Clinical response correlates with 4-week postinjection ustekinumab concentrations in patients with moderate-to-severe psoriasis.

BACKGROUND: Cost-effective use of biologicals is important. As drug concentrations have been linked to clinical outcomes, monitoring drug concentrations is a valuable tool to guide clinical decision-making. A concentration-response relationship for ustekinumab at trough is uncertain owing to the contradictory results reported. OBJECTIVES: To investigate the relationship between 4-week postinjection ustekinumab concentrations and clinical response in patients with psoriasis. METHODS: Forty-nine patients with moderate-to-severe psoriasis treated with 45 mg or 90 mg ustekinumab every 12 weeks for ≥ 16 weeks were included. Ustekinumab serum concentrations and anti-ustekinumab antibodies were measured at week 4 after injection and disease severity was assessed by Psoriasis Area and Severity Index (PASI). RESULTS: At week 4 after injection, a significantly negative correlation was observed between ustekinumab concentrations and absolute PASI score up to 5·9 µg mL-1 (ρ = -0·357, P = 0·032). Ustekinumab concentrations were higher in optimal responders (PASI ≤ 2) than in suboptimal responders (PASI > 2) (4·0 vs 2·8 µg mL-1 , P = 0·036). The ustekinumab concentration threshold associated with optimal response was determined to be 3·6 µg mL-1 (area under the curve 0·71, sensitivity 86%, specificity 63%). Only one patient (2%) had anti-ustekinumab antibodies. Psoriatic arthritis was identified as an independent predictor of higher PASI scores and higher ustekinumab concentrations (P = 0·003 and P = 0·048, respectively). CONCLUSIONS: A concentration-response relationship at week 4 after injection was observed for patients with psoriasis treated with ustekinumab. Monitoring 4-week postinjection ustekinumab concentrations could timely identify underexposed patients who might benefit from treatment optimization. What's already known about this topic? Monitoring drug concentrations is a valuable tool that can guide clinical decision-making when drug concentrations are linked to clinical outcomes. The presence of a concentration-response relationship for ustekinumab at trough is still debated owing to the contradictory results reported. What does this study add? A concentration-response relationship at week 4 after injection for ustekinumab-treated patients with psoriasis was demonstrated. Monitoring 4-week postinjection ustekinumab concentrations could timely identify underexposed patients who might benefit from treatment optimization. Based on the findings of this study, a treatment algorithm for patients with a suboptimal response is proposed.

Development and User Experiences of a Biopsychosocial Interprofessional Online Course on Persistent Somatic Symptoms.

Background: Communication between healthcare providers and patients with persistent somatic symptoms (PSS) is frequently hampered by mutual misunderstanding and dissatisfaction. Methods: We developed an online, interprofessional course to teach healthcare providers the knowledge, skills, and attitude they need to diagnose and treat PSS in a patient-centered manner based on the biopsychosocial model. The course consisted of six modules of 45-60 min. Each module contained different types of assignments, based on six cases: videos, discussion boards, reading assignments, polls, and quizzes. For this study, we included (1) medical residents, following the course as part of their residency training, and (2) healthcare providers (general practitioners, medical specialists, physiotherapists, nurses, and psychologists), following the course as continuing vocational training. Throughout the course, participants were asked to fill out online surveys, enquiring about their learning gains and satisfaction with the course. Results: The biopsychosocial approach was integrated across the modules and teached health care workers about recent insights on biological, psychological and social aspects of PSS. In total, 801 participants with a wide variety in clinical experience started the course; the largest groups of professionals were general practitioners (N = 400), physiotherapists (N = 124) and mental healthcare workers (N = 53). At the start of the course, 22% of the participants rated their level of knowledge on PSS as adequate. At the end of the course, 359 participants completed the evaluation questionnaires. Of this group, 81% rated their level of knowledge on PSS as adequate and 86% felt that following the course increased their competencies in communicating with patients with PSS (N = 359). On a scale from 1 to 10, participants gave the course a mean grade of 7.8 points. Accordingly, 85% stated that they would recommend the course to a colleague. Conclusion: Our course developed in a co-design process involving multiple stakeholders can be implemented, is being used, and is positively evaluated by professionals across a variety of health care settings.

Exploring temporal relationships among worrying, anxiety, and somatic symptoms.

