ALBERT

Description: We have previously designed and investigated novel allosteric effectors of hemoglobin (AEHs), as potential targeted treatment of sickle cell disease (SCD). In general, AEHs transiently covalently bind to hemoglobin (Hb), increase its affinity for O2, increasing the fraction of oxygenated sickle Hb (HbS), thus reducing HbS polymerization and countering red blood cell (RBC) sickling. In the current study, we designed a novel class of AEH molecules, incorporating a secondary mechanism of action (MOA), which is independent of Hb O2-affinity by interacting with the F-helix of deoxygenated HbS to directly destabilize its polymerization. Here, we report current results from our in-vitro and in-vivo studies with a representative AEH compound (PP-14). First, we assessed the anti-sickling properties in-vitro by incubating 0.5, 1, and 2 mM of PP-14 with whole blood suspensions from a subject with homozygous SCD (SS, hematocrit: 20%) under hypoxic conditions, with subsequent RBC sickling assessment by microscopy. Next, we subjected the samples to anoxia (100% N2 gas) to demonstrate the O2-affinity-independent antisickling mechanism. Subsequently, we tested residual samples for the degree of Hb modification (i.e., HbS-AEH adduct formation) and O2-affinity (p50) shifts. In a second experiment to further assess the secondary MOA, we subjected SS blood samples treated with various concentrations of PP-14 to hypoxia in the Hemox analyzer, which permitted us to obtain aliquot samples at defined pO2 values to establish pO2-dependent sickling. Additionally, we conducted in silico and in-vitro ADME studies to evaluate possible metabolic inhibition of a panel of CYP enzymes. Finally, we conducted a preliminary in-vivo PK/PD study in wild-type mice administered single doses of PP-14 via the oral (P.O.: 100-200 mg/kg) and intraperitoneal (I.P.: 75 mg/kg) routes. Serial blood samples were collected for up to 52 h after P.O., and up to 30 h after I.P. administration, and samples were assayed to quantify PP-14 concentrations. Residual blood samples were assayed for in-vivo Hb-AEH adduct formation, and the corresponding change in O2-affinity (Δp50, %). Our in-vitro studies demonstrated concentration-dependent inhibition of cell sickling of 25.5±11%, 44.4±3.8% and 90.8±1%, at 0.5, 1 and 2 mM of PP-14, respectively. HbS was modified correspondingly (38.9±9%, 55.7±4.9 %, and 92.4±9.8%), and was correlated linearly with the left-shift in OEC (Δp50 values of 11.3±5.1%, 29.0±13.2%, and 67.5±8.2%). Importantly, the antisickling effect was sustained under anoxic conditions (100% N2), strongly supporting the notion of a secondary, O2-affinity-independent MOA. Furthermore, we observed a dose-dependent delay in sickling, with initiation of sickling recorded at a pO2 level of 40 mmHg in absence of PP-14; and at 30 and 20 mmHg at 0.5, and 1 mM PP-14 concentrations, respectively. Complete inhibition of sickling was observed at 2 mM PP-14 through the lowest recorded pO2 level of 1.5 mmHg, a unique, effect not previously observed in any analogous AEH. In-vitro partitioning studies demonstrated that 〉90% of PP-14 partitioned into the RBC compartment when whole blood was incubated with 100-300 µM concentrations. Metabolic studies using pooled human liver microsomes (HLM) and isozyme-specific probe substrates suggested that up to 100 µM PP-14 did not inhibit CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 or CYP2B6. Wild-type mice after I.P. administration of PP-14 (75 mg/kg, n=5) showed peak concentrations in blood at 7 hrs (416.3±81.2 µM), with corresponding PD effects (Δp50 of 41.6±13.5%; modified Hb levels of 43.6±8.0%). Orally-treated mice had peak drug concentrations after 10-24 hrs, (~150 µM at 200 mg/kg, n= 2), with corresponding PD effects (Δp50 of 36.5±7.0%; modified Hb levels of 28.8±4.9%), which declined by 52 hrs. Overall, our data confirm that PP-14 is novel antisickling AEH with a secondary, O2-independent MOA in addition to the primary O2-dependent effect, as demonstrated by the inhibition of sickling under anoxic conditions. Additionally, PP-14 showed: excellent partitioning into the RBC compartment; acceptable in-silico ADME properties and in-vivo oral bioavailability; PD effects; and low liability for metabolic drug-drug interactions. Further studies to investigate formal detailed pharmacokinetic properties, and biological activity after single- or repeat-doses in a SCD mouse model are ongoing. Disclosures Safo: Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Pagare:Virginia Commonwealth University: Patents & Royalties. Ghatge:Virginia Commonwealth University: Patents & Royalties. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment. Zhang:Virginia Commonwealth University: Patents & Royalties. Venitz:Virginia Commonwealth University: Patents & Royalties. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent.

