Chromatin-independent binding of serum amyloid P component to apoptotic cells.

Human serum amyloid P component (SAP) is a glycoprotein structurally belonging to the pentraxin family of proteins, which has a characteristic pentameric organization. Mice with a targeted deletion of the SAP gene develop antinuclear Abs, which was interpreted as evidence for a role of SAP in controlling the degradation of chromatin. However, in vitro SAP also can bind to phosphatidylethanolamine, a phospholipid which in normal cells is located mainly in the inner leaflet of the cell membrane, to be translocated to the outer leaflet of the cell membrane during a membrane flip-flop. We hypothesized that SAP, because of its specificity for phosphatidylethanolamine, may bind to apoptotic cells independent of its nuclear binding. Calcium-dependent binding of SAP to early, nonpermeable apoptotic Jurkat, SKW, and Raji cells was indeed observed. Experiments with flip-flopped erythrocytes confirmed that SAP bound to early apoptotic cells via exposed phosphatidylethanolamine. Binding of SAP was stronger to late, permeable apoptotic cells. Experiments with enucleated neutrophils, with DNase/RNase treatment of late apoptotic Jurkat cells, and competition experiments with histones suggested that binding of SAP to late apoptotic cells was largely independent of chromatin. Confocal laser microscopic studies indeed suggested that SAP bound to these apoptotic cells mainly via the blebs. Thus, this study shows that SAP binds to apoptotic cells already at an early stage, which raises the possibility that SAP is involved in dealing with apoptotic cells in vivo.

The second exon-encoded factor XII region is involved in the interaction of factor XII with factor XI and does not contribute to the binding site for negatively charged surfaces.

Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-triangle up19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-triangle up19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-triangle up19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-triangle up19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-triangle up19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-triangle up19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-triangle up19 bound to kaolin, which binding was equally well inhibited by either, rFXII-triangle up19 or full-length FXII (IC50 = 7.2 +/- 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-triangle up19 generated a similar amount of FXIIa- and kallikrein-C1-inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1-inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-triangle up19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.

Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia.

Evidence of factor XI (FXI) activation in vivo is scarce. In addition, it remains uncertain whether thrombin, factor XIIa (FXIIa), or perhaps another protease is responsible for FXI conversion. We investigated the activation of FXI in eight healthy volunteers after infusion of a low dose of endotoxin (4 ng/kg of body weight). Activation of prekallikrein FXII, FXI, and prothrombin was measured with sensitive enzyme-linked immunosorbent assays (ELISAs), and FXI activation was measured with a novel enzyme capture assay that detects noncomplexed FXIa. Activation of FXI was apparent with a significant plasma peak level of noncomplexed FXIa of 10 to 11 pmol/L at 1 and 2 hours after endotoxin infusion, followed by a gradual increase in FXIa-FXIa inhibitor complexes, measured in the ELISAs, with a summit of 11 to 15 pmol/L at 6 and 24 hours, respectively. In accordance with previous studies, thrombin generation was detected 1 hour after endotoxin infusion to become maximal after 3 to 4 hours. In contrast, we did not find any evidence of contact activation, because markers of activation of prekallikrein and FXII remained undetectable. From the FXIa data a theoretical model was constructed which suggested that inhibition of FXIa does not take place in the plasma compartment, but is localized on a surface. These data provide the first evidence for FXI activation in low-grade endotoxemia and suggest that FXI is activated independently of FXII.

Inactivation of factor XIa in human plasma assessed by measuring factor XIa-protease inhibitor complexes: major role for C1-inhibitor.

