Human Platelet Function Research Articles

Pharmacological effects of small molecule BCR-ABL tyrosine kinase inhibitors on platelet function.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein, such as imatinib (Gleevec), have revolutionized targeted cancer therapies. However, drug resistance and side effects, particularly those affecting hemostasis, continue to pose significant challenges for TKI therapies. As tyrosine kinases serve pivotal roles in platelet hemostatic function, we investigated the potential impact of both established and emerging ABL TKIs on human platelet activities ex vivo Our study included standard-of-care agents (e.g., imatinib and nilotinib), and second-generation ABL inhibitors including ponatinib and bosutinib designed to mitigate drug resistance. Additionally, we explored the effects of allosteric inhibitors targeting the myristoyl pocket of ABL (e.g., asciminib and GNF-2), and novel agents in preclinical development, including ELVN-919, which uniquely exhibits high specificity for the ABL kinase active site. Our findings reveal that while ABL inhibitors such as ponatinib and bosutinib impede platelet activity, highly specific new-generation ABL inhibitors, including first-in-class therapeutics, do not impact platelet function ex vivo Overall, these new insights around the effects of ABL TKIs on platelet function could inform the development of targeted therapies with reduced hematologic toxicities. Significance Statement This study examines the effects of clinically relevant small molecule BCR-ABL tyrosine kinase inhibitors (TKIs) on platelet activity. This analysis includes first-time assessments of agents such as asciminib and ELVN-919 on human platelet function ex vivo, alongside established therapies (e.g., imatinib, ponatinib) with well-characterized effects on platelet function, to discern potential anti-platelet and other effects of BCR-ABL TKIs and inform clinical safety.

NADPH oxidase 1/4 dual inhibitor setanaxib suppresses platelet activation and thrombus formation

AimsThe production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways. Materials and methodsIn collagen-stimulated human platelets, aggregometry, assessment of ROS and Ca2+, immunoblotting, ELISA, flow cytometry, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study. Key findingsSetanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and Ca2+ mobilization. Setanaxib also inhibited collagen-induced activation of integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA2 and reduced TXA2 generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis. SignificanceThis study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases.

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Effect of toxins from different periodontitis-associated bacteria on human platelet function.

Periodontitis is caused by a dysbiosis of oral bacteria resulting in alveolar bone destruction and teeth loss. The role of platelets in pathogenesis of periodontitis is a subject of research. The release of toxins from periodontitis-associated bacteria may influence platelet function and contribute to the modulation of hemostatic or inflammatory responses. Therefore, we explored platelet function upon exposure to defined toxins: leukotoxin A from Aggregatibacter actinomycetemcomitans (LtxA), a synthetic version of the C14-Tri-LAN-Gly peptide from Fusobacterium nucleatum (C14), and lipopolysaccharides from Porphyromonas gingivalis (LPS). Light transmission aggregometry was performed after the addition of toxins to platelet-rich plasma in different doses. Flow cytometry was used to identify inhibitory effects of toxins by measuring phosphorylation of the vaso-dilator-stimulated phosphoprotein or to identify activating effects by the detection of CD62P expression. The release of chemokines derived from washed platelets was determined by immunoassays. Collagen-induced threshold aggregation values were diminished upon incubation with LtxA and C14, accompanied with an increase of vaso-dilator-stimulated phosphoprotein (VASP) phosphorylation, indicating platelet inhibition. In contrast, LPS did not affect aggregation but slightly enhanced CD62P expression under co-stimulation with low-dose thrombin pointing to slight platelet activation. The three toxins did not relevantly influence the secretion of chemokines. Although weak, the investigated toxins differently influenced human platelet function. LtxA and C14 mediated inhibitory effects, whereas LPS contributed to a slight activation of platelets. Further analysis of specific cellular responses mediated by bacterial toxins may render novel targets and suggestions for the treatment of periodontitis.

Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential.