OBJECTIVES: The role of anxiety symptoms in the development of functional somatic symptoms (FSS) is unknown. Somatic symptoms may be triggered by or give rise to anxiety symptoms. This study aimed to 1) explore interrelationships among within-day worrying, feeling anxious, and somatic symptoms, and 2) investigate the association between these interrelationships and overall level of FSS. METHODS: This study included 767 participants (83% females, mean age 39 years), who were recruited through an online crowdsourcing study in the Dutch general population. Somatic, and anxiety symptoms were reported thrice daily (6-h intervals) for 30 days using electronic diaries. FSS were assessed at baseline (PHQ-15). Temporal relationships among worrying, feeling anxious, and somatic symptoms were modeled using a multilevel vector autoregressive model. RESULTS: We observed large heterogeneity in the within-person interrelationships among worrying, feeling anxious and somatic symptoms. Averaged over participants, higher-than-usual somatic symptoms were associated with increases in levels of worrying six hours later (B = 0.017, 95% CI [0.006, 0.027]). At the between-person level, FSS levels predicted the persistence of feeling anxious (B = 0.230 95% CI [0.105, 0.350]) and the carry-over of worrying to feeling anxious over six-hours (B = 0.159, 95% CI [0.031, 0.283]). CONCLUSIONS: In contrast to what we expected, higher levels of somatic symptoms over multiple weeks were associated with the persistence and carry-over of within-day anxiety-related symptoms. One within-person association between psychological and somatic symptoms during the day was observed, suggesting that, over a time span of 6-h, anxiety symptoms relate to somatic symptoms only in a minority of people from the general population.

Development of sandwich-type ELISAs for the quantification of rat and murine thrombin activatable fibrinolysis inhibitor in plasma.

BACKGROUND: Altered plasma levels of thrombin activatable fibrinolysis inhibitor (TAFI) are associated with a large number of pathologies. Rat and murine models are frequently used to study the pathophysiological role of TAFI in vivo but immunological tools to quantify rat and murine TAFI are lacking. OBJECTIVE: The production of monoclonal antibodies (mAb) towards rat TAFI and the development of an ELISA for the quantification of rat and murine TAFI in plasma. METHODS AND RESULTS: Monoclonal antibodies were raised in TAFI-deficient mice towards (activated) recombinant rat TAFI. Pair-wise testing of the mAb revealed three suitable ELISA combinations, namely RT36A3F5/RT30D8-HRP, RT36A3F5/RT82F12-HRP and RT82F12/RT36A3F5-HRP. All three ELISAs are highly specific for rat and murine TAFI. TAFI concentrations in the lower ng mL(-1) range can be determined in plasma samples with a high reproducibility. Comparing TAFI antigen levels measured by these ELISAs with TAFIa activity values determined by activity based assays revealed excellent correlations (R(2) > 0.98). The average antigen levels of 20 individual rat plasma samples were 16 +/- 2 microg mL(-1) using the RT36A3F5-RT30D8-HRP, 12 +/- 2 microg mL(-1) using the RT36A3F5-RT82F12-HRP and 21 +/- 2 microg mL(-1) using the RT82F12-RT36A3F5-HRP ELISA. The determined antigen levels in rat plasma are similar to the levels reported for human plasma. CONCLUSIONS: We developed three highly specific and extremely sensitive sandwich-type ELISAs for the quantification of rat and murine TAFI in plasma. The described ELISAs will facilitate in vivo investigation on the pathophysiological role of TAFI.

Individual variation in temporal relationships between exposure to radiofrequency electromagnetic fields and non-specific physical symptoms: A new approach in studying 'electrosensitivity'.

