Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice.

Essentials Plasmin is able to proteolyse von Willebrand factor. It was unclear if plasmin influences acute thrombotic thrombocytopenic purpura (TTP). Plasmin levels are increased during acute TTP though suppressed via plasmin(ogen) inhibitors. Allowing amplified endogenous plasmin activity in mice results in resolution of TTP signs. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin-activatable fibrinolysis inhibitor activation and activated thrombin-activatable fibrinolysis inhibitor activity.

UNLABELLED: Essentials Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for cardiovascular disorders. TAFI inhibitory nanobodies represent a promising step in developing profibrinolytic therapeutics. We have solved three crystal structures of TAFI in complex with inhibitory nanobodies. Nanobodies inhibit TAFI through distinct mechanisms and represent novel profibrinolytic leads. SUMMARY: Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-Å-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-Å-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-Å-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.

Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated to form activated TAFI (TAFIa) (Ala93-Val401) through removal of the N-terminal activation peptide (Phe1-Arg92). TAFIa is thermally unstable, and the role of the activation peptide in the activity and stability of TAFI zymogen remains unclear. OBJECTIVES: To better understand the role of the activation peptide in the activity and stability of TAFI. METHODS: We constructed a deletion mutant, TAFI-CIIYQ-∆1-73 , in which the first 73 amino acids of the activation peptide are absent. The intrinsic activity and functional stability were determined with a chromogenic assay. The activation of TAFI-CIIYQ-∆1-73 by TAFI activators was evaluated with western blot analysis. RESULTS: In comparison with TAFI-CIIYQ, the deletion mutant exerted high intrinsic activity ('full' apparent TAFIa activity) without cleavage by TAFI activators. TAFI-CIIYQ-∆1-73 was cleavable by thrombin. However, in the presence of thrombomodulin, the thrombin-mediated cleavage of TAFI-CIIYQ-∆1-73 was not accelerated. TAFI-CIIYQ-∆1-73 showed a similar functional stability profile to that of TAFI-CIIYQ. Full cleavage by thrombin did not affect the apparent carboxypeptidase activity of TAFI-CIIYQ-∆1-73 , but resulted in a significant loss of functional stability. CONCLUSIONS: A stable deletion mutant of TAFI with full carboxypeptidase activity without activation is described. The segment Ala74-Arg92 in the activation peptide contributes significantly to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen.

Systemic inhibition and liver-specific over-expression of PAI-1 failed to improve survival in all-inclusive populations or homogenous cohorts of CLP mice.

BACKGROUND: The role of plasminogen activator inhibitor type-1 (PAI-1) in abdominal sepsis remains elusive. OBJECTIVES: To study the influence of inhibition and over-expression of PAI-1 upon survival in cecal ligation and puncture (CLP) sepsis. METHODS: (i) Mice underwent moderate CLP and received 10 mg kg(-1) of either monoclonal anti-PAI-1 (MA-MP6H6) or control (MA-Control) antibody intravenously at 0, 18 or 30 h post-CLP. The 30-h treatment group was additionally stratified into mice predicted to survive (P-SUR) or die (P-DIE) based on IL 6 measured at 24 h post-CLP. (ii) PAI-1 expression was induced with pLIVE.PAI-1 plasmid administered 72 h pre-CLP. Blood was sampled for 5 days and survival was monitored for 28 days. RESULTS: MA-MP6H6 effectively neutralized active PAI-1 and fully restored fibrinolysis while PAI-1 over-expression was liver-specific and correlated with PAI-1 increase in the blood. Without stratification, MA-MP6H6 co-/post-treatment conferred no survival benefit. Prospective stratification (IL-6 cut-off: 14 ng mL(-1) ) suggested increased mortality by MA-MP6H6 treatment in P-SUR that reached 30% difference (vs. MA-Control; P < 0.05) after a retrospective cut-off readjustment to 3.3 ng mL(-1) for better P-SUR homogeneity. Subsequent prospective anti-PAI-1 treatment in P-SUR mice with 3.3 ng mL(-1) cut-off demonstrated a negative but statistically insignificant effect: mortality was higher by 17% after MA-MP6H6 vs. MA-Control. Over-expression of PAI 1 did not alter post-CLP survival. Neither PAI-1 inhibition nor over-expression meaningfully modified inflammatory response and/or organ function. CONCLUSIONS: Restoration of fibrinolysis in early abdominal sepsis was not beneficial and it may prove detrimental in subjects with the lowest risk of death, while preemptive PAI-1 up-regulation at the current magnitude was not protective.