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Non-Enzymatic Role of Platelet 12-LOX in Platelet Activation

Background 12-lipoxygenase (12-LOX) plays an essential role in regulation of vascular disease and human platelet function through oxylipin generation such as 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE) and 12-hydroxyeicosatetraenoic acid (12-HETE). Previous literature has demonstrated that 12-LOX enzymatic function such as oxylipin generation is important for collagen- or thrombin-mediated signaling events. However, the addition of 12(S)-HETE does not completely restore the inhibitory effect of pharmacologically or genetically inhibiting 12-LOX on platelet activation, indicating that 12-LOX may have a non-enzymatic function in platelets. Therefore, we tested the non-enzymatic role of 12-LOX in platelet activation. Method 12 LOX was co-immunoprecipitated (co-IP) from washed human platelets following FcγRIIa or thrombin stimulation. The bound proteins were eluted by laemmli buffer and the elutes were analyzed on SDS-PAGE. Proteins co-IP with 12-LOX at various times were identified by mass spectrometry and confirmed by western blot analysis. Result Silver-stained gel of 12-LOX co-IP indicated the presence of several protein bands in complex association with 12-LOX at different time points. These bands were analyzed by mass spectroscopy and several proteins observed in complex with 12-LOX were identified including integrin β3, Talin-1, Myh9, Nexilin, Gelsolin, Actin, cytoplasmic 2, and Actin related protein 2/3 (ARP 2/3) . To confirm the mass spectroscopy identified protein, these proteins were immunoblotted following co-IP of 12-LOX. to confirm their association with 12-LOX prior to and following platelet activation. These interactions were further confirmed with a reverse co-IP. Finally, to determine if the association pattern of these proteins with 12-LOX is disrupted in the presence of a 12-LOX inhibitor already shown to limit platelet activation and which is currently in clinical trials for treatment of HIT (ML355/VLX-1005), we were able to demonstrate that the inhibitor appears to lock the protein complex into place. Conclusion Our study identified several proteins that are present in 12-LOX complex such as integrin β3, talin-1, Myh9 and ARP 2/3. 12-LOX interacts with Integrin β3, Talin-1 and cytoskeletal proteins. Furthermore, the dynamic movement of the proteins in this complex into and out of association with 12-LOX is essential for normal platelet activation suggesting 12-LOX plays an important non-enzymatic role in regulation of agonist-mediated platelet activation.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets

Objective Platelets for transfusion are stored for 5-7 days. During storage, platelets undergo numerous detrimental functional changes. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacologic inhibition of 12-lipoxygenase (12-LOX) affects platelets during storage and after transfusion. Approach and Results Platelets from12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using LC-MS/MS-MRM. Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, while oxylipin concentrations were significantly higher in WT platelets. Baseline αIIbβ 3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, after transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets in thrombocytopenic recipients challenged with a FeCl 3-induced carotid artery injury. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of cyclooxygenase-1 (COX-1) with acetylsalicylic acid abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting a critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with the 12-LOX inhibitor, VLX-1005, showed increased integrin activation compared to vehicle-treated platelets upon transfusion in NOD/SCID mice. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine and human platelets by increasing thromboxane generation through a COX-1-dependent mechanism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Platelets from Ehlers-Danlos Syndrome Patients Exhibit Reduced GPVI Levels and Impaired Integrin αIIbβ3 inside-out Signaling

