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Background: The pediatric direct oral anticoagulation (DOAC) trials provide an opportunity to evaluate and characterize challenges in their design and execution to inform future antithrombotic trials., Objectives: To perform a systematic review of pediatric DOAC trials for the treatment of venous thromboembolism to critically appraise their methodology and understand the feasibility and challenges., Methods: We performed a systematic search of MEDLINE, EMBASE, the Cochrane Library, and ClinicalTrials.gov (January 2002 to December 2022). Studies reporting the results of interventional trials of a DOAC for the treatment of acute venous thromboembolism in children and their respective design papers were included. Trial registration information was reviewed in ClinicalTrials.gov. Discrepancies in study design, targeted populations, sample size, and analyses between planned and actual trial conduct were examined qualitatively., Results: Five published studies and unpublished data for 2 additional trials were included. All trials had modifications to their design or methodology and discrepancies between the trial's registration and the final published study, suggesting feasibility challenges. Modifications to the eligibility criteria, changes in sample size, challenges with the recruitment of younger patients, and an enrolled population not matching the clinical target population were identified for all trials. Discrepancies in outcome reporting, particularly for secondary endpoints, were also common., Conclusion: DOAC trials experienced feasibility challenges that led to design or methodology modifications. Future pediatric antithrombotic trials will need to be adaptive in their design, prioritize enrollment of younger children and input from clinicians providing care to target populations, ensure that enrolled populations match the clinical population, and select clinically meaningful endpoints., Competing Interests: Declarations of competing interest M.B. receives salary support from an National Heart, Lung, and Blood Institute K23 award. M.A. is a member of the Pediatric Antithrombotic Trials Leadership and Steering (Pedi-ATLAS) Group. L.R.B. is part of the pediatric advisory group for both dabigatran etexilate for the treatment of acute venous thromboembolism in children and the long-term safety of dabigatran etexilate for the secondary prevention of venous thromboembolism in children study (stipends sent to the institution up to 2019), principal investigator site investigator for rivaroxaban compared with standard anticoagulants for the treatment of acute venous thromboembolism in children and apixaban for the acute treatment of venous thromboembolism in children (per patient reimbursements transferred to the institution), and currently, unpaid coordinator in the apixaban for the acute treatment of venous thromboembolism in children venous thromboembolism study. P.M. was an unpaid member of the rivaroxaban compared with standard anticoagulants for the treatment of acute venous thromboembolism in children (per patient reimbursements transferred to the institution) and apixaban for prevention of thromboembolism in pediatric heart disease (per patient reimbursements transferred to the institution) steering committees; he is an unpaid member of the advisory boards for Takeda, Jansen Pharmaceuticals, and Bayer; and a member of the Pedi-ATLAS Group. L.R. is a member of the Pedi-ATLAS Group. C.H.v.O. receives or has recently received consultancy fees from Bayer and Octapharma and is a member of the Pedi-ATLAS Group. N.A.G. receives or has recently received consultancy fees from Anthos Therapeutics, Bayer, and the University of Colorado-affiliated Academic Research Organization Colorado Prevention Center Clinical Research for roles in clinical trial planning or oversight committees in pharmaceutical industry-sponsored pediatric multicenter clinical trials of antithrombotic agents; he receives consultancy fees from Novartis, salary support from an National Heart, Lung, and Blood Institute K24, he is a member of the Pedi-ATLAS Group, and his employer receives salary support on his behalf from Boehringer-Ingelheim for data coordinating center leadership for a pediatric antithrombotic multicenter prospective observational study. C.M. has received consultancy fees from Anthos, AstraZeneca, Bayer, Chiesi, Janssen, and Norgine and is a member of the Pedi-ATLAS Group. H.W. received salary support from the National Bleeding Disorders Foundation-Takeda Clinical Fellowship. T.B., R.V.B., T.D., S.R.-v., and C.H.v.O. declare no conflict of interest related to this publication., (Copyright © 2025 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Clinical and analytical information on laboratory data of neonates in scientific publications is sparse and incomplete. Furthermore, interpreting neonatal laboratory data can be complex due to their time-dependent and developmental physiology, and paucity of well-established age-appropriate reference ranges for neonates. This study aims to develop publication recommendations to report laboratory data of neonates to enhance the quality of these data in research and clinical care., Methods: A modified Delphi approach was used to develop recommendations in cooperation with the International Neonatal Consortium. A Core Group, including different stakeholders, was responsible for developing the recommendations, in collaboration with a Reflection Group, responsible for providing additional input., Results: The recommendations were classified into three categories: 'Clinical Characteristics', 'Bio-analytical Information' and 'Data-analytical Information'. These were each divided into 'Core Data' (always to be reported) and 'Supplemental Considerations' (to be reported when considered relevant to the study)., Conclusion: Our recommendations provide guidance on standardization of neonatal laboratory data in publications. This will enhance the comparison, replication, and application of study results in research initiatives and clinical practice. Furthermore, these recommendations also serve as foundational work to develop reference ranges for neonatal laboratory values by standardizing the quality of information needed for such efforts., Impact: Standardized reporting of neonatal laboratory data in scientific publications will enhance the comparison, replication, and application of study results in research initiatives and clinical practice, as well as improve reporting to regulatory agencies. To integrate multistakeholder perspectives, a modified Delphi approach was used to develop publication recommendations which strengthens the applicability of the recommendations. Implementation of standardization will likely improve the overall quality of neonatal clinical research and neonatal healthcare. In addition, these recommendations are foundational to develop reference ranges for neonatal laboratory values by standardizing the quality of information needed for such efforts., (© 2024. The Author(s).)

