Coagulation and platelet biology at the intersection of health and disease: illustrated capsules of the 11th Symposium on Hemostasis at the University of North Carolina.

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19.

BACKGROUND: COVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. OBJECTIVES: From the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. METHODS: We utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. RESULTS: Compared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. CONCLUSION: We conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

The antioxidant and anti-inflammatory activities of avasopasem manganese in age-associated, cisplatin-induced renal injury.

PURPOSE: Cisplatin contributes to acute kidney injury (AKI) and chronic kidney disease (CKD) that occurs with greater frequency and severity in older patients. Age-associated cisplatin sensitivity in human fibroblasts involves increased mitochondrial superoxide produced by older donor cells. EXPERIMENTAL DESIGN: Young and old C57BL/6 J murine models of cisplatin-induced AKI and CKD were treated with the SOD mimetic avasopasem manganese to investigate the potential antioxidant and anti-inflammatory effects. Adverse event reporting from a phase 2 and a phase 3 randomized clinical trial (NCT02508389 and NCT03689712) conducted in patients treated with cisplatin and AVA was determined to have established the incidence and severity of AKI. RESULTS: Cisplatin-induced AKI and CKD occurred in all mice, however, was more pronounced in older mice. AVA reduced cisplatin-induced mortality, AKI, and CKD, in older animals. AVA also alleviated cisplatin-induced alterations in mitochondrial electron transport chain (ETC) complex activities and NADPH Oxidase 4 (NOX4) and inhibited the increased levels of the inflammation markers, TNFα, IL1, ICAM-1, and VCAM-1. Analysis of age-stratified subjects treated with cisplatin from clinical trials (NCT02508389, NCT03689712) also supported that the incidence of AKI increased with age and AVA reduced age-associated therapy-induced adverse events (AE), including hypomagnesemia, increased creatinine, and AKI. CONCLUSIONS: Older mice and humans are more susceptible to cisplatin-induced kidney injury, and treatment with AVA mitigates age-associated damage. Mitochondrial ETC and NOX4 activities represent sources of superoxide production contributing to cisplatin-induced kidney injury, and pro-inflammatory cytokine production and endothelial dysfunction may also be increased by superoxide formation.

NLRP3-Induced NETosis: A Potential Therapeutic Target for Ischemic Thrombotic Diseases?

Ischemic thrombotic disease, characterized by the formation of obstructive blood clots within arteries or veins, is a condition associated with life-threatening events, such as stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism. The conventional therapeutic strategy relies on treatments with anticoagulants that unfortunately pose an inherent risk of bleeding complications. These anticoagulants primarily target clotting factors, often overlooking upstream events, including the release of neutrophil extracellular traps (NETs). Neutrophils are integral components of the innate immune system, traditionally known for their role in combating pathogens through NET formation. Emerging evidence has now revealed that NETs contribute to a prothrombotic milieu by promoting platelet activation, increasing thrombin generation, and providing a scaffold for clot formation. Additionally, NET components enhance clot stability and resistance to fibrinolysis. Clinical and preclinical studies have underscored the mechanistic involvement of NETs in the pathogenesis of thrombotic complications, since the clots obtained from patients and experimental models consistently exhibit the presence of NETs. Given these insights, the inhibition of NETs or NET formation is emerging as a promising therapeutic approach for ischemic thrombotic diseases. Recent investigations also implicate a role for the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome as a mediator of NETosis and thrombosis, suggesting that NLRP3 inhibition may also hold potential for mitigating thrombotic events. Therefore, future preclinical and clinical studies aimed at identifying and validating NLRP3 inhibition as a novel therapeutic intervention for thrombotic disorders are imperative.

Endogenous SOD2 (Superoxide Dismutase) Regulates Platelet-Dependent Thrombin Generation and Thrombosis During Aging.

BACKGROUND: Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. METHODS: We studied littermates of platelet-specific SOD2-knockout (SOD2fl/flPf4Cre, pSOD2-KO) and control (SOD2fl/fl) mice at young (4-5 months) or old (18-20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. RESULTS: Platelet αIIbß3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. CONCLUSIONS: Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

The Role of Platelet-Derived Extracellular Vesicles in Immune-Mediated Thrombosis.