Background: Patients with Ehlers-Danlos syndrome (EDS) have a high susceptibility to bleeding. Easy bruising is included as a criterion for the classification of multiple types of EDS. Although the association between EDS and the tendency to bleed has been acknowledged for a long time, a definitive understanding of the underlying mechanisms contributing to bleeding diathesis in EDS patients is still lacking. Objective: To characterize platelet function in a cohort of EDS patients and a murine EDS model. Methods: A cohort of 53 patients with hypermobile (49%), classical (26%), classical-like (21%), or vascular (4%) types of EDS and a group of 53 healthy control subjects matched by age and gender were included in the study in accordance with the University of Iowa Institutional Review Board-approved protocol. The International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) was used to assess the bleeding tendency in study participants. Because a single mutation in hypermobile type is currently unknown, we used Col5a 1 +/− mice as a murine model of the classical type of EDS. The function of human and mouse platelets was evaluated using standardized agonist-induced in vitro assays. Results: The mean ISTH-BAT score was 0.1 in healthy controls and 9.2 in EDS patients ( P < 0.001). An abnormal ISTH-BAT score was observed in 33 out of 53 (62%) EDS patients and 0 of 53 healthy controls ( P < 0.001). Platelets from EDS patients exhibited reduced aggregation and αIIbβ3 activation (with normal αIIbβ3 surface exposure) in response to stimulation with collagen, collagen-related peptide, or thrombin receptor activator peptide-6, compared to healthy controls. Platelet secretion of dense and α-granules and surface phosphatidylserine exposure were comparable between EDS and healthy subjects. Evaluation of collagen receptors on the surface of resting platelets showed a mild deficiency of GPVI in EDS patients compared to healthy controls ( P < 0.001), while the integrin α2 levels were similar in both groups ( P = 0.152). Analysis of GPVI signaling revealed downregulation of Syk (Y525/526), PLCγ2 (Y1217), and talin (S425) phosphorylation in EDS patients ( P < 0.05 vs. healthy controls), suggesting a defect in collagen-induced integrin αIIbβ3 inside-out signaling. Consistent with our observations in EDS patients, Col5a1 +/− mice demonstrated a bleeding tendency characterized by prolonged tail-bleeding time ( P < 0.05 vs. wild-type control). Platelet function testing showed that Col5a 1 +/− mouse platelets recapitulated the abnormalities detected in human EDS platelets. Conclusions: Our data corroborate the observation that EDS patients exhibit a high risk of hemorrhagic complications.EDS platelets are characterized by reduced baseline GPVI expression that was associated with decreased agonist-induced platelet aggregation, αIIbβ3 activation, and impaired collagen-induced integrin αIIbβ3 inside-out signaling.

MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

Human MutT Homolog 1 (MTH1) is a nucleotide pool sanitization enzyme that hydrolyzes oxidized nucleotides to prevent their mis-incorporation into DNA under oxidative stress. Expression and functional roles of MTH1 in platelets are not known. Here, we show MTH1 expression in platelets and its deficiency impairs hemostasis and arterial/venous thrombosis in vivo. MTH1 deficiency reduced platelet aggregation, phosphatidylserine exposure and calcium mobilization induced by thrombin but not by collagen-related peptide (CRP) along with decreased mitochondrial ATP production. Thrombin but not CRP induced Ca2+-dependent mitochondria reactive oxygen species generation. Mechanistically, MTH1 deficiency caused mitochondrial DNA oxidative damage and reduced the expression of cytochrome c oxidase 1. Furthermore, MTH1 exerts a similar role in human platelet function. Our study suggests that MTH1 exerts a protective function against oxidative stress in platelets and indicates that MTH1 could be a potential therapeutic target for the prevention of thrombotic diseases.

RETRACTED: Severe fever with thrombocytopenia syndrome virus induces platelet activation and apoptosis via a reactive oxygen species-dependent pathway

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV) and with a high fatality rate. Thrombocytopenia is a major clinical manifestation observed in SFTS patients, but the underlying mechanism remains largely unclear. Here, we explored the effects of SFTSV infection on platelet function in vivo in severely infected SFTSV IFNar−/− mice and on mouse and human platelet function in vitro. Results showed that SFTSV-induced platelet clearance acceleration may be the main reason for thrombocytopenia. SFTSV-potentiated platelet activation and apoptosis were also observed in infected mice. Further investigation showed that SFTSV infection induced platelet reactive oxygen species (ROS) production and mitochondrial dysfunction. In vitro experiments revealed that administration of SFTSV or SFTSV glycoprotein (Gn) increased activation, apoptosis, ROS production, and mitochondrial dysfunction in separated mouse platelets, which could be effectively ameliorated by the application of antioxidants (NAC (N-acetyl-l-cysteine), SKQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) and resveratrol). In vivo experiments showed that the antioxidants partially rescued SFTSV infection-induced thrombocytopenia by improving excessive ROS production and mitochondrial dysfunction and down-regulating platelet apoptosis and activation. Furthermore, while SFTSV and Gn directly potentiated human platelet activation, it was completely abolished by antioxidants. This study revealed that SFTSV and Gn can directly trigger platelet activation and apoptosis in an ROS-MAPK-dependent manner, which may contribute to thrombocytopenia and hemorrhage during infection, but can be abolished by antioxidants.

