Characterization and Outcomes of Hospitalized Children With Coronavirus Disease 2019: A Report From a Multicenter, Viral Infection and Respiratory Illness Universal Study (Coronavirus Disease 2019) Registry.

OBJECTIVES: Multicenter data on the characteristics and outcomes of children hospitalized with coronavirus disease 2019 are limited. Our objective was to describe the characteristics, ICU admissions, and outcomes among children hospitalized with coronavirus disease 2019 using Society of Critical Care Medicine Discovery Viral Infection and Respiratory Illness Universal Study: Coronavirus Disease 2019 registry. DESIGN: Retrospective study. SETTING: Society of Critical Care Medicine Viral Infection and Respiratory Illness Universal Study (Coronavirus Disease 2019) registry. PATIENTS: Children (< 18 yr) hospitalized with coronavirus disease 2019 at participating hospitals from February 2020 to January 2021. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was ICU admission. Secondary outcomes included hospital and ICU duration of stay and ICU, hospital, and 28-day mortality. A total of 874 children with coronavirus disease 2019 were reported to Viral Infection and Respiratory Illness Universal Study registry from 51 participating centers, majority in the United States. Median age was 8 years (interquartile range, 1.25-14 yr) with a male:female ratio of 1:2. A majority were non-Hispanic (492/874; 62.9%). Median body mass index (n = 817) was 19.4 kg/m2 (16-25.8 kg/m2), with 110 (13.4%) overweight and 300 (36.6%) obese. A majority (67%) presented with fever, and 43.2% had comorbidities. A total of 238 of 838 (28.2%) met the Centers for Disease Control and Prevention criteria for multisystem inflammatory syndrome in children, and 404 of 874 (46.2%) were admitted to the ICU. In multivariate logistic regression, age, fever, multisystem inflammatory syndrome in children, and pre-existing seizure disorder were independently associated with a greater odds of ICU admission. Hospital mortality was 16 of 874 (1.8%). Median (interquartile range) duration of ICU (n = 379) and hospital (n = 857) stay were 3.9 days (2-7.7 d) and 4 days (1.9-7.5 d), respectively. For patients with 28-day data, survival was 679 of 787, 86.3% with 13.4% lost to follow-up, and 0.3% deceased. CONCLUSIONS: In this observational, multicenter registry of children with coronavirus disease 2019, ICU admission was common. Older age, fever, multisystem inflammatory syndrome in children, and seizure disorder were independently associated with ICU admission, and mortality was lower among children than mortality reported in adults.

Cognitive Dysfunction After Analgesia and Sedation: Out of the Operating Room and Into the Pediatric Intensive Care Unit.

In the midst of concerns for potential neurodevelopmental effects after surgical anesthesia, there is a growing awareness that children who require sedation during critical illness are susceptible to neurologic dysfunctions collectively termed pediatric post-intensive care syndrome, or PICS-p. In contrast to healthy children undergoing elective surgery, critically ill children are subject to inordinate neurologic stress or injury and need to be considered separately. Despite recognition of PICS-p, inconsistency in techniques and timing of post-discharge assessments continues to be a significant barrier to understanding the specific role of sedation in later cognitive dysfunction. Nonetheless, available pediatric studies that account for analgesia and sedation consistently identify sedative and opioid analgesic exposures as risk factors for both in-hospital delirium and post-discharge neurologic sequelae. Clinical observations are supported by animal models showing neuroinflammation, increased neuronal death, dysmyelination, and altered synaptic plasticity and neurotransmission. Additionally, intensive care sedation also contributes to sleep disruption, an important and overlooked variable during acute illness and post-discharge recovery. Because analgesia and sedation are potentially modifiable, understanding the underlying mechanisms could transform sedation strategies to improve outcomes. To move the needle on this, prospective clinical studies would benefit from cohesion with regard to datasets and core outcome assessments, including sleep quality. Analyses should also account for the wide range of diagnoses, heterogeneity of this population, and the dynamic nature of neurodevelopment in age cohorts. Much of the related preclinical evidence has been studied in comparatively brief anesthetic exposures in healthy animals during infancy and is not generalizable to critically ill children. Thus, complementary animal models that more accurately "reverse translate" critical illness paradigms and the effect of analgesia and sedation on neuropathology and functional outcomes are needed. This review explores the interactive role of sedatives and the neurologic vulnerability of critically ill children as it pertains to survivorship and functional outcomes, which is the next frontier in pediatric intensive care.

