Platelet Adhesion Research Articles

Elastic and recoverable sponges based on collagen/yeast β-glucan for quick hemostasis

In this investigation, we aimed to engineer sponges with exceptional mechanical and hemostatic capabilities for effective wound healing. By combining collagen, a stiff fibril protein in ECM, with β-glucan, an elastic and triple-helical polysaccharide from yeast cell wall, we prepared a series of composite sponges, designated as CY sponges. This material exhibited a uniform pore structure, displaying enhanced elasticity and shape recovery ability compared to pure collagen sponges. Also, the incorporation of Yeast β-glucan (YG) significantly improved the fluid absorption ability and stability of the sponges. In vitro hemostasis tests demonstrated that the CY sponges exhibited a notably lower in vitro coagulation index (19.21 %) compared to the collagen control (64.84 %), accompanied by superior erythrocyte (64.64 %) and platelet (64.95 %) adhesion properties. Animal studies further substantiated the sponge's hemostatic efficacy, as CY40 led to a reduction in average bleeding volume by 25.26 % and 28.97 %, and a shorter hemostatic time by 31.70 % and 30.77 % compared to collagen, indicating accelerated wound healing. These findings suggest that the addition of yeast β-glucan into collagen sponges can improve their elasticity, shape recovery ability, hemostatic performance and wound repair ability.

Inhibition of PCSK9 Protects against Cerebral Ischemia‒Reperfusion Injury via Attenuating Microcirculatory Dysfunction.

Proprotein convertase substilin/kexin type 9 (PCSK9), a pivotal protein regulating lipid metabolism, has been implicated in promoting microthrombotic formation and inflammatory cascades, thereby contributing to cardiovascular ischemia/reperfusion (I/R) injury. However, its involvement in cerebral I/R injury and its potential role in microcirculation protection remain unexplored. In this investigation, we utilized a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to simulate ischemic stroke. Different concentrations of evolocumab (1, 5, 10mg/kg, i.v.), a PCSK9 inhibitor, were administered to assess its impact. Immunofluorescence staining was employed to analyze changes in the expression of occludin, claudin-5, thrombocyte, ICAM-1, VCAM-1, and CD45, providing insights into blood-brain barrier integrity, platelet adhesion, and immune cell infiltration. Moreover, the Morris water maze and elevated plus maze were utilized to evaluate neurological and behavioral functions in MCAO/R mice, shedding light on the effects of PCSK9 inhibition. Our findings revealed a surge in plasma PCSK9 levels post-MCAO/R, peaking at 24h post-reperfusion. Evolocumab (10mg/kg) treatment significantly mitigated brain infarction and neurological deficits, evidenced by enhanced locomotor function and reduced post-stroke anxiety. However, it did not ameliorate cognitive impairment following MCAO/R. Additionally, evolocumab administration led to diminished leakage of evans blue solution and upregulated expression of occludin and claudin-5. Thrombocyte, ICAM-1, VCAM-1, and CD45 levels were notably reduced in the penumbral area post-evolocumab treatment. These protective effects are speculated to be mediated through the inhibition of the ERK/NF-κB pathway. The PCSK9 inhibitor evolocumab holds promise as a therapeutic agent during the acute phase of stroke, exerting its beneficial effects by modulating the ERK/NF-κB signaling pathway.

Small but mighty: Platelets as multifunctional architects of tumor metastasis and immune regulation

AbstractPlatelets play an irreplaceable role in hemostasis and wound healing. However, beyond these classical roles, as the smallest anucleate cells in the blood stream, they are crucial for immune response which have inflammatory functions through specialized receptors and different signaling pathways, influencing both innate and adaptive immune response. Furthermore, many research have proved that platelets significantly contribute to tumor metastasis and are associated with poor prognoses in cancer patients through its coagulability and supporting an immunosuppressive tumor microenvironment. When tumor cells detach from the primary tumor mass and enter the bloodstream, they rapidly initiate the direct activation and adhesion of platelets, forming a protective microenvironment. This environment shields circulating tumor cells (CTCs) from the mechanical shear forces of blood flow and immune surveillance. Here we delve into the interaction between platelets and immunomodulation and explore the multifaceted roles and underlying mechanisms by which platelets influence tumor cell metastasis and tumor growth. Furthermore, we also discussed the diagnostic role of platelets in cancer occurrence and progression, as well as the feasibility and prospects of targeting platelets for antitumor immunotherapy. This review provides a multidimensional perspective and reference for platelet‐related cancer treatment strategies and diagnosis.