Identification of a novel, nanobody-induced, mechanism of TAFI inactivation and its in vivo application.

BACKGROUND: Down-regulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (i.e. zymogen activity) was reported for the proenzyme (TAFI); however, there is some discussion about its ability to cleave high molecular weight substrates. OBJECTIVE: We aimed to identify and characterize nanobodies toward mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model. METHODS AND RESULTS: Screening of a library of nanobodies toward mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (< 0.35 U mg⁻¹) to 4.4 U mg⁻¹ (at a 16-fold molar ratio over mTAFI). The generated carboxypeptidase activity is unstable at 37 °C. Incubation of mTAFI with VHH-mTAFI-i49 revealed a time-dependent reduced activatability of mTAFI. Epitope mapping revealed that Arg227 and Lys212 are important for the nanobody/mTAFI interaction and suggest destabilization of mTAFI by disrupting the stabilizing interaction between the activation peptide and the dynamic flap region. In vitro clot lysis experiments revealed an enhanced clot lysis due to a reduced activation of mTAFI during clot formation. In vivo application of VHH-mTAFI-i49 in a mouse thromboembolism model decreased dose-dependently the fibrin deposition in the lungs of thromboembolism-induced mice. CONCLUSION: The novel, nanobody-induced, reduced activatability of mTAFI demonstrates to be a very potent approach to enhance clot lysis.

Monoclonal antibodies targeting the antifibrinolytic activity of activated thrombin-activatable fibrinolysis inhibitor but not the anti-inflammatory activity on osteopontin and C5a.

BACKGROUND: Recently, anti-thrombin-activatable fibrinolysis inhibitor (TAFI) mAbs selectively inhibiting plasmin-mediated TAFI activation were shown to stimulate fibrinolysis in vitro and in vivo, suggesting, in contrast to other findings, that plasmin-mediated TAFI activation plays an important role in fibrinolysis regulation. OBJECTIVE: To further characterize the effects of two plasmin-specific anti-TAFI mAbs (MA-TCK11A9 and MA-TCK26D6) on TAFI-dependent inhibition of fibrinolysis. METHODS AND RESULTS: Both mAbs inhibited plasmin-mediated but not thrombin/thrombomodulin-mediated TAFI activation, whereas neither inhibited the cleavage of hippuryl-arginine by activated TAFI (TAFIa). They stimulated tissue-type plasminogen activator-induced fibrinolysis in different clot lysis models through a TAFI-dependent mechanism, especially in the presence of thrombomodulin (TM), a condition in which TAFI is largely activated by the thrombin-TM complex. In a fibrinolysis-based TAFIa activity assay, both mAbs inhibited TAFIa, whereas other mAbs targeting thrombin-TM-mediated TAFI activation did not. The inhibition of TAFIa activity, however, was substrate-specific, because neither mAb inhibited the cleavage of thrombin-activated osteopontin and C5a by TAFIa, thus sparing the anti-inflammatory activity of TAFIa. CONCLUSIONS: Our anti-TAFI mAbs, by selectively inhibiting TAFIa activity on fibrin, may represent the prototype of a new class of TAFI inhibitors with improved pharmacologic activity.

The hyperfibrinolytic state of mice with combined thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 gene deficiency is critically dependent on TAFI deficiency.

