Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for coronary heart disease. TAFI is proteolytically activated by thrombin, the thrombin-thrombomodulin complex and plasmin. Once active, it dampens fibrinolysis and inflammation. The aim of this study was to generate TAFI-derived peptides that specifically modulate TAFI activation and activity. Methods: Thirty-four overlapping TAFI peptides, and modifications thereof, were synthesized. The effects of these peptides on TAFI activation and TAFIa activity were determined. In addition, the binding of the peptides to thrombin were determined. Results: Four peptides (peptides 2, 18, 19 and 34) inhibited TAFI activation and two peptides (peptides 14 and 24) inhibited TAFIa activity directly. Peptide 2 (Arg12-Glu28) and peptide 34 (Cys383-Val401) inhibited TAFI activation by the thrombin-thrombomodulin complex with IC50 values of 7.3 1.8 and 6.1 +/- 0.9 m, respectively. However, no inhibition was observed in the absence of thrombomodulin. This suggests that the regions Arg12-Glu28 and Cys383-Val401 in TAFI are involved in thrombomodulin-mediated TAFI activation. Peptide 18 (Gly205-Ser221) and peptide 19 (Arg214-Asp232) inhibited TAFI activation by thrombin and the thrombin-thrombomodulin complex. Furthermore, these peptides bound to thrombin (K-D: 1.5 +/- 0.4 and 0.52 +/- 0.07 m for peptides 18 and 19, respectively), suggesting that Gly205-Asp232 of TAFI is involved in binding to thrombin. Peptide 14 (His159-His175) inhibited TAFIa activity. The inhibition was TAFIa specific, because no effect on the homologous enzyme carboxypeptidase B was observed. Conclusions: Thrombin-activatable fibrinolysis inhibitor-derived peptides show promise as new tools to modulate TAFI activation and TAFIa activity. Furthermore, these peptides revealed potential binding sites on TAFI for thrombin and the thrombin-thrombomodulin complex.

Summary Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI binding characteristics to SclA/SclB. Thirty-four overlapping TAFI peptides of ∼20 amino acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232) specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner with TAFI binding to SclA/SclB. In another series of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities. The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/ SclB, but dissociated faster than TAFI-IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter the enzyme easily dissociates.

Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-beta 2-glycoprotein I (beta 2-GPI) autoantibodies. beta 2-GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. beta 2-GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes-derived proteins to induce anti-beta 2-GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen-like protein A [SclA], and streptococcal collagen-like protein B [SclB]) were found to interact with beta 2-GPI. Only binding to protein H induces a conformational change in beta 2-GPI, thereby exposing a cryptic epitope for APS-related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody-related epitope in domain I of beta 2-GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti-protein H antibodies also generated anti-beta 2-GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in beta 2-GPI, resulting in the formation of anti beta 2-GPI autoantibodies. This constitutes a novel mechanism for the formation of anti-beta 2-GPI autoantibodies.

Background. Ixodes ticks are the main vectors for Borrelia burgdorferi sensu lato. In the United States, B. burgdorferi is the sole causative agent of Lyme borreliosis and is transmitted by Ixodes scapularis. In Europe, 3 Borrelia species-B. burgdorferi, B. garinii, and B. afzelii-are prevalent, which are transmitted by Ixodes ricinus. The I. scapularis salivary protein Salp15 has been shown to bind to B. burgdorferi outer surface protein (Osp) C, protecting the spirochete from antibody-mediated killing.Methods and results. We recently identified a Salp15 homologue in I. ricinus, Salp15 Iric-1. Here, we have demonstrated, by solid-phase overlays, enzyme-linked immunosorbent assay, and surface plasmon resonance, that Salp15 Iric-1 binds to B. burgdorferi OspC. Importantly, this binding protected the spirochete from antibody-mediated killing in vitro and in vivo; immune mice rechallenged with B. burgdorferi preincubated with Salp15 Iric-1 displayed significantly higher Borrelia numbers and more severe carditis, compared with control mice. Furthermore, Salp15 Iric-1 was capable of binding to OspC from B. garinii and B. afzelii, but these Borrelia species were not protected from antibody-mediated killing.Conclusions. Salp15 Iric-1 interacts with all European Borrelia species but differentially protects B. burgdorferi from antibody-mediated killing, putatively giving this Borrelia species a survival advantage in nature.

Lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria, is a potent endotoxin that triggers cytokine-mediated systemic inflammatory responses in the host. Plasma lipoproteins are capable of LPS sequestration, thereby attenuating the host response to infection, but ensuing dyslipidemia severely compromises this host defense mechanism. We have recently reported that Escherichia coli J5 and Re595 LPS chemotypes that contain relatively short O-antigen polysaccharide side chains are efficiently redistributed from high-density lipoproteins (HDL) to other lipoprotein subclasses in normal human whole blood (ex vivo). In this study, we examined the role of the acute-phase proteins LPS-binding protein (LBP) and phospholipid transfer protein (PLTP) in this process. By the use of isolated HDL containing fluorescent J5 LPS, the redistribution of endotoxin among the major lipoprotein subclasses in a model system was determined by gel permeation chromatography. The kinetics of LPS and lipid particle interactions were determined by using Biacore analysis. LBP and PLTP were found to transfer LPS from HDL predominantly to low-density lipoproteins (LDL), in a time- and dose-dependent manner, to induce remodeling of HDL into two subpopulations as a consequence of the LPS transfer and to enhance the steady-state association of LDL with HDL in a dose-dependent fashion. The presence of LPS on HDL further enhanced LBP-dependent interactions of LDL with HDL and increased the stability of the HDL-LDL complexes. We postulate that HDL remodeling induced by LBP- and PLTP-mediated LPS transfer may contribute to the plasma lipoprotein dyslipidemia characteristic of the acute-phase response to infection.

