Platelet (Plt) derived-extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial ...cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular endothelial cell permeability, similar to fresh Plts. To investigate this hypothesis we utilized in vitro and in vivo models of vascular endothelial compromise and bleeding.
In vitro: Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function was assessed by trans - endothelial electrical resistance (TEER) measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts were assessed by multiple electrode Plt aggregometry. In vivo: The effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in NOD-SCID mice by an established Miles Assay of vascular permeability and a tail snip bleeding assay.
In vitro: Plt-EVs displayed exosomal size distribution and expressed Plt specific surface markers. Plts and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced Thrombin Receptor Activating Peptide (TRAP) mediated aggregation of whole blood, whereas Plts enhanced TRAP, Arachidonic Acid (ASPI), Collagen, and Adenosine Diphosphate (ADP) mediated aggregation. In vivo: Plt-EVs are equivalent to Plts in attenuating VEGF-A induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model.
Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability and mitigate the endotheliopathy of trauma (EOT).
Original Article LEVEL OF EVIDENCE: This is a pre-clinical study so it does not confirm to the level of evidence table for all clinical studies and case reports.
Platelet (Plt)-derived extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial ...cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular EC permeability, similar to fresh Plts. To investigate this hypothesis, we used in vitro and in vivo models of vascular endothelial compromise and bleeding.
In the vitro model, Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function were assessed by transendothelial electrical resistance measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts was assessed by multiple electrode Plt aggregometry. Using an in vivo model, the effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by an established Miles assay of vascular permeability and a tail snip bleeding assay.
In the in vitro model, Plt-EVs displayed exosomal size distribution and expressed Plt-specific surface markers. Platelets and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced thrombin receptor-activating peptide-mediated aggregation of whole blood, whereas Plts enhanced thrombin receptor-activating peptide-, arachidonic acid-, collagen-, and adenosine diphosphate-mediated aggregation. In the in vivo model, Plt-EVs are equivalent to Plts in attenuating vascular endothelial growth factor (VEGF)-A-induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model.
Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability, and mitigate the endotheliopathy of trauma.
Mobilization of intra and extracellular calcium is required for platelet activation, aggregation, and degranulation. However, the importance of alterations in the calcium-platelet axis after injury ...is unknown. We hypothesized that in injured patients, in vivo initial calcium concentrations (pretransfusion) predict ex vivo platelet activation and aggregation, viscoelastic clot strength, and transfusion of blood products. We additionally hypothesized that increasing calcium concentrations ex vivo increases the expression of platelet activation surface receptors and platelet aggregation responses to agonist stimulation in healthy donor blood.
Blood samples were collected from 538 trauma patients on arrival to the emergency department. Standard assays (including calcium), platelet aggregometry (PA) and thromboelastometry (ROTEM) were performed. In PA, platelet activation (prestimulation impedance Ω) and aggregation responses to agonist stimulation (area under the aggregation curve AUC) with adenosine diphosphate (ADP), thrombin receptor-activating peptide, arachidonic acid (AA), and collagen (COL) were measured. Multivariable regression tested the associations of calcium with PA, ROTEM, and transfusions. To further examine the calcium-platelet axis, calcium was titrated in healthy blood. Platelet aggregometry and ROTEM were performed, and expression of platelet glycoprotein IIb/IIIa and P-selectin was measured by flow cytometry.
The patients were moderately injured with normal calcium and platelet counts. Higher calcium on arrival (pretransfusion) was independently associated with increased platelet activation (prestimulation, Ω; p < 0.001), aggregation (ADP-stimulated, AUC; p = 0.002; thrombin receptor-activating peptide-stimulated, AUC; p = 0.038), and clot strength (ROTEM max clot firmness; p < 0.001), and inversely associated with 24-hour transfusions of blood, plasma, and platelets (all p < 0.005). Up-titrating calcium in healthy blood increased platelet activation (prestimulation, Ω; p < 0.001), aggregation (ADP, AA, COL-stimulated AUCs; p < 0.050), and expression of P-selectin (p = 0.003).
Initial calcium concentrations (pretransfusion) are independently associated with platelet activation, aggregation, clot-strength, and transfusions after injury. These changes may be mediated by calcium driven expression of surface receptors necessary for platelet activation and aggregation. However, the therapeutic benefit of early, empiric calcium repletion in trauma patients remains undefined.
Prognostic, level V.