BACKGROUND: Mice with single gene deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) or plasminogen activator inhibitor-1 (PAI-1) have an enhanced fibrinolytic capacity. OBJECTIVES: To unravel the function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI-1 gene deficiency. RESULTS: Mating of TAFI knockout (KO) mice with PAI-1 KO mice resulted in the production of TAFI/PAI-1 double-KO mice that were viable, were fertile, and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI-1 double-KO mice did not deviate significantly from those of the single-KO mice or of the wild-type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry measurements with whole blood samples, TAFI KO mice and TAFI/PAI-1 double-KO mice were more sensitive to fibrinolytic activation with tissue-type plasminogen activator than WT or PAI-1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model, as shown by decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI-1 double-KO mice as compared with WT or PAI-1 KO mice. CONCLUSIONS: TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI-1 double-gene-deficient mice, as observed in some basic thrombosis models.

Increased zymogen activity of thrombin-activatable fibrinolysis inhibitor prolongs clot lysis.

BACKGROUND AND OBJECTIVES: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated by proteolytic cleavage into the active enzyme TAFIa. Hydrolysis of the C-terminal lysines on fibrin by TAFIa results in a down-regulation of fibrinolysis. Recent studies demonstrated that the zymogen also exerts an intrinsic enzymatic activity. Our objective was to identify and characterize zymogen-stimulatory nanobodies. METHODS AND RESULTS: The screening of 24 nanobodies against TAFI revealed that two nanobodies (i.e. Vhh-TAFI-a51 and Vhh-TAFI-i103) were able to stimulate the zymogen activity 10- to 21-fold compared with the baseline zymogen activity of TAFI. The increase in catalytic efficiency can be attributed mainly to an increased catalytic rate, as no change in the K(M) -value was observed. The stability, the susceptibility towards PTCI and GEMSA and the kinetics of the stimulated zymogen activity differ significantly from those of TAFIa activity. Epitope mapping revealed that both Asp(75) and Thr(301) are major determinants in the binding of these nanobodies to TAFI. Localization of the epitope strongly suggests that this instability is as a result of a disruption of the stabilizing interactions between the activation peptide and the dynamic flap region (residues 296-350). In TAFI-depleted plasma reconstituted with a non-activatable variant of TAFI (TAFI-R92A), clot lysis could be prolonged by nanobody-induced stimulation of its zymogen activity as well as by increasing its concentration. CONCLUSIONS: Increasing the zymogen activity of TAFI results in an antifibrinolytic effect.

TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation.

BACKGROUND: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment. OBJECTIVE: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis. RESULTS: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A(147) -C(305) -I(325) -I(329) -Y(333) -Q(335) ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC(50) 's range between 0.38- and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their time-dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition. CONCLUSION: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.

Thrombin activatable fibrinolysis inhibitor.

Thrombin activatable fibrinolysis inhibitor (TAFI) was discovered two decades ago as a consequence of the identification of an unstable carboxypeptidase (CPU), which was formed upon thrombin activation of the respective pro-enzyme (proCPU). The antifibrinolytic function of the activated form (TAFIa, CPU) is directly linked to its capacity to remove C-terminal lysines from the surface of the fibrin clot. No endogenous inhibitors have been identified, but TAFIa activity is regulated by its intrinsic temperature-dependent instability with a half-life of 8 to 15 min at 37 °C. A variety of studies have demonstrated a role for TAFI/TAFIa in venous and arterial diseases. In addition, a role in inflammation and cell migration has been shown. Since an elevated level of TAFIa it is a potential risk factor for thrombotic disorders, many inhibitors, both at the level of activation or at the level of activity, have been developed and were proven to exhibit a profibrinolytic effect in animal models. Pharmacologically active inhibitors of the TAFI/TAFIa system may open new ways for the prevention of thrombotic diseases or for the establishment of adjunctive treatments during thrombolytic therapy.

Identification of a bacterial inhibitor against g-type lysozyme.

Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyze peptidoglycan. Bacteria have evolved protective mechanisms that contribute to lysozyme tolerance such as the production of lysozyme inhibitors, but only inhibitors of chicken (c-) and invertebrate (i-) type lysozyme have been identified. We here report the discovery of a novel Escherichia coli inhibitor specific for goose (g-) type lysozymes, which we designate PliG (periplasmic lysozyme inhibitor of g-type lysozyme). Although it does not inhibit c- or i-type lysozymes, PliG shares a structural sequence motif with the previously described PliI and MliC/PliC lysozyme inhibitor families, suggesting a common ancestry and mode of action. Deletion of pliG increased the sensitivity of E. coli to g-type lysozyme. The existence of inhibitors against all major types of animal lysozyme and their contribution to lysozyme tolerance suggest that lysozyme inhibitors may play a role in bacterial interactions with animal hosts.