Assessing mitochondria-targeted acyl hydroquinones on the mitochondrial platelet function and cytotoxic activity: Role of the linker length

IntroductionThe use of triphenylphosphonium cation (TPP+) linked to phenolic compounds by alkyl chains has a significant relevance as a mitochondrial delivery strategy in biomedicine because it affects mitochondrial bioenergetics in models of noncommunicable diseases such as cancer and cardiovascular-related conditions. Studies indicate that a long alkyl chain (10–12 carbon) increases the mitochondrial accumulation of TPP+-linked drugs. In contrast, other studies show that these compounds are consistently toxic to micromolar concentrations (as observed in platelets). In the present study, we evaluated the in vitro effect of three series of triphenylphosphonium-linked acyl hydroquinones derivates on the metabolism and function of human platelets using 3–9 carbons for the alkyl linker. Those were assessed to determine the role of the length of the alkyl chain linker on platelet toxicity. MethodsHuman platelets were exposed in vitro to different concentrations (2–40 μM) of every compound; cellular viability, phosphatidylserine exposition, mitochondrial membrane potential (ΔΨm), intracellular calcium release, and intracellular ROS generation were assessed by flow cytometry. An in silico energetic profile was generated with Umbrella sampling molecular dynamics (MD). Results and conclusionsThere was an increase in cytotoxic activity directly related to the length of the acyl chain and lipophilicity, as seen by three techniques, and this was consistent with a decrease in ΔΨm. The in silico energetic profiles point out that the permeability of the mitochondrial membrane may be involved in the cytotoxicity of phosphonium salts. This information may be relevant for the design of new TPP+ -based drugs with a safe cardiovascular profile.

Sodium bicarbonate as a local adjunctive agent for limiting platelet activation, aggregation, and adhesion within cardiovascular therapeutic devices

Cardiovascular therapeutic devices (CTDs) remain limited by thrombotic adverse events. Current antithrombotic agents limit thrombosis partially, often adding to bleeding. The Impella® blood pump utilizes heparin in 5% dextrose (D5W) as an internal purge to limit thrombosis. While effective, exogenous heparin often complicates overall anticoagulation management, increasing bleeding tendency. Recent clinical studies suggest sodium bicarbonate (bicarb) may be an effective alternative to heparin for local anti-thrombosis. We examined the effect of sodium bicarbonate on human platelet morphology and function to better understand its translational utility. Human platelets were incubated (60:40) with D5W + 25 mEq/L, 50 mEq/L, or 100 mEq/L sodium bicarbonate versus D5W or D5W + Heparin 50 U/mL as controls. pH of platelet-bicarbonate solutions mixtures was measured. Platelet morphology was examined via transmission electron microscopy; activation assessed via P-selectin expression, phosphatidylserine exposure and thrombin generation; and aggregation with TRAP-6, calcium ionophore, ADP and collagen quantified; adhesion to glass measured via fluorescence microscopy. Sodium bicarbonate did not alter platelet morphology but did significantly inhibit activation, aggregation, and adhesion. Phosphatidylserine exposure and thrombin generation were both reduced in a concentration-dependent manner—between 26.6 ± 8.2% (p = 0.01) and 70.7 ± 5.6% (p < 0.0001); and 14.0 ± 6.2% (p = 0.15) and 41.7 ± 6.8% (p = 0.03), respectively, compared to D5W control. Platelet aggregation via all agonists was also reduced, particularly at higher concentrations of bicarb. Platelet adhesion to glass was similarly reduced, between 0.04 ± 0.03% (p = 0.61) and 0.11 ± 0.04% (p = 0.05). Sodium bicarbonate has direct, local, dose-dependent effects limiting platelet activation and adhesion. Our results highlight the potential utility of sodium bicarbonate as a locally acting agent to limit device thrombosis.

