J. C. M. Meijers

Thrombin-mediated activation of factor XI results in a thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis.

Recently, it has been shown that Factor XI can be activated by thrombin, and that Factor XIa significantly contributes to the generation of thrombin via the intrinsic pathway after the clot has been formed. This additional thrombin, generated inside the clot, was found to protect the clot from fibrinolysis. A plausible mechanism for this inhibitory effect of thrombin involves TAFI (thrombin-activatable fibrinolysis inhibitor, procarboxypeptidase B) which, upon activation, may inhibit fibrinolysis by removing carboxy-terminal lysines from fibrin. We studied the role of Factor XI and TAFI in fibrinolysis using a clot lysis assay. The lysis time was decreased twofold when TAFI was absent, when TAFI activation was inhibited by anti-TAFI antibodies, or when activated TAFI was inhibited by the competitive inhibitor (2-guanidinoethylmercapto)succinic acid. Inhibition of either TAFI activation or Factor XIa exhibited equivalent profibrinolytic effects. In the absence of TAFI, no additional effect of anti-Factor XI was observed on the rate of clot lysis. We conclude that the mechanism of Factor XI-dependent inhibition of fibrinolysis is through the generation of thrombin via the intrinsic pathway, and is dependent upon TAFI. This pathway may play a role in determining the fate of in vivo formed clots.

Enhancement of rabbit jugular vein thrombolysis by neutralization of factor XI. In vivo evidence for a role of factor XI as an anti-fibrinolytic factor.

Recent in vitro studies have shown that fibrinolytic activity may be attenuated by a thrombin-activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin, generated via the intrinsic pathway of coagulation in a factor XI-dependent way. Thus factor XI may play a role in the regulation of endogenous fibrinolysis. The aim of this study was to investigate the effect of in vivo inhibition of factor XI and TAFI in an experimental thrombosis model in rabbits. Incorporation of anti-factor XI antibodies in jugular vein thrombi resulted in an almost twofold increase in endogenous thrombolysis compared with a control antibody. A similar effect was observed when the anti-factor XI antibody was administered systemically. Inhibition of TAFI activity also resulted in a twofold increase in clot lysis whereas inhibition of both factor XI and TAFI activity had no additional effect. Thus, we provide the first in vivo evidence for enhanced thrombolysis through inhibition of clotting factor XI, demonstrating a novel role for the intrinsic pathway of coagulation. Furthermore we demonstrate that inhibition of TAFI had a similar effect on thrombolysis. We postulate that inhibition of factor XI activity enhances thrombolysis because of diminished indirect activation of TAFI.

Lupus anticoagulants and the risk of a first episode of deep venous thrombosis

Summary.u2002 We have determined lupus anticoagulants, anti‐β2 glycoprotein I (β2GPI) and antiprothrombin antibodies in the Leiden Thrombophilia Study, a population‐based case–control study designed to determine risk factors for deep venous thrombosis (DVT). Lupus anticoagulant (LAC) was measured in 473 patients and 472 control subjects. Four control subjects (0.9%) and 14 patients (3.1%) had a positive LAC, resulting in a 3.6‐fold increased risk [odds ratio (OR) 3.6, 95% CI: 1.2–10.9]. Of the total population, 49 were positive for anti‐β2GPI antibodies: 15 controls (3.4%) and 34 patients (7.5%), implying a 2.4‐fold increased risk (95% CI: 1.3–4.2). Antiprothrombin antibodies were present in 114 subjects: 48 controls (11.0%) and 66 cases (14.6%) with an OR of 1.4 (95% CI: 1.0–2.1). When LAC was considered in the co‐presence of antiprothrombin or anti‐β2GPI antibodies the OR increased to 10.1 (95% CI: 1.3–79.8). A LAC without a positive anti‐β2GPI or antiprothrombin test was not associated with a risk for DVT (OR 1.3, 95% CI: 0.3–6.0). This study demonstrates that the presence of LAC, anti‐β2GPI antibodies and antiprothrombin antibodies are risk factors for DVT in a general population. The strongest association holds for the combination LAC and the presence of anti‐β2GPI or antiprothrombin antibodies.

Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia

Acute promyelocytic leukaemia (APL) is a disease that is distinguished from other leukaemias by the high potential for early haemorrhagic death. Several processes are involved, such as disseminated intravascular coagulation and hyperfibrinolysis. Recently, TAFI (thrombin‐activatable fibrinolysis inhibitor) was identified as a link between coagulation and fibrinolysis. TAFI can be activated by thrombin, and in its activated form potently attenuates fibrinolysis by removing C‐terminal lysine and arginine residues that are important for the binding and activation of plasminogen. Activation of TAFI by the coagulation system results in a down‐regulation of fibrinolytic activity and, thereby, prevents a rapid dissolution of the fibrin clot. To establish whether TAFI was involved in the severity of the bleeding complications in APL, the TAFI antigen and activity levels were determined in a group of 15 patients. The TAFI antigen concentration was normal, but the activity of TAFI was severely reduced in APL by ≈u200360%. The reduction of TAFI activity was most probably caused by the action of plasmin on TAFI because in vitro experiments revealed that plasmin slightly reduced antigen levels but severely reduced TAFI activity. The acquired functional TAFI deficiency in APL may contribute to the severity of the haemorrhagic diathesis because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis.

Recombinant factor VIIa reverses the in vitro and ex vivo anticoagulant and profibrinolytic effects of fondaparinux.

