Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Incorporating Diversity, Equity, and Inclusion Content Into Bioengineering Curricula: A Program-Level Approach.

Diversity, equity, and inclusion (DEI) are interconnected with bioengineering, yet have historically been absent from accreditation standards and curricula. Toward educating DEI-competent bioengineers and meeting evolving accreditation requirements, we took a program-level approach to incorporate, catalog, and assess DEI content through the bioengineering undergraduate program. To support instructors in adding DEI content and inclusive pedagogy, our team developed a DEI planning worksheet and surveyed instructors pre- and post-course. Over the academic year, 74% of instructors provided a pre-term and/or post-term response. Of responding instructors, 91% described at least one DEI curricular content improvement, and 88% incorporated at least one new inclusive pedagogical approach. Based on the curricular adjustments reported by instructors, we grouped the bioengineering-related DEI content into five DEI competency categories: bioethics, inclusive design, inclusive scholarship, inclusive professionalism, and systemic inequality. To assess the DEI content incorporation, we employed direct assessment via course assignments, end-of-module student surveys, end-of-term course evaluations, and an end-of-year program review. When asked how much their experience in the program helped them develop specific DEI competencies, students reported a relatively high average of 3.79 (scale of 1 = "not at all" to 5 = "very much"). Additionally, based on student performance in course assignments and other student feedback, we found that instructors were able to effectively incorporate DEI content into a wide variety of courses. We offer this framework and lessons learned to be adopted by programs similarly motivated to train DEI-competent engineering professionals and provide an equitable, inclusive engineering education for all students.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Distinct platelet F-actin patterns and traction forces on von Willebrand factor versus fibrinogen.

Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.

Black dots: High-yield traction force microscopy reveals structural factors contributing to platelet forces.

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed 'black dots') that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces. STATEMENT OF SIGNIFICANCE: Cells produce contractile forces during division, migration, or wound healing. Measuring cellular forces provides insight into their health, behavior, and function. We developed a technique that calculates cellular forces by seeding cells onto a pattern and quantifying how much each cell displaces the pattern. This technique is capable of measuring hundreds of cells without needing to detach them. Using this technique to evaluate human platelets, we find that platelets exerting more force tend to have more spread area, are more circular in shape, and have more uniformly distributed cytoskeletal filaments. Due to our high yield of data, we were able to apply statistical techniques that revealed combinatorial effects between these factors.

Storage temperature determines platelet GPVI levels and function in mice and humans.

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature-stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP; however, their rate of aggregation in response to the GPVI agonist collagen was significantly lower. In a mouse model of platelet transfusion, we found a significantly lower response rate to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.

(De)form and Function: Measuring Cellular Forces with Deformable Materials and Deformable Structures.

The ability for biological cells to produce mechanical forces is important for the development, function, and homeostasis of tissue. The measurement of cellular forces is not a straightforward task because individual cells are microscopic in size and the forces they produce are at the nanonewton scale. Consequently, studies in cell mechanics rely on advanced biomaterials or flexible structures that permit one to infer these forces by the deformation they impart on the material or structure. Herein, the scientific progression on the use of deformable materials and deformable structures to measure cellular forces are reviewed. The findings and insights made possible with these approaches in the field of cell mechanics are summarized.

Engineering Cell Surface Function with DNA Origami.

A specific and reversible method is reported to engineer cell-membrane function by embedding DNA-origami nanodevices onto the cell surface. Robust membrane functionalization across epithelial, mesenchymal, and nonadherent immune cells is achieved with DNA nanoplatforms that enable functions including the construction of higher-order DNA assemblies at the cell surface and programed cell-cell adhesion between homotypic and heterotypic cells via sequence-specific DNA hybridization. It is anticipated that integration of DNA-origami nanodevices can transform the cell membrane into an engineered material that can mimic, manipulate, and measure biophysical and biochemical function within the plasma membrane of living cells.

Effects of nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NONO-NSAIDs) on melanoma cell adhesion.

A new class of nitric oxide (NO•)-releasing nonsteroidal anti-inflammatory drugs (NONO-NSAIDs) were developed in recent years and have shown promising potential as NSAID substitutes due to their gentle nature on cardiovascular and gastrointestinal systems. Since nitric oxide plays a role in regulation of cell adhesion, we assessed the potential use of NONO-NSAIDs as anti-metastasis drugs. In this regard, we compared the effects of NONO-aspirin and a novel NONO-naproxen to those exerted by their respective parent NSAIDs on avidities of human melanoma M624 cells. Both NONO-NSAIDs, but not the corresponding parent NSAIDs, reduced M624 adhesion on vascular cellular adhesion molecule-1 (VCAM-1) by 20-30% and fibronectin by 25-44% under fluid flow conditions and static conditions, respectively. Only NONO-naproxen reduced (~56%) the activity of ß1 integrin, which binds to α4 integrin to form very late antigen-4 (VLA-4), the ligand of VCAM-1. These results indicate that the diazeniumdiolate (NO•)-donor moiety is critical for reducing the adhesion between VLA-4 and its ligands, while the NSAID moiety can impact the regulation mechanism of melanoma cell adhesion.