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The high affinity interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin is mediated by a multimotif glycosulfopeptide (GSP) recognition domain consisting of clustered tyrosine sulfates and a Core 2 O -glycan terminated with sialyl Lewis X (C2- O -sLe X ). These distinct GSP motifs are much more common than previously appreciated within a wide variety of functionally important domains involved in protein-protein interactions. However, despite the potential of GSPs to serve as tools for fundamental studies and prospects for drug discovery, their utility has been limited by the absence of chemical schemes for synthesis on scale. Herein, we report the total synthesis of GSnP-6, an analogue of the N-terminal domain of PSGL-1, and potent inhibitor of P-selectin. An efficient, scalable, hydrogenolysis-free synthesis of C2- O -sLe X -Thr-COOH was identified by both convergent and orthogonal one-pot assembly, which afforded this crucial building block, ready for direct use in solid phase peptide synthesis (SPPS). C2- O -sLe X -Thr-COOH was synthesized in 10 steps with an overall yield of 23% from the 4- O ,5- N oxazolidinone thiosialoside donor. This synthesis represents an 80-fold improvement in reaction yield as compared to prior reports, achieving the first gram scale synthesis of SPPS ready C2- O -sLe X -Thr-COOH and enabling the scalable synthesis of GSnP-6 for preclinical evaluation. Significantly, we established that GSnP-6 displays dose-dependent inhibition of venous thrombosis in vivo and inhibits vaso-occlusive events in a human sickle cell disease equivalent microvasculature-on-a-chip system. The insights gained in formulating this design strategy can be broadly applied to the synthesis of a wide variety of biologically important oligosaccharides and O -glycan bearing glycopeptides.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs . hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zheng, Ricci, Zhang, Alawieh, Yang, Nadig, He, Engel, Jin, Atkinson and Tomlinson.)

Cardiovascular disease (CAD) is the main cause of morbidity and deaths in the western world. The development of atherosclerosis underlying CAD development begins early in human life. There are numerous genetic and environmental risk factors accelerating its progression which then leads to the occurrence of acute events. Despite considerable progress in determining risk factors, there is still a lot of work ahead since identified determinants are responsible only for a part of overall CAD risk. Current therapies are insufficient to successfully reduce the risk of atherosclerosis development. Therefore, there is a need for effective preventive measures of clinical manifestations of atherosclerosis since the currently available drugs cannot prevent the occurrence of even 70% of clinical events. The shift of the target from lipid metabolism has opened the door to many new therapeutic targets. Currently, the majority of known targets for anti-atherosclerotic drugs focus also on inflammation (a common mediator of many risk factors), mechanisms of innate and adaptive immunity in atherosclerosis, molecule scavengers, etc. The therapeutic potential of cyclodextrins, protein kinase inhibitors, colchicine, inhibitors of p38 mitogen-activated protein kinase (MAPK), lipid dicarbonyl scavengers, a monoclonal antibody targeting interleukin-1β, and P-selectin inhibitors is still not fully confirmed and requires confirmation in large clinical trials. The preliminary results look promising.

Events mediated by the P-selectin/PSGL-1 pathway play a critical role in the initiation and propagation of venous thrombosis by facilitating the accumulation of leukocytes and platelets within the growing thrombus. Activated platelets and endothelium express P-selectin, which binds P-selectin glycoprotein ligand-1 (PSGL-1) that is expressed on the surface of all leukocytes. We developed a pegylated glycomimetic of the N terminus of PSGL-1, PEG40-GSnP-6 (P-G6), which proved to be a highly potent P-selectin inhibitor with a favorable pharmacokinetic profile for clinical translation. P-G6 inhibits human and mouse platelet-monocyte and platelet-neutrophil aggregation in vitro and blocks microcirculatory platelet-leukocyte interactions in vivo. Administration of P-G6 reduces thrombus formation in a nonocclusive model of deep vein thrombosis with a commensurate reduction in leukocyte accumulation, but without disruption of hemostasis. P-G6 potently inhibits the P-selectin/PSGL-1 pathway and represents a promising drug candidate for the prevention of venous thrombosis without increased bleeding risk., (© 2021 by The American Society of Hematology.)

Introduction: Previously, in a murine model of blunt thoracic trauma, we provided evidence of primary pulmonary thrombosis associated with increased expression of the cell adhesion molecule, P-selectin. In this study, mice are treated with P-selectin blocking antibody after injury to investigate the clinical viability of this antibody for the prevention of pulmonary thrombosis. In addition, viscoelastic testing is performed to investigate if P-selectin inhibition has a detrimental impact on normal hemostasis., Methods: A murine model of thoracic trauma was used. Mice were divided into sham control and experimental injury groups. Thirty minutes after trauma, mice were treated with the following: P-selectin blocking antibody, isotype control antibody, low-dose heparin, high-dose heparin, or normal saline. At 90 minutes, whole blood was collected for characterization of coagulation by viscoelastic coagulation monitor (VCM Vet; Entegrion, Durham, NC). Mean clotting time, clot formation time, clot kinetics (α angle), and maximum clot firmness were compared between each treatment group., Results: Mice that received P-selectin antibody 30 minutes after blunt thoracic trauma had four- to fivefold less (p < 0.001) arterial fibrin accumulation than those that received the isotype control. In both sham and trauma groups, compared with vehicle (normal saline) alone, no statistical difference was noted in any coagulation parameters after injection with P-selectin antibody, isotype control, or low-dose heparin. In addition, blinded histopathological evaluation yielded no difference in hemorrhage scores between injured mice treated with P-selectin blocking antibody and those treated with isotype antibody control., Conclusion: This study supports the clinical use of P-selectin blocking antibody for the prevention of pulmonary thrombosis by confirming its efficacy when given after a blunt thoracic trauma. In addition, we demonstrated that the administration of P-selectin antibody does not adversely affect systemic coagulation as measured by viscoelastic testing, suggesting that P-selectin antibody can be safely given during the acute posttraumatic period., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: To examine the current molecular therapeutics in the medical treatment of recurrent ischemic priapism (RIP). To propose a stepwise clinical management paradigm for the treatment of RIP., Methods: We performed a literature search using the PubMed database for the terms 'recurrent ischemic priapism' and 'stuttering priapism' up until December 2020. We assessed pre-clinical and clinical studies regarding medical management of RIP and molecular pathophysiology. Case series and randomized trials were evaluated by study quality and patient outcomes to determine a potential clinical management scheme., Results: Recent research has fostered an improved understanding of the underlying molecular pathophysiology of RIP that has paved the way forward for developing new therapeutic agents. Medications targeting neurovascular, hormonal and haematological mechanisms associated with RIP show great promise towards remedying this condition. A host of therapeutic agents operating across different mechanistic directions may be implemented according to a clinical management scheme to potentially optimize RIP outcomes., Conclusion: RIP remains a medically neglected condition with current management focused on treating the acute condition rather than modulating the course of disease. Continued research into the molecular mechanisms of RIP and standardized clinical pathways can improve the quality of care for patients suffering from this condition., (© 2021 The Authors BJU International © 2021 BJU International.)

Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

Acute kidney injury (AKI) is a life-threatening disease without effective treatment. The utilization of curcumin (Cur) for the treatment of AKI is still facing challenges due to its poor water-solubility and low bioavailability. Herein, kidney-targeted octenyl succinic anhydride-grafted fucoidan loaded with Cur (OSA-Fucoidan/Cur) was fabricated for synergistic treatment of AKI. It was found that OSA-Fucoidan/Cur micelles had a sustained drug release behavior and excellent physicochemical stability. Cellular uptake studies demonstrated that the specific binding between fucoidan and P-selectin overexpressed on H 2 O 2 -stimulated HUVECs contributed to the higher internalization of OSA-Fucoidan/Cur micelles by the cells. In addition, OSA-Fucoidan micelles exhibited an ideal kidney-targeted characteristic in lipopolysaccharide (LPS)-induced AKI mice. In vivo studies showed that the combination of Cur and OSA-Fucoidan endowed the OSA-Fucoidan/Cur micelles with synergistically anti-inflammatory and antioxidant abilities, thereby largely enhancing the therapeutic efficacy of AKI. Therefore, OSA-Fucoidan/Cur micelles may represent a potential kidney-targeted nanomedicine for effective treatment of AKI., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fucoidan, a type of sulfated polysaccharide known for its anticoagulant, anti-tumor and anti-inflammatory effects, has been reported to have strong affinity towards P-selectin. P-selectin, which plays an important role in metastasis by enhancing the adhesion of cancer cells to endothelium and activated platelets in distant organs, is overexpressed on many cancer types. This study demonstrates the synthesis of a fucoidan-based drug delivery system for minimizing the side effects of doxorubicin (Dox) with the help of active targeting toward P-selectin. Fucoidan-doxorubicin nanoparticles (FU-Dox NPs), developed by direct conjugation of Dox to the fucoidan backbone, showed a well-controlled size distribution and sustained release. The active targeting capability of FU-Dox NPs toward P-selectin resulted in enhanced cellular uptake and cytotoxicity against the MDA-MB-231 cell line with high P-selectin expression compared to the MDA-MB-468 cell line with low P-selectin expression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Two studies evaluated the effects of renal and hepatic impairment on pharmacokinetics and safety of rivipansel (NCT02813798, NCT02871570). A single intravenous 840-mg rivipansel dose was administered to subjects with renal impairment or normal renal function in study 1005 and subjects with moderate hepatic impairment or normal hepatic function in study 1006. Plasma (both studies) and urine (study 1005) samples were collected for 96 hours postdose. All subjects in studies 1005 (n = 28) and 1006 (n = 16) completed all study procedures. Rivipansel exposure (AUC inf ) was 47%, 124%, and 437% higher and total clearance 30%, 57%, and 82% lower in the mild, moderate, and severe renal impairment groups, respectively, than in the normal renal function group. Overall rivipansel exposure was 20% lower and total clearance 31% higher in the moderate hepatic impairment group than in the normal hepatic function group. Ten treatment-emergent adverse events occurred in studies 1005 and 1006; no event was considered treatment related. As expected, clearance of rivipansel decreased with increasing renal impairment. The difference observed between rivipansel pharmacokinetics in subjects with moderate hepatic impairment and subjects with normal hepatic function was not considered clinically significant. Single doses of rivipansel were well tolerated in subjects with either renal or hepatic impairment., (© 2020, The American College of Clinical Pharmacology.)

Upregulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of vaso-occlusive crises (VOC), a major cause of morbidity in sickle cell disease (SCD). Crizanlizumab (ADAKVEO®), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin, and has been shown to decrease the frequency of VOCs in clinical trials. However, the lack of reliable in vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to evaluating and improving the therapeutic treatment in SCD. Here, we present a standardized microfluidic BioChip whole blood adhesion assay to assess leukocyte adhesion to P-selectin under physiologic flow conditions. Our results demonstrated heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to Crizanlizumab. Importantly, treatment with Crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes. Taken together, our results suggest that the microfluidic BioChip system is a promising in vitro assay with which to screen patients, monitor treatment response, and guide current and emerging anti-adhesive therapies in SCD., Competing Interests: Declaration of competing interest YM, UG, EK, JAL, and UAG are inventors of intellectual property (PCT/US2018/022888 filed on March 16, 2018 and provisional patent application 62/928,109 filed on November 1, 2019) on the microfluidic BioChip technology presented in this manuscript. Competing interests of Case Western Reserve University employees are overseen and managed by the Conflict of Interests Committee according to a Conflict of Interest Management Plan., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hydroxyurea, indicated for managing sickle cell anemia (SCA), and L-glutamine, indicated for treating sickle cell disease (SCD), were the only pharmacotherapeutic options in this patient population before the approval of crizanlizumab by the U.S. Food and Drug Administration in November 2019 to reduce vaso-occlusive crisis (VOC) frequency. This article reviews the evidence pertaining to crizanlizumab in SCD by searching records in Medline, Embase, and International Pharmaceutical Abstracts. Crizanlizumab, a P-selectin inhibitor, mitigates the microvascular vaso-occlusion in SCD. In the multicenter randomized double-blind SUSTAIN trial, a higher dose of crizanlizumab decreased the incidence of VOCs by 45% and prolonged the median time to the first and second VOC. A post hoc subgroup analysis demonstrated that the proportion of patients who had no VOC incidence during the study period was greater in the crizanlizumab group, and this benefit was consistent regardless of concomitant hydroxyurea use, prior categorized history of VOC frequency, or SCD genotype. Crizanlizumab had a safety profile comparable with placebo. Multiple ongoing clinical trials are trying to establish its roles in pediatric patients with SCD and its effects on alleviating other SCD-related complications. As the first parenteral option for SCD, providers need to formulate administration logistics to improve patients' access to crizanlizumab. Current available data suggest crizanlizumab is a promising agent to reduce VOC in patients with SCD., (© 2020 Pharmacotherapy Publications, Inc.)