Brighter Days May Be Ahead: Continuous Measurement of Pediatric Intensive Care Unit Light and Sound.

Objective: To describe light and sound characteristics in the rooms of critically ill children. Design: Prospective observational cohort study, with continuously measured light and sound levels. Setting: Tertiary care pediatric intensive care unit (PICU), with a newly constructed expansion and an older, pre-existing section. Patients: Critically ill patients 0-18 years old, requiring respiratory or cardiovascular support. Patients with severe cognitive pre-conditions were excluded. Measurements and Main Results: One hundred patients were enrolled, totaling 602 patient-days. The twenty-four hour median illuminance was 16 (IQR 5-53) lux (lx). Daytime (07:00-21:00) median light level was 27 lx (IQR 13-82), compared with 4 lx (IQR 1-10) overnight (22:00-06:00). Peak light levels occurred midday between 11:00 and 14:00, with a median of 48 lx (IQR 24-119). Daytime median illuminance trended higher over the course of admission, whereas light levels overnight were consistent. Midday light levels were higher in newly constructed rooms: 78 lx (IQR 30-143) vs. 26 lx (IQR 20-40) in existing rooms. The twenty-four hour median equivalent sound level (LAeq) was 60 (IQR 55-64) decibels (dB). Median daytime LAeq was 62 dB (IQR 58-65) and 56 dB (IQR 52-61) overnight. On average, 35% of patients experienced at least one sound peak >80 dB every hour from 22:00 to 06:00. Overnight peaks, but not median sound levels nor daytime peaks, decreased over the course of admission. There was no difference in sound between new and pre-existing rooms. Conclusions: This study describes continuously measured light and sound in PICU rooms. Light levels were low even during daytime hours, while sound levels were consistently higher than World Health Organization hospital room recommendations of <35 dB. Given the relevance of light and sound to sleep/wake patterns, and evidence of post-intensive care syndromes, the clinical effects of light and sound on critically ill children should be further explored as potentially modifiable environmental factors.

Potential Neurodevelopmental Effects of Pediatric Intensive Care Sedation and Analgesia: Repetitive Benzodiazepine and Opioid Exposure Alters Expression of Glial and Synaptic Proteins in Juvenile Rats.

Sedatives are suspected contributors to neurologic dysfunction in PICU patients, to whom they are administered during sensitive neurodevelopment. Relevant preclinical modeling has largely used comparatively brief anesthesia in infant age-approximate animals, with insufficient study of repetitive combined drug administration during childhood. We hypothesized that childhood neurodevelopment is selectively vulnerable to repeated treatment with benzodiazepine and opioid. We report a preclinical model of combined midazolam and morphine in early childhood age-approximate rats. DESIGN: Animal model. SETTING: Basic science laboratory. SUBJECTS: Male and female Long-Evans rats. INTERVENTIONS: Injections of morphine + midazolam were administered twice daily from postnatal days 18-22, tapering on postnatal days 23 and 24. Control groups included saline, morphine, or midazolam. To screen for acute neurodevelopmental effects, brain homogenates were analyzed by western blot for synaptophysin, drebrin, glial fibrillary acidic protein, S100 calcium-binding protein B, ionized calcium-binding adaptor molecule 1, and myelin basic proteins. Data analysis used Kruskal-Wallis with Dunn posttest, with a p value of less than 0.05 significance. MEASUREMENTS AND MAIN RESULTS: Morphine + midazolam and morphine animals gained less weight than saline or midazolam (p ≤ 0.01). Compared with saline, morphine + midazolam expressed significantly higher drebrin levels (p = 0.01), with numerically but not statistically decreased glial fibrillary acidic protein. Similarly, morphine animals exhibited less glial fibrillary acidic protein and more S100 calcium-binding protein B and synaptophysin. Midazolam animals expressed significantly more S100 calcium-binding protein B (p < 0.001) and 17-18.5 kDa myelin basic protein splicing isoform (p = 0.01), with numerically increased synaptophysin, ionized calcium-binding adaptor molecule 1, and 21.5 kDa myelin basic protein, and decreased glial fibrillary acidic protein. CONCLUSIONS: Analysis of brain tissue in this novel rodent model of repetitive morphine and midazolam administration showed effects on synaptic, astrocytic, microglial, and myelin proteins. These findings warrant further investigation because they may have implications for critically ill children requiring sedation and analgesia.

