonitis (SBP). During the infection, activation of the alternative complement pathway in ascitic fluid was manifested by a 35% reduction in functional activity and depression of both properdin and factor B concentrations to nondetectable levels. Activation of the complement cascade was also demonstrated by a 50% reduction in the C3 concentration and depression of total hemolytic complement. There was no evidence of complement activation of a functionally intact complement system in the ascitic fluid of cirrhotic patients. Complement consumption in ascitic fluid may predispose the cirrhotic to SBP.     

Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparin

Complement (C')-mediated haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) is mainly due to the deficiency of glycosyl phosphatidylinositol-anchored membrane proteins with C'-regulatory activities CD55 and CD59 in PNH-affected red blood cells (RBCs). Hydrophobic insertion of C5b-7 to RBC membranes, initiating the formation of a membrane attack complex, readily results in lysis of PNH RBCs due to the deficiency of CD59. We studied the significance of the electrostatic interactions between C5b-6 and RBC membranes preceding the insertion of C5b-7. In vitro, C'-mediated lysis of PNH RBCs (assessed by sucrose haemolytic assay) was inhibited by heparin, low-molecular weight heparin (LMWH) or protamine, indicating the significance of the electrostatic interactions between C' components and RBC membranes in the process of C'-mediated haemolysis. Neuraminidase-treated PNH RBCs became resistant to C' activation, suggesting that the sialic acid moieties on RBC membranes are involved in the interactions of RBC with C' components. By using biotin-labelled C7, we demonstrated that LMWH as well as heparin inhibited the insertion of C5b-7 to RBCs, although they did not inhibit the incorporation of C7 into membrane-associated C5b-6. Neither heparin nor LMWH could inhibit the procoagulant alteration of PNH RBC membranes induced by C' activation even at concentrations which inhibited the haemolysis completely. Because LMWH inhibited the C'-mediated lysis of PNH RBCs in vitro at the range which induced a limited prolongation of activated partial thromboplastin time of normal plasma, we consider that LMWH may be useful for both the inhibition of haemolysis and the prevention of thrombosis, which often follow a haemolytic attack in PNH.