In an immunocompetent host, excess infiltration of immune cells in the lung is a key factor in infection-induced severe acute lung injury. Kidney transplant patients are immunocompromised by the use of immunosuppressive drugs. Immune cell infiltration in the lung in a renal transplant recipient suffering from pulmonary infection is significantly less than that in an immunocompetent host; however, the extent of lung injury in renal transplant patients is more serious than that in immunocompetent hosts. Therefore, we explored the role of platelet activation in a Klebsiella pneumoniae-induced lung injury model with P-selectin gene knockout mice or wild-type mice. Our study suggested that the inhibition of platelets reduced severe acute lung injury and increased survival after acute lung infection in mice. In addition, P-selectin expression on the surface of platelets in mice increased after administration of immunosuppressive drugs, and the extent of lung injury induced by infection decreased in P-selectin gene knockout mice. In conclusion, p-selectin plays a key role in severe acute lung injury in immunocompromised mice by reducing platelet activation and inflammatory processes., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Yang Liu et al.)

Background: Platelets play important roles in cancer progression and metastasis, as well as in cancer-associated thrombosis (CAT). Tyrosine kinases are implicated in several intracellular signaling pathways involved in tumor biology, thus tyrosine kinase inhibitors (TKIs) represent an important class of anticancer drugs, based on the concept of targeted therapy., Purpose: The objective of this study is the design and synthesis of analogues of the TKIs imatinib and nilotinib in order to develop tyrosine kinase inhibitors, by investigating their molecular requirements, which would express antiplatelet properties., Methods: Based on a recently described by us improved approach in the preparation of imatinib and/or nilotinib analogues, we designed and synthesized in five-step reaction sequences, 8 analogues of imatinib ( I - IV ), nilotinib ( V, VI ) and imatinib/nilotinib ( VI I, VIII ). Their inhibitory effects on platelet aggregation and P-selectin membrane expression induced by arachidonic acid (AA), adenosine diphosphate (ADP) and thrombin receptor activating peptide-6 (TRAP-6), in vitro, were studied. Molecular docking studies and calculations were also performed., Results: The novel analogues V - VIII were well established with the aid of spectroscopic methods. Imatinib and nilotinib inhibited AA-induced platelet aggregation, exhibiting IC 50 values of 13.30 μΜ and 3.91 μΜ, respectively. Analogues I and II exhibited an improved inhibitory activity compared with imatinib. Among the nilotinib analogues, V exhibited a 9-fold higher activity than nilotinib. All compounds were less efficient in inhibiting platelet aggregation towards ADP and TRAP-6. Similar results were obtained for the membrane expression of P-selectin. Molecular docking studies showed that the improved antiplatelet activity of nilotinib analogue V is primarily attributed to the number and the strength of hydrogen bonds., Conclusion: Our results show that there is considerable potential to develop synthetic analogues of imatinib and nilotinib, as TKIs with antiplatelet properties and therefore being suitable to target cancer progression and metastasis, as well as CAT by inhibiting platelet activation., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Pantazi et al.)

Inflammatory processes contribute to intimal hyperplasia (IH) and long-term failure of vein grafts used in bypass surgery. Leukocyte recruitment on endothelial cells of vessels during inflammation is regulated by P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), which also mediates the interaction between platelets and endothelial cells in vein grafts transferred to arteries. However, how this pathway causes IH in vein grafts is unclear. In this study, we used a murine model of vein grafting to investigate P-selectin-mediated platelet adhesion, followed by IH. On the luminal surface of the vein graft, leukocyte recruitment occurred mainly in areas with adhered platelets rather than on endothelial cells without adherent platelets 1 hour after vein grafting. Blockage of either P-selectin or PSGL-1 reduced platelet adhesion and leukocyte recruitment on the luminal surface of vein grafts. Inhibition of the P-selectin pathway in vein grafts significantly reduced platelet-mediated leukocyte recruitment and IH of vein grafts 28 days after surgery. The study demonstrates that functional blockage of the P-selectin/PSGL-1 pathway in the early inflammatory phase after vein grafting reduced leukocyte invasion in the vein graft wall and later IH development. The findings imply an attractive early time window for prevention of vein graft failure by manipulating platelet adhesion., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Hepatocellular carcinoma (HCC) with metastatic disease is associated with a low survival in clinical practice. Many curative options including liver resection, transplantation, and thermal ablation are effective in local but limited for patients with distant metastasis. In this study, the efficacy, specificity, and safety of P-selectin targeted delivery and microwave (MW) responsive drug release is investigated for development of HCC therapy. By encapsulating doxorubicin (DOX) and MW sensitizer (1-butyl-3-methylimidazolium-l-lactate, BML) into fucoidan conjugated liposomal nanoparticles (TBP@DOX), specific accumulation and prominent release of DOX in orthotopic HCC and lung metastasis are achieved with adjuvant MW exposure. This results in orthotopic HCC growth inhibition that is not only 1.95-fold higher than found for nontargeted BP@DOX and 1.6-fold higher than nonstimuli responsive TP@DOX but is also equivalent to treatment with free DOX at a 10-fold higher dose. Furthermore, the optimum anticancer efficacy against distant lung metastasis and effective prevention of widespread dissemination with a prolonged survival is described. In addition, no adverse metabolic events are identified using the TBP@DOX nanodelivery system despite these events being commonly observed with traditional DOX chemotherapy. Therefore, administering TBP@DOX with MW exposure could potentially enhance the therapeutic efficacy of thermal-chemotherapy of HCC, especially those in the advanced stages.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD., (© 2018 Wiley Periodicals, Inc.)

The cell adhesion molecule P-selectin plays a key role in the pathogenesis of a vaso-occlusive crisis (VOC) in patients with sickle cell disease (SCD). In the double-blind, placebo-controlled phase 2 SUSTAIN study, crizanlizumab (humanized, anti-P-selectin monoclonal antibody) 5 mg/kg significantly lowered the rate of VOC in patients with SCD by 45% vs placebo. In SUSTAIN, patients with SCD were randomized to crizanlizumab 2.5 mg/kg, crizanlizumab 5 mg/kg, or placebo intravenously 14 times over 52 weeks. The primary endpoint was the annual rate of VOC with crizanlizumab vs placebo. This post hoc descriptive analysis evaluated the proportion of patients who did not experience a VOC during the study in the following subgroups: VOCs in the year prior to study entry (2-4/5-10), SCD genotype (HbSS/non-HbSS), and concomitant hydroxyurea use (yes/no). More patients were VOC event-free in the crizanlizumab 5 mg/kg arm than in the placebo arm, including those with more frequent prior VOCs (ie, 5-10; 28.0% vs 4.2%), the HbSS genotype (31.9% vs 17.0%) and/or using concomitant hydroxyurea (33.3% vs 17.5%). Further analyses of secondary endpoints demonstrated that crizanlizumab treatment significantly increased time-to-first VOC vs placebo in these subgroups. The rates of treatment-emergent adverse events were similar between treatment arms across all subgroups. This post hoc analysis of SUSTAIN shows that in patients with a high number of prior VOCs, on concomitant hydroxyurea and/or with the HbSS genotype, crizanlizumab treatment increases the likelihood of patients being VOC event-free and delays time-to-first VOC., (© 2018 Wiley Periodicals, Inc.)

