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Journal of Thrombosis and Haemostasis :... Aug 2023Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG...
BACKGROUND
Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported.
OBJECTIVES
Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples.
METHODS
Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer.
RESULTS
We confirmed the requirement of platelets, platelet contraction, and αIIbβ3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4 samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4 mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk.
CONCLUSION
Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.
Topics: Animals; Humans; Mice; Blood Platelets; Glycoproteins; Hemostatics; Integrins; Receptors, Proteinase-Activated; Receptors, Thrombin; Thrombelastography
PubMed: 37068592
DOI: 10.1016/j.jtha.2023.04.008 -
Arteriosclerosis, Thrombosis, and... Sep 2023Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic...
BACKGROUND
Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches.
METHODS
Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbβ3.
RESULTS
We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca] rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide.
CONCLUSIONS
Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.
Topics: Blood Platelets; Platelet Glycoprotein GPIIb-IIIa Complex; Chemokines; Thrombosis; Neutrophil Activation; CD40 Ligand; Neutrophils; Cell Adhesion; Humans; Arteries
PubMed: 37409530
DOI: 10.1161/ATVBAHA.122.318767 -
Platelets Dec 2023Although the presence of glycogen in platelets was established in the 1960s, its importance to specific functions (., activation, secretion, aggregation, and clot...
Although the presence of glycogen in platelets was established in the 1960s, its importance to specific functions (., activation, secretion, aggregation, and clot contraction) remains unclear. Patients with glycogen storage disease often present with increased bleeding and glycogen phosphorylase (GP) inhibitors, when used as treatments for diabetes, induce bleeding in preclinical studies suggesting some role for this form of glucose in hemostasis. In the present work, we examined how glycogen mobilization affects platelet function using GP inhibitors (CP316819 and CP91149) and a battery of assays. Blocking GP activity increased glycogen levels in resting and thrombin-activated platelets and inhibited platelet secretion and clot contraction, with minimal effects on aggregation. Seahorse energy flux analysis and metabolite supplementation experiments suggested that glycogen is an important metabolic fuel whose role is affected by platelet activation and the availability of external glucose and other metabolic fuels. Our data shed light on the bleeding diathesis in glycogen storage disease patients and offer insights into the potential effects of hyperglycemia on platelets.
Topics: Humans; Blood Platelets; Glycogenolysis; Glucose; Glycogen; Thrombosis; Glycogen Storage Disease
PubMed: 37292023
DOI: 10.1080/09537104.2023.2222184 -
Blood Nov 2023Genetic studies of platelet reactivity (PR) phenotypes may identify novel antiplatelet drug targets. However, such studies have been limited by small sample sizes (n <...
Genetic studies of platelet reactivity (PR) phenotypes may identify novel antiplatelet drug targets. However, such studies have been limited by small sample sizes (n < 5000) because of the complexity of measuring PR. We trained a model to predict PR from complete blood count (CBC) scattergrams. A genome-wide association study of this phenotype in 29 806 blood donors identified 21 distinct associations implicating 20 genes, of which 6 have been identified previously. The effect size estimates were significantly correlated with estimates from a study of flow cytometry-measured PR and a study of a phenotype of in vitro thrombus formation. A genetic score of PR built from the 21 variants was associated with the incidence rates of myocardial infarction and pulmonary embolism. Mendelian randomization analyses showed that PR was causally associated with the risks of coronary artery disease, stroke, and venous thromboembolism. Our approach provides a blueprint for using phenotype imputation to study the determinants of hard-to-measure but biologically important hematological traits.
Topics: Humans; Platelet Aggregation Inhibitors; Genome-Wide Association Study; Blood Platelets; Thrombosis; Blood Cell Count
PubMed: 37647652
DOI: 10.1182/blood.2023021100 -
Arteriosclerosis, Thrombosis, and... Oct 2023ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation...
BACKGROUND
ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y and P2Y receptors in ADP-induced TF exposure; (2) modulation of TF-platelets in anti-P2Y-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets.
METHODS
The effects of P2Y or P2Y antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y, and patients with gray platelet syndrome. Ex vivo, P2Y inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization.