Advances and Future Directions of Diagnosis and Management of Pediatric Abusive Head Trauma: A Review of the Literature.

Abusive head trauma (AHT) is broadly defined as injury of the skull and intracranial contents as a result of perpetrator-inflicted force and represents a persistent and significant disease burden in children under the age of 4 years. When compared to age-matched controls with typically single occurrence accidental traumatic brain injury (TBI), mortality after AHT is disproportionately high and likely attributable to key differences between injury phenotypes. This article aims to review the epidemiology of AHT, summarize the current state of AHT diagnosis, treatment, and prevention as well as areas for future directions of study. Despite neuroimaging advances and an evolved understanding of AHT, early identification remains a challenge for contemporary clinicians. As such, the reported incidence of 10-30 per 100,000 infants per year may be a considerable underestimate that has not significantly decreased over the past several decades despite social campaigns for public education such as "Never Shake a Baby." This may reflect caregivers in crisis for whom education is not sufficient without support and intervention, or dangerous environments in which other family members are at risk in addition to the child. Acute management specific to AHT has not advanced beyond usual supportive care for childhood TBI, and prevention and early recognition remain crucial. Moreover, AHT is frequently excluded from studies of childhood TBI, which limits the precise translation of important brain injury research to this population. Repeated injury, antecedent abuse or neglect, delayed medical attention, and high rates of apnea and seizures on presentation are important variables to be considered. More research, including AHT inclusion in childhood TBI studies with comparisons to age-matched controls, and translational models with clinical fidelity are needed to better elucidate the pathophysiology of AHT and inform both clinical care and the development of targeted therapies. Clinical prediction rules, biomarkers, and imaging modalities hold promise, though these have largely been developed and validated in patients after clinically evident AHT has already occurred. Nevertheless, recognition of warning signs and intervention before irreversible harm occurs remains the current best strategy for medical professionals to protect vulnerable infants and toddlers.

Elevated endocan levels and its association with clinical severity in Stevens-Johnson Syndrome and toxic epidermal necrolysis.

