Automated (Centrifugal) therapeutic plasma exchange option for guillain-barre syndrome: A report from Calabar, Nigeria.

Therapeutic plasma exchange (TPE) is performed frequently and effectively in developed countries, whereas the reverse is the case in developing countries. Guillain-Barre syndrome (GBS), synonymous with acute inflammatory demyelinating polyneuropathy, is an important indication for TPE, but this is rarely administered in the treatment of such patients in Nigeria due to lack of such automated facility, limited expertise, and high cost. This report therefore presents an uncommon case of GBS in which automated TPE was utilized in the management, with the aims of highlighting the current status and challenges of therapeutic apheresis services in Nigeria. A 42-year-old male presented with rapidly progressive (in an ascending fashion) paralysis of all four limbs within 24 h without any preceding history of fever or other symptoms. Clinical examination revealed a young man, afebrile, not pale, and also not dehydrated. Central nervous system examination showed a conscious man, alert, and oriented in time, person, and place. There were no signs of meningeal irritation and the cranial nerves were grossly intact. There was no power in the limbs: global hypotonia and areflexia were noted on examination. However, he had intact sensory perceptions to touch and pain. Following a diagnosis of GBS, he was treated with four sessions of plasmapheresis and TPE. The TPE session was done using a discontinuous flow apheresis machine which exchanged one plasma volume (3 L of plasma) and 5% albumin used for replacement. The patient made gradual but steady recovery as return of power to the upper limbs and trunk started by the 2nd week of treatment. TPE is an important treatment modality in the management of GBS as well as several other conditions, and it is becoming increasingly available in Nigeria. However, it is still grossly underutilized, thus the need for more therapeutic apheresis facilities and trained personnel, in addition to concerted efforts to subsidize the cost of accessing the treatment.

The lipid-lowering effects of lomitapide are unaffected by adjunctive apheresis in patients with homozygous familial hypercholesterolaemia - a post-hoc analysis of a Phase 3, single-arm, open-label trial.

OBJECTIVE: Lomitapide (a microsomal triglyceride transfer protein inhibitor) is an adjunctive treatment for homozygous familial hypercholesterolaemia (HoFH), a rare genetic condition characterised by elevated low-density lipoprotein-cholesterol (LDL-C), and premature, severe, accelerated atherosclerosis. Standard of care for HoFH includes lipid-lowering drugs and lipoprotein apheresis. We conducted a post-hoc analysis using data from a Phase 3 study to assess whether concomitant apheresis affected the lipid-lowering efficacy of lomitapide. METHODS: Existing lipid-lowering therapy, including apheresis, was to remain stable from Week -6 to Week 26. Lomitapide dose was escalated on the basis of individual safety/tolerability from 5 mg to 60 mg a day (maximum). The primary endpoint was mean percent change in LDL-C from baseline to Week 26 (efficacy phase), after which patients remained on lomitapide through Week 78 for safety assessment and further evaluation of efficacy. During this latter period, apheresis could be adjusted. We analysed the impact of apheresis on LDL-C reductions in patients receiving lomitapide. RESULTS: Of the 29 patients that entered the efficacy phase, 18 (62%) were receiving apheresis at baseline. Twenty-three patients (13 receiving apheresis) completed the Week 26 evaluation. Of the six patients who discontinued in the first 26 weeks, five were receiving apheresis. There were no significant differences in percent change from baseline of LDL-C at Week 26 in patients treated (-48%) and not treated (-55%) with apheresis (p = 0.545). Changes in Lp(a) levels were modest and not different between groups (p = 0.436). CONCLUSION: The LDL-C lowering efficacy of lomitapide is unaffected by lipoprotein apheresis.

Transfusion of 28-day-old leucoreduced or non-leucoreduced stored red blood cells induces an inflammatory response in healthy dogs.