Multi-omics approaches are being used increasingly to study physiological and pathophysiologic processes. Proteomics specifically focuses on the study of proteins as functional elements and key contributors to, and markers of the phenotype, as well as targets for diagnostic and therapeutic approaches. Depending on the condition, the plasma proteome can mirror the platelet proteome, and hence play an important role in elucidating both physiologic and pathologic processes. In fact, both plasma and platelet protein signatures have been shown to be important in the setting of thrombosis-prone disease states such as atherosclerosis and cancer. Plasma and platelet proteomes are increasingly being studied as a part of a single entity, as is the case with patient-centric sample collection approaches such as capillary blood. Future studies should cut across the plasma and platelet proteome silos, taking advantage of the vast knowledge available when they are considered as part of the same studies, rather than studied as distinct entities.

Importance: Genomic testing in infancy guides medical decisions and can improve health outcomes. However, it is unclear whether genomic sequencing or a targeted neonatal gene-sequencing test provides comparable molecular diagnostic yields and times to return of results., Objective: To compare outcomes of genomic sequencing with those of a targeted neonatal gene-sequencing test., Design, Setting, and Participants: The Genomic Medicine for Ill Neonates and Infants (GEMINI) study was a prospective, comparative, multicenter study of 400 hospitalized infants younger than 1 year of age (proband) and their parents, when available, suspected of having a genetic disorder. The study was conducted at 6 US hospitals from June 2019 to November 2021., Exposure: Enrolled participants underwent simultaneous testing with genomic sequencing and a targeted neonatal gene-sequencing test. Each laboratory performed an independent interpretation of variants guided by knowledge of the patient's phenotype and returned results to the clinical care team. Change in clinical management, therapies offered, and redirection of care was provided to families based on genetic findings from either platform., Main Outcomes and Measures: Primary end points were molecular diagnostic yield (participants with ≥1 pathogenic variant or variant of unknown significance), time to return of results, and clinical utility (changes in patient care)., Results: A molecular diagnostic variant was identified in 51% of participants (n = 204; 297 variants identified with 134 being novel). Molecular diagnostic yield of genomic sequencing was 49% (95% CI, 44%-54%) vs 27% (95% CI, 23%-32%) with the targeted gene-sequencing test. Genomic sequencing did not report 19 variants found by the targeted neonatal gene-sequencing test; the targeted gene-sequencing test did not report 164 variants identified by genomic sequencing as diagnostic. Variants unidentified by the targeted genomic-sequencing test included structural variants longer than 1 kilobase (25.1%) and genes excluded from the test (24.6%) (McNemar odds ratio, 8.6 [95% CI, 5.4-14.7]). Variant interpretation by laboratories differed by 43%. Median time to return of results was 6.1 days for genomic sequencing and 4.2 days for the targeted genomic-sequencing test; for urgent cases (n = 107) the time was 3.3 days for genomic sequencing and 4.0 days for the targeted gene-sequencing test. Changes in clinical care affected 19% of participants, and 76% of clinicians viewed genomic testing as useful or very useful in clinical decision-making, irrespective of a diagnosis., Conclusions and Relevance: The molecular diagnostic yield for genomic sequencing was higher than a targeted neonatal gene-sequencing test, but the time to return of routine results was slower. Interlaboratory variant interpretation contributes to differences in molecular diagnostic yield and may have important consequences for clinical management.