Background: The impact of upregulation of platelet membrane glycoprotein (GP)IIb/IIIa and P-selectin on the onset of arterial thrombosis, venous thrombosis, and cancer encourages to hypothesize that dual inhibitor of GPIIb/IIIa and P-selectin receptors should simultaneously inhibit arterial thrombosis, block venous thrombosis, and slow tumor growth., Methods: For this reason, the structural characteristics and the CDOCKER interaction energies of 12 carbolines were analyzed. This led to the design of 1-(4-isopropyl-phenyl)- β -carboline-3-carboxylic acid (ICCA) as a promising inhibitor of GPIIb/IIIa and P-selectin receptors., Results: The synthetic route provided ICCA in 48% total yield and 99.6% high-performance liquid chromatography purity. In vivo 5 μmol/kg oral ICCA downregulated GPIIb/IIIa and P-selectin expression thereby inhibited arterial thrombosis, blocked venous thrombosis, and slowed down tumor growth, but did not damage the kidney and the liver., Conclusion: Therefore, ICCA could be a promising candidate capable of downregulating GPIIb/IIIa and P-selectin receptors, inhibiting arterial thrombosis, blocking venous thrombosis, and slowing down tumor growth., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Aptamers which bind to proteins involved in cell-cell interactions could have significant value to directly affect cancer cell adhesion or for directed cargo delivery. Here, I discuss two aptamers: aptamer SDA which binds to E- and P-selectin, and aptamer IDA which binds to α6β4 integrin. Both aptamers (SDA 91 nt and IDA 77 nt) bind their target proteins with dissociation constants in the 100-150 nM range and substantially inhibit special cellular adhesion, possibly a first and pivotal step in transendothelial migration during metastasis formation. The aptamers' half-lives in cell culture media are between two and six hours. IDA is internalized by integrin presenting cells within minutes thus possibly serving as vehicle for directed cargo delivery., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Injection of recombinant tissue plasminogen activator (rt-PA) is the standard drug treatment for thrombolysis. However, rt-PA shows risk of hemorrhages and limited efficiency even at high doses. Polysaccharide-poly(isobutylcyanoacrylate) nanoparticles functionalized with fucoidan and loaded with rt-PA were designed to accumulate on the thrombus. Fucoidan has a nanomolar affinity for the P-selectin expressed by activated platelets in the thrombus. Solid spherical fluorescent nanoparticles with a hydrodynamic diameter of 136 ± 4 nm were synthesized by redox radical emulsion polymerization. The clinical rt-PA formulation was successfully loaded by adsorption on aminated nanoparticles and able to be released in vitro. We validated the in vitro fibrinolytic activity and binding under flow to both recombinant P-selectin and activated platelet aggregates. The thrombolysis efficiency was demonstrated in a mouse model of venous thrombosis by monitoring the platelet density with intravital microscopy. This study supports the hypothesis that fucoidan-nanoparticles improve the rt-PA efficiency. This work establishes the proof-of-concept of fucoidan-based carriers for targeted thrombolysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To overcome the harmful side effects, low tolerance, and undesirable outcomes of the anticancer drugs, we used ethane-1,2-diamine to bridge antitumoral ( S )-3-acetyl-4-oxo-tetrahydroindolo[2,3-a]quinolizine-6-carboxylic acid (ATIQC) and tumor-targeting d-glucuronic acid, thereby providing (6 S )-3-acetyl-4-oxo- N -(2-(3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)ethyl)-4,6,7,12-tetrahydroindolo[2,3-a]quinolizine-6-carboxamide (ATIQCTPC). Atomic force microscopy images visualized, that in serum, ATIQCTPC formed particles of height <81 nm. These particles effectively avoided phagocytosis of macrophages and were stable in blood circulation. Distribution analysis indicated that ATIQCTPC accumulated and released ATIQC in the tumor tissue through a targeting manner. Thus, the antitumor and the anti-thrombotic activities of ATIQCTPC were 100-fold higher than those of ATIQC, and ATIQCTPC was able to prevent cancer patients from suffering from thrombosis. Based on the observation that ATIQCTPC decreased serum tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in S180 mice, we hypothesized that this is the mechanism that ATIQCTPC utilized to slow tumor growth. Additionally, we observed that ATIQCTPC inhibited thrombosis by decreasing serum P-selectin of thrombotic rats. The intermolecular association and the hexamerization manner of ATIQCTPC were experimentally evidenced and correlated with the formation of the nanoparticles., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: The up-regulation of P-selectin in endothelial cells and platelets contributes to the cell-cell interactions that are involved in the pathogenesis of vaso-occlusion and sickle cell-related pain crises. The safety and efficacy of crizanlizumab, an antibody against the adhesion molecule P-selectin, were evaluated in patients with sickle cell disease., Methods: In this double-blind, randomized, placebo-controlled, phase 2 trial, we assigned patients to receive low-dose crizanlizumab (2.5 mg per kilogram of body weight), high-dose crizanlizumab (5.0 mg per kilogram), or placebo, administered intravenously 14 times over a period of 52 weeks. Patients who were receiving concomitant hydroxyurea as well as those not receiving hydroxyurea were included in the study. The primary end point was the annual rate of sickle cell-related pain crises with high-dose crizanlizumab versus placebo. The annual rate of days hospitalized, the times to first and second crises, annual rates of uncomplicated crises (defined as crises other than the acute chest syndrome, hepatic sequestration, splenic sequestration, or priapism) and the acute chest syndrome, and patient-reported outcomes were also assessed., Results: A total of 198 patients underwent randomization at 60 sites. The median rate of crises per year was 1.63 with high-dose crizanlizumab versus 2.98 with placebo (indicating a 45.3% lower rate with high-dose crizanlizumab, P=0.01). The median time to the first crisis was significantly longer with high-dose crizanlizumab than with placebo (4.07 vs. 1.38 months, P=0.001), as was the median time to the second crisis (10.32 vs. 5.09 months, P=0.02). The median rate of uncomplicated crises per year was 1.08 with high-dose crizanlizumab, as compared with 2.91 with placebo (indicating a 62.9% lower rate with high-dose crizanlizumab, P=0.02). Adverse events that occurred in 10% or more of the patients in either active-treatment group and at a frequency that was at least twice as high as that in the placebo group were arthralgia, diarrhea, pruritus, vomiting, and chest pain., Conclusions: In patients with sickle cell disease, crizanlizumab therapy resulted in a significantly lower rate of sickle cell-related pain crises than placebo and was associated with a low incidence of adverse events. (Funded by Selexys Pharmaceuticals and others; SUSTAIN ClinicalTrials.gov number, NCT01895361 .).