From experiments with purified proteins, it has been concluded that factor XIa (FXIa) is inhibited in plasma mainly by alpha 1-antitrypsin (a1AT), followed by antithrombin III (ATIII), C1-inhibitor (C1Inh), and alpha 2-antiplasmin (a2AP). However, the validity of this concept has never been studied in plasma. We established the relative contribution of different inhibitors to the inactivation of FXIa in human plasma, using enzyme-linked immunosorbent assays (ELISAs) for the quantification of complexes of FXIa with a1AT, C1Inh, a2AP, and ATIII. We found that 47% of FXIa added to plasma formed complexes with C1Inh, 24.5% with a2AP, 23.5% with a1AT, and 5% with ATIII. The distribution of FXIa between these inhibitors in plasma was independent of whether FXIa was added to plasma, or was activated endogenously by kaolin, celite, or glass. However, in the presence of heparin (1 or 50 U/mL), C1Inh appeared to be the major inhibitor of FXIa, followed by ATIII. Furthermore, at lower temperatures, less FXIa-C1Inh and FXIa-a1AT complexes but more FXIa-a2AP complexes were formed. These data demonstrate that the contribution of the different inhibitors to inactivation of FXIa in plasma may vary, but C1Inh is the principal inhibitor under most conditions.

COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization.

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema. Equivalent substitutions have been described in alpha 1-antitrypsin and antithrombin III. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional. Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor. A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val451-->Met and Pro476-->Ser mutant proteins, and to a lesser extent Phe455-->Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val451-->Met and Pro476-->Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val451-->Met or Pro476-->Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxylterminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.

Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model.

Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis.

Activation of the complement system in baboons challenged with live Escherichia coli: correlation with mortality and evidence for a biphasic activation pattern.

Activation of the complement system was studied in baboons that were challenged with live Escherichia coli. In the group challenged with a lethal dose (n = 4), the complement activation parameters C3b/c, C4b/c, and C5b-9 increased 13, 5, and 12 times the baseline value, respectively, during the first 6 h after the E. coli infusion, whereas in the group challenged with a sublethal dose (n = 10), they increased only moderately, by 2 to 3 times the baseline value. However, in this latter group, a more pronounced activation occurred at 24 h. Subsequent experiments showed that this second phase in complement activation started at 6 h after the challenge, at which time infused microorganisms had been cleared from the circulation. The simultaneous increase in C-reactive protein with this second phase suggested an endogenous activation mechanism involving this acute-phase protein. Levels of inactivated (modified) C1 inhibitor also increased in both groups, with peak levels of 2.5 times the baseline value at 24 h in the sublethal group and of 4 times at 6 h after the challenge in the lethal group. Thus, activation of complement in this animal model for sepsis occurs in a biphasic pattern, the initial phase mediated by the bacteria and the later phase mediated by an endogenous mechanism possibly involving C-reactive protein. The differences in complement activation between animals with lethal or sublethal sepsis support the hypothesis that complement activation contributes to the lethal complications of sepsis.

Controlled insect-sting challenge in 55 patients: correlation between activation of plasminogen and the development of anaphylactic shock.

The pathogenesis of anaphylactic shock is not completely understood. Mast cell degranulation products may stimulate endothelial cells, leading to activation of fibrinolytic and coagulation systems. We investigated the activation of these systems in insect-sting anaphylaxis. Fifty-five patients with a previous insect-sting anaphylactic reaction and 8 volunteers were challenged with an in-hospital sting. Plasma levels of von Willebrand factor (vWF), coagulation, and fibrinolytic parameters were assessed. After the sting challenge, 20 patients developed anaphylactic symptoms, 7 of whom developed hypotension. In only these 7 patients, but not in the volunteers or in the other patients with no or mild anaphylactic symptoms, vWF levels increased from 107% +/- 33% (mean +/- SD) before, to 235% +/- 134% 60 minutes after the onset of clinical symptoms. This increase of vWF was accompanied by an increase of circulating tissue-type plasminogen-activator (tPA) levels from 5 +/- 3 micrograms/L to 50 +/- 59 micrograms/L and of plasminogen-alpha 2-antiplasmin complex (PAP-c) levels from 6 +/- 3 nmol/L to 297 +/- 225 nmol/L. Both tPA and PAP-c levels peaked 5 minutes after the onset of clinical symptoms. Such increases of tPA and PAP-c were not observed in the volunteers or in the patients who did not develop shock. The increase of tPA and PAP-c levels in the hypotensive patients correlated positively with the degree of mast cell degranulation and inversely with the mean arterial pressure. We conclude that activation of plasminogen may be involved in the pathogenesis of anaphylactic shock induced by insect venom.