Importance: A targeted genomic sequencing platform focused on diseases presenting in the first year of life may minimize financial and ethical challenges associated with rapid whole-genomic sequencing., Objective: To report interim variants and associated interpretations of an ongoing study comparing rapid whole-genomic sequencing with a novel targeted genomic platform composed of 1722 actionable genes targeting disorders presenting in infancy., Design, Setting, and Participants: The Genomic Medicine in Ill Neonates and Infants (GEMINI) study is a prospective, multicenter clinical trial with projected enrollment of 400 patients. The study is being conducted at 6 US hospitals. Hospitalized infants younger than 1 year of age suspected of having a genetic disorder are eligible. Results of the first 113 patients enrolled are reported here. Patient recruitment began in July 2019, and the interim analysis of enrolled patients occurred from March to June 2020., Interventions: Patient (proband) and parents (trios, when available) were tested simultaneously on both genomic platforms. Each laboratory performed its own phenotypically driven interpretation and was blinded to other results., Main Outcomes and Measures: Variants were classified according to the American College of Medical Genetics and Genomics standards of pathogenic (P), likely pathogenic (LP), or variants of unknown significance (VUS). Chromosomal and structural variations were reported by rapid whole-genomic sequencing., Results: Gestational age of 113 patients ranged from 23 to 40 weeks and postmenstrual age from 27 to 83 weeks. Sixty-seven patients (59%) were male. Diagnostic and/or VUS were returned for 51 patients (45%), while 62 (55%) had negative results. Results were concordant between platforms in 83 patients (73%). Thirty-seven patients (33%) were found to have a P/LP variant by 2 or both platforms and 14 (12%) had a VUS possibly related to phenotype. The median day of life at diagnosis was 22 days (range, 3-313 days). Significant alterations in clinical care occurred in 29 infants (78%) with a P/LP variant. Incidental findings were reported in 7 trios. Of 51 positive cases, 34 (67%) differed in the reported result because of technical limitations of the targeted platform, interpretation of the variant, filtering discrepancies, or multiple causes., Conclusions and Relevance: As comprehensive genetic testing becomes more routine, these data highlight the critically important variant detection capabilities of existing genomic sequencing technologies and the significant limitations that must be better understood.

Essentials An optimal therapeutic strategy has yet to be established to prevent early shunt thrombosis. A phase 1 study of cangrelor was performed in neonates after palliation of congenital heart disease. PD endpoint of >90% platelet inhibition in 60% of patients was achieved at 0.5 µg/kg/min dosing. No serious adverse events related to drug administration were observed, including bleeding. ABSTRACT: Background Systemic-to-pulmonary artery shunt thrombosis is a significant cause of early postoperative mortality in neonates after palliation of congenital heart disease. In the context of thromboprophylaxis, an optimal therapeutic strategy has yet to be established before aspirin administration. Cangrelor, a fast-acting, reversible P2Y 12 inhibitor, may fill this unmet need. Objectives To evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and safety of cangrelor in neonates undergoing stage 1 palliation. Methods This prospective, open-label, single-arm study evaluated two cangrelor dosing cohorts following placement of a systemic-to-pulmonary artery shunt, right ventricle-to-pulmonary artery shunt, or ductal stent. Drug concentrations and platelet reactivity, assessed by light transmission aggregometry and in microfluidic assays (MF), were measured. Results Twenty-two patients were consented and 15 received a 1-hour infusion of cangrelor at either 0.5 µg/kg/min (cohort 1) or 0.25 µg/kg/min (cohort 2). Whereas the primary PD endpoint was achieved at the higher dose (ie, reduction in maximal platelet aggregation by ≥90% in 60% of participants), only 29% of those in cohort 2 attained this goal. Comparable and statistically significant results were obtained in MF assays (P < .0001 vs. baseline). Drug levels during infusion were 3-fold higher in cohort 1 vs. cohort 2 (P < .001). Most participants (70%) had undetectable drug levels by 10 minutes postinfusion with full recovery in platelet function at 1 hour. No drug-related bleeding events occurred. Conclusions Favorable PK/PD properties of cangrelor 0.5 µg/kg/min dosing and safety profile warrant further evaluation in neonates following palliative cardiac procedures., (© 2020 International Society on Thrombosis and Haemostasis.)