Background: During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS., Methods: ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera., Results: The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating neutrophils. Both inhibiting the endothelial activation with an anti-toll like receptor (TLR) antibody and inhibiting the interaction of the endothelium with neutrophil using an anti-P-selectin antibody decreased the degree of neutrophil activation., Conclusions: Extracellular histones are pro-inflammatory mediators in LPS-induced ARDS in mice. In addition to direct action to neutrophils, extracellular histones promote neutrophil adhesion and subsequent activation by first activating the pulmonary endothelium via TLR signaling. Thus, endothelial activation is important for extracellular histone-induced inflammatory injury.

Background: The Effects of the P-Selectin Antagonist Inclacumab on Myocardial Damage After Percutaneous Coronary Intervention for Non-ST-Segment Elevation Myocardial Infarction (SELECT-ACS) trial suggested beneficial effects of inclacumab, a monoclonal antibody directed against P-selectin, on periprocedural myocardial damage. This study evaluated the effect of inclacumab on myocardial damage according to varying time intervals between study drug infusion and percutaneous coronary intervention (PCI)., Methods and Results: Patients (n=544) enrolled in the SELECT-ACS trial and randomized to receive 1 infusion of placebo or inclacumab (5 or 20 mg/kg, administered between 1 and 24 hours before PCI) were divided according to the time interval between study drug infusion and PCI. The primary end point was the change in troponin I from baseline at 16 and 24 hours after PCI. In patients receiving inclacumab 20 mg/kg with a short (less than median) time interval between infusion and PCI, placebo-adjusted geometric mean percent changes in troponin I, creatine kinase-myocardial band, and peak troponin I at 24 hours were -45.6% (P=0.005), -30.7% (P=0.01), and -37.3% (P=0.02), respectively. No significant changes were observed in patients with a long (greater than median) time interval between infusion and PCI. Placebo-adjusted geometric mean percent changes in troponin I and creatine kinase-myocardial band were -43.5% (P=0.02) and -26.0% (P=0.07), respectively, when inclacumab 20 mg/kg was administered between 1 and 3 hours before PCI, whereas the drug had no effect with longer intervals., Conclusions: Inclacumab 20 mg/kg significantly reduces myocardial damage after PCI in patients with non-ST-segment elevation myocardial infarction, and benefits are larger when the infusion is administered <3 hours before PCI., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01327183., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

By docking 126 derivatives of β-carboline-3-carboxylic acid, tetrahydro-β-carboline-3-carboxylic acid and indoloquinolizine into the active pocket of P-selectin (2-(3-(hydroxymethyl)-9H-pyrido[3,4-b]indol-1-yl)ethyl)-l-phenylalanine (HMCEF) was assigned a novel inhibitor. ELISA and flow cytometry experiments showed that HMCEF effectively down-regulated P-selectin expression and supported the rationality of the computer assistant screening, while UV spectrum experiments demonstrated that HMCEF directly bound to P-selectin. In vivo HMCEF dose dependently inhibited the rats and mice to form thrombus and had a minimal effective dose of 20nmol/kg, dose dependently inhibited inflammatory response of mice and had a minimal effective dose of 20nmol/kg. The decrease of serum TNFα and IL-8 of the treated mice was proposed to be the action mechanism of HMCEF inhibiting thrombosis and inflammation. All data imply that HMCEF is a novel lead of P-selectin inhibitor., (Copyright © 2016. Published by Elsevier Ltd.)

Unlabelled: Platelet P-selectin and activated glycoprotein IIb-IIIa (GPIIb-IIIa) are markers of platelet activation and mediates platelet aggregation. Prasugrel (Pras) 5 mg may be used in very elderly (VE) acute coronary syndrome (ACS) patients undergoing PCI, but its effect on platelet P-selectin and activated GPIIb-IIIa in those patients is not known. Stable ACS patients, VE (78 ± 5 years, n = 23) and non-elderly (NE) (55 ± 5 years, n = 22) were randomized to Pras (5 or 10 mg) or clopidogrel (Clop) 75 mg during three 12-day periods. Platelet activation markers were measured by flow cytometry on unstimulated or stimulated (adenosine diphosphate (ADP) 20 μM) platelets, before and after each dosing period., Results: At baseline there was no difference in platelet activation markers, either unstimulated or ADP-stimulated, between NE and VE. Pras 5 mg reduced both ADP-stimulated platelet P-selectin and activated GPIIb-IIIa in VE (p < 0.01 for both analyses) and NE (p < 0.001 and p < 0.05, respectively). Clop 75 mg had a similar effect as Pras 5 mg but did not significantly reduce activated GPIIb-IIIa in VE. Prasugrel 10 mg resulted in decreased platelet activation in both age groups compared to Clop 75 mg (p < 0.01)., Conclusions: In VE and NE-patients, Pras 5 mg inhibited platelet P-selectin expression similar to Clop 75 mg and Pras 10 mg. Prasugrel 10 mg inhibited platelet P-selectin expression better than Clop 75 mg. Prasugrel 10 mg and 5 mg, but not Clop 75 mg, significantly inhibited activated GPIIb-IIIa in VE. This platelet reactivity data support the use of Pras 5 mg for VE patients.

A study in mice indicates that the adhesion molecule P-selectin, which is expressed on the endothelium during inflammation, may serve as a target for the delivery of drug-filled nanoparticles to tumors. Antitumor efficacy was achieved in several mouse models of cancer, and the researchers successfully used this approach even against noninflammatory tumors, by first using ionizing radiation to trigger P-selectin's expression on the tumor vasculature., (©2016 American Association for Cancer Research.)