Abstract 4134384: Sotagliflozin, a Dual Inhibitor of Sodium-Glucose Transporters 1 and 2, Elicits Cardioprotective Effects Through Attenuation of Platelet Activation and Thrombosis

Sodium-glucose cotransporter (SGLT) inhibitors are used in the treatment of type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD). In the SCORED trial, sotagliflozin (SOTA), a dual SGLT1/2 inhibitor, demonstrated significant reductions in both myocardial infarction (MI) and stroke in patients with T2DM and CKD. These results suggest a cardiovascular (CV) benefit of SOTA in patients with T2DM and CKD who are at increased risk of thromboembolic disease. To investigate the underlying mechanism of CV protection, we evaluated the effects of SOTA on platelet activation and thrombus formation, in both in vivo and ex vivo models, to understand its effects on MI and stroke. Washed human platelets treated with SOTA were stimulated with various agonists and platelet activation was assessed via lumi-aggregometry and flow cytometry. In whole blood, SOTA was evaluated for its potential to reduce platelet adhesion and thrombus formation in the perfusion flow chamber and Total Thrombus formation Analysis System (TTAS) assays. In vivo , mice dosed with SOTA were assessed for thrombus formation at the site of injury in real-time in the cremaster arteriole injury model. To determine the effect of SOTA on hemostasis, we assessed coagulation parameters ex vivo in human whole blood using thromboelastography. Finally, bleeding time was measured in vivo in mice dosed with SOTA using the tail bleeding assay. In washed platelets, SOTA inhibits platelet activation and aggregation in a dose-dependent manner. Similarly, a dose-dependent decrease in platelet adhesion and thrombus formation was observed in whole blood assays. In vivo , platelet accumulation and fibrin formation were decreased in mice dosed with SOTA, however no effect is observed in coagulation parameters or bleeding time. SOTA inhibits platelet activation and thrombus formation with no impact on coagulation parameters or bleeding potential. These findings highlight a potential mechanism through which SOTA reduces the risk of stroke and MI in patients with T2DM and CKD, underscoring the importance of further investigation into the role of SOTA in CV protection, particularly in patients suffering from both T2DM and CKD at high risk of thromboembolic events.

Abstract 4134630: Focal Delivery of Antiplatelet Therapy to Prevent Post-Stent Bleeding in High-Risk Patients

Background: Over 3 million percutaneous coronary interventions (PCI) are performed yearly to treat coronary artery stenosis. Stent thrombosis is a catastrophic complication associated with high morbidity and mortality, and its prevention requires the use of prolonged dual antiplatelet therapy (DAPT), which increases bleeding risk. Within the first 30 days after PCI, the mortality of DAPT-associated major bleeding is on par with recurrent myocardial infarction. Research question: No stent system that provides focal antiplatelet activity to prevent stent thrombosis exists, eliminating the need for systemic DAPT and subsequent bleeding risk. Aim: To address this unmet need, we have developed such a stent, termed “the ticagrelor coated stent” (TCS). Methods: Self-assembled monolayers (SAMs) of 12-aminododecylphosphonic acid (ADPA) were formed on cobalt-chromium stents. The amine tail group of ADPA was used to link the ticagrelor molecule through a Mitsunobu reaction and confirmed via infrared spectroscopy. Coating uniformity was validated via atomic force microscopy. In an ex-vivo porcine arterio-venous fistula model, the ticagrelor-coated stents (TCS) were placed in alternating series adjacent to uncoated bare metal stents (BMS). Similarly, TCS and everolimus-eluting stents were placed in the porcine left circumflex arteries for acute (7 days) and chronic (35 days) studies. Results: ( Figure 1 ): Grossly, no thrombus was seen on the TCS compared to the BMS. Platelet and micro-thrombi adherence were significantly reduced on TCS. Notably, inflammation, measured by neutrophil and monocyte adherence, was also reduced by approximately 10-fold on the TCS vs the BMS. Angiography, optical coherence tomography (OCT), and histopathology results show the TCS widely patent without systemic DAPT. Conclusion: These findings show that TCS prevents stent thrombosis through focal anti-platelet action and may reduce the bleeding risk associated with prolonged use of systemic DAPT. Long-term safety and efficacy studies are underway.