AIM: The aim of this study was to determine the levels of endocan and other biomarkers of inflammation in the systemic circulation of three groups of patients: 1) biopsy confirmed Stevens Johnson Syndrome, Toxic Epidermal Necrolysis (SJS/TEN) subjects; 2) patients with allergic skin reactions but biopsy negative for SJS/TEN; and 3) normal controls. Besides, this paper aims to investigate the association of endocan levels with the extent of the skin lesions, the presence of purpura, and the degree of acute renal insufficiency, as well as to investigate endocan as a marker of clinical severity by correlating endocan levels with the SCORTEN results (a prognositic score for SJS/TEN). METHODS: Sixteen patients over the age of 18 years who were referred to Loyola University Medical Center with severe allergic skin reactions were recruited over a two-year period from May 2012 to May 2014. A diagnosis of SJS or TEN was confirmed in 7 subjects by skin biopsy. Citrated plasma samples were assayed for endocan, tumor necrosis factor-α (TNFα), vascular endothelial growth factor (VEGF), and C-reactive protein (CRP). The differences between SJS/TEN subjects, biopsy negative subjects, and normal controls (N.=23) were explored using ANOVA and Tukey's post-hoc test. Associations with other clinical variables were identified using linear and logistic regression. RESULTS: Biopsy positive SJS/TEN subjects and biopsy negative subjects had higher endocan levels than normal controls (SJS/TEN: 3.01 ng/mL [IQR: 2.15-8.11]; biopsy negative: 3.96 ng/mL [IQR: 1.54-4.85]; normal controls: 1.79 ng/mL [IQR: 1.67-1.98]; ANOVA P=0.0038). Endocan levels were more strongly associated with SCORTEN in SJS/TEN subjects than in biopsy negative subjects (R2 SJS/TEN=0.5110; biopsy negative=0.0317). SJS/TEN subjects exhibited significantly higher levels of TNF-α compared to normal controls (P=0.0267). The TNF-α levels were significantly lower compared to biopsy negative subjects (P=0.0052). VEGF levels were also elevated among SJS/TEN and biopsy negative subjects compared to normal controls (SJS/TEN: 12.04 pg/mL: [IQR: 7.64-52.7]; biopsy negative: 10.54 pg/mL [IQR: 4.17-6.46]; normal controls: 4.94 pg/mL [IQR: 4.17-6.46]; ANOVA P<0.0001). There was no significant difference in VEGF levels between SJS/TEN and biopsy negative subjects (P=0.7110). Similarly, CRP levels were elevated among SJS/TEN patients and biopsy negative subjects compared to normal controls (SJS/TEN: 32.09 µg/mL [IQR: 31.49-52.08]; biopsy negative: 83.38 µg/mL [IQR: 44.74-145.38]; healthy normal: 1.08 µg/mL [IQR: 0.73-2.03]; ANOVA P<0.0001). There was no significant difference in CRP levels between SJS/TEN and biopsy negative subjects (P=0.2416). CONCLUSION: To our knowledge, this is the first study to evaluate enodcan, a marker of endothelial dysfunction, in the systemic circulation of SJS/TEN patients. Elevated endocan levels were more strongly associated with disease severity among SJS/TEN subjects than among less severe allergic reactions with skin involvement.

Note: Simulation and test of a strip source electron gun.

We present simulation and test of an indirectly heated strip source electron beam gun assembly using Stanford Linear Accelerator Center (SLAC) electron beam trajectory program. The beam is now sharply focused with 3.04 mm diameter in the post anode region at 15.9 mm. The measured emission current and emission density were 1.12 A and 1.15 A/cm(2), respectively, that corresponds to power density of 11.5 kW/cm(2), at 10 kV acceleration potential. The simulated results were compared with then and now experiments and found in agreement. The gun is without any biasing, electrostatic and magnetic fields; hence simple and inexpensive. Moreover, it is now more powerful and is useful for accelerators technology due to high emission and low emittance parameters.

MRI assessment of cerebral blood flow after experimental traumatic brain injury combined with hemorrhagic shock in mice.

Secondary insults such as hypotension or hemorrhagic shock (HS) can greatly worsen outcome after traumatic brain injury (TBI). We recently developed a mouse combined injury model of TBI and HS using a controlled cortical impact (CCI) model and showed that 90 minutes of HS can exacerbate neuronal death in hippocampus beneath the contusion. This combined injury model has three clinically relevant phases, a shock, pre hospital, and definitive care phases. Mice were randomly assigned to four groups, shams as well as a CCI only, an HS only, and a CCI+HS groups. The CCI and HS reduced cerebral blood flow (CBF) in multiple regions of interest (ROIs) in the hemisphere ipsilateral and contralateral to injury. Hemorrhagic shock to a level of ∼30 mm Hg exacerbated the CCI-induced CBF reductions in multiple ROIs ipsilateral to injury (hemisphere and thalamus) and in the hemisphere contralateral to injury (hemisphere, thalamus, hippocampus, and cortex, all P<0.05 versus CCI only, HS only or both). An important effect of HS duration was also seen after CCI with maximal CBF reduction seen at 90 minutes (P<0.0001 group-time effect in ipsilateral hippocampus). Given that neuronal death in hippocampus is exacerbated by 90 minutes of HS in this model, our data suggest an important role for exacerbation of posttraumatic ischemia in mediating the secondary injury in CCI plus HS. In conclusion, the serial, non invasive assessment of CBF using ASL-MRI (magnetic resonance imaging with arterial spin labeling) is feasible in mice even in the complex setting of combined CCI+HS. The impact of resuscitation therapies and various mutant mouse strains on CBF and other outcomes merits investigation in this model.

