Synthesis and Evaluation of Glycosyl Luciferins.

Measuring glycosidase activity is important to monitor any aberrations in carbohydrate hydrolase activity, but also for the screening of potential glycosidase inhibitors. To this end, synthetic substrates are needed which provide an enzyme-dependent read-out upon hydrolysis by the glycosidase. Herein, we present two new routes for the synthesis of caged luminescent carbohydrates, which can be used for determining glycosidase activity with a luminescent reporter molecule. The substrates were validated with glycosidase and revealed a clear linear range and enzyme-dependent signal upon the in situ generation of the luciferin moiety from the corresponding nitrile precursors. Besides, we showed that these compounds could directly be synthesized from unprotected glycosyl-α-fluorides in a two-step procedure with yields up to 75 %. The intermediate methyl imidate appeared a key intermediate which also reacted with d-cysteine to give the corresponding d-luciferin substrate rendering this a highly attractive method for synthesizing glycosyl luciferins in good yields.

Solid-Phase Synthesis of Caged Luminescent Peptides via Side Chain Anchoring.

The synthesis of caged luminescent peptide substrates remains challenging, especially when libraries of the substrates are required. Most currently available synthetic methods rely on a solution-phase approach, which is less suited for parallel synthesis purposes. We herein present a solid-phase peptide synthesis (SPPS) method for the synthesis of caged aminoluciferin peptides via side chain anchoring of the P1 residue. After the synthesis of a preliminary test library consisting of 40 compounds, the synthetic method was validated and optimized for up to >100 g of resin. Subsequently, two separate larger peptide libraries were synthesized either having a P1 = lysine or arginine residue containing in total 719 novel peptide substrates. The use of a more stable caged nitrile precursor instead of caged aminoluciferin rendered our parallel synthetic approach completely suitable for SPPS and serine protease profiling was demonstrated using late-stage aminoluciferin generation.

Activity Sensing of Coagulation and Fibrinolytic Proteases.

The blood coagulation cascade is a complex physiological process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.

Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors.

Since the outbreak of SARS-CoV-2 in December 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (MPro ) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential MPro inhibitors we developed a luminescent assay using a peptide based probe containing a cleavage site specific for MPro . This assay was validated showing IC50 values similar to those reported in the literature for known MPro inhibitors and can be used to screen new inhibitors.

Thrombin and plasmin generation in patients with plasminogen or plasminogen activator inhibitor type 1 deficiency.

INTRODUCTION: Deficiencies of plasminogen and plasminogen activator inhibitor type 1 (PAI-1) are rare disorders of fibrinolysis. Current laboratory assays for analysis of activity of plasminogen and PAI-1 do not provide an accurate correlation with clinical phenotype. METHODS: The Nijmegen Hemostasis Assay (NHA) was used to simultaneously measure thrombin and plasmin generation in 5 patients with plasminogen deficiency (PLGD) and 10 patients with complete PAI-1 deficiency. Parameters analysed included: lag time ratio, thrombin peak time ratio, thrombin peak height, thrombin potential (AUC), fibrin lysis time, plasmin peak height and plasmin potential. Parameters were expressed as a percentage compared to a reference value of 53 healthy normal controls. RESULTS: Patients with PLGD demonstrated a short lag time and thrombin peak time, with normal thrombin peak height but an increased AUC. Plasmin generation was able to be detected in only one (23% plasminogen activity) of the five PLGD patients. All ten PAI-1 deficient patients demonstrated a short lag and thrombin peak time, low thrombin peak height with normal AUC. Plasmin generation revealed an increased plasmin peak and plasmin potential; interestingly, there was a large variation between individual patients despite all patients having the same homozygous defect. CONCLUSION: Patients with either PLGD or PAI-1 deficiency show distinct abnormalities in plasmin and thrombin generation in the NHA. The differences observed in the propagation phase of thrombin generation may be explained by plasmin generation. These results suggest that disorders of fibrinolysis also influence coagulation and a global assay measuring both activities may better correlate with clinical outcome.

Epilepsy under my skin?

A 44-year-old male patient was admitted to the hospital for observation after an unwitnessed syncope. Physical examination revealed skin purpura and bilateral tongue haematoma. Laboratory studies were unremarkable. Radiological imaging showed no abnormalities of the vasculature, signs of thrombosis or brain anomalies. Biopsy of a purpuric lesion revealed extravasation of erythrocytes. After excluding several causes of both syncope and purpura, the typical location of these thoracocervicofacial purpura, the tongue haematoma and an elevated prolactin level (which came back later) led to the diagnosis of an epileptic seizure. The patient was referred to the neurology department for follow-up. Within 3 weeks, the purpura were completely resolved, and the patient remained free of seizures during follow-up. In case of an unwitnessed syncope, an epileptic seizure should be carefully considered and thoracocervicofacial purpura can be the pivotal manifestation leading to this diagnosis.