Platelet-derived extracellular vesicles (PEVs) play important roles in hemostasis and thrombosis. There are three major types of PEVs described based on their size and characteristics, but newer types may continue to emerge owing to the ongoing improvement in the methodologies and terms used to define various types of EVs. As the literature on EVs is growing, there are continuing attempts to standardize protocols for EV isolation and reach consensus in the field. This review provides information on mechanisms of PEV production, characteristics, cellular interaction, and their pathological role, especially in autoimmune and infectious diseases. We also highlight the mechanisms through which PEVs can activate parent cells in a feedback loop.

Redox Mechanisms of Platelet Activation in Aging.

Aging is intrinsically linked with physiologic decline and is a major risk factor for a broad range of diseases. The deleterious effects of advancing age on the vascular system are evidenced by the high incidence and prevalence of cardiovascular disease in the elderly. Reactive oxygen species are critical mediators of normal vascular physiology and have been shown to gradually increase in the vasculature with age. There is a growing appreciation for the complexity of oxidant and antioxidant systems at the cellular and molecular levels, and accumulating evidence indicates a causal association between oxidative stress and age-related vascular disease. Herein, we review the current understanding of mechanistic links between oxidative stress and thrombotic vascular disease and the changes that occur with aging. While several vascular cells are key contributors, we focus on oxidative changes that occur in platelets and their mediation in disease progression. Additionally, we discuss the impact of comorbid conditions (i.e., diabetes, atherosclerosis, obesity, cancer, etc.) that have been associated with platelet redox dysregulation and vascular disease pathogenesis. As we continue to unravel the fundamental redox mechanisms of the vascular system, we will be able to develop more targeted therapeutic strategies for the prevention and management of age-associated vascular disease.

Heavy lone coronary artery thrombosis treated by stent retriever, in the setting of COVID-19 infection.

We present a case of heavy lone coronary thrombosis in the setting of COVID-19 infection. We highlight the special angiographic, ultrasonographic, and histological features of this thrombus, and we describe the application of carotid stent retriever for its removal.

DNase 1 Protects From Increased Thrombin Generation and Venous Thrombosis During Aging: Cross-Sectional Study in Mice and Humans.

Background Human aging is associated with increased risk of thrombosis, but the mechanisms are poorly defined. We hypothesized that aging induces peroxide-dependent release of neutrophil extracellular traps that contribute to thrombin generation and thrombosis. Methods and Results We studied C57BL6J mice and littermates of glutathione peroxidase-1 transgenic and wild-type mice at young (4 month) and old (20 month) ages and a healthy cohort of young (18-39 years) or middle-aged/older (50-72 years) humans. In plasma, we measured thrombin generation potential and components of neutrophil extracellular traps (cell-free DNA and citrullinated histone). Aged wild-type mice displayed a significant increase in thrombin generation that was decreased in aged glutathione peroxidase-1 transgenic mice. Both aged wild-type and aged glutathione peroxidase-1 transgenic mice demonstrated similar elevation of plasma cell-free DNA compared with young mice. In contrast, plasma levels of citrullinated histone were not altered with age or genotype. Release of neutrophil extracellular traps from neutrophils in vitro was also similar between young and aged wild-type or glutathione peroxidase-1 transgenic mice. Treatment of plasma or mice with DNase 1 decreased age-associated increases in thrombin generation, and DNase 1 treatment blocked the development of experimental venous thrombi in aged C57BL6J mice. Similarly, thrombin generation potential and plasma cell-free DNA, but not citrullinated histone, were higher in middle-aged/older humans, and treatment of plasma with DNase 1 reversed the increase in thrombin generation. Conclusions We conclude that DNase 1 limits thrombin generation and protects from venous thrombosis during aging, likely by hydrolyzing cell-free DNA.

Thrombotic potential during pediatric acute lymphoblastic leukemia induction: Role of cell-free DNA.

BACKGROUND: Thromboembolism affects up to 30% of children undergoing treatment for acute lymphoblastic leukemia (ALL). Increased thrombin generation has been reported in ALL, but the mechanisms remain elusive. OBJECTIVE: We aimed to show that extracellular traps and cell-free DNA (cfDNA) promote thrombin generation in pediatric ALL. METHODS: In a longitudinal single-center study, we recruited 17 consecutive pediatric ALL patients. Serial blood samples were collected at diagnosis and weekly during the 4-week induction phase of antileukemic chemotherapy. Healthy children (n = 14) and children with deep vein thrombosis (DVT; n = 7) or sepsis (n = 5) were recruited as negative and positive controls, respectively. In plasma, we measured endogenous thrombin generation potential (ETP) and components of extracellular traps, including cfDNA. RESULTS: In patients with ALL, ETP was increased at baseline and remained significantly elevated throughout the induction therapy. Plasma levels of cfDNA were increased at baseline and during the first 3 weeks of induction therapy. The extent of enhancement of ETP and plasma cfDNA in patients with ALL was similar to that seen in patients with DVT or sepsis. Treatment of plasma with DNase 1 lowered ETP in patients with ALL at each time point but did not affect ETP in healthy controls. CONCLUSION: We conclude that childhood ALL is associated with a prothrombotic milieu at the time of diagnosis that continues during induction chemotherapy, and cfDNA contributes to increased thrombogenic potential.