Abstract 4137045: Sustained Anti-Thrombotic Efficacy of CS585, a Novel Prostacyclin Receptor Agonist, Demonstrates Therapeutic Potential

The formation of occlusive thrombi resulting in myocardial infarction or stroke present a significant challenge for the healthcare community. Activation of the prostacyclin (IP) receptor has been shown to decrease platelet reactivity, however current IP agonists lack a sustained effect in the blood. We have developed CS585, an IP receptor agonist with sustained anti-thrombotic effects in the blood, which could represent a novel prevention strategy in targeting thrombosis. We sought to assess the anti-thrombotic efficacy and pharmacodynamic stability of IP agonists CS585, iloprost and selexipag, in both ex vivo and in vivo models. We evaluated the timeframe of effect of CS585, iloprost, and selexipag in mice following a single IV dose. Inhibition of thrombus formation was measured ex vivo in whole blood under arterial shear rates. In vivo , CS585, iloprost, or selexipag, were administered prior to labeling of platelets and fibrin. Thrombus formation at the site of injury was measured using the cremaster arteriole injury thrombosis assay. CS585 administered to mice prior to blood draw decreases platelet adhesion and blood clot formation under arterial shear conditions. These effects are observed up to 24 hours post-administration; however, the effects of iloprost and selexipag return to pre-treatment levels by 24 hours. In vivo , mice administered iloprost or selexipag demonstrated a decrease in platelet accumulation and fibrin formation, however the effects were abrogated post-administration by 10 minutes and 4 hours, respectively. Administration of CS585, however, demonstrated sustained inhibition of thrombus formation at the site of injury, with inhibitory effects observed at 18 hours post-administration. We have used both in vivo and ex vivo models to demonstrate the anti-thrombotic efficacy of IP receptor agonists. Our results suggest that CS585, a novel IP receptor agonist, sustainably inhibits platelet activation and clot formation for extended periods, in contrast to existing alternatives. This demonstrates a significant improvement in the pharmacodynamic effects of IP receptor agonists in the blood, highlighting CS585 as a novel anti-platelet therapeutic with the potential to treat thrombotic diseases.

Heparin Immobilization Enhances Hemocompatibility, Re-Endothelization, and Angiogenesis of Decellularized Liver Scaffolds

Bioengineered livers are currently an acceptable alternative to orthotopic liver transplants to overcome the scarcity of donors. However, the challenge of using a bioengineered liver is the lack of an intact endothelial layer in the vascular network leading to thrombosis. Heparin-modified surfaces have been demonstrated to decrease thrombogenicity in earlier research. However, in our study, we aimed to apply heparin immobilization to enhance the hemocompatibility, endothelial cell (EC) adhesion, and angiogenesis of rat decellularized liver scaffolds (DLS). Heparin was immobilized on the DLS by the end-point attachment technique. The scaffold’s hemocompatibility was assessed using ex vivo blood perfusion and platelet adhesion studies. The heparinized scaffold (HEP-DLS) showed a significantly reduced thrombogenicity and platelet aggregation. HEP-DLS was recellularized with EA.hy926 cells via the portal vein and maintained in the bioreactor for 7 days, showing increased EC adhesion and coverage within the blood vessels. The Resazurin reduction assay confirmed the presence of actively proliferating cells in the HEP-DLS. The scaffolds were implanted subcutaneously into the dorsum of mice for 21 days to evaluate cell migration and angiogenesis. The results showed significant increases in the number of blood vessels in the HEP-DLS group. Our results demonstrated that heparin immobilization reduces thrombosis, promotes re-endothelialization, and enhances angiogenesis in DLS. The research provides insight into the potential use of heparin in the formation of a functioning vasculature.

Construction of a Carbon Monoxide-Releasing Bioactive Hydrogel Coating on the Magnesium Alloy Surface for Better Corrosion Resistance, Anticoagulant Properties, and Endothelial Cell Growth