Molecular profiling of heparinase-I resistant glycosaminoglycans in contaminated heparins. Comparative studies with uncontaminated heparin and porcine oversulfated chondroitin sulfate.

AIM: Heparin is a widely used anticoagulant which is usually obtained from porcine mucosal tissue. The structure of heparin is comparable to other naturally occurring glycosaminoglycans such as chondroitin sulfate and dermatan sulfate. The commercially available heparin preparations may contain small amounts of dermatan sulfate as a carry-over impurity. More recently (November 2007 to April 2008), an increased incidence of adverse events and deaths associated with the use of heparin alerted regulatory agencies to investigate the composition of heparin. As a result, oversulfated chondroitin sulfate was found to be the main determinant of the observed adverse reactions. This glycosaminoglycan is not usually found in the mammalian tissues. METHODS: This investigation reports on the comparison of contaminant free and contaminated heparins and their digestion by heparinase-I. It also describes the molecular profile of the contaminant isolated from the recalled heparin preparations in comparison to oversulfated chondroitin sulfate. The anticoagulant and anti-Xa activities are also reported. RESULTS: The contaminant is found to be comparable to the synthesized OSCS as both were resistant to heparinase-I digestion. The contaminant and OSCS exhibited weaker anticoagulant activities than heparin and did not have any anti-Xa effects. CONCLUSION: This data strongly suggests that such glycosaminoglycans as chondroitin sulfate can be structurally modified to exhibit anticoagulant activities and their molecular weight can be adjusted to mimic heparin.

Changing trends in anti-coagulant therapies. Are heparins and oral anti-coagulants challenged?

The conventional management of thrombotic and cardiovascular disorders is based on the use of heparin, oral anticoagulants and aspirin. Despite progress in the sciences, these drugs still remain a challenge and mystery. The development of low molecular weight heparins (LMWHS) and the synthesis of heparinomimetics represent a refined use of heparin. Additional drugs will continue to develop. However, none of these drugs will ever match the polypharmacology of heparin. Aspirin still remains the leading drug in the management of thrombotic and cardiovascular disorders. The newer antiplatelet drugs such as adenosine diphosphate receptor inhibitors, GPIIb/IIIa inhibitors and other specific inhibitors have limited effects and have been tested in patients who have already been treated with aspirin. Warfarin provides a convenient and affordable approach in the long-term outpatient management of thrombotic disorders. The optimized use of these drugs still remains the approach of choice to manage thrombotic disorders. The new anticoagulant targets, such as tissue factor, individual clotting factors, recombinant forms of serpins (antithrombin, heparin co-factor II and tissue factor pathway inhibitors), recombinant activated protein C, thrombomodulin and site specific serine proteases inhibitors complexes have also been developed. There is a major thrust on the development of orally bioavailable anti-Xa and IIa agents, which are slated to replace oral anticoagulants. Both the anti-factor Xa and anti-IIa agents have been developed for oral use and have provided impressive clinical results. However, safety concerns related to liver enzyme elevations and thrombosis rebound have been reported with their use. For these reasons, the US Food and Drug Administration did not approve the orally active antithrombin agent Ximelagatran for several indications. The synthetic pentasaccharide (Fondaparinux) has undergone clinical development. Unexpectedly, Fondaparinux also produced major bleeding problems at minimal dosages. Fondaparinux represents only one of the multiple pharmacologic effects of heparins. Thus, its therapeutic index will be proportionately narrower. The newer antiplatelet drugs have added a new dimension in the management of thrombotic disorders. The favorable clinical outcomes with aspirin and clopidogrel have validated COX-1 and P2Y12 receptors as targets for new drug development. Prasugrel, a novel thienopyridine, Cangrelor and AZD 6140 represent newer P2Y12 antagonists. Cangrelor and AZD 6140 are direct inhibitors, whereas Prasugrel requires metabolic activation. While clinically effective, recent results have prompted a closure of a clinical trial with Prasugrel due to bleeding. The newer anticoagulant and antiplatelet drugs are attractive, however, none of these are expected to replace the conventional drugs in polytherapeutic approaches. Heparins, warfarin and aspirin will continue to play a major role in the management of thrombotic and cardiovascular disorders for years to come.