Platelet behavior in trauma-induced coagulopathy is poorly understood. Injured patients have impaired platelet aggregation (dysfunction) in ex vivo agonist-stimulated platelet aggregometry (PA). ...However, PA assumes that platelets are inactivated before ex vivo stimulated aggregation, which may be altered by injury. We hypothesized that following trauma, platelet aggregation (area under the curve) is decreased regardless of injury burden, but that (1) minor injury is associated with an increased baseline electrical impedance, characteristic of a functional platelet phenotype (platelets that activate in response to injury), and that (2) severe injury is not associated with an increased baseline electrical impedance, characteristic of a dysfunctional phenotype (platelets that do not activate well in response to injury) compared with healthy controls.
Blood from 458 trauma patients and 30 healthy donors was collected for PA. Baseline electrical impedance (Ω); platelet aggregation stimulated by adenosine diphosphate, collagen, thrombin, and arachidonic acid; and rotational thromboelastometry were measured. Multivariate regression was performed to identify associations of PA measures with blood transfusion.
Compared with healthy controls, injured patients had impaired platelet aggregation in response to ex vivo stimulation, regardless of injury burden. However, minorly injured patients had increased endogenous platelet activation (baseline electrical impedance, Ω: with shock, p = 0.012; without shock, p = 0.084), but severely injured patients did not have significant increases in endogenous platelet activation (baseline electrical impedance, Ω: with shock, p = 0.86; without shock, p = 0.37). For every 10 Ω increase in baseline electrical impedance, there was an 8% decrease in units of blood transfused in the first 24 h (-0.08; confidence interval, -0.14 to -0.02; p = 0.015).
Injury and shock confer differential patterns of platelet aggregation in PA. Minor injury overestimates the presence of platelet dysfunction, while severe injury induces a truly dysfunctional phenotype-platelets that do not activate nor aggregate appropriately after injury. This is consequential in improving accurate phenotyping of postinjury platelet behavior for platelet-based therapeutics.
Prognostic, level IV.
Autopsy and biomarker studies suggest that endotheliopathy contributes to coronavirus disease (COVID-19)-associated acute respiratory distress syndrome. However, the effects of COVID-19 on the lung ...endothelium are not well defined. We hypothesized that the lung endotheliopathy of COVID-19 is caused by circulating host factors and direct endothelial infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
We aimed to determine the effects of SARS-CoV-2 or sera from patients with COVID-19 on the permeability and inflammatory activation of lung microvascular endothelial cells.
Human lung microvascular endothelial cells were treated with live SARS-CoV-2; inactivated viral particles; or sera from patients with COVID-19, patients without COVID-19, and healthy volunteers. Permeability was determined by measuring transendothelial resistance to electrical current flow, where decreased resistance signifies increased permeability. Inflammatory mediators were quantified in culture supernatants. Endothelial biomarkers were quantified in patient sera.
Viral PCR confirmed that SARS-CoV-2 enters and replicates in endothelial cells. Live SARS-CoV-2, but not dead virus or spike protein, induces endothelial permeability and secretion of plasminogen activator inhibitor 1 and vascular endothelial growth factor. There was substantial variability in the effects of SARS-CoV-2 on endothelial cells from different donors. Sera from patients with COVID-19 induced endothelial permeability, which correlated with disease severity. Serum levels of endothelial activation and injury biomarkers were increased in patients with COVID-19 and correlated with severity of illness.
SARS-CoV-2 infects and dysregulates endothelial cell functions. Circulating factors in patients with COVID-19 also induce endothelial cell dysfunction. Our data point to roles for both systemic factors acting on lung endothelial cells and viral infection of endothelial cells in COVID-19-associated endotheliopathy.
BACKGROUND:Altered post-injury platelet behavior is recognized in the pathophysiology of trauma-induced coagulopathy (TIC), but the mechanisms remain largely undefined. Studies suggest that soluble ...factors released by injury may inhibit signaling pathways and induce structural changes in circulating platelets. Given this, we sought to examine the impact of treating healthy platelets with plasma from injured patients. We hypothesized that healthy platelets treated ex-vivo with plasma from injured patients with shock would impair platelet aggregation, while treatment with plasma from injured patients with significant injury burden, but without shock, would enhance platelet aggregation.
METHODS:Plasma samples were isolated from injured patients (pre-transfusion) and healthy donors at a Level I trauma center and stored at −80°C. Plasma samples from four separate patients in each of the following stratified clinical groups were usedmild injury/no shock (injury severity score ISS 2–15, base excess BE>-6), mild injury/with shock (ISS 2–15, BE≤-6), severe injury/no shock (ISS>25, BE>-6), severe injury/with shock (ISS>25, BE≤-6), minimal injury (ISS 0/1, BE>-6), and healthy. Platelets were isolated from three healthy adult males and were treated with plasma for 30 min. Aggregation was stimulated with a thrombin receptor agonist and measured via multiple-electrode platelet aggregometry. Data were normalized to HEPES Tyrodeʼs (HT) buffer-only treated platelets. Associations of plasma treatment groups with platelet aggregation measures were tested with Mann-Whitney U Tests.