In this study, we first fabricated a crosslinked hydrogel coating by polymerizing methacryloyloxyethyl sulfonyl betaine and acrylamide (SBMA) on the magnesium (Mg) alloy surface employing ultraviolet (UV) polymerization. Bivalirudin and CO-releasing molecules (CORM-401) were further grafted onto the hydrogel coating surface to acquire a multifunctional biocompatible coating capable of releasing CO to augment corrosion-resisting properties and biocompatibility. The findings verified that the bioactive hydrogel coating significantly increased the corrosion potential and reduced the corrosion current, thereby improving the anticorrosion performance. Meanwhile, owing to the excellent hydrophilicity, the antifouling performance of the hydrogel coating, and the excellent anticoagulant performance of bivalirudin, the hydrogel coating significantly reduced the fibrinogen adsorption, platelet adhesion and activation, and hemolysis occurrence, displaying excellent ability to inhibit blood clotting. Moreover, endothelial cell (EC) experimental results demonstrated that the hydrogel coating could significantly promote EC growth, displaying great potential to induce re-endothelialization after implantation. Specifically, in the presence of cysteine capable of catalyzing CO release, the anticoagulant performance and ability to promote EC growth were further improved significantly. Therefore, the study offers an effective strategy to prepare a hydrogel coating capable of releasing CO to improve the corrosion-resisting performance and biocompatibility of Mg alloys, which is anticipated to be applied in the surface modification of Mg alloy intravascular stents.

Agarose Micro/Nanostructured Surfaces: A Step Toward an Innovative Solution for Platelet Storage Bags

AbstractIn addressing the critical limitations associated with platelet storage, the study investigates an innovative solution aimed at preventing the unwanted activation of platelets within storage bags. This activation is a key factor that currently restricts the shelf life of platelet products to only 72 h, leading to extreme waste production and high costs. Here, highly effective surfaces are identified for minimizing surface‐induced platelet activation. Using thermal nanoimprint lithography (T‐NIL), a new method is demonstrated for patterning reproducible agarose micro/nanostructures (a natural hydrogel with anti‐platelet adhesive properties) including dots, chains, pills, and squares. The agarose (3%) structured surfaces displayed outstanding flexibility and hydrophilic behavior that prevented platelet adhesion as confirmed by confocal microscopy. Importantly, pill‐shaped structures effectively maintained their ability to prevent platelet adhesion, even after a long cell‐contacting duration. Atomic force microscopy indicated that the effectiveness of dampening platelet adherence is determined by the shape, size, height, and aspect ratio of the structures. A model is provided to explain how the different shapes affect wettability and thereby hinder platelet adherence. The developed anti‐adhesive agarose structured surfaces show promise to revolutionize platelet storage, provide vital insights into biomaterials research, and demonstrate the potential of tailored agarose surfaces in biomedical applications.

A biomimetic approach towards a universal slippery liquid infused surface coating.

One biomimetic approach to surface passivation involves a series of surface coatings based on the slick surfaces of carnivorous pitcher plants (Nepenthes), termed slippery liquid-infused porous surfaces (SLIPS). This study introduces a simplified method to produce SLIPS using a polydopamine (PDA) anchor layer, inspired by mussel adhesion. SLIPS layers were formed on cyclic olefin copolymer, silicon, and stainless steel substrates, by first growing a PDA film on each substrate. This was followed by a hydrophobic liquid anchor layer created by functionalizing the PDA film with a fluorinated thiol. Finally, perfluorodecalin was applied to the surface immediately prior to use. These biomimetic surface functionalization steps were confirmed by several complimentary surface analysis techniques. The wettability of each surface was probed with water contact angle measurements, while the chemical composition of the layer was determined by X-ray photoelectron spectroscopy. Finally, ordering of specific chemical groups within our PDA SLIPS layer was determined via sum frequency generation spectroscopy. The hemocompatibility of our new PDA-based SLIPS coating was then evaluated by tracking FXII activation, fibrin generation time, clot morphology, and platelet adhesion to the surface. This hemocompatibility work suggests that PDA SLIPS coatings slow or prevent clotting, but the observation of both FXII activation and the presence of adherent and activated platelets at the PDA SLIPS samples imply that this formulation of a SLIPS coating is not completely omniphobic.

A novel stent flow chamber system demonstrates reduced thrombogenicity of bioresorbable magnesium scaffolds