Standard prophylactic versus intermediate dose enoxaparin in adults with severe COVID-19: A multi-center, open-label, randomized controlled trial.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with coagulopathy but the optimal prophylactic anticoagulation therapy remains uncertain and may depend on COVID-19 severity. OBJECTIVE: To compare outcomes in hospitalized adults with severe COVID-19 treated with standard prophylactic versus intermediate dose enoxaparin. METHODS: We conducted a multi-center, open-label, randomized controlled trial comparing standard prophylactic dose versus intermediate dose enoxaparin in adults who were hospitalized with COVID-19 and admitted to an intensive care unit (ICU) and/or had laboratory evidence of coagulopathy. Patients were randomly assigned in a 1:1 ratio to receive standard prophylactic dose enoxaparin or intermediate weight-adjusted dose enoxaparin. The primary outcome was all-cause mortality at 30 days. Secondary outcomes included arterial or venous thromboembolism and major bleeding. RESULTS: A total of 176 patients (99 males and 77 females) underwent randomization. In the intention-to-treat population, all-cause mortality at 30 days was 15% for intermediate dose enoxaparin and 21% for standard prophylactic dose enoxaparin (odds ratio, 0.66; 95% confidence interval, 0.30-1.45; P = .31 by Chi-square test). Unadjusted Cox proportional hazards modeling demonstrated no significant difference in mortality between intermediate and standard dose enoxaparin (hazard ratio, 0.67; 95% confidence interval, 0.33-1.37; P = .28). Arterial or venous thrombosis occurred in 13% of patients assigned to intermediate dose enoxaparin and 9% of patients assigned to standard dose enoxaparin. Major bleeding occurred in 2% of patients in each arm. CONCLUSION: In hospitalized adults with severe COVID-19, standard prophylactic dose and intermediate dose enoxaparin did not differ significantly in preventing death or thrombosis at 30 days.

Glutathione peroxidase-1 overexpression reduces oxidative stress, and improves pathology and proteome remodeling in the kidneys of old mice.

This study investigated the direct roles of hydrogen peroxide (H2 O2 ) in kidney aging using transgenic mice overexpressing glutathione peroxidase-1 (GPX1 TG). We demonstrated that kidneys in old mice recapitulated kidneys in elderly humans and were characterized by glomerulosclerosis, tubular atrophy, interstitial fibrosis, and loss of cortical mass. Scavenging H2 O2 by GPX1 TG significantly reduced mitochondrial and total cellular reactive oxygen species (ROS) and mitigated oxidative damage, thus improving these pathologies. The potential mechanisms by which ROS are increased in the aged kidney include a decreased abundance of an anti-aging hormone, Klotho, in kidney tissue, and decreased expression of nuclear respiratory factor 2 (Nrf2), a master regulator of the stress response. Decreased Klotho or Nrf2 was not improved in the kidneys of old GPX1 TG mice, even though mitochondrial morphology was better preserved. Using laser capture microdissection followed by label-free shotgun proteomics analysis, we show that the glomerular proteome in old mice was characterized by decreased abundance of cytoskeletal proteins (critical for maintaining normal glomerular function) and heat shock proteins, leading to increased accumulation of apolipoprotein E and inflammatory molecules. Targeted proteomic analysis of kidney tubules from old mice showed decreased abundance of fatty acid oxidation enzymes and antioxidant proteins, as well as increased abundance of glycolytic enzymes and molecular chaperones. GPX1 TG partially attenuated the remodeling of glomerular and tubule proteomes in aged kidneys. In summary, mitochondria from GPX1 TG mice are protected and kidney aging is ameliorated via its antioxidant activities, independent and downstream of Nrf2 or Klotho signaling.

Memantine Protects From Exacerbation of Ischemic Stroke and Blood Brain Barrier Disruption in Mild But Not Severe Hyperhomocysteinemia.