Summary.u2002 Background:u2002Fondaparinux is a synthetic pentasaccharide, which selectively inhibits coagulation factor (F) Xa, and is registered for prevention of venous thromboembolism following hip fracture, hip replacement, and knee replacement surgery. Recently, it was shown that recombinant FVIIa (rFVIIa) reverses anticoagulant effects of fondaparinux in healthy volunteers. Objectives:u2002In this study, we have explored the in vitro and ex vivo effects of rFVIIa on clot formation and thrombin‐activatable fibrinolysis inhibitor (TAFI)‐mediated down‐regulation of fibrinolysis after fondaparinux administration. Methods:u2002In vitro clot lysis assays were performed in pooled normal plasma from healthy volunteers to which fondaparinux was added, and in serial samples from healthy volunteers who received a single bolus dose of fondaparinux, a single bolus dose of rFVIIa, or both. Results and conclusions:u2002Fondaparinux significantly delayed clot formation, and clot lysis was significantly increased due to decreased activation of TAFI. Addition of recombinant FVIIa corrected the inhibited clot formation induced by fondaparinux, and the acceleration of clot lysis was partially reversed. In vivo administration of fondaparinux (10u2003mg) to healthy volunteers similarly resulted in accelerated plasma clot lysis. Subsequent administration of rFVIIa (90u2003µgu2003kg−1) normalized the clot lysis time up to 6u2003h postadministration. rFVIIa might be a good therapeutic option in patients treated with fondaparinux who develop bleeding complications, since both clot formation as well as fibrinolytic resistance are improved.

Complexes of anti‐prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces

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.

MAPPING OF THE DISCONTINUOUS H-KININOGEN BINDING SITE OF PLASMA PREKALLIKREIN : EVIDENCE FOR A CRITICAL ROLE OF APPLE DOMAIN-2

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K D = 1.2 × 10−8 m). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 ≈ A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC50 of 8 nm mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.

The effect of initiating combined antiretroviral therapy on endothelial cell activation and coagulation markers in South African HIV-infected individuals.

An increased incidence of venous thromboembolism (VTE) is observed in human immunodeficiency virus (HIV)-infected patients. Only a limited number of studies described the effect of combined antiretroviral therapy (cART) on coagulation markers. In a prospective cohort study in cART-naive South African HIV-infected individuals the effect of initiating cART on markers of endothelial cell activation, coagulation and natural anticoagulation was studied. These markers were compared to the reference ranges for an HIV-uninfected control population recruited from hospital staff. A venous ultrasound of both legs was performed to detect asymptomatic deep venous thrombosis (DVT). A total number of 123 HIV-infected participants were included. The patients were predominantly black and severely immuno-compromised. The CD4 cell count increased and the HIV viral load decreased significantly after the initiation of cART (p<0.001). The median follow-up period was 7.2 (± 1.6) months. Laboratory testing before and after initiation of cART was completed by 86 patients. Before initiating cART significantly elevated von Willebrand factor and D-dimer levels, increased activated protein C sensitivity ratio (APCsr) and decreased total and free protein S and protein C levels were observed compared to HIV-negative controls. At follow-up all markers, except APCsr, improved towards the normal range for controls without showing complete normalisation. In a subgroup of 57 patients no asymptomatic DVT was found. Compared to the controls, abnormal levels of coagulation markers were observed in HIV-infected individuals before and after the initiation of cART. Most markers improved after starting cART, but remained significantly different from the controls, indicating a persistent disturbed haemostatic balance.

Plasminogen activator inhibitor type I contributes to protective immunity during experimental Gram-negative sepsis (melioidosis)

Summary.u2002 Background:u2002Melioidosis is a frequent cause of sepsis in Southeast Asia caused by the Gram‐negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of plasminogen activator inhibitor type 1 (PAI‐1), an important regulator of inflammation and fibrinolysis. Objectives:u2002In this study, we aimed to investigate the role of PAI‐1 during melioidosis. Methods:u2002Wild‐type (WT) and PAI‐1‐deficient (PAI‐1–/1−/−) mice were intranasally infected with B. pseudomallei. Mice were killed after 24, 48 or 72u2003h. Lungs, liver and blood were harvested for measurement of bacterial loads, cytokines, clinical chemistry, histopathology, and coagulation parameters. Additionally, survival studies were performed. Results:u2002PAI‐1−/− mice demonstrated enhanced susceptibility to B. pseudomallei infection, as shown by a strongly increased mortality rate (100% vs. 58% among WT mice, Pu2003<u20030.001), associated with enhanced bacterial loads in lungs, liver, and blood. Additionally, PAI‐1−/− mice showed elevated levels of proinflammatory cytokines in lungs and plasma, accompanied by enhanced local and systemic coagulation activation (thrombin–antithrombin complexes and D‐dimer), increased hepatocellular injury (plasma aspartate aminotransferase and alanine aminotransferase), and renal failure (plasma creatinine and urea). Conclusions:u2002PAI‐1 has a protective role during severe Gram‐negative sepsis caused by B. pseudomallei by limiting bacterial growth, inflammation, and coagulation, and probably, as a consequence thereof, distant organ injury.

Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats

Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical α-chains and a single β-chain (C4BPβ), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPβ. To further localize the protein S binding site, we constructed chimeras containing C4BPβ SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, β-chain SCR-2 contributes to the interaction of C4BP with protein S.