Intentional overdose of dolutegravir/abacavir/lamivudine (Triumeq) in a 26-year-old man.

Triumeq is a single-tablet regimen for patients with HIV infection comprising dolutegravir, abacavir and lamivudine. Overdoses with Triumeq have not been reported previously. We present a case of a 26-year-old man who presented to our hospital after intentionally ingesting 30 tablets of Triumeq. An intoxication with Triumeq can lead to several side effects. An overdose of abacavir and lamivudine can cause mitochondrial toxicity and lactic acidosis. An intoxication with dolutegravir appears to be relatively harmless. As Triumeq will be used on a regular basis as treatment for patients with HIV-1 infection, these intoxications are expected to be encountered more often.

Pharmacodynamics of recombinant activated factor VII and plasma-derived factor VII in a cohort of severe FVII deficient patients.

Recombinant activated factor VII (rFVIIa) and plasma-derived factor VII (pdFVII) are used to prevent bleedings in severe FVII deficient patients, despite their short half-lifes. It is suggested that FVII levels of 15-20 IU/dL are sufficient to maintain hemostasis. We analyzed the pharmacodynamic effects of FVII substitution therapy in the Nijmegen Hemostasis Assay (NHA) that simultaneously measures thrombin and plasmin generation. Ten severe FVII deficient patients were treated with 20 µg/kg rFVIIa or 25 IU/kg pdFVII in a cross-over design. Thrombin generation lag-time (TG-LT) was identified as an effect-response parameter. Pharmacodynamic analysis using a maximum effect model showed 50% reduction of the TG-LT effect at ~2 IU/dL FVII activity for both rFVIIa and pdFVII. The FVII activity to obtain TG-LT comparable to the upper limit of normal range in healthy controls (4 min) was given by the effective concentration (ECnormal), showing sufficient hemostasis at 3-4 IU/dL FVII activity. No association was seen between FVII activity and other thrombin or plasmin generation parameters as measured by NHA. In conclusion, 3-4 IU/dL FVII activity seems sufficient to maintain hemostasis in patients with severe FVII deficiency during prophylaxis. These data may suggest a potential value for measurement of TG-LT in the monitoring of FVII(a) therapy.

Global haemostasis assays, from bench to bedside.

Bleeding and thrombosis are the ultimate clinical outcomes of aberrations in the haemostatic process. Haemostasis prevents excessive blood loss due to the effort of various compartments like the vasculature, blood cells, coagulation and fibrinolysis. The complexity of all processes involved makes the diagnosis of aberrations difficult, cumbersome and expensive. A single assay to detect any factor disturbing this haemostatic balance with high sensitivity and specificity would be of great value, especially if the outcome of this assay correlates well with clinical outcome. Despite years of research, such an assay is not yet available; however, some interesting candidates are under development and combine the effects of various compartments. This review describes the development of global haemostasis assays and summarizes the current state of the art of these haemostasis assays covering thrombin and plasmin generation, turbidity and thromboelastography/thromboelastometry. Finally, we discuss the applicability of global assays in clinical practice and we provide a future perspective on the ongoing development of automation and miniaturisation as it is our belief that these developments will benefit the standardization of global haemostasis assays.

A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were < 9% and 6-25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC-MS.

A method for high-resolution proteomics analyses of complex protein mixtures is presented using multidimensional HPLC coupled to MS (MDLC-MS). The method was applied to identify proteins that are differentially expressed during fruit ripening of tomato. Protein extracts from red and green tomato fruits were digested by trypsin. The resulting highly complex peptide mixtures were separated by strong cation exchange chromatography (SCX), and subsequently analyzed by RP nano-LC coupled to quadrupole-TOF MS. For detailed quantitative comparison, triplicate RP-LC-MS runs were performed for each SCX fraction. The resulting data sets were analyzed using MetAlign software for noise and data reduction, multiple alignment and statistical variance analysis. For each RP-LC-MS chromatogram, up to 7000 mass components were detected. Peak intensity data were compared by multivariate and statistical analysis. This revealed a clear separation between the green and red tomato samples, and a clear separation of the different SCX fractions. MS/MS spectra were collected using the data-dependent acquisition mode from a selected set of differentially detected peptide masses, enabling the identification of proteins that were differentially expressed during ripening of tomato fruits. Our approach is a highly sensitive method to analyze proteins in complex mixtures without the need of isotope labeling.