Biology subjects

Passam, F. H.

Publications and source records attributed to Passam, F. H..

5 recordsLinked to original sources

bioRxiv2026

Patient-specific "Physical Twin" artery-on-chip platform reveals complex flow-dependent VWF mechanobiology and guides personalized antithrombotic therapy

Ischemic stroke triggered by carotid atherosclerosis remains unpredictable because the thrombosis embolization is governed by patient-specific vascular geometry and hemodynamics that cannot be recapitulated in conventional models. Here, we engineered "Physical Twin" artery-on-chip platforms that reproduce individualized carotid anatomy with humanized subendothelial matrix composition and arterial endothelial phenotype under physiological flow patterns. We then established thrombotic microenvironments following laser-induced injury. Computational fluid dynamics-guided experiments across distinct patient geometries reveal that local shear dictates a mechanobiological hierarchy: high-shear bifurcations ([~]3,000 s-{superscript 1}) produce von Willebrand factor (VWF) A1-dependent thrombi suppressible by conformationally sensitive inhibitors, whereas low-shear stenoses generate fragile aggregates where VWF-integrin IIb{beta} coupling governs embolization overgrowth. Pulsatile flow suppresses thrombotic growth independent of mean shear. Molecular dynamics simulations reveal why conformationally sensitive nanobodies (caplacizumab) outperform shear-independent aptamers (ARC1172) in high-shear bifurcation flow zones. This Physical Twin platform provides a mechanistic blueprint for geometry-informed, personalized antithrombotic therapy selection.

bioengineering↗

bioRxiv2026

Iterative structural homology search identifies new substrates of the protein O-fucosyltransferases POFUT3 and POFUT4

O-fucosylation plays an essential role in controlling protein folding, secretion and protein-protein interactions within the extracellular space. Recently, we identified a new form of protein O-fucosylation occurring on the N-terminal Elastin Microfibril Interaction (EMI) domain of several secreted proteins, mediated by two previously uncharacterized protein O-fucosyltransferases, POFUT3 (FUT10) and POFUT4 (FUT11). As all POFUT enzymes (POFUT1-4) are highly specific for the three-dimensional (3D) structure of their substrate protein domains, we postulated that structural homologues of these domains in other proteins may also be O-fucosylated. Here, we employed iterative protein structural homology searches as a novel strategy for identifying EMI-like domains that may serve as potential substrates for POFUT3/4. We discovered that microfibrillar-associated protein 2 and 5 (MFAP2/MFAP5) contain EMI-like domains and are O-fucosylated at high stoichiometry in human tissues. Unexpectedly, we showed that only POFUT3 is both necessary and sufficient for MFAP2/MFAP5 O-fucosylation, despite POFUT4 also having strong protein-protein interactions with MFAP2/MFAP5. Finally, we determined that O-fucosylation of MFAP2/MFAP5 is required for their efficient secretion, similar to other EMI domain-containing proteins. Together, these data demonstrate the power of sensitive structural homology analysis in identifying new enzyme-substrate relationships and protein-protein interactions.

biochemistry↗

bioRxiv2024

Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

High platelet reactivity is associated with adverse clinical events and is more frequent in people with diabetes mellitus (DM). To better understand platelet dysfunction in DM, we performed a proteomic analysis in platelets from a matched cohort of 34 people without, and 42 people with type 2 DM. The cohorts were matched by clinical characteristics including age, sex, and coronary artery disease burden. Using high sensitivity unbiased proteomics, we consistently identified over 2,400 intracellular proteins, and detected proteins that are differentially released by platelets from people with diabetes in response to low dose thrombin. Importantly, we identified the endoplasmic reticulum (ER) protein SEC61 translocon subunit beta (SEC61B) was increased in platelets from humans and mice with in vivo hyperglycemia. SEC61B was increased in megakaryocytes in mouse models of diabetes, in association with megakaryocyte ER stress. A rise in cytosolic calcium is a key aspect in platelet activation, and the SEC61 translocon is known to act as a channel for ER calcium leak. We demonstrate that cultured cells overexpressing SEC61B have increased calcium flux and decreased protein synthesis. In accordance, hyperglycemic mouse platelets mobilized more calcium to the cytosol and had lower protein synthesis compared with normoglycemic platelets. Independently, in vitro induction of ER stress increased platelet SEC61B expression and markers of platelet activation. We propose a mechanism whereby ER stress-induced upregulation of platelet SEC61B leads to increased cytosolic calcium, potentially contributing to platelet hyperactivity in people with diabetes. Key PointsO_LIPlatelet SEC61B is increased in hyperglycemia and contributes to increased endoplasmic reticulum (ER) calcium leak C_LIO_LIIncreased ER calcium leak is associated with ER stress and platelet hyperactivity C_LI

cell biology↗

bioRxiv2022

Ultra-sensitive platelet proteome maps the O-glycosylation landscape and charts the response to thrombin dosage

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets tendency to pre-activate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1,300 proteins. Unbiased scanning for post-translational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterised sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal EMI domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new consensus sequence. Our data suggest that Protein O-fucosyltransferase 1 (POFUT1) is responsible for this modification. Secretion of MMRN1 was reduced in cells lacking POFUT1, supporting a key role of O-fucosylation in MMRN1 function. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3,800 proteins, which confirmed the platelet origin of releasate proteins by anti-correlation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The comprehensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis. Key PointsO_LIHigh-sensitivity proteome mapping of human platelets identifies O-glycosylation of platelet proteins at key functional sites C_LIO_LIPlatelet O-fucosyltransferase POFUT1 regulates the secretion of adhesive protein multimerin-1 (MMRN1) C_LI

biochemistry↗

bioRxiv2022

Mechano-redox control of Mac-1 de-adhesion from ICAM-1 by protein disulfide isomerase promotes directional movement of neutrophils under flow

Macrophage-1 antigen or Mac-1 (CD11b/CD18, M{beta}2) is a leukocyte integrin essential for firm adhesion of neutrophils, lymphocytes and monocytes against flow when recruited to the endothelium. To migrate to the site of inflammation, leukocytes require coordinated adhesion and de-adhesion for directional movement. The vascular thiol isomerase, protein disulfide isomerase (PDI), was found by fluorescence microscopy to colocalize with high affinity Mac-1 at the trailing edge of stimulated neutrophils when adhered to ICAM-1 under fluid shear. From differential cysteine alkylation and mass spectrometry studies, PDI cleaves two allosteric disulfide bonds, C169-C176 and C224-C264, in the {beta}I domain of the {beta}2 subunit, and in mutagenesis and cell transfection studies, cleavage of the C224-C264 disulfide bond was shown to selectively control Mac-1 dis-engagement from ICAM-1 under fluid shear. Molecular dynamics simulations and binding of conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the {beta}I domain that allosterically alters exposure of an I domain epitope and shifts Mac-1 to a lower affinity state. From studies of neutrophil adherence to ICAM-1 under fluid shear, these molecular events promote neutrophil motility in the direction of flow at high shear stress. In summary, shear-dependent PDI cleavage of neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

biochemistry↗