BACKGROUND: Everyday exposure to radiofrequency electromagnetic fields (RF-EMF) emitted from wireless devices such as mobile phones and base stations, radio and television transmitters is ubiquitous. Some people attribute non-specific physical symptoms (NSPS) such as headache and fatigue to exposure to RF-EMF. Most previous laboratory studies or studies that analyzed populations at a group level did not find evidence of an association between RF-EMF exposure and NSPS. OBJECTIVES: We explored the association between exposure to RF-EMF in daily life and the occurrence of NSPS in individual self-declared electrohypersensitive persons using body worn exposimeters and electronic diaries. METHODS: We selected seven individuals who attributed their NSPS to RF-EMF exposure. The level of and variability in personal RF-EMF exposure and NSPS were determined during a three-week period. Data were analyzed using time series analysis in which exposure as measured and recorded in the diary was correlated with NSPS. RESULTS: We found statistically significant correlations between perceived and actual exposure to wireless internet (WiFi - rate of change and number of peaks above threshold) and base stations for mobile telecommunications (GSM + UMTS downlink, rate of change) and NSPS scores in four of the seven participants. In two persons a higher EMF exposure was associated with higher symptom scores, and in two other persons it was associated with lower scores. Remarkably, we found no significant correlations between NSPS and time-weighted average power density, the most commonly used exposure metric. CONCLUSIONS: RF-EMF exposure was associated either positively or negatively with NSPS in some but not all of the selected self-declared electrohypersensitive persons.

Anti-infliximab antibody concentrations can guide treatment intensification in patients with Crohn's disease who lose clinical response.

BACKGROUND: The presence of antibodies towards infliximab (ATI) is associated with lower infliximab (IFX) trough concentrations and loss of response. IFX treatment intensification is effective for restoring response in most, but not all patients with Crohn's disease (CD). AIM: To compare outcome, pharmacokinetics and immunogenicity of treatment intensification strategies in patients with CD who lost clinical response to IFX. METHODS: A retrospective cohort study was conducted, including 103 patients with CD who lost clinical response during IFX maintenance therapy and therefore received a double dose IFX (10 mg/kg) and/or a next infusion after a shortened interval. IFX and ATI concentrations were measured in consecutive trough samples, just before (T0) and after (T+1) treatment intensification. RESULTS: Clinical response (physicians' global assessment) and biological response and remission (CRP) were restored in 63%, 42% and 24% of patients (evaluated at T+1). Treatment intensification increased IFX trough concentrations from 1.2 µg/mL [0.3-3.6] at T0 to 3.6 µg/mL [0.5-10.2] at T+1 (P < .0001). Using a drug tolerant assay, ATI were detected in the T0 sample of 47% of patients. ATI negatively impacted the achieved IFX trough concentration (Spearman r -0.57, P < .0001) and the probability of clinical response (P = 0.034) at T+1. When ATI were quantifiable but <282 ng/mL eq. at T0, combined interval shortening and dose doubling was more effective for restoring therapeutic IFX trough concentrations (≥3 µg/mL at T+1) than dose doubling alone, which in turn was more effective than interval shortening alone (P < .001). CONCLUSION: Antibodies towards infliximab can guide clinical decision-making on treatment intensification.

Efficacy of switching to infliximab in patients with Crohn's disease with loss of response to adalimumab.

BACKGROUND AND STUDY AIMS: Anti-TNF monoclonal antibodies are a cornerstone in the treatment of Crohn's disease. Prospective data on switching from the subcutaneous and human adalimumab (ADM) to the intravenous and chimeric infliximab (IFX) are scarce. PATIENTS AND METHODS: In this prospective, observational, multicentre cohort study we included 21 patients with loss of response to ADM despite at least 4 consecutive weekly injections. Clinical response (CDAI drop≥70 points) and remission (CDAI≤150) were assessed after switching from ADM to IFX after 10 weeks, 6 and 12 months. Predictive factors of response/remission, the need for therapy intensification, discontinuation and safety were investigated. RESULTS: Short-term response and remission (10 weeks) were seen in 57% and 48% respectively. Mid- and long-term clinical response and remission were achieved in 40% and 25% after 6 months and in 45% and 20% after 12 months respectively. At 12 months, 81% still were on IFX. IFX therapy intensification was needed in half of the patients at 6 months and three quarter of patients at 12 months. Undetectable ADM trough levels (despite weekly injections) were a predictive factor for short-term response and remission to IFX. About half of the patients with response at week 10 maintained response at 6 and 12 months. CONCLUSIONS: Switching from ADM to IFX can be efficacious in patients with loss of response, in particular in case of undetectable ADM trough levels. The majority of patients however will need IFX therapy intensification during their first year of treatment.

Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability.

BACKGROUND: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11. OBJECTIVES: To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant. RESULTS: The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity. CONCLUSIONS: Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.

Dose de-escalation to adalimumab 40 mg every 3 weeks in patients with Crohn's disease - a nested case-control study.