Coronary artery disease (CAD) is characterized by narrowing and subsequent blockade of coronary arteries, and imposes a significant health and economic burden. Stent and scaffold devices are introduced in advanced CAD to improve vascular stability and restore blood flow. Although in vitro flow systems like the Chandler loop have been developed to enhance the understanding of interactions between device materials, their coatings, and vascular cells, imaging-based in vitro analysis of device performance is limited. In this study, we established a novel stent flow chamber system designed to assess the thrombogenicity of bioresorbable magnesium scaffold (RMS) and stent materials in vitro. Additionally, we compared the thrombogenicity – an important clinical parameter in stent performance – of the Magmaris-316 L stainless steel stent with its predecessors, Magmaris RMS and a prototype of the third-generation RMS (DREAMS 3G). Analysis of platelet adhesion and coverage of the different devices under flow conditions demonstrated that the Magmaris RMS exhibits reduced thrombogenicity compared to the Magmaris-316 L stainless steel stent. Moreover, thrombogenicity of the DREAMS 3G prototype, composed of BIOmag material, is further decreased compared to its predecessors. The observed reduction in thrombogenicity of the DREAMS 3G prototype in vitro suggests additional improvements in clinical safety and efficacy, highlighting its promise for treating CAD. Future research on this prototype may thus open avenues for analyzing other blood components and patient-derived endothelial cells. In line with the 3R principles, this approach may also help reduce the need for animal testing.

Shore hardness of bulk polyurethane affects the properties of nanofibrous materials differently

The present study shows the effect of the hardness of bulk polyurethane on the properties of nanofibrous materials produced in the solution blow spinning process. This study focuses on nanofibrous materials made from medical-grade polyurethanes with different hardness values on the Shore scale, from 75A to 75D. We aimed to determine the effect of the intrinsic properties of polyurethane used to produce nanofibers on the tensile properties of the resulting nanofibrous materials and in vitro platelet adhesiveness. This study used a solution blow spinning process to produce nanofibrous materials from polyurethane solutions. It evaluates their properties using scanning electron microscopy, followed by porosity determination, tensile testing, and platelet adhesion assays. Generally, the bulk polymer's Shore hardness affects nanofibrous products' porosity and tensile properties. In the tested Shore hardness range, the most visible differences in material properties were observed for the fibers produced from the hardest (75D) and softest (75A) polyurethanes. The nanofibrous material produced using 75D polyurethane exhibited the highest porosity, up to approximately 0.87, owing to the low packing density of the stiff nanofibers. It also remained the stiffest, with the highest Young’s modulus. On the other hand, the softest 75A polyurethane produced a less porous nanofibrous mat with the highest tensile strength among the tested polyurethanes. All tested nanofibrous materials retained their platelet adhesion resistance upon processing into nanofibers, with a mean platelet coverage below 1 % of the nanofibrous mat surface. The study results provide insights into the relationship between the hardness of bulk polyurethane and the properties of nanofibrous materials, which can be useful in various biomedical applications, particularly in producing tissue-engineered vascular grafts.

Zirconia encrusted ceramic composite membrane for uremic toxins removal: Fabrication and assessment of biocompatibility

In this study, tubular zirconia composite membranes (ZM1-ZM3) were developed using low-cost tubular substrate which was prepared utilizing naturally available clay materials by an extrusion approach. The zirconia nanoparticles were encrusted on a low-cost tubular substrate using spray pyrolysis technique. The membranes were investigated for their biocompatibility in addition to pore size, chemical stability, water permeability, porosity, contact angle, thermogravimetric (TGA), and X-ray diffraction (XRD). Water permeability, pore size and porosity of optimized membrane (ZM3) were 3.05 × 10-4 ± 0.03 L.m-2.h-1.Pa-1, 119 ± 0.57 nm and 36 ± 0.12 %, respectively. Platelets adhesion and protein adsorption were measured to be 4630 ± 46 platelets.mm-2, and 1.05 ± 0.02 μg.cm-2 respectively, while the activated partial thromboplastin time (APTT) and prothrombin time (PT) were 80 ± 0.51 s and 27 ± 0.26 s, respectively. Complement activation C3 and C4 concentrations were assessed to be 76.2 ± 0.88 mg.dL-1 and 21.57 ± 0.80 mg.dL-1, respectively and hemolysis ratio was 0.31 ± 0.01 %. Moreover, the membrane had a considerable antifouling nature with a flux recovery ratio of 89.22 ± 0.58 %. Protein retention was found to be 81.14 ± 0.58 %, in addition to outstanding sieving coefficients of uremic toxins like urea (0.95 ± 0.005), creatinine (0.93 ± 0.004), and phosphate (0.90 ± 0.004). These findings suggest that the produced tubular zirconia composite membrane has the potency for hemofiltration.