CARD29: Sodium Bicarbonate as an Adjunctive Local Anti-Thrombotic Agent for Cardiovascular Therapeutic Devices

Purpose: Despite clinical efficacy, mechanical circulatory support (MCS) systems remain challenged in maintaining physiologic hemostatic balance. The Impella heart pump is a micro-axial percutaneous MCS device that utilizes a purge flow for maintaining bearing lubrication, purge patency, and anti-thrombosis purposes, forming a purge gap micro-environment wherein purge fluid and blood may mix. During operation, a continuous 5% dextrose in water (D5W) + heparin purge solution flows through the Impella catheter to inhibit protein adsorption and thrombosis in internal purge gaps. This exogenous heparin can increase bleeding risk and complicate systemic heparin and anticoagulation management. Recent FDA approval has made available sodium bicarbonate (SB) as an alternative to purge heparin. Despite promising early clinical results, the influence of SB on blood platelets, central mediators of thrombosis and bleeding, remains unclear. Here, we investigated the effect of SB on human platelet function (adhesion, aggregation, activation) in vitro following incubation with relevant concentrations of heparin or SB. Methods: Human gel-filtered platelets (20,000/µL) or platelet rich plasma (100,000/µL) were obtained from ACD-A anticoagulated whole blood. Platelets were incubated (6:4 platelets: purge) at 37°C for 15 min with purge solutions (D5W; 50U/mL heparin + D5W; 25mEq/L SB + D5W; 50mEq/L SB + D5W; 100mEq/L SB + D5W). Platelet activation was measured as thrombin generation rate utilizing a previously-described modified prothrombinase assay. Platelet adhesion was measured via imaging (Nikon DS-Qi2; 100X) of fluorescent-labelled platelets (BioTracker490) adhered to glass. Platelet aggregation was quantified from agonist-mediated aggregation using a light-transmission aggregometer (PAP8E). All experiments were performed in duplicate with N≥3 donors. Results: SB altered overall platelet function in a concentration-dependent manner. 100mEq/L SB led to a significant decrease in platelet adhesion (0.11% decrease; Fig 1A), whereas only 25mEq/L SB led to a significant reduction in thrombin generation rate (14.0% decrease; Fig 1B). Collagen-mediated aggregation was significantly reduced after addition of 100mEq/L SB (42.2% decrease; Fig 1C). Traditional D5W+heparin solution significantly reduced platelet function, in all categories, to a greater degree than 25mEq/L SB, but had a similar effect to 50 or 100mEq/L SB when comparing platelet adhesion and activation metrics. Conclusion: SB in the concentrations tested led to altered platelet function, modulating surface adhesion, activation, and collagen-mediated aggregation –useful effects to limit device fouling and purge gap thrombosis. Use of SB as a localized anti-thrombotic agent, where its concentration and effect are not rapidly diluted, is promising for MCS such as the Impella system. SB use offers the potential for locally limiting thrombogenicity at the purge gap, while avoiding the need for exogenous heparin.

Mitochondrial Respiratory Function in Human Platelets: Influence of Sample Preparation, Assay Buffer and Instrumental Platform

