Research Institute for Chromatography

By proteolyzing prothrombotic von Willebrand factor (VWF) multimers, ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type-1 repeats, member 13) ensures balanced hemostasis and prevents microvascular thrombosis. ADAMTS13's conformational regulation is not only crucial for its enzymatic function, but also for the pathophysiology of thrombotic thrombocytopenic purpura (TTP). In the first part of this review, the unique structural features that keep ADAMTS13 in its closed, latent conformation are explored. Moreover, the recent structure predictions that propose a compactly folded model for closed ADAMTS13, and the molecular mechanisms involved in ADAMTS13's opening by its VWF substrate and other allosteric activators are discussed. Over the last decade, the changes in ADAMTS13's conformation in the context of immune-mediated TTP (iTTP) were increasingly characterized, with open ADAMTS13 having emerged as a novel specific biomarker for acute and subclinical iTTP. Furthermore, open ADAMTS13 is gaining clinical attention to improve the prediction of early relapses during follow-up of iTTP patients in remission. The specificity of the open ADAMTS13 biomarker for iTTP was retrospectively validated in patient cohorts with various thrombotic microangiopathies or hemostatic disorders, all dominantly presenting closed ADAMTS13. Hence, this review summarizes the molecular mechanisms that regulate ADAMTS13's conformation and links these with the clinical implications of ADAMTS13's open and closed conformations.

The use of comprehensive two-dimensional liquid chromatography (LC×LC) coupled to mass spectrometry (MS) for characterizing glycosylation of therapeutic enzymes is presented. Recombinant human acid α-glucosidase (rhGAA) was digested and resulting peptides were separated by reversed-phase LC (RPLC) at high and low pH in, respectively, the first and second dimension. Glycopeptide peaks were then selectively detected and identified by MS operated in all-ion fragmentation mode. The study of first generation rhGAA (myozyme), expressed in Chinese hamster ovary (CHO) cells, and next-generation glyco-engineered rhGAA, produced in yeast cells to fine-tune the mannose-6-phosphate (M6P) content, is described.

Resistance to r-tPA (recombinant tissue-type plasminogen activator) is a well-known but poorly understood phenomenon that hampers successful recanalization in patients with acute ischemic stroke. Using clinically relevant thrombi from patients with acute ischemic stroke, we investigated if and how thrombus composition impacts r-tPA-mediated lysis. In addition, we explored strategies to overcome r-tPA resistance. Thrombi were split into 2 parts, 1 of which was used for thrombolysis and the other for detailed histological analysis. Thrombolysis was performed in normal human plasma using r-tPA alone, using r-tPA in combination with DNase-1 or using r-tPA in combination with N,N'-diacetyl-l-cystine. Thrombus lysis was calculated as the percentage of residual thrombus weight compared with its initial weight and the degree of lysis was linked to thrombus composition determined via histology. Interestingly, we found that the efficacy of r-tPA-mediated thrombolysis was strongly correlated with the composition of the thrombi. Thrombi containing high amounts of red blood cells and low amounts of DNA and von Willebrand Factor were efficiently degraded by r-tPA, whereas thrombi containing low amounts of red blood cells and higher amounts of DNA and von Willebrand Factor were resistant to r-tPA. Importantly, combination of r-tPA with DNase-1 or N,N'-diacetyl-l-cystine significantly and specifically improved the lysis of these r-tPA-resistant thrombi. Using patient thrombus material, our results for the first time show that the composition of stroke thrombi largely determines their susceptibility to r-tPA-mediated thrombolysis. Red blood cell-poor thrombi have a specific resistance to r-tPA, which can be overcome by targeting nonfibrin components using DNase-1 or N,N'-diacetyl-l-cystine.

A case is presented of an 83-year-old female patient with a strong suspicion of active bleeding, but no diagnostic contrast blush could be seen on the original computed tomography (CT) scan. Teaching point: When performing CT angiography in veno-arterial extracorporeal membrane oxygenation (VA-ECMO), it is important to understand the altered haemodynamics, as flow-related artefacts such as the vascular watershed phenomenon can obscure bleeding.

Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.

Pre-vaccination SARS-CoV-2 infection can boost protection elicited by COVID-19 vaccination and post-vaccination breakthrough SARS-CoV-2 infection can boost existing immunity conferred by COVID-19 vaccination. Such ‘hybrid immunity’ is effective against SARS-CoV-2 variants. In order to understand ‘hybrid immunity’ at the molecular level we studied the complementarity determining regions (CDR) of anti-RBD (receptor binding domain) antibodies isolated from individuals with ‘hybrid immunity’ as well as from ‘naive’ (not SARS-CoV-2 infected) vaccinated individuals. CDR analysis was done by liquid chromatography/mass spectrometry-mass spectrometry. Principal component analysis and partial least square differential analysis showed that COVID-19 vaccinated people share CDR profiles and that pre-vaccination SARS-CoV-2 infection or breakthrough infection further shape the CDR profile, with a CDR profile in hybrid immunity that clustered away from the CDR profile in vaccinated people without infection. Thus, our results show a CDR profile in hybrid immunity that is distinct from the vaccination-induced CDR profile.