BACKGROUND AND OBJECTIVES: Studies in mice suggest that rapid transfusions of red blood cells (RBCs), refrigerator stored for longer durations, induce a pro-inflammatory cytokine response. Studies in human neonates confirm these findings; however, to date, adult human studies have failed to replicate these findings. We used healthy research dogs to begin to examine the factors affecting the cytokine response to transfusion. MATERIALS AND METHODS: In a prospective study, healthy dogs were randomized for two autologous packed RBC transfusions after 7 (i.e. 'fresh') and 28 (i.e. 'old') days of storage, or after 28 and 7 days of storage, with or without prestorage leucoreduction (LR). RESULTS: No significant differences were observed between LR and non-LR transfusions for all circulating analytes measured following transfusion. A pro-inflammatory cytokine response, exemplified by monocyte chemoattractant protein-1, was observed 6 h after only old RBC transfusions, irrespective of infusion rate (P < 0·001). This response was accompanied by increased neutrophil counts (P < 0·001) and decreased platelet counts (P < 0·001). CONCLUSION: In healthy dogs, old RBC transfusions induce inflammation, which is unaffected by infusion rate.

Cryopreservation of canine platelets.

BACKGROUND: Platelet cryopreservation allows long-term storage and immediate availability of transfusion products. HYPOTHESIS: The addition of a preparation inhibiting platelet activation (Thrombosol, in 2% dimethyl sulfoxide [DMSO]) will enhance in vitro function and prolong in vivo survival of cryopreserved platelets compared with those preserved in 6% DMSO. ANIMALS: Thirty-three research dogs. METHODS: Prospective study. Eleven fresh canine apheresis platelet concentrates (PCs) were each split into 3 units: fresh and cryopreserved in 6% DMSO or Thrombosol. Platelet analysis, performed 1-10 weeks postfreezing, included in vitro functional testing and in vivo survival assessed by administration of biotinylated platelets. RESULTS: Platelet aggregation was diminished in cryopreserved PC. Cryopreserved platelets could be activated, as based on mean thrombin-stimulated P-selectin expression (6% DMSO, 23.0%; Thrombosol, 18.4%), although to a lesser extent than fresh PC (49.1%) (P < .0001). The mean maximum in vivo platelet recovery for fresh PC was 80.3%, significantly greater than recovery for 6% DMSO (49.2%) and Thrombosol PC (43.7%) (P< or = .001). The half-life (days) of fresh PC (3.8 +/- 0.4) was significantly (P < .002) greater than that of 6% DMSO (1.9 +/- 1.0) and Thrombosol (2.4 +/- 1.1) PC, with no difference (P= .3) between cryopreserved PC. CONCLUSIONS AND CLINICAL IMPORTANCE: Cryopreservation of canine platelets using Thrombosol did not provide any advantage over preservation using 6% DMSO. Cryopreserved platelets can be activated in vitro and provide therapeutic benefit when fresh platelets are unavailable. Further studies are needed to assess their in vivo hemostatic function.

Prothrombotic factors enhance heparin-induced thrombocytopenia and thrombosis in vivo in a mouse model.

BACKGROUND: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common cause of life- and limb-threatening thrombosis. The development of antibodies that react with complexes of heparin and platelet factor 4 (PF4) is fundamental to the development of the disease. However, anti-PF4/heparin antibodies are far more common than is HIT/T and there is less understanding of the factors that contribute to thrombosis in only a subset of patients. OBJECTIVES: Both qualitative and quantitative differences in multiple factors (e.g. antibodies, heparin and platelets) may influence the clinical course of patients who develop anti-PF4/heparin antibodies. We examined the hypothesis that host-specific factors, such as comorbid prothrombotic conditions, would exacerbate the pathologic effects of anti-PF4/heparin antibodies. METHODS AND RESULTS: A mouse model transgenic for human Fcgamma RIIa and PF4 and null for mouse PF4 was used to study the influence of prothrombotic conditions on the effects of anti-PF4/heparin antibodies in vivo. To simulate a prothrombotic milieu, mice were fed a hypercholesterolemic diet (HD). HD-fed mice had elevated plasma cholesterol, increased platelet reactivity and increased endothelial activation relative to mice fed a standard diet (SD). Age- and sex-matched mice from each diet group were treated with an anti-PF4/heparin antibody and heparin. HD-fed mice developed more severe thrombocytopenia than similarly treated SD-fed mice. Mice with moderate to severe thrombocytopenia had elevated plasma levels of thrombin-antithrombin complexes, indicative of increased thrombin generation in vivo. Platelet-fibrin thrombi were observed in multiple organs of HD-fed mice that developed severe thrombocytopenia. CONCLUSIONS: Host-specific factors, such as prothrombotic changes in platelet reactivity and/or endothelial activation, may influence the development of thrombosis in a subset of patients who develop anti-PF4/heparin antibodies.

Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcgammaRIIA.

Heparin-induced thrombocytopenia/thrombosis (HIT/HITT) is a severe, life-threatening complication that occurs in 1% to 3% of patients exposed to heparin. Interactions between heparin, human platelet factor 4 (hPF4), antibodies to the hPF4/heparin complex, and the platelet Fc receptor (FcR) for immunoglobulin G, FcgammaRIIA, are the proposed primary determinants of the disease on the basis of in vitro studies. The goal of this study was to create a mouse model that recapitulates the disease process in humans in order to understand the factors that predispose some patients to develop thrombocytopenia and thrombosis and to investigate new therapeutic approaches. Mice that express both human platelet FcgammaRIIA and hPF4 were generated. The FcgammaRIIA/hPF4 mice and controls, transgenic for either FcgammaRIIA or hPF4, were injected with KKO, a mouse monoclonal antibody specific for hPF4/heparin complexes, and then received heparin (20 U/d). Nadir platelet counts for KKO/heparin-treated FcgammaRIIA/hPF4 mice were 80% below baseline values, significantly different (P <.001) from similarly treated controls. FcgammaRIIA/hPF4 mice injected with KKO and 50 U/d heparin developed shock and showed fibrin-rich thrombi in multiple organs, including thrombosis in the pulmonary vasculature. This is the first mouse model of HIT to recapitulate the salient features of the human disease and demonstrates that FcgammaRIIA and hPF4 are both necessary and sufficient to replicate HIT/HITT in an animal model. This model should facilitate the identification of factors that modulate disease expression and the testing of novel therapeutic interventions.

Platelet-endothelial interactions in atherosclerosis.

The pathogenesis of atherosclerosis, the leading cause of morbidity and mortality in the United States, is multifactorial. Many factors that have been shown to influence the development of atherosclerosis also affect the function of the endothelium through soluble or cell-cell interactions. Among these, interactions between platelets and endothelial cells have only recently begun to receive systematic study. This article reviews recent evidence showing how the interaction between platelets and endothelial cells may play a important role in the pathogenesis of atherosclerosis, suggesting an underappreciated potential locus for pharmacologic intervention.

Blood transfusion costs by diagnosis-related groups in 60 university hospitals in 1995.

BACKGROUND: Transfusion services are frequently challenged to initiate efforts to reduce blood transfusion costs. One approach is to analyze blood transfusion costs for individual medical and surgical Diagnosis-Related Groups (DRGs). Rank ordering of DRGs by transfusion costs and interinstitutional comparisons of these costs may lead to the selection of DRGs for further analysis of the process of blood transfusion. STUDY DESIGN AND METHODS: Common DRGs (n = 486) that were related to discharges in 1995 were analyzed from 60 university hospital members of the University HealthSystems Consortium (UHC). Cost data were tabulated by using cost-to-charge ratios reflecting all aspects of blood transfusion-related costs of participating institutions. RESULTS: Of these 486 DRGs, 471 had identifiable mean blood costs, and 34 had median blood costs, mostly for surgical conditions. Transfusion costs represented a small proportion (< or = 1%) of total hospitalization costs for most DRGS: Nonetheless, millions of dollars were spent on blood transfusion, and for the most expensive DRGs, the costs ranged from 5.0 to 8.6 percent of total hospitalization costs. Transfusion costs are more variable for the DRGs with the lowest transfusion costs than for those with the highest transfusion costs. CONCLUSION: Members of the UHC may utilize such analyses to identify surgical or medical diagnoses with transfusion costs at variance with the group norm. These DRGs could then be targeted for further evaluation of components contributing to high costs, for possible alterations in physician or clinical laboratory practices. Considering those conditions with the highest cumulative transfusion costs (e.g., BMT, liver transplant, acute leukemia, and cardiothoracic procedures), changes in transfusion practices that affect these particular patient categories may have a significant impact on global blood transfusion costs.