Sulfonated polyphenylene oxide-based artificial lung membrane with prominent selectivity of CO2

The primary issues impeding oxygenation efficacy in extracorporeal membrane oxygenation (ECMO) are low CO2/O2 selectivity and insufficient permeability of CO2. Herein, sulfonated polyphenylene oxide (SPPO) was chosen as a candidate for the oxygenation membrane material. The CO2-affinitive sulfonic acid groups on the molecular chain of SPPO enhance the interaction between CO2 and the SPPO membrane; thereby, the CO2/O2 selectivity is effectively enhanced. When the solid content of the casting solution is 23%, the SPPO membrane achieves a CO2/O2 selectivity of 17.0, almost as selective as human alveoli. The blood clotting time, red blood cell adhesion, platelet adhesion, and hemolysis ratio were employed to evaluate the blood compatibility of SPPO materials. The results demonstrated that the manufactured SPPO membranes exhibited good hemocompatibility. This work offers novel thinking for researching and applying effective artificial lung membranes.

Serotonin-functionalized starch-based hemostatic sponges enhance platelet activation in the management of non-compressible hemorrhage

Massive hemorrhage poses a serious threat to human health and even life. Access to rapid and potent hemostatic materials is crucial for lowering mortality rates. Starch-based materials exhibit good biocompatibility and are extensively utilized in hemostatic. However, existing starch-based hemostatic products suffer from limited hemostatic efficacy. Serotonin, an indoleamine naturally occurring in the human body, is recognized for its potent platelet-activating properties. Therefore, this study focused on enhancing the procoagulant activity of starch by incorporating serotonin into the starch backbone via esterification and amidation reactions, yielding a novel serotonin-loaded starch-based hemostatic sponge (SLS sponge). The SLS sponges featured exceptional porosity (≥80 %) and water absorption capacity (≥2000 %), which rapidly initiated the coagulation cascade reaction, promoted the adhesion and aggregation of red blood cells and platelets, and intensified platelet activation. This multifaceted approach synergistically enhanced hemostasis via both active and passive coagulation mechanisms. Notably, the SLS sponges adhered firmly to the wound surfaces without pressure. Compared with gelatin sponges, the SLS sponges significantly reduced blood loss by approximately 40.5 % and shortened the time to achieve hemostasis by approximately 28.9 %. These findings indicate that the newly developed SLS sponges are a promising active hemostatic material, particularly effective in managing non-compressible hemorrhages.

Antimicrobial peptide-based pH-responsive Polyvinyl alcohol hydrogel artificial vascular grafts with excellent antimicrobial and hematologic compatibility

Thrombosis and infection are the primary challenges limiting the clinical application of small-diameter artificial blood vessels. Numerous studies have focused on improving the hemocompatibility of artificial vascular materials, addressing properties such as anticoagulant activity, antithrombogenicity, low hemolysis rates, and reduced platelet adhesion. However, there has been comparatively less attention given to the antibacterial properties of these materials. This study utilizes polyester braided tubes and PVA/PAAm hydrogel materials, combined with surface modification techniques, to develop a pH-responsive antibacterial hydrogel artificial blood vessel graft. The hydrogel surface was grafted with a polymer brush hierarchical structure, and antimicrobial peptide MLT was immobilized to confer antibacterial properties. Additionally, 2,3-dimethylmaleic anhydride (DMMA) was covalently attached as a shielding group to achieve an environment-responsive strategy. The research demonstrates that this artificial blood vessel graft exhibits structural stability, excellent mechanical properties, and good cellular and hemocompatibility. Furthermore, it exhibits pH-responsive capabilities, triggering efficient antibacterial activity in low pH environments. The modified antibacterial hydrogel artificial blood vessel material shows low hemolysis rates, excellent anticoagulant properties, and reduced platelet adhesion. Therefore, the strategy of combining MLT with DMMA may effectively enhance the hemocompatibility and responsive antibacterial performance of artificial blood vessel materials.