Assessment of the relative contribution of different protease inhibitors to the inhibition of plasmin in vivo.

It has been shown that the most important inhibitor of plasmin is alpha 2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed. To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin. It was confirmed that alpha 2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than alpha 2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-alpha 1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with alpha 2-macroglobulin and with antithrombin III were significantly elevated. In conclusion, we confirmed the important role of alpha 2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation patterns of coagulation and fibrinolysis in baboons following infusion with lethal or sublethal dose of Escherichia coli.

Administration of low doses endotoxin or tumor necrosis factor (TNF) in human experimental models for sepsis results in transient activation of both coagulation and fibrinolysis and subsequent inhibition of the fibrinolytic system by plasminogen activator inhibitor type 1 (PAI-1). We have investigated in a baboon model for sepsis, whether administration of a lethal or sublethal dose of living E. coli could induce similar activation patterns. Levels of thrombin-antithrombin III (TAT) complexes increased significantly to zeniths of 425 and 33 times the baseline values at t+360 in the lethal and sublethal group, respectively. Activation of fibrinolysis, as reflected by plasmin-alpha 2 antiplasmin (PAP) complexes, in the sublethal group was maximal at t+60 and was increasingly inhibited thereafter in spite of a sustained increase of tissue type plasminogen activator (t-PA) levels. In the lethal group PAP complexes increased to a zenith of 38 times the baseline values at t+240. PAI-1 levels increased to 15 times the baseline values at t+360 in the sublethal group, whereas in the lethal group they increased almost linearly to 20 times the baseline values at t+360. Despite high levels of PAI-1, effective inhibition of the fibrinolysis was not established until at T+240 in the lethal group. The difference in activation patterns of both mediator systems in the sublethal and lethal group of baboons indicate that extensive activation of coagulation contributes to the lethal complications in sepsis.

Interleukin-2 induces activation of coagulation and fibrinolysis: resemblance to the changes seen during experimental endotoxaemia.

The administration of Interleukin-2 (IL-2) causes the release or generation of other cytokines such as tumour necrosis factor (TNF) which, by disturbing the anticoagulant properties of the endothelium, may induce a procoagulant state in patients receiving this drug. We therefore evaluated the effects of IL-2 on coagulation and fibrinolysis in 14 patients receiving 12 or 18 x 10(6) IU/m2/d of IL-2 given as a 15 min infusion for 5 d. Blood samples were drawn at short intervals after the first IL-2 infusion. The parameters were analysed by way of analysis for repeated measures (F tests rather than t tests). During the first day, thrombin-antithrombin (TAT) complexes started to increase 2 h after the IL-2 infusion, reaching peak levels at 4 h (n = 14; 11.2 +/- 6.4 micrograms/l v 49.8 +/- 49.2 micrograms/l, P < 0.01). Plasma alpha 2 antiplasmin (PAP) complexes showed a similar pattern rising from a mean baseline value of 17.5 +/- 7.6 nmol/l to 66.8 +/- 47.7 nmol at 4 h (P < 0.01). In four patients the peak of PAP preceeded that of TAT. Tissue plasminogen activator (tPA) rose from a mean baseline value of 4.9 +/- 3.7 micrograms/l to 26.3 +/- 13.5 micrograms/l at 4 h (P < 0.01). Plasminogen-activator-inhibitor-1 (PAI-1) levels increased from 59 +/- 35 micrograms/l to 113 +/- 39 micrograms/l at 6 h (P < 0.01). tPA PAI-1 complexes increased from 0.15 +/- 0.07 to 0.69 +/- 0.21 nmol/l at 6 h (P < 0.01). Our study indicates that IL-2 activates the coagulation and fibrinolytic systems in vivo. The changes resemble the perturbations observed after endotoxin/TNF administration. These abnormalities may play a role in the side-effects induced by IL-2 therapy.

