Biology subjects

Ahnstroem, J.

Publications and source records attributed to Ahnstroem, J..

3 recordsLinked to original sources

bioRxiv2025

Pro-coagulant lipids in physiological ratios found in the activated platelet membrane do not impact clot structure or fibrinolysis in purified assays.

PurposeA central role for the pro-coagulant membrane comprising aminophospholipids (aPL) and enzymatically oxidized phospholipids (eoxPL) in promoting hemostasis via interaction with coagulation factor Gla domains is well established. However, little is known about their interactions with the fibrinolytic pathway, their ability to alter clot structure or to support the activated protein C (APC) pathway. Previous studies used membrane liposome compositions that differ from those expected physiologically and/or generated inconsistent findings. To address this, pro-coagulant membranes comprising physiological proportions of aPL and eoxPL will be tested for their ability to support fibrinolysis using standard assays. MethodsThe impact of phospholipids on clot structure and clot lysis was tested using absorbance-based assays. To investigate the impact of PS or eoxPL on fibrinolysis, plasmin was monitored chromogenically, and clot dissolution measured in a purified lysis system activated by tissue plasminogen activator or urokinase. To determine the impact of eoxPL on APC/protein S, FVa was incubated with APC (+/-protein S) in a purified prothrombinase assay. ResultsAt the concentrations of lipids tested in our study, PS did not significantly impact clot structure or fibrinolysis. Similarly, eoxPL did not impact either fibrinolysis or activity of APC/Protein S. ConclusionUsing liposome compositions that approximate activated blood cells, we found that the pro-coagulant membrane is unlikely to influence either clot structure or fibrinolytic activity directly, beyond its well characterized role in supporting Gla dependent coagulation factors and the actions of platelet associated proteins/receptors.

pharmacology and toxicology↗

bioRxiv2025

A Microfluidic Blood Vessel-On-Chip Model of Thrombosis

BackgroundThrombus formation is regulated by the interplay between endothelial cells (EC), platelets and coagulation factors. However, most in vitro assays used to study thrombosis and develop anti-thrombotic therapies do not include the endothelium. ObjectiveTo develop a thrombus-on-chip model that includes endothelium and whole blood and allows for manipulation of extracellular matrix (ECM), shear stress and the addition of multiple cell types. MethodsA cylindrical vessel was created in a collagen matrix using the needle-based fabrication technique in a microfluidic device. Human umbilical vein EC (HUVEC) or endothelial colony-forming cells (ECFC) were cultured in the channel, with continuous monodirectional media turnover. Immunostaining and permeability measurement validated confluence and integrity of the monolayer. To investigate thrombosis, whole blood from healthy donors was perfused through TNF--activated or untreated EC-lined vessels. Image analysis and time-lapse microscopy were used to quantify labelled platelet adhesion and fibrin deposition. ResultsTNF- treatment resulted in increased platelet adhesion and fibrin deposition compared to control vessels. TNF-induced endothelial activation was confirmed by upregulation of adhesion molecules ICAM-1, E-selectin and tissue factor (TF). Thrombus formation in TNF- treated vessels was inhibited by an anti-TF antibody. In ECFC vessels, platelet adhesion and fibrin deposition were comparable to HUVEC, supporting feasibility of patient-based studies. ConclusionsWe developed a perfused thrombus-on-chip model that combines key elements of thrombus formation including endothelium. The model is amenable to independent control of microenvironmental stimuli, crosstalk with tissue-specific cells, and the inclusion of patients own cells and blood for precision medicine studies. ESSENTIALS- The endothelium is a key contributor to thrombosis - Standard in vitro methods to study thrombosis do not include the endothelium - We have developed a thrombus-on-a-chip model of thromboinflammation to measure live platelet adhesion and fibrin deposition on activated endothelium - Using endothelial colony forming cells (ECFC), we show that the method is suitable for patient studies with autologous endothelium and blood

bioengineering↗

bioRxiv2020

Exosite inhibition of A Disintegrin And Metalloproteinase with Thrombospondin motif (ADAMTS)-5 by a glycoconjugated arylsulfonamide

ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Its aggrecanase activity has been directly linked to the etiology of osteoarthritis (OA), identifying ADAMTS-5 as a pharmaceutical target for OA treatment. However, most existing ADAMTS-5 inhibitors target its active site and therefore suffer from poor selectivity. Here, using a novel approach, we have designed a new class of sugar-based arylsulfonamide inhibitors, which are selective for ADAMTS-5 through binding to a previously unknown substrate-binding site (exosite). Docking calculations combined with molecular dynamics simulations demonstrated that our lead compound is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Targeted mutagenesis identified disintegrin-like domain residues K532 and K533 as an exosite which is critical for substrate recognition. Furthermore, we show that this exosite acts as major determinant for inhibitor binding and, therefore, can be targeted for development of selective ADAMTS-5 inhibitors.

biochemistry↗