CS585, a novel prostacyclin receptor agonist, demonstrates sustained efficacy in vivo in the prevention of thrombosis

Abstract Background Uncontrolled platelet activity resulting from cardiovascular disease can lead to occlusive thrombi, culminating in myocardial infarction or stroke. Despite major advances in anti-platelet therapeutics, many patients remain at risk. Although prostacyclin (IP) receptor agonists are currently used for the treatment of pulmonary arterial hypertension (PAH), activation of the IP receptor has been shown to decrease platelet reactivity. However, engaging the IP receptor as a therapeutic target was previously considered unviable due to the lack of a sustained effect of current IP agonists in the blood. The development of an IP agonist with sustained effects in the blood would represent a novel prevention strategy in targeting platelet hyperreactivity and thrombosis. Purpose Assess the stability profile of CS585, an IV and orally bioavailable IP agonist, compared to FDA-approved IP agonists iloprost and selexipag, with regards to sustained activity in vivo in the blood in limiting platelet activation and thrombosis. Methods Using ex vivo and in vivo mouse models, we assessed the timeframe of effect of CS585, iloprost and selexipag in blood. Blood was drawn from wild-type mice 24 hours following a single IV dose of CS585, iloprost or selexipag. Platelet adhesion and blood clotting were measured using perfusion flow chamber at arterial shear and total thrombus activation system (T-TAS), respectively. In vivo, platelets and fibrin were labelled 24 hours post IV or oral administration of CS585, iloprost or selexipag, and accumulation at the site of injury was measured using the cremaster arteriole injury thrombosis assay. Results Blood drawn from mice administered CS585 was observed to show a decrease in platelet adhesion and clot formation in the perfusion flow chamber and T-TAS assays. The effects of iloprost and selexipag are no longer observed at 24 hours post-administration. Administration of CS585 resulted in sustained inhibition of platelet accumulation and fibrin formation in the laser-induced cremaster arteriole thrombosis assay up to 18 hours post-administration. In mice administered iloprost, a decrease in thrombus formation was observed 10 minutes post-administration, but thrombus size returned to vehicle levels by 4 hours. Selexipag-dosed mice demonstrated inhibition of thrombus formation up to 4 hours, but effects were no longer observed at 18 hours. Conclusions CS585, a novel IP agonist, inhibits platelet activation and clot formation up to 24 hours post-administration. In both in vivo and ex vivo models, we demonstrate the sustained effects of CS585. In contrast, current FDA-approved IP agonists do not possess an effect profile sufficient to target the IP receptor in blood. By overcoming the sustainability challenge facing IP agonists, CS585 represents a novel approach to anti-platelet treatment of thrombotic diseases, which could include thrombosis, VTE, secondary prevention after MI and stroke, and PAH.

GJA4 gene inhibition may represent a potential target for novel antithrombotic therapies

Abstract The GJA4 gene encodes a protein called connexin 37, a component of gap junctions in vascular endothelial cells, crucial in regulating endothelial function and influencing inflammation, platelet adhesion and thrombus formation. This gene deletion in mice diminished thrombus formation in vitro in human blood. In these circumstances, connexin inhibition with pharmacological agents may represent potential avenues for developing novel antithrombotic agents. Aim To investigate whether the GJA4 rs618675 T>C variant is a risk factor for progressing atherosclerosis and cardiovascular (CV) events in an asymptomatic free of apparent CAD from a Portuguese population. Methods A prospective study was performed with 1421 individuals without apparent CV disease (73.6% male, aged 52.2±8.3 years) followed during an extended period of 7.2±5.1 years). GJA4 rs618675 T>C variant was genotyped by TaqMan real-time PCR technique (Applied Biosystems). Conventional, anthropometric, biochemical and clinical risk factors were studied. Bivariate analysis and multivariate Cox regression adjusted for age, gender, traditional risk factors, biochemical markers, and the genotypes under study was performed to determine which variables were, significantly and independently, associated with CV events. Kaplan-Meier's estimate assessed the prognosis. Results Bivariate analysis showed that 50.6% of wild genotype (TT) carriers had CV events vs. 66.2% without events. Of the risk genotype (CC) carriers, 10.1% had CV events, and 3.4% did not. After the Kaplan-Meier analysis, the TT carriers had 90% CV event-free survival time and the CC risk carriers had only 64% of event-free survival. Finally, after adjustment, CC genotype remained in the equation with an HR of 3.1 (p=0.003) and CT heterozygous with HR of 1.6 (p=0.043), together with Hypertension (HR 3.0; p<0.0001), Smoking habits (HR 2.3; p=0.001) and Diabetes (HR 1.9; p=0.015). Conclusion: The CC risk genotype of the GJA4 rs618675 represented an independent risk factor for progressing atherosclerosis and CV events in a Portuguese Population. Thrombus formation often begins with the adhesion of platelets to the endothelium, and connexin 37 may influence this process. The inhibition of connexin 37 emerges as a promising candidate for future cardiovascular prevention as well as for the development of new antithrombotic drugs.