BACKGROUND: Data on dose de-escalation in patients with Crohn's disease (CD) are limited. AIM: To evaluate outcomes of dose de-escalation from adalimumab (ADM) every other week (EOW) to every three weeks (ETW). METHODS: We selected patients with CD receiving maintenance therapy with ADM 40 mg ETW with serum levels (SL) available before and after dose de-escalation. Sex- and age-matched controls continuing ADM 40 mg EOW were identified. Patient reported outcome, C-reactive protein (CRP) and serum albumin were collected. RESULTS: Out of 898 patients, we identified 40 (11 male, median 37 years) who de-escalated to ADM 40 mg ETW for ADM-related adverse events (AE, n = 1), ADM SL >7 µg/mL (n = 8), or both (n = 31). Compared to controls, ADM SL dropped significantly within 4 months, without associated clinical or biochemical changes. In 53% of patients, dose de-escalation was associated with disappearance of AE (8/16 skin manifestation, 3/6 arthralgia, 5/7 frequent infectious episodes). During a median follow-up of 24 months, 65% of patients maintained clinical response, but 35% needed dose escalation back to ADM 40 mg EOW because of clinical relapse (n = 8), ADM SL <4 µg/mL (n = 2), or both (n = 4). CRP <3.5 mg/L at dose de-escalation was independently associated with dose escalation-free survival [odds ratio 6.28 (95% CI 1.83-21.59), P = 0.004]. We could not define a minimal ADM SL to consider or maintain dose de-escalation. CONCLUSIONS: Overall, 65% of patients who de-escalated to adalimumab 40 mg every 3 weeks remained in clinical remission for a median of 24 months. In 53% of patients, adalimumab-related adverse events disappeared after dose de-escalation. Regardless of adalimumab SL, disease remission should be assessed objectively prior to dose de-escalation.

Thrombin-activatable fibrinolysis inhibitor in human abdominal aortic aneurysm disease.

Essentials Patients with abdominal aortic aneurysms (AAA) develop dense clots that are resistant to lysis. This study explores the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in human AAA. There is evidence of chronically increased TAFI activation in patients with AAA. TAFI may represent a pharmacological target for cardiovascular risk reduction in AAA. SUMMARY: Background Intra-luminal thrombosis is a key factor in growth of abdominal aortic aneurysms (AAAs). Patients with AAA form dense clots that are resistant to fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) has been shown to influence AAA development in murine models. Objective The aim of this study is to characterize the role of TAFI in human AAA. Methods Plasma levels of TAFI, TAFI activation peptide (TAFI-AP), activated/inactivated TAFI (TAFIa/ai) and plasmin-α2-antiplasmin complex were measured by ELISAs in patients with AAA (n = 202) and controls (n = 188). Results TAFIa/ai and TAFI-AP levels were higher in patients than controls (median [IQR], 20.3 [14.6-32.8] ng mL-1 vs. 14.2 [11.2-19.3] ng mL-1 and 355.0 [232.4-528.1] ng mL-1 vs. 248.6 [197.1-328.1] ng mL-1 ). TAFIa/ai was positively correlated with TAFI-AP (r = 0.164). Intact TAFI levels were not different between patients and controls (13.4 [11.2-16.1] µg mL-1 vs. 12.8 [10.6-15.4] µg mL-1 ). Plasmin-α2-antiplasmin was higher in AAA patients than controls (690.0 [489.1-924.3] ng mL-1 vs. 480.7 [392.6-555.3] ng mL-1 ). Conclusions The increase in TAFIa/ai and TAFI-AP suggests an increased TAFI activation in patients with AAA. Prospective studies are required to further elucidate the role of TAFI and fibrinolysis in AAA pathogenesis.

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.

Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI)--a comparative study assessing the biological equivalence of rat, murine and human TAFI.