Successful use of recombinant hirudin and its monitoring by ecarin clotting time in patients with heparin-induced thrombocytopenia undergoing off-pump coronary artery revascularization.

Refludan (lepirudin-rDNA for injection) is the first direct thrombin inhibitor approved by the United States FDA for anticoagulation to patients with heparin-induced thrombocytopenia (HIT). It was monitored by ecarin clotting time (ECT) assay in patients with HIT. Case histories and clotting parameters for three patients undergoing off-pump coronary artery revascularization procedure are discussed. The first patient received r-hirudin at a dose of 0.2 mg/kg intravenous (IV) bolus followed by 0.15 mg/kg/hour infusion. The second patient received 0.4 mg/kg IV bolus followed by infusion of 0.15 mg/kg/hour infusion. The third patient with renal failure received 0.2 mg/kg IV bolus followed by an infusion of 0.02 mg/kg/hour. Blood samples were drawn at baseline, 5 minutes post bolus and every 15 minutes during the coronary artery revascularization procedure. ECT was performed immediately on the citrated whole blood samples using the ECT cards in conjunction with the point-of-care, the thrombolytic assessment system (TAS) Analyzer (Pharmanetics, Raleigh, NC). The plasma samples were then analyzed for APTT and liquid ECT assay performed on a kinetic centrifugal analyzer (ACL 300 Plus). The ECT by cards was ideally maintained above 600 seconds during the surgical procedure. Additional boluses of Refludan were given as and when necessary (ECT < 600 sec) in order to maintain adequate anticoagulation. The calculated circulating concentrations of Refludan, following a bolus administration, based on the ECT cards, liquid ECT and APTT were 3.20 +/- 1.3, 3.51 +/- 1.35 and 2.02 +/- 1.19 microg/mL, respectively.

Comparative tissue factor pathway inhibitor release potential of heparins.

Tissue factor pathway inhibitor (TFPI) is released following the administration of unfractionated heparin, low-molecular-weight heparins, defibrotide and PI-88. In this study, the comparative effects of heparin, a low-molecular-weight heparin-gammaparin and a heparin-derived oligosaccharide mixture-subeparin (C3) were studied on functional and immunologic tissue factor pathway inhibitor activity levels in a non-human primate (Macaca mulatta) model. The dose-dependent effect was studied following intravenous and subcutaneous administration. Following the administration of 1 mg/kg of heparin, gammaparin, and C3, the functional levels of TFPI at 5 minutes were 2.40, 2.56, and 1.08 U/mL and the corresponding TFPI immunologic levels were 4.3-, 4.0-, and 2.1-fold, increased, respectively, over the baseline value. From these results, it can be concluded that heparin and gammaparin produced similar levels of TFPI release. Hence, gammaparin and heparin have similar TFPI release potential despite their differences in molecular weight. The influence of molecular weight, charge density, and interactions with heparin cofactor II on TFPI release are also discussed.

Blood levels of nitric oxide, C-reactive protein, and tumor necrosis factor-alpha are upregulated in patients with malignancy-associated hypercoagulable state: pathophysiologic implications.