Analysis of intraarticular fibrinolytic pathways in patients with inflammatory and noninflammatory joint diseases.

OBJECTIVE: Intraarticular activation of the fibrinolytic system has been suspected to occur in patients with arthritis. We undertook the present study to investigate the relation of this activation to clinical symptoms, and the molecular pathways involved. METHODS: We quantitatively assessed levels of plasmin-alpha 2-antiplasmin (PAP) complexes in synovial fluid (SF) from 25 patients with rheumatoid arthritis (RA), 7 with seronegative spondylarthropathy (SSA), and 10 with osteoarthritis (OA), and conducted an analysis to determine the plasminogen-activating pathway via which these complexes were generated. In addition, we studied the relationship of intraarticular fibrinolysis to clinical and biochemical parameters. RESULTS: All patients studied had increased SF levels of PAP complexes. Levels in patients with RA and SSA were slightly higher than those in patients with OA. These complexes were probably formed by activation of urokinase-type plasminogen activator (u-PA), and not tissue-type plasminogen activator (t-PA), since SF levels of both u-PA antigen and u-PA-plasminogen activator inhibitor (PAI) complexes were increased in 27 of the 42 patients. Conversely, SF levels of t-PA were below normal in all but 1 patient. In some patients, activation of factor XII presumably also contributed to plasminogen activation in SF, since levels of factor XIIa-C1 inhibitor in SF were increased in 8 of the 42 patients and correlated, as did u-PA-PAI levels, with levels of PAP complexes. Several of the parameters of fibrinolysis in SF, particularly u-PA antigen and u-PA-PAI-1 complexes, were found to correlate with clinical and biochemical parameters. CONCLUSION: Our results suggest that plasminogen is frequently activated in the joints of patients with inflammatory or noninflammatory arthropathy and that this activation mainly occurs via a u-PA-, and in some cases also via a factor XII-, dependent pathway. The possible relation of this activation process to stimulation of synovial cells by cytokines is discussed.

Plasminogen activation in vivo upon intravenous infusion of DDAVP. Quantitative assessment of plasmin-alpha 2-antiplasmin complex with a novel monoclonal antibody based radioimmunoassay.

Infusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established. A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor alpha 2-antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated alpha 2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system. Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex. We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

PAI-1 synthesis in the human hepatoma cell line HepG2 is increased by cytokines--evidence that the liver contributes to acute phase behaviour of PAI-1.

The acute phase behaviour of the fast inhibitor of tissue-type plasminogen activator (PAI-1) in vivo has been attributed to increased synthesis by endothelial cells. However, most other acute phase proteins in vivo are synthesized in the liver, which process is regulated by cytokines and can be studied in the hepatoma derived cell line HepG2. In this study, we investigated whether the synthesis of PAI-1 by HepG2 cells is regulated by the cytokines recombinant IL-1, rIL-6 and rTNF. Recombinant IL-1 and rTNF each increased PAI-1 synthesis by HepG2 cells two to three fold, whereas rIL-6 hardly had an effect. Mixtures of rIL-1, rIL-6 and rTNF increased PAI-1 synthesis up to eleven fold. The effects observed were not due to non-specific effects on HepG2 cell metabolism, since synthesis of alpha-2-antiplasmin was not effected by any of those cytokines, whereas fibrinogen synthesis was increased three to four fold by rIL-6, but was unaffected by rIL-1. Thus, our results demonstrate that synthesis of PAI-1 by HepG2 cells is regulated by cytokines and implicate that the acute phase behaviour of PAI-1 in vivo at least in part may be due to an increased synthesis by the liver.