BACKGROUND AND OBJECTIVES: Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis. Although rat models to study the role of TAFI are available, the biochemical properties of rat TAFI are not well investigated and immunologic tools are lacking. Therefore, we have characterized recombinant rat TAFI-6His and compared its properties with those of human TAFI as well as of murine TAFI-V5-6His. METHODS AND RESULTS: TAFI from all three species is activatable by the thrombin-thrombomodulin complex, generating a highly unstable protein (TAFIa). Half-lives at 37 degrees C are 8.5+/-0.6 min, 3.4+/-0.4 min and 2.2+/-0.2 min for human, rat and murine TAFIa, respectively. The 50% clot lysis times are 6+/-1 min for TAFI-depleted rat plasma and 137+/-34 min, 62+/-9 min and 50+/-8 min when TAFI-depleted rat plasma is supplemented with 0.02 U of human, rat or murine TAFIa, respectively, which correlates with their half-lives. Upon incubation with the thrombin-thrombomodulin complex, the 36-kDa fragment of rat and murine TAFI is not cleaved into 25-kDa and 11-kDa fragments. Upon incubation of rat TAFI and murine TAFI with plasmin, a 32-kDa fragment is formed due to cleavage at Arg147, in contrast to the formation of a 36-kDa fragment for human TAFI. Concomitantly, activity levels upon plasmin incubation are drastically reduced for rat and murine TAFI. CONCLUSIONS: Recombinant human, rat and murine TAFI have similar but not identical biochemical characteristics, suggesting a similar role during fibrinolysis in vivo.

The pathophysiological relevance of PAI-1 in cardiovascular diseases and the development of monoclonal antibodies as PAI-1 inhibitors.

Plasminogen activator inhibitor- (PAI-1) is an important component of the plasminogen/plasmin system as it is the main inhibitor of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). Consequently, PAI-1 plays an important role in cardiovascular diseases (mainly through inhibition of t-PA) and in cell migration and tumor development (mainly through inhibition of u-PA and interaction with vitronectin). As a member of the serpin superfamily, PAI-1 shares important structural properties with other serpins. However, PAI-1 also exhibits unique conformational and functional properties. The current review provides an overview of the knowledge on PAI-1 gathered since its discovery two decades ago. We are discussing (a) its structural properties of the protein and their subsequent relation to functional activities, (b) its role in a wide variety of (patho)physiological processes and (c) the development of monoclonal antibodies aiming to modulate pharmacologically this risk factor.

Prognostic factors for long-term infliximab treatment in Crohn's disease patients: a 20-year single centre experience.

BACKGROUND: The long-term efficacy of infliximab in patients with Crohn's disease is suboptimal. AIM: To study prognostic factors for real-life long-term effcacy of infliximab in Crohn's disease. METHODS: All consecutive Crohn's disease patients treated with infliximab at a tertiary centre were retrospectively analysed. Only patients who received scheduled infliximab maintenance treatment were considered. Patient- and disease-related factors were used to identify independent predictors of infliximab failure-free survival using Cox proportional hazards regression. RESULTS: Of 1031 patients with Crohn's disease, 261 were eligible for inclusion. Median time on infliximab was 2.4 [IQR 1.4-4.7] years, and 65 (24.9%) patients experienced infliximab failure. Estimated 5-year infliximab failure-free survival was 65.9% (95% CI 58.3-73.5). Multivariate Cox regression identified disease duration ≥1 year (HR 2.5 (95% CI 1.2-5.2), P = 0.02), L1 disease location [HR 2.0 (1.1-3.5), P = 0.02], prior anti-TNF use [HR 2.3 (1.1-4.8), P = 0.03], haemoglobin <13.5 g/dL [HR 2.3 (1.2-4.4), P = 0.02], not using therapeutic drug monitoring [HR 8.0 (4.1-15.6), P = 1 × 10(-9) ], and first dose optimisation within first year [HR 3.7 (2.1-6.6), P = 5 × 10(-6) ] as independent predictors of infliximab failure-free survival. Stratifying patients into risk groups resulted in estimated 3-year infliximab failure-free survival rates ranging from 95.3% (94.2-96.4) to 26.3% (8.6-44.0) depending on the number of risk factors (P = 8 × 10(-13) ). CONCLUSIONS: This study identified several easy to obtain predictors of infliximab failure in patients with Crohn's disease, and these are in line with previous reports. Those with a high-risk profile for infliximab failure in whom infliximab initiation is considered, should be treated as early as possible making use of therapeutic drug monitoring.