Endogenous generation of nitric oxide (NO) plays an important role in the regulation of cardiovascular and inflammatory responses. This mediator is synthesized by a family of enzymes collectively known as NO synthase. Several isoforms of this enzyme have been identified and can be grouped as constitutive or inducible. Increased production of NO is reported in several inflammatory disorders, such as sepsis, arthritis, thrombotic thrombocytopenic purpura (TTP), and antiphospholipid syndrome. In addition, NO upregulates cyclo-oxygenase-2 and synthesis of several other inflammatory cytokines. Inflammation and thrombotic complications are usually associated with malignancy. Earlier reports indicate the upregulation of tumor necrosis factor-alpha (TNF-alpha), C-reactive protein (CRP), and tissue factor (TF) in patients with malignancy. To determine the relationship between inflammatory cytokines and NO in cancer patients with hypercoagulable states, baseline plasma samples from 160 patients with confirmed malignancy and hypercoagulable state were analyzed for NO levels. A chemical method based on a chemiluminescent reaction between NO and ozone using a highly sensitive gas phase NO analyzer was used. CRP, TF, and TNF-alpha were measured using enzyme-linked immunosorbent assay methods. Of the 160 patients who were plasma tested, the baseline NO levels ranged from 13.7 to 98.6 microM (63.1+/-15.9 microM, mean+/-SD) in contrast to age-matched control, which ranged from 9.1 to 34.6 microM (19.8+/-6.2 microM, mean+/-SD, n=138). Cancer patients also showed marked variations in the NO levels. Eighteen of 60 cancer patients exhibited greater than 60 microM NO levels. The CRP, TNF-alpha and TF were also significantly elevated. A correlation between CRP (r(2)=0.73) and NO levels was noted in cancer patients with hypercoagulable state. These data suggest that the pathogenesis associated with malignancy/hypercoagulable state is associated with an inflammatory component. In addition, the observed hemodynamic changes in some of the cancer patients may be due to increased NO production.

Influence of different anticoagulant agents on fibrinopeptide a generation.

The purpose of this study was to determine the in vitro effects of different anticoagulant drugs on fibrinopeptide A (FPA) generation inhibition and to identify whether there is any correlation between FPA generation, Hemochron ACT, global clotting assays, and chromogenic assays. Unfractionated heparin is a conventionally used anticoagulant. New anticoagulant drugs such as low molecular weight heparins (LMWHs), pentasaccharide, and antithrombin drugs are now approved for various indications. Anti-Xa drugs are in various phases of clinical development. The influence of different anticoagulant agents has been studied on fibrinopeptide A generation, Hemochron celite ACT, global clotting assays, and chromogenic anti-Xa and anti-IIa assays. Different LMWHs (Clivarin, Dalteparin, Enoxaparin, and Tinzaparin), anti-Xa agents (Pentasaccharide, DX-9065a and unfractionated heparin), and anti-IIa agents (PEG-Hirudin, Hirudin, Efegatran and Argatroban) were studied. The blood from healthy volunteers (n=4) was drawn for each drug. Imuclone FPA enzyme-linked immunosorbent kit assay, Hemochron celite ACT assay, global clotting assays (PT, APTT, Heptest-HI, thrombin time), and Loyola chromogenic anti-Xa and anti-IIIa assays were studied. Pentasaccharide demonstrated minimal effects on the whole blood clotting time such as ACT and on inhibition of FPA generation (IC50 > 25 microg/mL). DX-9065a exhibited a significant prolongation of ACT and marked inhibition of FPA generation (IC50 = 4.12 microg/mL). Unfractionated heparin showed a marked inhibition of FPA generation (IC50 = 5.16 microg/mL). Pentasaccharide, DX-9065a and UFH showed a marked correlation between ACT and inhibition of FPA generation. LMWHs demonstrated concentration-dependent inhibition of FPA generation. LMWHs studied showed good correlation between FPA generation inhibition and ACT test. Similar correlation was seen between FPA generation inhibition and the APTT, anti Xa (heptest-HI assay) and anti-IIa activity. Anti-IIa drugs demonstrated concentration-dependent inhibition of FPA generation. Their FPA generation inhibition potency is correlated with the ACT assay. A strong correlation between Hemochron ACT and FPA generation inhibition was observed. Based on this significant correlation, the FPA generation inhibition can be predicted by point-of-care ACT assay.

Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88).

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-II) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprotease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase I, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r2 = 0.94; TT, r2 = 0.84; APTT, r2 = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacologic action of PI-88, this agent represents an attractive pharmacologic agent for the control of thrombotic and proliferative disorders.

Global anticoagulant effects of a novel sulfated pentomanan oligosaccharide mixture.

PI-88 is a potent antiproliferative agent, which is developed for various indications in cancer. This agent is obtained from yeast fermentation and is primarily composed of pentamannose and tetramannose oligosaccharide units. PI-88 is capable of producing anticoagulant effects, which are mediated by heparin cofactor II. The purpose of this study was to determine the anticoagulant properties of PI-88 in native whole blood, freshly drawn from human volunteers, supplemented with PI-88 at various concentrations (0-100 microg/mL). Whole blood activated clotting time (ACT) was measured using Hemochron instruments. PI-88 produced a strong anticoagulant effect at 100 microg/mL (479.0+/-59.5 sec). This anticoagulant effect was comparable to that observed in interventional cardiology and open-heart surgery. At the lower level, PI-88 produced concentration-dependent effects on ACT. Using thromboelastographic techniques (TEG), the effect of PI-88 was measured in terms of various parameters. PI-88 produced potent anticoagulant effects in the TEG studies. At the concentration of 25 microg/mL, it produced a complete anticoagulant effect in whole blood. Whole blood samples supplemented with PI-88 showed a concentration-dependent decrease in the generation of various markers of clotting activation. These results clearly suggest that PI-88 exerts an anticoagulant effect in whole blood. Because of the low-molecular-weight nature and a novel mechanism of action, this new drug may be considered for further development, particularly in cancer patients.

Ecarin clotting time is sensitive to heparinoids: comparison of two different techniques.

Ecarin clotting time (ECT) is currently developed for the specific monitoring of antithrombin drugs, such as hirudin, argatroban, and hirulog. Aqueous reagent and dry chemistry technology have become available for ECT monitoring of antithrombin agents. Currently, many heparinoids and heparinomimetic drugs are being developed. These agents activate heparin cofactor II (HCII) and primarily mediate their effects by inhibiting thrombin. Although the test is specific for antithrombin agents, heparin cofactor II-mediated thrombin inhibitors are capable of prolonging the ECT. In order to study the relative effects of some of these agents, ECT was measured in human plasma supplemented with Pl-88 (a sulfated pentomanose; Progen Industries Limited, Sydney, Australia), aprosulate, pentosan polysulfate, dermatan sulfate, unfractionated heparin (UFH), and recombinant hirudin (r-hirudin). All agents were supplemented to the citrated-pooled plasma prepared from 10 healthy volunteers at a graded dosage of 0 to 100 microg/ml. These techniques gave comparable results for all of the agents used (PI-88, r2 = 0.99; r-hirudin, r2 = 0.98; UFH, r2 = 0.98; dermatan sulfate, r2 = 0.95; aprosulate, r2 = 0.95; pentosan polysulfate, r2 = 0.94). The relative anticoagulant effects of various agents used on ECT varied widely, exhibiting their potency in the following order: r-hirudin = pentosan polysulfate > dermatan sulfate > PI-88 > aprosulate > UFH. The sensitivity of ECT was adjusted by varying the concentration of the ecarin reagent. The results suggest that HCII-mediated inhibition of thrombin can be detected by using ECT reagents.

Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I.

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.

Synthetic heparin pentasaccharide depolymerization by heparinase I: molecular and biological implications.

A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 IU/mg) and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase I. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540, graded concentrations (1.25-100 microg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was employed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37 degrees C) at 1 mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide. SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.