BackgroundUp to 90% of all drugs used in neonatal intensive care units (NICUs) have not been clinically tested for safety and efficacy. To promote drug development for neonates, the pharmaceutical industry is moving toward rigorous testing, necessitating the need to development, and validating biomarkers in neonates to predict their response. The objective of this review is to evaluate the quality of the response biomarker reporting in neonatal clinical trials.MethodsA validated literature search strategy was applied. Prospective neonatal intervention studies reporting response biomarkers published in 2014 were included. The data were extracted independently and in duplicate using a data-extraction form.ResultsFollowing the full-text review, 167 published prospective neonatal trials were included; 35% (59/167) reported the use of response biomarkers. In these 59 trials, we identified 275 biomarkers used to measure the response (pharmacodynamics and safety) reported as primary or secondary outcomes. Heart rate and oxygen saturation were the most commonly reported. Measurement and instrumentation data were often not provided.ConclusionWe identified a huge variability in the selection, measurement, and reporting of neonatal response biomarkers in prospective intervention studies. Reporting initiatives are needed to reduce research waste and improve the reproducibility of biomarker use in neonatal intervention studies.

We report a case of neonatal generalized erythema and epidermolysis resulting from a novel mutation in the junctional plakoglobin gene causing truncation of the plakoglobin protein. Expedited genetic testing enabled diagnosis while the patient was in the neonatal intensive care unit, providing valuable information for the clinicians and family., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: In vivo testing of novel antiplatelet agents requires informative biomarkers. By genetically modifying mouse von Willebrand factor (VWF(R1326H)), we have developed a small animal model that supports human but not mouse platelet-mediated thrombosis. Here, we evaluate the use of this biological platform as a pharmacodynamic biomarker for antithrombotic therapies., Methods and Results: The antithrombotic effects of several αIIbβ3 inhibitors were determined in VWF(R1326H) mutant mice infused with human platelets. Administration of abciximab, eptifibatide, or tirofiban at doses recommended for percutaneous coronary intervention (per 1 kg of body weight) significantly reduced human platelet-mediated thrombus formation in laser-injured arterioles by > 75% (P < 0.001). In contrast, clot size in wild-type control animals remained essentially unchanged (P > 0.05), results consistent with observed species differences in IC₅₀ values obtained by aggregometry. To further demonstrate that our biological platform is unique among standard mouse models, we evaluated the thrombogenic potential of platelets from healthy volunteers before and after clopidogrel therapy. Consistent with the antithrombotic effect of this agent, platelets postdrug administration formed smaller thrombi than cells before therapy and were less responsive to ADP-induced aggregation (P < 0.001)., Conclusions: The ability of αIIbβ3 and P2Y₁₂ inhibitors to limit human platelet clot formation at doses recommended by the American College of Cardiology/American Heart Association suggests that VWF(R1326H) mutant mice can serve as both a pharmacodynamic and a functional response biomarker, attributes essential for not only expediting drug development but also designing clinical studies.

The tumor suppressor in lung cancer-1 (TSLC1) gene is frequently silenced in human lung carcinomas, and its expression suppresses tumorigenesis in nude mice. TSLC1 encodes a cell-surface protein called Necl-2 that belongs to the Nectin and Nectin-like (Necl) family of molecules. Necl-2 mediates epithelial cell junctions by homotypic contacts and/or heterotypic interactions with other Nectins and Necls. Thus, it inhibits tumorigenesis by ensuring that epithelial cells grow in organized layers. Here, we demonstrate that natural killer (NK) cells and CD8+ T cells recognize Necl-2 through a receptor known as class I-restricted T-cell-associated molecule (CRTAM), which is expressed only on activated cells. CRTAM-Necl-2 interactions promote cytotoxicity of NK cells and interferon gamma (IFN-gamma) secretion of CD8+ T cells in vitro as well as NK cell-mediated rejection of tumors expressing Necl-2 in vivo. These results provide evidence for an additional mechanism of tumor suppression mediated by TSLC1 that involves cytotoxic lymphocytes. Furthermore, they reveal Necl-2 as one of the molecular targets that allows the immunosurveillance network to distinguish tumor cells from normal cells.