Background: Immune rejection continues to threaten all tissue transplants. Here we sought to determine whether platelet (P)- and endothelial (E)-selectin mediate T cell recruitment in corneal transplantation and whether their blockade can reduce T cell graft infiltration and improve long-term corneal allograft survival., Methods: In a murine model of allogeneic corneal transplantation, we used PCR and immunohistochemistry to investigate expression of P- and E-selectin in rejected versus accepted allografts and lymph node flow cytometry to assess expression of selectin ligands by effector T cells. Using P- and E-selectin neutralizing antibodies, we evaluated the effect of blockade on CD4 T cell recruitment, as well as the effect of anti-E-selectin on long-term allograft survival., Results: The P- (93.3-fold, P < 0.05) and E-selectin (17.1-fold, P < 0.005) are upregulated in rejected versus accepted allogeneic transplants. Type 1 T helper cells from hosts with accepted and rejected grafts express high levels of P-selectin glycoprotein ligand 1 and glycosylated CD43. In vivo blockade of P (0.47 ± 0.03, P < 0.05) and E selectin (0.49 ± 0.1, P < 0.05) reduced the number of recruited T cells compared with IgG control (0.98 ± 0.1). Anti-E-selectin reduced the number of mature antigen-presenting cells trafficking to lymphoid tissue compared with control (6.96 ± 0.9 vs 12.67 ± 0.5, P < 0.05). Anti-E-selectin treatment delayed graft rejection and increased survival compared with control, although this difference did not reach statistical significance., Conclusions: In a model of corneal transplantation, P- and E-selectin mediate T cell recruitment to the graft, E-selectin mediates APC trafficking to lymphoid tissue, and blockade of E-selectin has a modest effect on improving long-term graft survival.

Introduction: Ischemia-reperfusion injury (IRI) involves a complex sequence of events and limits the outcome of various surgical interventions. Clinical trials, based on the data of experimental models, aim to prove whether a pharmacological or technical approach could be suitable to provide a beneficial effect in humans. Due to the complexity of IRI, few pharmacological treatments have been investigated in clinical Phase III., Areas Covered: In this review we report clinical trials that test specific drugs in clinical trials of organ transplantation. These studies form part of Phase II trials and examine the administration of caspase inhibitors, P-selectin antagonist or an antioxidant component in order to attenuate cold IRI during transplantation. Moreover, we provide a brief description of drugs tested on trials of different clinical situations associated to IRI, such as the coronary artery bypass graft surgery and percutaneous coronary intervention., Expert Opinion: Future clinical trials could be centered on the application of techniques suitable for organs with increased vulnerability toward IRI. Furthermore, the standardization of reliable biomarkers and a careful estimation of the impact of high risk factors may be the key in order to achieve a more critical evaluation of the obtained results.

P- and E-selectins are expressed on the surface of endothelial cells and may contribute to neutrophil recruitment following injurious lengthening contractions of skeletal muscle. Blunting neutrophil, but not macrophage, accumulation after lengthening contractions may provide a therapeutic benefit as neutrophils exacerbate damage to muscle fibers, while macrophages promote repair. In this study, we tested the hypothesis that P- and E-selectins contribute to neutrophil, but not macrophage, accumulation in muscles after contraction-induced injury, and that reducing neutrophil accumulation by blocking the selectins would be sufficient to reduce damage to muscle fibers. To test our hypothesis, we treated mice with antibodies to block P- and E-selectin function and assessed leukocyte accumulation and damage in muscles 2 days after lengthening contractions. Treatment with P/E-selectin blocking antibodies reduced neutrophil content by about half in muscles subjected to lengthening contractions. In spite of the reduction in neutrophil accumulation, we did not detect a decrease in damage 2 days after lengthening contractions. We conclude that P- and/or E-selectin contribute to the neutrophil accumulation associated with contraction-induced muscle damage and that only a portion of the neutrophils that typically accumulate following injurious lengthening contractions is sufficient to induce muscle fiber damage and force deficits. Thus, therapeutic interventions based on blocking the selectins or other adhesion proteins will have to reduce neutrophil numbers by more than 50% in order to provide a benefit., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

P-selectin-mediated tumor cell adhesion to platelets is a well-established stage in the process of tumor metastasis. Through computerized structural analysis, we found a marine-derived polysaccharide, holothurian glycosaminoglycan (hGAG), behaved as a ligand-competitive inhibitor of P-selectin, indicating its potential to disrupt the binding of P-selectin to cell surface receptor and activation of downstream regulators of tumor cell migration. Our experimental data demonstrated that hGAG significantly inhibited P-selectin-mediated adhesion of tumor cells to platelets and tumor cell migration in vitro and reduced subsequent pulmonary metastasis in vivo. Furthermore, abrogation of the P-selectin-mediated adhesion of tumor cells led to down-regulation of protein levels of integrins, FAK and MMP-2/9 in B16F10 cells, which is a crucial molecular mechanism of hGAG to inhibit tumor metastasis. In conclusion, hGAG has emerged as a novel anti-cancer agent via blocking P-selectin-mediated malignant events of tumor metastasis.

Purpose: Inclacumab, a novel monoclonal antibody against P-selectin, is in development for the treatment and prevention of atherosclerotic cardiovascular diseases. This study was conducted to investigate potential differences in the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single intravenous doses of inclacumab between Japanese and Caucasian healthy volunteers., Method: Sixty-two subjects (31 Japanese and 31 Caucasian) were enrolled in a single-center, open-label, parallel, three dose groups (0.3, 3.0, and 20 mg/kg), single-dose study in Japanese and Caucasian healthy volunteers. Inclacumab concentrations, platelet-leukocyte aggregates (PLA), free/total soluble P-selectin (sP-selectin) ratio, and antibody formation were measured along with routine safety monitoring during the conduct of the study., Results: The PK profiles of inclacumab in Caucasian and Japanese subjects were similar following single-dose intravenous infusion. The statistical analysis of peak (C max) and total exposure (AUClast) indicated that bioavailability was similar for both races when corrected for body weight. The geometric mean ratios for AUClast and C max in the Japanese versus Caucasian cohort were 101 and 111%, respectively, in 0.3 mg/kg dose group, 108 and 107%, respectively, in 3.0 mg/kg dose group, and 97 and 96%, respectively, in 20 mg/kg dose group. No differences were observed in the level of PLA inhibition and mean free/total soluble P-selectin ratio between Japanese and Caucasian subjects. PK/PD relationship between the free/total sP-selectin ratio or PLA and plasma concentration of inclacumab appeared to be similar in both Japanese and Caucasian populations. The effect of race as a covariate was explored on both PK/PD models for PLA and free/total sP-selectin ratio and did not have a significant effect over the reduced model without race as a covariate., Conclusions: Ethnicity had no clinically relevant influence on inclacumab pharmacokinetics or pharmacodynamics. No dose adjustment of inclacumab is required for differences in race.