Generation and characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor.

BACKGROUND AND OBJECTIVE: As activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelid-derived single-domain antibodies towards TAFI. METHODS AND RESULTS: This study reports the generation and characterization of a panel of 22 inhibitory nanobodies. This panel represents a wide diversity in mechanisms for interference with the functional properties of TAFI as the nanobodies interfere with various modes of TAFI activation, TAFIa activity and/or TAFI zymogen activity. Nanobodies inhibiting TAFIa activity and thrombin/thrombomodulin-mediated TAFI activation revealed profibrinolytic properties in a clot lysis experiment with exogenously added thrombomodulin (TM), whereas nanobodies inhibiting plasmin-mediated TAFI activation only revealed profibrinolytic properties in a clot lysis experiment without TM. The results of in vitro clot lysis experiments provided evidence that inhibitory nanobodies penetrate the clot better compared with inhibitory monoclonal antibodies. CONCLUSIONS: These data suggest that the generated nanobodies are potent TAFI inhibitors and are a step forward in the development of a profibrinolytic drug. They might also be an excellent tool to unravel the role of the physiological activators of TAFI in various pathophysiological processes.

Lysozyme inhibitor conferring bacterial tolerance to invertebrate type lysozyme.

Invertebrate (I-) type lysozymes, like all other known lysozymes, are dedicated to the hydrolysis of peptidoglycan, the major bacterial cell wall polymer, thereby contributing to the innate immune system and/or digestive system of invertebrate organisms. Bacteria on the other hand have developed several protective strategies against lysozymes, including the production of periplasmic and/or membrane-bound lysozyme inhibitors. The latter have until now only been described for chicken (C-) type lysozymes. We here report the discovery, purification, identification and characterization of the first bacterial specific I-type lysozyme inhibitor from Aeromonas hydrophila, which we designate PliI (periplasmic lysozyme inhibitor of the I-type lysozyme). PliI has homologs in several proteobacterial genera and contributes to I-type lysozyme tolerance in A. hydrophila in the presence of an outer membrane permeabilizer. These and previous findings on C-type lysozyme inhibitors suggest that bacterial lysozyme inhibitors may have an important function, for example, in bacteria-host interactions.

The role of thrombin activatable fibrinolysis inhibitor in arterial thrombosis at a young age: the ATTAC study.

BACKGROUND AND OBJECTIVES: Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis and may therefore contribute to the pathophysiology of arterial thrombosis. The aim of the present study was to elucidate the pathogenetic role of TAFI levels and genotypes in young patients with arterial thrombosis. PATIENTS AND METHODS: In a case-control study, 327 young patients with a recent first-ever event of coronary heart disease (CHD subgroup) or cerebrovascular disease (ischemic stroke subgroup) and 332 healthy young controls were included. TAFI levels [intact TAFI, activation peptide (TAFI-AP) and (in)activated TAFI (TAFIa(i)] and TAFI activity were measured and genetic variations in the TAFI gene (-438G/A, 505G/A and 1040C/T) were determined. RESULTS: In the total group of patients, TAFIa(i) levels were higher (145.1 +/- 37.5%) than in controls (137.5 +/- 31.3%, P = 0.02). Plasma levels of intact TAFI, TAFI-AP and TAFI activity were similar in patients and controls. In the CHD subgroup (n = 218), intact TAFI levels were higher (109.4 +/- 23.0%) than in controls (102.8 +/- 20.7%, P = 0.02). In 325Ile/Ile homozygotes, lower TAFI levels and a decreased risk of arterial thrombosis were observed (OR 0.58, 95% CI 0.34-0.99) compared with patients with the common 325Thr/Thr genotype. This association was most evident in CHD patients (OR 0.48, 95% CI 0.26-0.90). Haplotype analyses supported a role for the Thr325Ile polymorphism. CONCLUSIONS: TAFIa(i) levels were higher in patients with cardiovascular disease. Furthermore, the TAFI 325Thr/Ile polymorphism was associated with lower TAFI levels and with the risk of cardiovascular disease in young patients, especially in CHD.