Background Hyperhomocysteinemia is a risk factor for ischemic stroke; however, a targeted treatment strategy is lacking partly because of limited understanding of the causal role of homocysteine in cerebrovascular pathogenesis. Methods and Results In a genetic model of cystathionine beta synthase (CBS) deficiency, we tested the hypothesis that elevation in plasma total homocysteine exacerbates cerebrovascular injury and that memantine, a N-methyl-D-aspartate receptor antagonist, is protective. Mild or severe elevation in plasma total homocysteine was observed in Cbs+/- (6.1±0.3 µmol/L) or Cbs-/- (309±18 µmol/L) mice versus Cbs+/+ (3.1±0.6 µmol/L) mice. Surprisingly, Cbs-/- and Cbs+/- mice exhibited similar increases in cerebral infarct size following middle cerebral artery ischemia/reperfusion injury, despite the much higher total homocysteine levels in Cbs-/- mice. Likewise, disruption of the blood brain barrier was observed in both Cbs+/- and Cbs-/- mice. Administration of the N-methyl-D-aspartate receptor antagonist memantine protected Cbs+/- but not Cbs-/- mice from cerebral infarction and blood brain barrier disruption. Our data suggest that the differential effect of memantine in Cbs+/- versus Cbs-/- mice may be related to changes in expression of N-methyl-D-aspartate receptor subunits. Cbs-/-, but not Cbs+/- mice had increased expression of NR2B subunit, which is known to be relatively insensitive to homocysteine. Conclusions These data provide experimental evidence that even a mild increase in plasma total homocysteine can exacerbate cerebrovascular injury and suggest that N-methyl-D-aspartate receptor antagonism may represent a strategy to prevent reperfusion injury after acute ischemic stroke in patients with mild hyperhomocysteinemia.

Modulators of platelet function in aging.

Platelets are small, anucleated effector cells that play an important role in linking the hemostatic and inflammatory processes in the body. Platelet function is known to be altered under various inflammatory conditions including aging. A gain in platelet function during aging can increase the risk of thrombotic events, such as stroke and acute myocardial infarction. Anti-platelet therapy is designed to reduce risk of serious cerebrovascular and cardiovascular events, but the adverse consequences of therapy, such as risk for bleeding increases with aging as well. Age-associated comorbidities such as obesity, diabetes, and hyperlipidemia also contribute to increased platelet activity and thus can enhance the risk of thrombosis. Therefore, identification of unique mechanisms of platelet dysfunction in aging and in age-associated comorbidities is warranted to design novel antiplatelet drugs. This review outlines some of the current areas of research on aging-related mechanisms of platelet hyperactivity and addresses the clinical urgency for designing anti-platelet therapies toward novel molecular targets in the aging population.

Nox2 NADPH oxidase is dispensable for platelet activation or arterial thrombosis in mice.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome.

The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.

Dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, inhibits platelet aggregation and arterial thrombosis.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLCγ2 in response to collagen or convulxin in human and mouse platelets (P < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s-1 (P < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models (P < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function.

Helicopter "Drip and Ship" Flights Do Not Alter the Pharmacological Integrity of rtPA.

INTRODUCTION: Rural and critical access hospitals rely on the "drip and ship" practice using helicopter emergency medical services (HEMS). But those helicopter flights are an unusual environment with physical factors such as vibration and accelerations that could potentially affect the stability, and pharmacological properties of IV rtPA, an issue that has not been previously addressed. MATERIALS AND METHODS: This was a prospective cohort study of consecutive acute ischemic stroke patients receiving IV rtPA through a Comprehensive Stroke Center from November 2015 to February 2017 to measure the effects of HEMS on the integrity and activity of rtPA by collecting residual medication left in the vial. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. NCT02752256 RESULTS: A total of 33 patients and rtPA samples were included; 18 patients who presented directly to the Comprehensive Stroke Center emergency department and 15 patients who received rtPA during air ambulance transfer. The median rtPA antigen concentration in the residual medication vial was 3.04 mg/mL (IQR: 1.24-3.87) in the HEMS group and 1.91 mg/mL (IQR: 1.33-2.60) in the controls (P = .168). There were no significant differences in rtPA activity or specific activity between the HEMS and control groups and there was no association between total HEMS flight time on overall rtPA specific activity. CONCLUSIONS: In summary, this study provides supportive evidence of the lack of a detrimental effect of the HEMS physical environment on the integrity of rtPA, therefore endorsing current drip and ship practices without infusion adjustments.