Polysulfonated macromolecules are known to bind selectins, adhesion membrane proteins which are broadly implicated in inflammation. Commercially available branched polyethylenimine (PEI) was reacted with chlorosulfonic acid to generate sulfonated PEI with varying degrees of sulfonation. Remaining unreacted amine groups were then used for straightforward conjugation with pyropheophoribide-a, a near-infrared photosensitizer. Photosensitizer-labeled sulfonated PEI conjugates inhibited blood coagulation and were demonstrated to specifically bind to cells genetically programmed to overexpress L-selectin (CD62L) or P-selectin (CD62P). In vitro, following targeting, selectin-expressing cells could be destroyed via photodynamic therapy.

Platelet markers [soluble CD40 ligand (sCD40L) and soluble p selectin (sPselectin)] are associated with platelet activation and cardiovascular events. We sought to investigate the reproducibility of these markers over time and the effect of low-dose aspirin on sCD40L and sPselectin in plasma and serum. Following an overnight fast, 40 healthy volunteers had weekly phlebotomy and were administered aspirin 81 mg/day between weeks 3 and 4. Reproducibility over time was assessed by coefficient of variation (CV) and inter-class correlation coefficient. Correlation between markers was assessed using Pearson r statistic. Difference between levels pre- and post-aspirin was measured with Wilcoxon signed-rank test. Data are presented as median (interquartile range). sCD40L and sPselectin measurements were reproducible over time in plasma and serum (CV < 10 %). Measurement of sCD40L and sPselectin in plasma correlated with levels in serum before aspirin and after aspirin. There was no significant correlation between sCD40L and sPselectin. After 1-week of aspirin 81 mg/day, there was a reduction in sCD40L and sPselectin in serum and plasma, respectively. Soluble CD40L and sPselectin are independent markers that are reproducible over time in both plasma and sera and are reduced by 1-week of low-dose aspirin.

Inclacumab, a novel monoclonal antibody against P-selectin in development for the treatment and prevention of atherosclerotic cardiovascular diseases, was administered in an ascending single-dose study as intravenous infusion to evaluate safety, pharmacokinetics, and pharmacodynamics. Fifty-six healthy subjects were enrolled in this randomized, double-blind placebo-controlled study. Each dose level (0.03-20 mg/kg) was investigated in separate groups of 8 subjects (6 on inclacumab, 2 on placebo). Platelet-leukocyte aggregates, free/total soluble P-selectin concentration ratio, drug concentrations, bleeding time, platelet aggregation, antibody formation, and routine laboratory parameters were measured frequently until 32 weeks. Pharmacokinetic profiles were indicative of target-mediated drug disposition. Platelet-leukocyte aggregate inhibition and soluble P-selectin occupancy showed dose dependency and were strongly correlated to inclacumab plasma concentrations, with IC50 of 740 and 4600 ng/mL, respectively. Inclacumab was well tolerated by the majority of subjects and did neither affect bleeding time nor platelet aggregation. These findings allowed the investigation of the potential beneficial therapeutic use of inclacumab in patient study.

Inflammation and cancer are related pathologies acting synergistically to promote tumor progression. In both, hematogenous metastasis and inflammation, P-selectin participates in interactions involving tumor cells, platelets, leukocytes and endothelium. Heparin has been shown to inhibit P-selectin and as a consequence it blunts metastasis and inflammation. Some heparin analogs obtained from marine invertebrates are P-selectin inhibitors and do not induce bleeding effects. The present work focuses on the P-selectin blocking activity of a unique heparan sulfate (HS) from the bivalve mollusk Nodipecten nodosus. Initially, we showed that the mollusk HS inhibited LS180 colon carcinoma cell adhesion to immobilized P-selectin in a dose-dependent manner. In addition, we demonstrated that this glycan attenuates leukocyte rolling on activated endothelium and inflammatory cell recruitment in thioglycollate-induced peritonitis in mice. Biochemical analysis indicated that the invertebrate glycan also inhibits heparanase, a key player in cell invasion and metastasis. Experimental metastasis of Lewis lung carcinoma cells was drastically attenuated by the mollusk HS through a mechanism involving inhibition of platelet-tumor-cell complex formation in blood vessels. These data suggest that the mollusk HS is a potential alternative to heparin for inhibiting P-selectin-mediated events such as metastasis and inflammatory cell recruitment., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inclacumab is a novel monoclonal antibody directed against P-selectin in development for the prevention and treatment of atherosclerotic cardiovascular diseases. It is likely to be used concomitantly with heparin in patients undergoing percutaneous coronary intervention. Coadministration of both drugs may potentially increase the bleeding risk associated with heparin. This crossover study evaluated the potential pharmacodynamic interaction between inclacumab and unfractionated heparin in 18 healthy smokers. Owing to the long elimination of inclacumab (half-life of approximately 18 days), a 2-period, one-sequence study design was used. Subjects received an intravenous bolus injection of unfractionated heparin (5000 IU) on days 1 and 8 and an intravenous infusion of inclacumab (20 mg/kg) on day 8. Blood samples were collected on days 1 and 8 for pharmacodynamic effects of unfractionated heparin (anti-FXa and anti-FIIa activities, activated partial thromboplastin time and tissue factor pathway inhibitor) and over 6 months for pharmacokinetics of inclacumab. Sixteen subjects were eligible for pharmacodynamic analysis. Inclacumab had no clinically significant pharmacodynamic interaction with unfractionated heparin. With the exception of the minor but statistically significant increase of the maximum effect [Emax] of anti-FIIa activity, pharmacodynamic parameters (areas under the effect curve [AUElast] and Emax of anti-FXa) were almost similar on days 1 and 8. The 90% confidence intervals of geometric mean ratios of day 8 to day 1 for AUElast and Emax were however all contained within bioequivalence boundaries. The data demonstrate that the anticoagulant effect of unfractionated heparin was not affected by the administration of inclacumab.

Objective: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT., Approach and Results: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed., Conclusions: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials., (© 2015 American Heart Association, Inc.)