The importance of helix F in plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile serpin, as it converts spontaneously into a non-reactive 'latent' conformation. Several studies have suggested an important role for helix F in the functional behavior and stability of the serpins, especially for PAI-1. We constructed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs3A) were deleted. Whereas wild-type PAI-1 (wtPAI-1) exhibits inhibitory properties towards t-PA and u-PA to an extent of 60-80% of the theoretical maximum, PAI-1-delhF did not exert any detectable inhibitory properties, but behaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those conditions converts to the latent conformation. In contrast to active wtPAI-1 and other substrate-type PAI-1 mutants, PAI-1-delhF showed a 3000-fold decreased binding to vitronectin. The obtained results clearly show the importance of helix F in the inhibitory activity of PAI-1. The absence of helix F apparently leads to an impaired kinetics of insertion of the reactive site loop upon interaction with its target proteinase resulting in the inability to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin.

Characterization of plasminogen activator inhibitor 1 mutants containing the P13 to P10 region of ovalbumin or antithrombin III: evidence that the P13 residue contributes significantly to the active to substrate transition.

The serpin plasminogen activator inhibitor 1 (PAI-1) can occur, in vitro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly; PAI-1-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala-Ala-Ala; PAI-1-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val-->Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80+/-9% (mean+/-S.D., n=4) of the theoretical maximum value towards t-PA. PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu) revealed specific activities of 86+/-15%, 77+/-11%, and 100+/-30% respectively, towards t-PA and similar inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37 degreesC, the active conformation of the PAI-1 mutants converted partly into a substrate conformation (i.e. 52+/-5.2%, 55+/-8.2% and 46+/-4.6% for PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86+/-1.0%). In conclusion, even though replacement of the P13 to P10 region of PAI-1 by the corresponding region of a non-inhibitory serpin or of an inhibitory serpin, does not directly affect its inhibitory properties, the nature of the amino acids in this region and of P13 in particular, contributes to its conformational transitions.

Modulation of TAFI function through different pathways--implications for the development of TAFI inhibitors.

OBJECTIVE: To elucidate the mechanism and the binding regions of monoclonal antibodies (MA) that interfere with thrombin-activatable fibrinolysis inhibitor (TAFI)/activated thrombin-activatable fibrinolysis inhibitor (TAFIa) activity. RESULTS: Of 42 MA, 19 interfere with the TAFI activation/TAFIa activity resulting in an inhibition of up to 92%. Characterization of the mechanism of inhibition revealed that 14 MA blocked the activation of TAFI by thrombin/thrombomodulin completely whereas five MA interfered directly with the enzymatic activity of TAFIa. Surprisingly, the former, except one, induced a significant reduction of clot lysis time whereas the latter did not. Affinity studies using a human/murine TAFI chimer revealed that the binding region of the 14 activation blocking MA is located between AA1 and AA67. MA that inhibit exclusively the activation of TAFI by thrombin/thrombomodulin bind to Gly66. A MA that inhibits the activation of TAFI by both thrombin/thrombomodulin and plasmin binds to Val41. The MA that interfere with the enzymatic activity bind to the TAFIa moiety. CONCLUSIONS: The current study reveals at least three different putative molecular targets in the search for pharmacologically active compounds to modulate TAFIa activity.

Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation.

The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A. The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered. The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A. The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.

Ara h 1 protein-antibody dissociation study: evidence for binding inhomogeneities on a molecular scale.

The characterization of biomolecular interactions is essential when designing novel biosensors, since the interaction between the bioreceptor and the ligand determines important biosensing parameters such as sensitivity and selectivity. In this paper we study the interaction of the trimeric Ara h 1 protein with a monoclonal anti-Ara h 1 antibody by means of magnetic force-induced dissociation. The proteins were bound to magnetic particles and polystyrene surfaces by EDC/NHS reaction chemistry and by physisorption, respectively. Two different molecular configurations have been investigated, with either the Ara h 1 protein on the particles or the Ara h 1 protein on the polystyrene surface. A model with a Gaussian distribution of energy barriers for dissociation gives an adequate description for the measured multi-exponential decays. We hypothesize that distributions of molecular orientations as well as experimentally induced variations may underlay the observed distributions. The two molecular configurations show a different peak value of the energy distribution. Similarly, SPR experiments for two distinct configurations (either Ara h 1 protein on the surface, or anti-Ara h 1 antibody on the surface) also show clear differences in dissociation behavior. We hypothesize that the multivalency of the involved molecules leads to different modes of binding. The results of this work highlight the importance of molecular inhomogeneities when studying the interaction processes of biomolecular complexes.