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is ∼40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293–333) and TAFI-CPB-(293–401). TAFI-CPB-(293–333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFI-deficient plasma. Binding of TAFI-CPB-(293–333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293–401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293–401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.

Summary. Protein C inhibitor (PCI), also known as plasminogen activator inhibitor‐3, is a serine proteinase inhibitor that can inhibit enzymes in blood coagulation, fibrinolysis and fertility. The role of PCI in regulating the blood coagulation mechanism is not known, as it can inhibit both procoagulant (thrombin, factor Xa, factor XIa) and anticoagulant (activated protein C, thrombin‐thrombomodulin, urokinase) enzymes. To determine the relevance of this inhibitor in thrombosis, PCI levels were assessed in the Leiden Thrombophilia Study, a case–control study of venous thrombosis in 473 patients with a first deep‐vein thrombosis and 474 age‐ and sex‐matched control subjects. PCI levels above the 95th percentile of the controls (136·1%) increased the risk 1·6‐fold compared with PCI levels below the 95th percentile (95% confidence interval 0·9–2·8). There was a gradual increase in risk of thrombosis with further increasing levels of PCI. Adjustment for a number of possible confounders led to a reduction of the risk estimates associated with PCI. However, it is unclear whether adjustment for such factors in the risk models is justified. These results indicate that high levels of PCI may constitute a mild risk factor for venous thrombosis.

We investigated the mechanism by which anti‐prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity‐purified anti‐prothrombin antibodies from LAC‐positive plasma samples (α‐FII‐LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified α‐FII‐LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when α‐FII‐LAC+ were added to prothrombin‐deficient plasma or when purified anti‐prothrombin antibodies from LAC‐negative plasma samples (α‐FII‐LAC−) were added. When a combination of prothrombin and α‐FII‐LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The α‐FII‐LAC+ alone or a combination of prothrombin and α‐FII‐LAC− did not show inhibition. Ellipsometry studies showed that, in the presence of α‐FII‐LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with α‐FII‐LAC−. Our results show that complexes of prothrombin and anti‐prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti‐β2‐glycoprotein I antibodies.

Summary Binding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the threedimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997; 5: 1147). We hypothesized that the top face of this domain might be the collagen-binding site. Based on this hypothesis, we made seven vWF mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, and R1074A). Collagen binding of these mutants was investigated in ELISA and with Surface Plasmon Resonance (BIAcore). In addition, we studied collagen binding of mutants lacking the A2 or D4 domains, which flank the A3 domain. In ELISA, all point mutants and deletion mutants bound to collagen in amounts similar to wild type (WT)-vWF. In the BIAcore we found that WT-vWF has an apparent KD for collagen of 1-7 nM on a subunit base. The apparent kinetic parameters of the point mutants and deletion mutants were not significantly different from WT-vWF, except for DA2-vWF, which had a lower KD, indicating that the A2 domain somehow modulates binding of vWF to collagen type III. Based on our results, we conclude that the amino acid residues mutated by us are not critically involved in the interaction between vWF and collagen type III, which suggests that the collagen binding site is not located on the top face of the A3 domain.

Protein C inhibitor (PCI) is a heparin-binding plasma serine protease inhibitor that was originally identified as an inhibitor of activated protein C. PCI has a broad protease specificity, inhibiting several proteases in hemostasis and fibrinolysis by acting as a suicide substrate. Recently it has been reported that proteases of the reproductive system, such as acrosin, prostate-specific antigen, and tissue kallikrein, can also be effectively inhibited by PCI. However, a direct relation between PCI and physiological events during fertilization has not yet been established. An attempt was made to monitor and localize the inhibition of the sperm protease acrosin by PCI. Localization experiments for PCI on epididymal spermatozoa showed that PCI is present on the acrosomal cap of human spermatozoa, which demonstrates the early presence of PCI in the male reproductive tract. Induction of the acrosome reaction in ejaculated human spermatozoa resulted in the disappearance of PCI from the plasma membrane overlying the acrosomal head and the appearance of a strict distribution at the equatorial segment of human spermatozoa. The activity of acrosin in sperm extracts could be effectively inhibited by PCI. Zona-binding assays showed that active PCI is able to block sperm-egg binding in a concentration-dependent manner. The combination of the potent inhibition of acrosin and sperm-egg binding by PCI and the localization studies suggested that PCI may protect spermatozoa against premature acrosome reaction and degradation, thereby modulating the acrosin activity so that it can coincide with binding to the oocyte.