The alpha-defensins stimulate proteoglycan-dependent catabolism of low-density lipoprotein by vascular cells: a new class of inflammatory apolipoprotein and a possible contributor to atherogenesis.

Inflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain alpha-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of (125)I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the alpha(2)-macroglobulin receptor/LDL-R-related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate-containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The alpha-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R-independent, proteoglycan-dependent pathway. (Blood. 2000;96:1393-1398)

The costs of disease.

BACKGROUND: To date there have been no studies identifying and comparing the component costs to treat a large number of diseases for hospitalized inpatients. METHODS: Hospital costs were analyzed for 486 diagnosis-related groups (DRGs) relating to >1.3 million patient discharges from 60 University Hospital members of the University HealthSystems Consortium. For each DRG, length of stay, total cost, and key cost components were analyzed, including accommodation, intensive care, and surgery. RESULTS: In general, total costs of diseases classified as surgical exceeded those classified as medical. Diseases involving organ transplantation typically cost more than other diseases. However, within the studied population, the two DRGs accounting for most total healthcare dollars were percutaneous cardiovascular procedures and management of neonates with immaturity or respiratory failure. CONCLUSIONS: Considering six key cost components, as well as disease complexity and length of stay, the best predictors of total costs for medical conditions were the length of stay and accommodation (housing, meals, nursing services) costs, whereas for surgical conditions, the best predictor of total costs was laboratory costs. This analysis may be used within an individual institution to identify surgical or medical diagnoses with total or component costs at variance with the group mean. A hospital may focus its cost reduction efforts to make decisions to expand, alter, or eliminate particular clinical programs based on comparison of its own total and component costs with those from other hospitals in the database.

Laboratory costs in the context of disease.

BACKGROUND: To determine the contribution of laboratory costs to the overall costs of managing hospital patients with different diseases, we studied the costs of laboratory testing overall and in relation to the other costs incurred during hospitalization. METHODS: We used a database developed by the University HealthSystems Consortium containing >1 million patients in 60 University Hospitals with diseases included in 486 diagnosis-related groups (DRGs). Laboratory costs included in the database comprised those associated with testing in the clinical laboratory together with those incurred in point-of-care testing and anatomic pathology but not those involving blood products and their transfusion. RESULTS: The mean laboratory costs to manage surgical patients were greater than those to manage medical patients in 19 of the 25 major diagnostic categories. The median laboratory costs for patients with liver transplants exceeded $8000, and the laboratory costs to support other organ transplants were among the highest. The highest proportion of total costs attributable to the laboratory was 18.3% for acute leukemia and kidney and urinary tract signs and symptoms, both in children. Laboratory costs were <1.0% of the total costs for only 15 DRGs. The highest median daily laboratory cost, $416, was attributable to liver transplant patients. Several conditions had median laboratory costs less than $30 per day, in spite of lengths of stay that exceeded 10 days in some cases. CONCLUSIONS: Although laboratory costs generally average 6% of the total costs for surgical conditions and 9% of the total costs for medical conditions, there is considerable variability. In general, laboratory costs were relatively poorly correlated with total costs. However, observation of high daily laboratory costs for many DRGs suggests that reducing length of stay would reduce both laboratory and total costs.

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo.

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.

A region in domain II of the urokinase receptor required for urokinase binding.

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.

Defensin promotes the binding of lipoprotein(a) to vascular matrix.

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Identification of the site in the substance P (NK-1) receptor for modulation of peptide binding by sulfhydryl reagents.