In humans, circulating blood cells such as platelets are being increasingly used as a readily available and minimally invasive biospecimen to determine mitochondrial respiratory function. Here, we set out to determine the influence of sample preparation, assay buffer composition, and instrumental platform, on the respiratory function of platelets isolated from human blood.Approximately 50 mL of whole blood was collected from healthy adults (n=16) following an overnight (&gt;12 hr.) fast. Platelets were immediately isolated from whole blood by centrifugation. Respiratory function was assayed in intact and permeabilized platelets using an Oxygraph-2K (O2K) high-resolution respirometer (Oroboros Instruments). Respiratory function was assayed in intact platelets suspended in either culture (RPMI) or respiration (MIR05) media (both supplemented with 5 mM glucose, 1 mM pyruvate and 2 mM glutamine), or the participants own plasma. Respiratory function was also assayed in digitonin-permeabilized platelets suspended in both RPMI and MIR05. In addition, respiratory function was determined in intact platelets using a Seahorse Extra-Cellular Flux analyzer (XFe) in RPMI buffer containing 5 mM glucose, 1 mM pyruvate and 2 mM glutamine.In intact platelets assayed in suspension using an O2K, routine and ATP-linked respiration were 35 and 34% greater in cells assayed in RPMI compared to MIR05, respectively (P&lt;0.001). However, coupling control in response to oligomycin was comparable in cells assayed in RPMI and MIR05 (92±4 vs. 94±4%). Compared to cells assayed in RPMI (0.19±0.01 pmol/s/100 million cells) or MIR05 (0.14±0.02 pmol/s/100 million cells), ATP-linked respiration was greater in intact cells assayed in their own plasma (0.24±0.02 pmol/s/100 million cells, both P&lt;0.001). In digitonin-permeabilized platelets, ATP-linked respiration was greater when assayed in MIR05 compared to RPMI (0.36±0.08 vs. 0.30±0.07 pmol/s/100 million cells, P&lt;0.05). Across instrumental platforms, routine and leak respiration were lower in intact platelets assayed on an O2K vs. XFe (P&lt;0.05), whereas ATP-linked respiration was greater in cells assayed on an O2K. (P&lt;0.001), due to a diminished coupling response to oligomycin in cells assayed in the XFe (P&lt;0.001). Platelet respiratory function is influenced by sample preparation, assay buffer and protocol, and the instrumental platform used. Consideration of these factors is necessary when using platelet respiratory function as a readout of cellular energetics. Supported by USDA-ARS Project 6026-51000-012-06S, NIH 5P20GM109096-07 and 5R35GM142744-02. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbβ3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

Aluminum Nanoparticles Affect Human Platelet Function In Vitro.

Endoprostheses are prone to tribological wear and biological processes that lead to the release of particles, including aluminum nanoparticles (Al NPs). Those particles can diffuse into circulation. However, the toxic effects of NPs on platelets have not been comprehensively analyzed. The aim of our work was to investigate the impact of Al NPs on human platelet function using a novel quartz crystal microbalance with dissipation (QCM-D) methodology. Moreover, a suite of assays, including light transmission aggregometry, flow cytometry, optical microscopy and transmission electron microscopy, were utilized. All Al NPs caused a significant increase in dissipation (D) and frequency (F), indicating platelet aggregation even at the lowest tested concentration (0.5 µg/mL), except for the largest (80 nm) Al NPs. A size-dependent effect on platelet aggregation was observed for the 5-20 nm NPs and the 30-50 nm NPs, with the larger Al NPs causing smaller increases in D and F; however, this was not observed for the 20-30 nm NPs. In conclusion, our study showed that small (5-50 nm) Al NPs caused platelet aggregation, and larger (80 nm) caused a bridging-penetrating effect in entering platelets, resulting in the formation of heterologous platelet-Al NPs structures. Therefore, physicians should consider monitoring NP serum levels and platelet activation indices in patients with orthopedic implants.

Deletion of 12-Lipoxygenase Improves Platelet Function after Storage and Transfusion in Thrombocytopenic Mice

