Isoelectric focusing (IEF) of alpha(1)-proteinase inhibitor (A1PI) shows that commercial products and plasma have different glycoisoform band patterns. Those in Aralast (Grifols Biologicals) reflect an anodal shift of glycoisoforms, which has caused concern. The protein, including glycoproteomic analyses, and structural features of A1PI products were investigated by state-of-the-art techniques.Batches from Aralast, Prolastin (Bayer), and Zemaira (Aventis Behring LLC) were analyzed by high-resolution IEF and high-performance size-exclusion chromatography (HP-SEC). Preparative separated isoforms from IEF were further purified by chromatography and subjected to mass spectrometry for sequence analyses, peptide mapping, and glycosylation analysis. Deamidation was quantified by enzymatic isoaspartate detection. Multiple sequence alignments and structural bioinformatics analyses were performed.In HP-SEC, Prolastin had the highest aggregate content at approximately 30 percent. Isoforms from all products purified by high-resolution IEF were sequenced with an amino acid coverage of more than 98 percent. Deamidation of Asn116 and Asn314 in A1PI was to found to some extent in all products and confirmed quantitatively by enzymatic analysis. There were no signs of methionine oxidation. Cys232 was found to be cysteinylated in A1PI in Prolastin and Aralast as in plasma, but not in Zemaira. All products showed truncation of the C-terminal lysine. Intact A1PI concentrates contained mainly diantennary, disialylated and smaller amounts of triantennary, trisialylated N-glycans. The percentage of fucosylation was similar in all products. Site-specific glycan analysis revealed bands M6 contained only diantennary glycans, whereas the more acidic bands M4 and M2 also carried triantennary structures. The most acidic isoforms, M2 in Prolastin and Zemaira and M0 in Aralast, additionally exhibited tetraantennary N-glycans.Protein chemical characterization of A1PI showed that all A1PI products to some extent differ from A1PI circulating in human plasma. Bioinformatic analysis indicated that removal of C-terminal Lys394 and cysteinylation of Cys232 are unlikely to affect structure and/or function of A1PI but cysteinylation may influence interaction between A1PI and its physiologic ligands. Aralast, Prolastin, and Zemaira contain the same set of N-glycans in the same ratios as those in normal human plasma A1PI. Tri- and tetraantennary structures are responsible for the partitioning into IEF isoforms, with the migration shift of Aralast not being due to any difference in the N-glycosylation, but to the partial loss of the C-terminal lysine.
Abstract Heparins represent one of the most frequently used pharmacotherapeutics. Discovered around 1926, routine clinical anticoagulant use of heparin was initiated only after the publication of several seminal papers in the early 1970s by the group of Kakkar. It was shown that heparin prevents venous thromboembolism and mortality from pulmonary embolism in patients after surgery. With the subsequent development of low-molecular-weight heparins and synthetic heparin derivatives, a family of related drugs was created that continues to prove its clinical value in thromboprophylaxis and in prevention of clotting in extracorporeal devices. Fundamental and applied research has revealed a complex pharmacodynamic profile of heparins that goes beyond its anticoagulant use. Recognition of the complex multifaceted beneficial effects of heparin underscores its therapeutic potential in various clinical situations. In this review we focus on the anticoagulant and nonanticoagulant activities of heparin and, where possible, discuss the underlying molecular mechanisms that explain the diversity of heparin's biological actions.
ABSTRACT Background The course of the disease in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mechanically ventilated patients is unknown. To unravel the clinical heterogeneity of the SARS-CoV-2 infection in these patients, we designed the prospective observational Maastricht Intensive Care COVID cohort; MaastrICCht . We incorporated serial measurements that harbour aetiological, diagnostic and predictive information. The study aims to investigate the heterogeneity of the natural course of critically ill patients with SARS-CoV-2 infection. Study population Mechanically ventilated patients admitted to the Intensive Care with SARS- CoV-2 infection. Main message We will collect clinical variables, vital parameters, laboratory variables, mechanical ventilator settings, chest electrical impedance tomography, electrocardiograms, echocardiography as well as other imaging modalities to assess heterogeneity of the natural course of SARS-CoV-2 infection in critically ill patients. The MaastrICCht cohort is, also designed to foster various other studies and registries and intends to create an open-source database for investigators. Therefore, a major part of the data collection is aligned with an existing national Intensive Care data registry and two international COVID-19 data collection initiatives. Additionally, we create a flexible design, so that additional measures can be added during the ongoing study based on new knowledge obtained from the rapidly growing body of evidence. Conclusion The spread of the COVID-19 pandemic requires the swift implementation of observational research to unravel heterogeneity of the natural course of the disease of SARS- CoV-2 infection in mechanically ventilated patients. Our design is expected to enhance aetiological, diagnostic and prognostic understanding of the disease. This paper describes the design of the MaastrICCht cohort. Strengths and limitations of this study Serial measurements that characterize the disease course of SARS-CoV-2 infection in mechanically ventilated patients Data collection and analysis according to a predefined protocol Flexible, evolving design enabling the study of multiple aspects of SARS-CoV-2 infection in mechanically ventilated patients Single centre, including only ICU patients
Virtual ligand screening (VLS) is an in silico technology used for the productive and cost effective search for novel hit or lead compounds. VLS can be divided into rigid body docking (e.g. FRED) and flexible body docking (e.g. Surflex) procedures. Theoretically, rigid body docking VLS is fast but less accurate whereas flexible docking VLS is more accurate but time consuming. The target (e.g. protein) for VLS is dynamic in nature and can adopt different conformations.