RESULTS
In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TF-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TF-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TF-platelets similar to the poor-responder group. Indeed, a stronger P2Y inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TF-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome.
CONCLUSIONS
Data show that TF expression is regulated by P2Y and not P2Y; P2Y antagonists downregulate the percentage of TF-platelets. In clopidogrel good-responder patients, assessment of TF-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.
Topics: Humans; Blood Platelets; Clopidogrel; Coronary Artery Disease; Gray Platelet Syndrome; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Prasugrel Hydrochloride; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y12; Thromboplastin; Ticagrelor
PubMed: 37589138
DOI: 10.1161/ATVBAHA.123.319099 -
Frontiers in Immunology 2023Thrombocytopenia, characterized by a decrease in platelet count, is commonly observed in sepsis and COVID-19. In sepsis, thrombocytopenia can result from various... (Review)
Review
Thrombocytopenia, characterized by a decrease in platelet count, is commonly observed in sepsis and COVID-19. In sepsis, thrombocytopenia can result from various mechanisms, including impaired platelet production in the bone marrow, accelerated platelet destruction due to increased inflammation, sequestration of platelets in the spleen, immune-mediated platelet destruction, or dysregulated host responses. Similarly, thrombocytopenia has been reported in COVID-19 patients, but the immune-related mechanisms underlying this association remain unclear. Notably, interventions targeting thrombocytopenia have shown potential for improving outcomes in both sepsis and COVID-19 patients. Understanding these mechanisms is crucial for developing effective treatments.
Topics: Humans; COVID-19; Thrombocytopenia; Blood Platelets; Platelet Count; Sepsis; Anemia
PubMed: 37841241
DOI: 10.3389/fimmu.2023.1213510 -
Frontiers in Immunology 2023Cachexia, a complex wasting syndrome, significantly affects the quality of life and treatment options for cancer patients. Studies have reported a strong correlation...
Cachexia, a complex wasting syndrome, significantly affects the quality of life and treatment options for cancer patients. Studies have reported a strong correlation between high platelet count and decreased survival in cachectic individuals. Therefore, this study aimed to investigate the immunopathogenesis of cancer cachexia using the Apc mouse model of spontaneous colorectal cancer. The research focused on identifying cellular elements in the blood at different stages of cancer cachexia, assessing inflammatory markers and fibrogenic factors in the skeletal muscle, and studying the behavioral and metabolic phenotype of Apc mice at the pre-cachectic and severely cachectic stages. Platelet measurements were also obtained from other animal models of cancer cachexia - Lewis Lung Carcinoma and Colon 26 adenocarcinoma. Our study revealed that platelet number is elevated prior to cachexia development in Apc mice and can become activated during its progression. We also observed increased expression of TGFβ2, TGFβ3, and SMAD3 in the skeletal muscle of pre-cachectic Apc mice. In severely cachectic mice, we observed an increase in Ly6g, CD206, and IL-10 mRNA. Meanwhile, IL-1β gene expression was elevated in the pre-cachectic stage. Our behavioral and metabolic phenotyping results indicate that pre-cachectic Apc mice exhibit decreased physical activity. Additionally, we found an increase in anemia at pre-cachectic and severely cachectic stages. These findings highlight the altered platelet status during early and late stages of cachexia and provide a basis for further investigation of platelets in the field of cancer cachexia.
Topics: Animals; Mice; Blood Platelets; Cachexia; Quality of Life; Colonic Neoplasms; Disease Models, Animal
PubMed: 37701438
DOI: 10.3389/fimmu.2023.1253587 -
Platelets Dec 2023Many patients with coronary artery disease (CAD) have reduced the effect of aspirin, which may partly be explained by immature platelets. We aimed to investigate whether...