Substance P (SP) is a peptide neurotransmitter that is involved in multiple responses in both the central and the peripheral nervous systems through a G-protein-coupled contains a number of conserved cysteine residues. To localize and identify the cysteine residues that participate in receptor binding, intact Chinese hamster ovary cells expressing the SP receptor were treated with various sulfhydryl reagents and the effect of these reagents on radioiodinated SP binding affinity and dissociation rate was determined. We used a series of amphiphilic maleimide derivatives in which the reactive maleimide group penetrates to different depths within the plane of membrane. Only the maleimide derivatives with intermediate chain lengths modified receptor binding properties, indicating that the reactive sulfhydryl group is located within a transmembrane domain of the receptor close (within 1.7 nm) to the extracellular border. Since peptide binding to a mutant receptor C199S, in which Cys-199 was replaced by a serine, was found to be insensitive to modulation by sulfhydryl reagents, this reactive sulfhydryl group is on Cys-199 of the receptor. Receptor occupancy by SP protects Cys-199 from modification and thus this residue is either located at or conformationally linked to the SP binding site.

Residue 78 in the second transmembrane domain of the neurokinin-1 receptor is important in coupling high affinity agonist binding to multiple second messenger responses.

The neurokinin-1 tachykinin receptor is a member of the G protein-coupled receptor superfamily. An unusual feature of the neurokinin-1 receptor is the presence of glutamic acid (residue 78) in the second putative transmembrane domain, at the location of a highly conserved aspartate residue in the G protein-coupled receptor superfamily. The rat neurokinin-1 receptor cDNA was mutated to lysine, aspartate, and glutamine at this site and functionally expressed in Chinese hamster ovary cells, and clonal cell lines were isolated and characterized. Radioligand binding demonstrated that the Asp78 and Lys78 receptors have substance P binding affinities indistinguishable from those of the wild-type receptor and are expressed at roughly the same number of receptors per cell. The Gln78 receptor variant, on the other hand, exhibited no detectable agonist binding. Although wild-type and Asp78 receptors have essentially the same ability to stimulate inositol phospholipid turnover, cAMP production, and arachidonic acid release, the Lys78 variant is markedly attenuated in its ability to activate any of these pathways. These data indicate that residue 78 plays a role in the coupling of the rat neurokinin-1 receptor to cellular effectors. In addition, both Asp78 and Lys78 receptors show a greater percentage of high affinity binding that is resistant to guanosine-5'-O-(3-thio)triphosphate than does the wild-type receptor, indicating a potential difference in G protein coupling between wild-type and mutated receptors.

Both extracellular and transmembrane residues contribute to the species selectivity of the neurokinin-1 receptor antagonist WIN 51708.

WIN 51708 is a nonpeptide antagonist of the neurokinin (NK)-1 (substance P) receptor that possesses a dramatically higher affinity for the rat NK-1 receptor, compared with the human NK-1 receptor. This selectivity is the opposite of the selectivity displayed by CP-96,345 and is much greater in magnitude than the selectivity of RP 67580. The naturally occurring peptide agonist substance P shows no such species selectivity. To determine the molecular basis for the species selectivity of WIN 51708, a series of chimeric and point-mutated NK-1 receptors were created and functionally expressed in Chinese hamster ovary cells. Residue 97 in the first extracellular loop and residue 290 in the seventh putative transmembrane domain are critical determinants for the selectivity of WIN 51708 for the rat over the human NK-1 receptor. Although mutation of either residue 97 or residue 290 in the rat NK-1 receptor is sufficient for a low, human-like affinity for WIN 51708, both of these residues must be simultaneously mutated in the human NK-1 receptor to allow nearly rat wild-type affinity for this antagonist. This suggests that the binding environment for WIN 51708 in the rat NK-1 receptor differs, at least in part, from the binding environment in the human NK-1 receptor. In addition, although residue 290 is critical for the species selectivity of WIN 51708 and CP-96,345, residue 97 does not play a role in the species selectivity of CP-96,345. These data support a model in which the binding environments for WIN 51708 and CP-96,345 in part differ.