The C-terminal C domains of activated coagulation factor VIII (FVIIIa) are essential to membrane binding of this crucial coagulation cofactor protein. To provide an overall membrane binding mechanism for FVIII, we performed simulations of membrane binding through coarse-grained molecular dynamics simulations of the C1 and C2 domain, and the combined C-domains (C1+C2). We found that the C1 and C2 domain have different membrane binding properties. The C1 domain uses hydrophobic spikes 3 and 4, of its total of four spikes, as major loops to bind the membrane, whereas all four of its hydrophobic loops of the C2 domain appear essential for membrane binding. Interestingly, in the C1+C2 system, we observed cooperative binding of the C1 and C2 domains such that all four C2 domain spikes bound first, after which all four loops of the C1 domain inserted into the membrane, while the net binding energy was higher than that of the sum of the isolated C domains. Several residues, mutations of which are known to cause haemophilia A, were identified as key residues for membrane binding. In addition to these known residues, we identified residues from the C1 and C2 domains, which are involved in the membrane binding process, that have not been reported before as a cause for haemophilia A, but which contribute to overall membrane binding and which are likely candidates for novel causative missense mutations in haemophilia A.
Atherosclerosis is a lipid-driven chronic inflammatory disease of the arterial wall. Current treatment of atherosclerosis is focused on limiting its risk factors, such as hyperlipidemia or hypertension. However, treatments that target the inflammatory nature of atherosclerosis are still under development. Discovery of novel targets involved in the inflammation of the arterial wall creates opportunities to design new therapeutics that successfully modulate atherosclerosis. Here, we review drug targets that have proven to play pivotal roles in the adaptive immune system in atherosclerosis, and we discuss their potential as novel therapeutics.
Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.
Epidemiological studies have shown that women who use third-generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate have a higher risk of venous thrombosis than women who use second-generation OC containing levonorgestrel. It is also known that a mutation in factor V (factor V(Leiden)), which results in resistance to activated protein C (APC) and which is the most common cause of hereditary thrombophilia, potentiates the prothrombotic effect of OC. Effects of APC on thrombin generation in the plasma of women using OC were compared to the response to APC in non-OC users and in individuals that were heterozygous or homozygous for factor V(Leiden). The response towards APC was evaluated on basis of the ratio (APC-sr) of the time integrals of thrombin formation determined in the presence and absence of APC. Compared with women not using OC, women who used OC exhibited a significantly decreased sensitivity to APC (P<0.001), independent of the kind of OC used. Women who used third-generation monophasic OC were significantly less sensitive to APC than women using second-generation OC (P<0.001) and had APC-sr that did not significantly differ from heterozygous female carriers of factor V(Leiden) who did not use OC. Women who were heterozygous for factor V(Leiden) and used OC had APC-sr in the range of homozygous carriers of factor V(Leiden). Two women who started OC therapy had significantly elevated APC-sr within 3 d. Acquired APC resistance may explain the epidemiological observation of increased risk for venous thrombosis in OC users, especially in women using third-generation OC.
Human monoclonal antibodies (hmAbs) that protect against all influenza A and B strains are considered the road to universal influenza vaccines. Based on publicly-available data, we analyze the mechanistic and structural basis of pan-influenza protection by CR9114, a hemagglutinin (HA) stem-reactive antibody that protects against influenza subtypes from groups A1, A2, and B. The mechanistic basis of CR9114’s universal protection is not limited to in vitro neutralization, as CR9114 also protects in vivo from strains that escape its neutralizing activity: some H2 strains and influenza B. Fusion inhibition, viral egress inhibition, and activation of Fc-mediated effector functions are key contributors to CR9114’s universal protection. A comparative analysis of paratopes – between CR9114 (pan-influenza protection) and structurally similar V H 1-69 hmAb CR6261 (influenza A1 protection) – pinpoints the structural basis of pan-influenza protection. CR9114’s heterosubtypic binding is conferred by its ability to bind HA with multiple domains: three HCDR loops and FR3. In contrast to other V H 1-69 hmAbs, CR9114 uses a long and polar side chain of tyrosine (Y) residues on its HCDR3 for crucial H-bonds with H3, H5, and B HA. The recognition of a highly conserved epitope by CR9114 results in a high genetic barrier for escape by influenza strains. The nested, hierarchical structure of the mutations between the germline ancestor and CR9114 demonstrates that it is the result of a narrow evolutionary pathway within the B cell population. This rare evolutionary pathway indicates an immuno-recessive epitope and limited opportunity for vaccines to induce a polyclonal CR9114-like response.