Activated thrombin activatable fibrinolysis inhibitor levels are associated with the risk of cardiovascular death in patients with coronary artery disease: the AtheroGene study.

BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis. Results on the association between TAFI levels and the risk of coronary artery disease (CAD) are inconsistent. OBJECTIVES: We investigated the association between TAFI levels and the risk of cardiovascular events in CAD. PATIENTS/METHODS: 1668 individuals with angiographically proven CAD at baseline were followed for a median of 2.3 years, as part of the prospective AtheroGene cohort. Fifty-six deaths from cardiovascular (CV) causes and 35 non-fatal CV events were observed. RESULTS: At baseline, three TAFI measurements were available: one evaluating the total amount of TAFI (t-TAFI), one measuring the TAFIa/TAFIai amount, and the last the released activated peptide (TAFI-AP). TAFIa/TAFIai levels were associated with increased risk of CV death [hazard ratio (HR) for one tertile increase, 2.38 (1.56-3.63); P < 10(-4)]. This association remained significant after adjustment for conventional risk factors, CRP levels, white blood count and markers of thrombin generation and fibrinolysis [HR = 1.69 (1.07-2.67); P = 0.01]. In addition, CPB2 gene polymorphisms explained 12%, 6%, and 3% of t-TAFI, TAFIa/TAFIai and TAFI-AP levels, respectively, but none was associated with CV events. CONCLUSIONS: The amount of activated TAFI, measured by TAFIa/TAFIai ELISA, but not of the t-TAFI is independently associated with the risk of CV death.

High quality structure of cleaved PAI-1-stab.

Here we report the crystal structure of a stablilized plasminogen activator inhibitor-1 variant (PAI-1-N150H-K154T-Q301P-Q319L-M354I (PAI-1-stab)) that shows a cleavage within the reactive centre loop. The new structure is of superior quality compared to the previously determined structure of the cleaved PAI-1-A335P mutant. We present a detailed comparison of the two structures and also compare them with the structure of the active PAI-1-stab. The structural data give important insights into the working mechanism of PAI-1 and also explain the role of various stabilizing mutations.

Bispecific targeting of thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 by a heterodimer diabody.

BACKGROUND AND OBJECTIVES: Thrombin activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) play important roles in fibrinolysis. Both reduce plasmin generation, but they exert their antifibrinolytic effects via different mechanisms. This study reports the cloning and characterization of a heterodimer diabody that inhibits TAFI and PAI-1 simultaneously. METHODS AND RESULTS: The diabody was derived from two inhibiting monoclonal antibodies, i.e. MA-33H1F7, an anti-PAI-1 antibody that induces non-inhibitory substrate behavior of PAI-1, and MA-T12D11, an anti-TAFI antibody that inhibits activation of TAFI by the thrombin-thrombomodulin complex. A single-chain variable fragment (scFv) was derived from MA-T12D11 that displayed slightly reduced binding and inhibitory properties as compared to MA-T12D11. Characterization of the diabody revealed a similar affinity for TAFI and PAI-1 as that of the parental antibodies. Furthermore, the inhibitory properties of MA-33H1F7 and MA-T12D11 were fully preserved in the diabody format. In platelet-free plasma (PFP) clots, addition of the diabody had a stronger effect in shortening lysis times than either MA-T12D11 or MA-33H1F7. A similar reduction in clot lysis time was observed in platelet-rich plasma (PRP) clots. The same effect on clot lysis times in PFP and PRP was also achieved by the combined addition of MA-T12D11 and MA-33H1F7. The lysis rate of human model thrombi, made from whole blood, was approximately doubled after addition of the diabody. Moreover, this effect was significantly better than after the combined addition of the individual antibodies. CONCLUSIONS: These observations demonstrate that simultaneous inhibition of TAFI and PAI-1 results in faster lysis of the formed thrombus.