Many patients with coronary artery disease (CAD) have reduced the effect of aspirin, which may partly be explained by immature platelets. We aimed to investigate whether immature platelet markers can predict cardiovascular events in a large cohort of stable CAD patients. A total of 900 stable CAD patients were included and followed for a median of 3 years. We measured markers of immature platelets (platelet count, immature platelet count, immature platelet fraction, mean platelet volume, platelet distribution width, platelet mass, and thrombopoietin) using automated flow cytometry and studied their relation to cardiovascular events. Our primary endpoint was a composite of acute myocardial infarction (MI), ischemic stroke, and cardiovascular death. A composite of MI, ischemic stroke, stent thrombosis and all-cause mortality was analyzed as a secondary endpoint. We found no difference in immature platelet markers between CAD patients with or without cardiovascular events. Regression analysis using hazards rates showed that markers of immature platelets did not have any predictive value for endpoints (p-values >.05). Markers of immature platelets did not predict future cardiovascular events during a 3-year follow-up period in CAD patients. This suggests that immature platelets measured in a stable phase does not have a major role in predicting future cardiovascular events.
Topics: Humans; Coronary Artery Disease; Blood Platelets; Myocardial Infarction; Aspirin; Ischemic Stroke; Platelet Aggregation Inhibitors
PubMed: 37246549
DOI: 10.1080/09537104.2023.2217960 -
Circulation Research Oct 2023Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence...
BACKGROUND
Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients.
METHODS
In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice.
RESULTS
In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (=0.00013, <0.0001, and <0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; =0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; <0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets.
CONCLUSIONS
ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.
Topics: Humans; Animals; Mice; von Willebrand Factor; Prospective Studies; Mice, Inbred NOD; Mice, SCID; Blood Platelets; Thrombocytopenia
PubMed: 37883587
DOI: 10.1161/CIRCRESAHA.123.322752 -
Blood Transfusion = Trasfusione Del... Nov 2023Most public cord blood (CB) banks currently discard more than 80% of umbilical CB units not suitable for hemopoietic stem cell transplant due to low stem cell count....
BACKGROUND
Most public cord blood (CB) banks currently discard more than 80% of umbilical CB units not suitable for hemopoietic stem cell transplant due to low stem cell count. Although CB platelets, plasma, and red blood cells have been used for experimental allogeneic applications in wound healing, corneal ulcer treatment, and neonatal transfusion, no standard procedures for their preparation have been defined internationally.
MATERIALS AND METHODS
A network of 12 public CB banks in Spain, Italy, Greece, the UK, and Singapore developed a protocol to validate a procedure for the routine production of CB platelet concentrate (CB-PC), CB platelet-poor plasma (CB-PPP), and CB leukoreduced red blood cells (CB-LR-RBC) using locally available equipment and the commercial BioNest ABC and EF medical devices. CB units with >50 mL volume (excluding anticoagulant) and ≥150×10/L platelets were double centrifuged to obtain CB-PC, CB-PPP, and CB-RBC. The CB-RBC were diluted with saline-adenine-glucose-mannitol (SAGM), leukoreduced by filtration, stored at 2-6°C, and tested for hemolysis and potassium (K+) release over 15 days, with gamma irradiation performed on day 14. A set of acceptance criteria was pre-defined. This was for CB-PC: volume ≥5 mL and platelet count 800-1,200×10/L; for CB-PPP: platelet count <50×10/L; and for CB-LR-RBC: volume ≥20 mL, hematocrit 55-65%, residual leukocytes <0.2×10/unit, and hemolysis ≤0.8%.
RESULTS
Eight CB banks completed the validation exercise. Compliance with acceptance criteria was 99% for minimum volume and 86.1% for platelet count in CB-PC, and 90% for platelet count in CB-PPP. Compliance in CB-LR-RBC was 85.7% for minimum volume, 98.9% for residual leukocytes, and 90% for hematocrit. Compliance for hemolysis ≤0.8% decreased from 89.0 to 63.2% from day 0 to 15. K+ release increased from 3.0±1.8 to 25.0±7.0 mmol/L from day 0 to 15, respectively.
DISCUSSION
The MultiCord12 protocol was a useful tool to develop preliminary standardization of CB-PC, CB-PPP, and CB-LR-RBC.
Topics: Infant, Newborn; Humans; Hemolysis; Blood Banking; Erythrocytes; Blood Banks; Blood Platelets
PubMed: 37146297
DOI: 10.2450/BloodTransfus.492