RESULTS:Platelets treated with plasma from patients with shock (regardless of degree of injury) had significantly impaired thrombin stimulated aggregation compared to platelets treated with plasma from patients without shock (p = 0.002). Conversely, platelets treated with plasma from patients with severe injury, but without shock, had amplified thrombin stimulated aggregation (p = 0.030).
CONCLUSION:Shock mediated soluble factors impair platelet aggregation, and tissue injury mediated soluble factors amplify platelet aggregation. Future characterization of these soluble factors will support development of novel treatments of TIC.
Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host ...defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming.
Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to trauma platelet releasates (TPR) and plasma (TP) was assessed via resistance measurement by Electric Cell-substrate Impedance Sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics.
TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared to untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared to TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms.
TPRs provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma.
Prognostic/Epidemiological, Level III.
BACKGROUND
Posttraumatic venous thromboembolism (VTE) remains prevalent in severely injured patients despite chemoprophylaxis. Importantly, although platelets are central to thrombosis, they are not ...routinely targeted in prevention of posttraumatic VTE. Furthermore, platelets from injured patients show ex vivo evidence of increased activation yet impaired aggregation, consistent with functional exhaustion. However, the relationship of this platelet functional phenotype with development of posttraumatic VTE is unknown. We hypothesized that, following injury, impaired ex vivo platelet aggregation (PA) is associated with the development of posttraumatic VTE.
METHODS
We performed a secondary analysis of 133 severely injured patients from a prospective observational study investigating coagulation and inflammation (2011–2019). Platelet aggregation in response to stimulation with adenosine diphosphate (ADP), collagen, and thrombin was measured at presentation (preresuscitation) and 24 hours (postresuscitation). Viscoelastic clot strength and lysis were measured in parallel by thromboelastography. Multivariable regression examined relationships between PA at presentation, 24 hours, and the change (δ) in PA between presentation and 24 hours with development of VTE.
RESULTS
The 133 patients were severely injured (median Injury Severity Score, 25), and 14% developed VTE (all >48 hours after admission). At presentation, platelet count and PA were not significantly different between those with and without incident VTE. However, at 24 hours, those who subsequently developed VTE had significantly lower platelet counts (126 × 10
9
/L vs. 164 × 10
9
/L,
p =
0.01) and lower PA in response to ADP (
p
< 0.05), collagen (
p
< 0.05), and thrombin (
p
= 0.06). Importantly, the magnitude of decrease in PA (δ) from presentation to 24 hours was independently associated with development of VTE (adjusted odds ratios per 10 aggregation unit decrease: δ-ADP, 1.31
p
= 0.03; δ-collagen, 1.36
p
= 0.01; δ-thrombin, 1.41
p
< 0.01).
CONCLUSION
Severely injured patients with decreasing ex vivo measures of PA despite resuscitation have an increased risk of developing VTE. This may have implications for predicting development of VTE and for studying platelet targeted chemoprophylaxis regimens.
LEVEL OF EVIDENCE
Prognostic/Epidemiological; Level III.
There remains a need to better identify patients at highest risk for developing severe Coronavirus Disease 2019 (COVID-19) as additional waves of the pandemic continue to impact hospital systems. We ...sought to characterize the association of receptor for advanced glycation end products (RAGE), SARS-CoV-2 nucleocapsid viral antigen, and a panel of thromboinflammatory biomarkers with development of severe disease in patients presenting to the emergency department with symptomatic COVID-19.
Blood samples were collected on arrival from 77 patients with symptomatic COVID-19, and plasma levels of thromboinflammatory biomarkers were measured.
Differences in biomarkers between those who did and did not develop severe disease or death 7 days after presentation were analyzed. After adjustment for multiple comparisons, RAGE, SARS-CoV-2 nucleocapsid viral antigen, interleukin (IL)-6, IL-10 and tumor necrosis factor receptor (TNFR)-1 were significantly elevated in the group who developed severe disease (all
<0.05). In a multivariable regression model, RAGE and SARS-CoV-2 nucleocapsid viral antigen remained significant risk factors for development of severe disease (both
<0.05), and each had sensitivity and specificity >80% on cut-point analysis.
Elevated RAGE and SARS-CoV-2 nucleocapsid viral antigen on emergency department presentation are strongly associated with development of severe disease at 7 days. These findings are of clinical relevance for patient prognostication and triage as hospital systems continue to be overwhelmed. Further studies are warranted to determine the feasibility and utility of point-of care measurements of these biomarkers in the emergency department setting to improve patient prognostication and triage.
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