The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here.

Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.

Sand fly saliva contains maxadilan, a peptide that causes vasodilation and modifies the secretion of pro-inflammatory cytokines by macrophages. We show that 1 to 10 microg maxadilan protected BALB/c mice against a lethal dose of LPS. Maxadilan reduced serum levels of TNF-alpha by approximately tenfold, while it caused a threefold increase in IL-6 and IL-10. The protective effect of maxadilan is partially dependent on its ability to induce IL-10 production since maxadilan did not prevent death from endotoxic shock in IL-10(-/-) mice. Finally, maxadilan is a selective agonist of the pituitary adenylate cyclase-activating peptide (PACAP) type I receptor, and we found that the natural ligand of this receptor (PACAP 38) also protected mice against lethal endotoxemia. These results indicate that activation of the PACAP type I receptor may contribute to the control of systemic inflammation by a mechanism that is partially dependent on IL-10.

Peripheral lymph nodes (PLN) are critical for immunologic memory formation in response to antigens that penetrate the skin. Blood-borne lymphocytes first encounter such antigens after they home to PLN through a multi-step adhesion process that is normally initiated by L-selectin (CD62L) in high endothelial venules (HEV). Since naive T cells can not enter PLN normally in L-selectin-deficient mice, a delayed type hypersensitivity response to cutaneously applied antigen cannot be mounted. In this study, we report that the administration of activated platelets into the systemic circulation of L-selectin knockout mice restores lymphocyte trafficking to PLN, and reconstitutes T cell-mediated immunity in response to a cutaneous antigen. These effects required platelet-expressed P-selectin that allows activated platelets to transiently form a bridge between lymphocytes and HEV, thereby enabling lymphocytes to undergo subsequent beta2 integrin-dependent firm adhesion. These profound effects of platelet-mediated cell-cell interactions on lymphocyte trafficking and formation of immunologic memory may impact on a variety of autoimmune and inflammatory conditions.

Two subpopulations of human T lymphocytes expressing different antigen receptors, alpha/beta and gamma/delta, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of alpha/beta and gamma/delta T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta stimulated similar, dose-dependent chemotaxis of purified gamma/delta T cells, whereas MCP-1, RANTES, and MIP-1alpha produced greater chemotaxis of purified alpha/beta T cells than MIP-1beta. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-gamma inducible protein-10 (IP-10) did not promote chemotaxis of either alpha/beta or gamma/delta T cells. Three gamma/delta T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mononuclear cells confirmed the results with purified gamma/delta T cells. Our data demonstrate that human peripheral blood alpha/beta and gamma/delta T cells can transmigrate to MCP-1, RANTES, MIP-1alpha, and MIP-1beta, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.

Circulating lymphocytes gain access to lymph nodes owing to their ability to initiate rolling along specialized high endothelial venules (HEVs). One mechanism of rolling involves L-selectin binding to peripheral node addressin (PNAd) on HEVs. Activated platelets are shown to bind to circulating lymphocytes and to mediate rolling in HEVs, in vivo, through another molecule, P-selectin, which also interacts with PNAd. In vitro, activated platelets enhanced tethering of lymphocytes to PNAd and sustained lymphocyte rolling, even in the absence of functional L-selectin. Thus, a platelet pathway operating through P-selectin provides a second mechanism for lymphocyte delivery to HEVs.