Introduction Human platelets are stored for up to 7 days for transfusion. During storage, platelets undergo numerous detrimental functional changes. Results from two recent randomized clinical trials challenge the hemostatic efficacy of stored platelets for transfusion. In a recent study in healthy humans, increased 12-HETE levels were associated with reduced platelet performance in vivo. In the current study, we sought to understand how genetic deletion of 12-LOX (the enzyme that generates 12-HETE) affects platelets during storage, before, and after transfusion in vitro and in mouse models. Methods and Results: We obtained platelets from wild-type (WT) and 12-LOX-/- mice and performed storage studies for 24 and 48 hours. Using LC-MS/MS-MRM, we showed that ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) increased significantly in stored platelets from 12-LOX-/- mice, while oxylipins were significantly lower in 12-LOX-/- platelets than in WT platelets. The ω-6/ω-3 PUFA ratio decreased significantly in stored 12-LOX-/- compared to stored WT platelets, highlighting a pronounced increase in ω-3 PUFAs. The circulation time of stored 12-LOX-/- platelets did not differ from stored WT platelets. Baseline, pre-transfusion αIIbβ3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, once stored platelets were transfused, we observed significantly increased baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. To test whether this translates into better in vivo function, we exposed 24-hour stored platelets to an in vivo model of thrombosis and hemostasis. In brief, hIL4R-TG mice express the receptor for human interleukin 4 instead of GPIbα on the platelet surface. hIL4R-TG mice were rendered thrombocytopenic with an anti-human IL4R antibody. We transfused 24-hour stored WT, or 12-LOX-/- platelets, to thrombocytopenic hIL4R-TG mice, injured the carotid artery by FeCl3, and recorded the arterial blood flow. Indeed, stored 12-LOX-/- platelets had better in vivo function, as evidenced by more frequent and significantly faster vessel occlusions with stored and transfused 12-LOX-/- platelets than transfusion of stored WT platelets. Importantly, stored and transfused 12-LOX-/- occluded vessels in a similar time as normocytic WT mice. Untransfused, thrombocytopenic hIL4R-TG mice and untransfused, normocytic hIL4R-TG mice did not show vessel occlusion. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine platelets. Pharmacologic inhibition of 12-LOX or dietary alterations of ω-3 and ω-6 PUFAs could significantly enhance human platelet quality and function after storage. Future studies must determine the feasibility and safety of 12-LOX inhibition in stored and transfused human platelets.

Coenzyme Q10 Attenuates Human Platelet Aggregation Induced by SARS-CoV-2 Spike Protein via Reducing Oxidative Stress In Vitro.

Platelet hyperreactivity and oxidative stress are the important causes of thrombotic disorders in patients with COVID-19. Oxidative stress, induced by the excessive generation of reactive oxygen species (ROS), could increase platelet function and the risk of thrombus formation. Coenzyme Q10 (CoQ10), exhibits strong antioxidative activity and anti-platelet effect. However, the effects and mechanisms of CoQ10 on attenuating platelet aggregation induced by spike protein have never been studied. This study aims to investigate whether the SARS-CoV-2 spike protein potentiates human platelet function via ROS signaling and the protective effect of CoQ10 in vitro. Using a series of platelet function assays, we found that spike protein potentiated platelet aggregation and oxidative stress, such as ROS level, mitochondrial membrane potential depolarization, and lipid damage level (MDA and 8-iso-PGF2α) in vitro. Furthermore, CoQ10 attenuated platelet aggregation induced by spike protein. As an anti-platelet mechanism, we showed that CoQ10 significantly decreased the excess production of ROS induced by spike protein. Our findings show that the protective effect of CoQ10 on spike protein-potentiated platelet aggregation is probably associated with its strong antioxidative ability.

Bioactive food components and their inhibitory actions in multiple platelet pathways.

In addition to hemostasis and thrombosis, blood platelets are involved in various processes such as inflammation, infection, immunobiology, cancer metastasis, wound repair and angiogenesis. Platelets' hemostatic and non-hemostatic functions are mediated by the expression of various membrane receptors and the release of proteins, ions and other mediators. Therefore, specific activities of platelets responsible for the non-hemostatic disease are to be inhibited while leaving the platelet's hemostatic function unaffected. Platelets' anti-aggregatory property has been used as a primary criterion for antiplatelet drugs/bioactives; however, their non-hemostatic activities are not well known. This review describes the hemostatic and non-hemostatic function of human blood platelets and the modulatory effects of bioactive food components. PRACTICAL APPLICATIONS: In this review, we have discussed the antiplatelet effects of several food components. These bioactive compounds inhibit both hemostatic and non-hemostatic pathways involving blood platelet. Platelets have emerged as critical biological factors of normal and pathologic vascular healing and other diseases such as cancers and inflammatory and immune disorders. The challenge for therapeutic intervention in these disorders will be to find drugs and bioactive compounds that preferentially block specific sites implicated in emerging roles of platelets' complicated contribution to inflammation, tumour growth, or other disorders while leaving at least some of their hemostatic function intact.