Objective: We tested the hypothesis that endothelial peroxisome proliferator-activated receptor-γ protects against vascular thrombosis using a transgenic mouse model expressing a peroxisome proliferator-activated receptor-γ mutant (E-V290M) selectively in endothelium., Approach and Results: The time to occlusive thrombosis of the carotid artery was significantly shortened in E-V290M mice compared with nontransgenic littermates after either chemical injury with ferric chloride (5.1 ± 0.2 versus 10.1 ± 3.3 minutes; P=0.01) or photochemical injury with rose bengal (48 ± 9 versus 74 ± 9 minutes; P=0.04). Gene set enrichment analysis demonstrated the upregulation of NF-κB target genes, including P-selectin, in aortic endothelial cells from E-V290M mice (P<0.001). Plasma P-selectin and carotid artery P-selectin mRNA were elevated in E-V290M mice (P<0.05). P-selectin-dependent leukocyte rolling on mesenteric venules was increased in E-V290M mice compared with nontransgenic mice (53 ± 8 versus 25 ± 7 per minute; P=0.02). The shortened time to arterial occlusion in E-V290M mice was reversed by administration of P-selectin-blocking antibodies or neutrophil-depleting antibodies (P=0.04 and P=0.02, respectively) before photochemical injury., Conclusions: Endothelial peroxisome proliferator-activated receptor-γ protects against thrombosis through a mechanism that involves downregulation of P-selectin expression and diminished P-selectin-mediated leukocyte-endothelial interactions., (© 2015 American Heart Association, Inc.)

Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

Context: P-selectin is a promising target for inflammatory-related diseases. Polysaccharides are the active ingredients of Sanguisorba officinalis L. (Rosaceae) responsible for its anti-inflammatory activities; however, the molecular mechanism is not clear yet., Objective: This study evaluates the effects of polysaccharides (SOPs) from Sanguisorba officinalis on their antagonistic function against P-selectin-mediated leukocyte adhesion., Materials and Methods: The antagonistic function of SOPs was investigated by flow cytometry and static adhesion assay at the concentrations of 25 and 100 μg/ml. The dynamic interaction between HL-60 cells and CHO-P cell monolayer treated with SOPs (25 and 100 μg/ml) was analyzed in a parallel plate flow chamber, and quantitatively calculated by ImageJ software (NIH, Bethesda, MD). In vitro protein binding assay was carried out to evaluate the blocking effects of SOPs (25 and 100 μg/ml) on the interaction between P-selectin and PSGL-1., Results: SOPs-treatment (100 μg/ml) significantly reduced the percentage of HL-60 cells binding to P-selectin (p < 0.01) determined by flow cytometry. In addition, SOPs (25 and 100 μg/ml) markedly blocked the adhesion between HL-60 cells and CHO-P cells under static condition, and the inhibitory rates reached 39.9% and 71.2%, respectively. Compared with the positive control group, SOPs-treatment (25 and 100 μg/ml) significantly reduced the percentage of HL-60 cells rolling on CHO-P cell monolayers by 43.5% and 75.2%, respectively. Protein binding assay showed the interaction between P-selectin and PSGL-1 was significantly blocked by SOPs., Discussion and Conclusion: SOPs possess a significant antagonistic function against P-selectin-mediated leukocyte adhesion, and SOPs could be considered as a promising candidate for amelioration of inflammation-related diseases.

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

Escin, a natural mixture of triterpenoid saponin isolated from the seed of the horse chestnut, is reported to have a potent antiulcer activity against ethanol-induced gastric mucosal lesions. This study investigated the possible mechanisms underlying the gastroprotective effect of escin against indomethacin-induced gastric ulcer in mice. Gastric ulceration was induced by a single intragastric administration of indomethacin (18 mg/kg). The mice underwent intragastric treatment with escin at doses of 0.45, 0.9 or 1.8 mg/kg. Gastric lesion was estimated morphometrically and histopathologically 6 h after the indomethacin administration. The antioxidative parameters in gastric mucosa were measured. Moreover, the activity of myeloperoxidase and the contents of TNF-α, P-selectin and VCAM-1 in gastric tissues were determined. The results showed that escin protected gastric tissues against indomethacin-induced gastropathy as demonstrated from a reduction in the ulcer index and an attenuation of histopathologic changes. Escin caused significant reductions of the contents of malondialdehyde, TNF-α, P-selectin, VCAM-1 and myeloperoxidase activity. The altered activities of superoxide dismutase, catalase and glutathione peroxidase in the stomach tissues were also ameliorated by escin treatment. The present study demonstrated that escin had a protective effect against indomethacin-induced gastric ulcer in mice, not only by virtue of its antioxidant potential, but also due to its anti-inflammatory effect.

Background: The heparan sulfate proteoglycan syndecan-1 (CD138) was shown to regulate inflammatory responses by binding chemokines and cytokines and interacting with adhesion molecules, thereby modulating leukocyte trafficking to tissues. The objectives of this study were to examine the expression of syndecan-1 and its role in leukocyte recruitment and chemokine presentation in the microcirculation underlying the parietal peritoneum., Methods: Wild-type BALB/c and syndecan-1 null mice were stimulated with an intraperitoneal injection of Staphylococcus aureus LTA, Escherichia coli LPS or TNFα and the microcirculation of the parietal peritoneum was examined by intravital microscopy after 4 hours. Fluorescence confocal microscopy was used to examine syndecan-1 expression in the peritoneal microcirculation using fluorescent antibodies. Blocking antibodies to adhesion molecules were used to examine the role of these molecules in leukocyte-endothelial cell interactions in response to LTA. To determine whether syndecan-1 co-localizes with chemokines in vivo, fluorescent antibodies to syndecan-1 were co-injected intravenously with anti-MIP-2 (CXCL2), anti-KC (CXCL1) or anti-MCP-1 (CCL2)., Results and Conclusion: Syndecan-1 was localized to the subendothelial region of peritoneal venules and the mesothelial layer. Leukocyte rolling was significantly decreased with LPS treatment while LTA and TNFα significantly increased leukocyte adhesion compared with saline control. Leukocyte-endothelial cell interactions were not different in syndecan-1 null mice. Antibody blockade of β2 integrin (CD18), ICAM-1 (CD54) and VCAM-1 (CD106) did not decrease leukocyte adhesion in response to LTA challenge while blockade of P-selectin (CD62P) abrogated leukocyte rolling. Lastly, MIP-2 expression in the peritoneal venules was not dependent on syndecan-1 in vivo. Our data suggest that syndecan-1 is expressed in the parietal peritoneum microvasculature but does not regulate leukocyte recruitment and is not necessary for the presentation of the chemokine MIP-2 in this tissue.