Platelets bound to thrombogenic surfaces have been shown to support activation-dependent firm adhesion of neutrophils in flow following selectin-mediated tethering and rolling. The specific receptor(s) responsible for mediating adhesion-strengthening interactions between neutrophils and platelets has not previously been identified. Furthermore, the ability of adherent platelets to support the migration of bound neutrophils has not been tested. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte binding in vascular shear flow and emigration at thrombogenic sites. Our results demonstrate that the beta 2-integrin Mac-1 (CD11b/CD18) is required for both firm attachment to and transmigration of neutrophils across surface-adherent platelets. In flow assays, neutrophils from patients with leukocyte adhesion deficiency-1 (LAD-I), which lack beta 2-integrin receptors, formed P-selectin-mediated rolling interactions, but were unable to develop firm adhesion to activated platelets, in contrast to healthy neutrophils, which developed firm adhesion within 5 to 30 seconds after initiation of rolling. Furthermore, the adhesion-strengthening interaction observed for healthy neutrophils could be specifically inhibited by monoclonal antibodies (mAbs) to Mac-1, but not to lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) or intercellular adhesion molecule-2 (ICAM-2; CD102). Further evidence for a beta 2-integrin-dependent neutrophil/platelet interaction is demonstrated by the complete inhibition of interleukin (IL)-8-induced neutrophil transmigration across platelets bound to fibronectin-coated polycarbonate filters by mAbs to Mac-1. Thus, Mac-1 is required for firm adhesion of neutrophils to activated, adherent platelets and may play an important role in promoting neutrophil accumulation on and migration across platelets deposited at sites of vascular injury.

We have prepared a series of novel double-chain amphiphiles with protonatable head groups, including acylated derivatives of various 2-substituted palmitic acids, amino acid conjugates of these species, and 1,2-dioleoyl-3-succinylglycerol. These species can be combined with phosphatidylethanolamine (PE) to prepare reverse-phase evaporation vesicles that are stable and trap hydrophilic solutes at pH 7. At weakly acidic pH values (as high as 6.5, depending on the titratable amphiphilic component), these pH-sensitive vesicles exhibit fusion, with a limited extent of contents mixing and extensive mixing of lipids, accompanied by leakage of aqueous contents. Protons and divalent cations show strong synergistic effects in promoting mixing of both lipids and aqueous contents between pH-sensitive vesicles prepared with any of a variety of double-chain titratable amphiphiles. Calorimetric results indicate that the relative stabilities of different types of pH-sensitive liposomes at low pH cannot be simply correlated with the propensity of the lipids to form a hexagonal II phase under these conditions. Fluorescence measurements demonstrate that single-chain fatty acids, but not double-chain titratable amphiphiles such as N-acyl-2-aminopalmitic acids, are rapidly removed from pH-sensitive vesicles in the presence of other lipid vesicles, serum albumin, or serum. Additionally, pH-sensitive liposomes containing double-chain titratable amphiphiles retain their aqueous contents better than do those containing single-chain amphiphiles in the presence of lipid membranes or albumin. Surprisingly, however, pH-sensitive vesicles of either type show retention of contents in the presence of serum that is comparable to that observed with vesicles composed purely of phospholipids. A model is proposed to explain these latter findings.

The structure and physical properties of aqueous dispersions of 1,2-diacyl-sn-glycero-3-phosphoethanolamines (PE's) and their N-methylated analogues have been studied by scanning calorimetry, 31P nuclear magnetic resonance, and freeze-fracture electron microscopy. While successive N-methylations of a diacylphosphatidylethanolamine cause only modest decreases in its gel to liquid-crystalline phase transition temperature, the introduction of even a single N-methyl group sharply increases the temperature at which the lipid forms a hexagonal II phase. However, 31P nuclear magnetic resonance and electron microscopy show that unlike pure PE species, N-methylated PE's can form a variety of irregular nonlamellar structures at temperatures well below that at which a well-defined hexagonal II phase is formed. The rate of calcium-induced leakage of encapsulated carboxyfluorescein from large unilamellar vesicles composed of dioleoyl- or dielaidoylphosphatidylserine and the corresponding PE is strongly reduced when PE is replaced by N-methylated derivatives. The rate of calcium-induced intermixing of lipids of PE/phosphatidylserine (PS) vesicles steadily decreases as the PE component is successively replaced by its mono-, di-, and tri-N-methylated (phosphatidylcholine) derivatives. By correlating calorimetrically obtained phase diagrams with measurements of vesicle lipid intermixing, we conclude that dielaidoyl-N-methylphosphatidylethanolamine, like PE, can support direct interactions between the surfaces of PS/N-methyl-PE vesicles without lateral separation of a PS(Ca2+)-rich phase, while dielaidoyl-N,N-dimethyl-PE (and phosphatidylcholine) cannot.(ABSTRACT TRUNCATED AT 250 WORDS)