Sphingosine-1-phosphate reduces arterial thrombosis via enhanced endothelial thrombomodulin expression by S1PR1 signaling

Abstract Background Sphingosine 1-phosphate (S1P) is an important lipid mediator in the cardiovascular system. Numerous links between S1P and blood coagulation have been described. However, results regarding the role of S1P in coagulation are inconclusive. A physiological antithrombotic regulator expressed by endothelial cells (EC) is thrombomodulin (TM). To date, the impact of S1P on TM-expression is unknown. Methods We evaluated S1P effects on TM-expression and subsequent platelet adhesion and thrombus formation. For this, we conducted cell culture with human umbilical cord venous endothelial cells (HUVECs), flow cytometry and flow chamber experiments. Additionally, in vivo arterial thrombus formation in mice was analyzed. Results Sphingosine kinase-1 deficient mice (SphK1-/-) have about 70% less circulating plasma S1P and show a significant reduction in TM expression on the aorta as compared to their littermates (WT: 207.2 ± 45.8 pg/g Aorta vs. SphK1-/- 124.5 ± 40.2 pg/g Aorta, p<0.0001). In contrast, S1P incubation of 24 hours significantly increased TM-expression on HUVECs by about 30 percent. This effect was abolished by S1PR1-inhibition with W146. Treatment with S1P reduced platelet adhesion from whole blood to endothelial cells in the flow chamber experiments (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). This effect could be reversed both by S1PR1 inhibition and with a TM-neutralizing antibody. P-selectin-expression on platelets was decreased after incubation with TM suggesting (Ctrl: 62.79 ± 13.30% vs. TM: 43.47 ± 11.36%, p=0.0222). Finally, to assess the in vivo effects of our findings, arterial thrombus formation in mice was performed. TM inhibited in vivo thrombus formation. Occlusion of the carotid artery could not be detected during the 30 minutes recording. In contrast, SphK1-/- mice with low S1P levels showed decreased times to first occlusion. Treatment with TM in these animals was able to reverse this effect. Conclusion We can conclude that the main findings of our study were (i) S1P increased endothelial TM-expression, (ii) which subsequently leads to reduced platelet adhesion and (iii) inhibition of arterial thrombus formation in vivo.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic disorder, diagnosed according to the presence of phenotypical traits of the myocardium. A specific biomarker which can associate with the severity of HCM is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as accessible diagnostic tools, as their abundance and molecular composition can reflect the severity of cardiac diseases (Rizzuto 2024). We aim at characterising plasma-derived EVs isolated from 28 HCM patients and 18 healthy volunteers (CTR). Methods The whole cohort underwent transthoracic echocardiography, whereas magnetic resonance and exome sequencing was performed on HCM patients. EVs were isolated via ultracentrifugation from plasma of both groups. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis, transmission electron microscopy, Western blot, targeted (Olink) and untargeted (nLC-MS/MS) proteomics and FACS analysis. Plasma-derived EV subpopulations were characterised according to their cellular origin. Data are expressed as median and interquartile range (25th, 75th). Results Most patients were male (68% HCM and 66% CTR) with a median age of 58 (49-69) years (HCM) and 47 (44-52) (CTR). Among HCM patients, missense mutations in the MYH7 gene were the most identified. The median maximum wall thickness (WTMax) in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. The isolated EVs expressed tetraspanins (CD9, CD63 and CD81), Alix and β-integrin and showed an intact phospholipid bilayer of 100 nm in size. EV concentration was reduced in HCM vs CTRL, respectively 2.8*109 EV/ml/cell count (2*109-4*109) and 4.6*10⁹ EV/ml/cell count (3*109-6*109). Among 15 plasma-derived EV subpopulations studied, those released from platelets (CD41a) and activated platelets (CD62P and CD40L) were increased in HCM patients vs CTR by 1.7 fold. Negative associations were found between E/e’, parameter of diastolic function, and cardiomyocyte-derived EVs (r= -0.2658), and between left ventricle ejection fraction and platelet-derived EVs (r=-0.2189); a positive correlation was found between WTMax and EVs derived from activated endothelial cells (r=0.2330). The proteomic analysis of EVs shows an enrichment in proteins related to platelets adhesion and inflammation in HCM patients. Conclusions HCM patients present a peculiar phenotypic pattern of EVs that may associate with diagnostic clinical features of HCM.