Description: Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive complications (VOCs); however, there are no objective measures for VOC as a clinical endpoint. Vaso-occlusion results from processes that reduce blood flow in the microvasculature, including red cell stickiness and erythrocyte sickling. These processes lead to pain, chronic organ damage, and decreased life expectancy. The decision to seek medical contact varies amongst patients. When VOCs are managed at home valuable information remains unknown. We designed a longitudinal, observational study to capture adhesion data at home and in a hospital setting. The objective of this study was to determine whether a standardized, flow-based adhesion bioassay is capable of identifying VOCs occurring in SCD patients with varying degrees of medical contact. SCD patients (n=33) were evaluated over a 6-month period. Blood samples were collected every 3 weeks; when patients report a VOC corresponding blood samples are collected and steady state samples are resumed. During 6 months of evaluation, longitudinal measures of pain and clinical laboratory and biomarker samples from SCD patients (+/- hydroxyurea therapy) in steady state to VOC were studied. Blood samples were collected in sodium citrate from SCD subjects at steady state and during VOCs. Blood samples were perfused through VCAM-1-coated microchannels at standard physiologic flow conditions (1dyne/cm2, 1.67Hz). An adhesion index was established by quantifying adherent cells within a standard viewing area (cells/mm2), and could be obtained within 6-9 min. Adhesion indices varied from sample-to-sample at baseline (n=289; mean = 355 ± 235; median = 297 cells/mm2) and during VOC (n=59, mean=416±233, median=390). Repeated measures of adhesion over 6 months reveals significant intra-patient associations with C-reactive protein (CRP, n=335, r=0.16; p=0.006), lactose dehydrogenase (LDH, n=336, r=0.12; p=0.032), white blood cells (WBC, n=341, r=0.13; p=0.019), and reticulocyte percent (n=336, r=0.37, p

Description: Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the 1, 2, β1, and γ1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin 5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin 5 chain. © 1998 by The American Society of Hematology.

Description: Background: Sickle erythrocyte adhesion and membrane fragility contribute to vaso-occlusion and downstream tissue and organ ischemia in sickle cell disease (SCD). Vepoloxamer is an amphipathic triblock copolymer with multi-mechanistic properties believed to improve these pathophysiologic consequences, by sealing damaged cell membranes and inhibiting hydrophobic cellular adhesive interactions. Vepoloxamer reduced both acute vaso-occlusive crisis duration and total opioid analgesic requirements in previous clinical studies. A phase 3 clinical trial of vepoloxamer in acute vaso-occlusive crises is ongoing. Currently, there are no standardized clinical assays to assess membrane properties such as adhesion and fragility that vepoloxamer is believed to target. We evaluated if and to what extent vepoloxamer affected adhesion, thrombosis, and membrane fragility in individual patient samples in our standardized microfluidic flow-based whole blood assays. Methods: Blood was obtained from pediatric homozygous sickle cell patients at steady state (n=12). Patients were first characterized by measuring the extent of whole bloodadhesion to vascular cell adhesion molecule (VCAM-1) in a standardized microfluidic flow-based assay. Dynamic adhesive interactions (rolling or sliding) of whole blood and isolated leukocytes to P-selectin were assessed by measuring absolute rolling cell number, cell rolling flux (number of rolling cells per total number of flowing cells), and cell rolling velocity at 0.3dyn/cm2. Detachment assays were performed by exposing adherent cells to vepoloxamer (10 mg/mL) under physiological flow conditions. Erythrocyte membrane fragility was evaluated based on mechanical stress-induced hemolysis at 3 min (Hem3min, %). Results: Vepoloxamer reduced whole blood adhesion to VCAM-1 at 0.1 mg/mL by 18% (p = 0.0015), at 1.0 mg/mL by 69% and at 10.0 mg/mL by 79% (in both cases, p 〈 0.001), and to HUVECs by 〉50% 0.1mg/mL (p=0.003, n=10). Vepoloxamer (10mg/mL) did not significantly affect cell-rolling flux in dynamic adhesion assays to P-selectin (p = 0.51 and 0.94, isolated leukocytes and whole blood, respectively). Vepoloxamer reversed established cell adhesion by 〉 30% in 3 or 10 patient samples tested, although the total average difference in percent detachment did not reach statistical significance (16.3%vs 29.6%, p=0.42, n=10). Finally, vepoloxamer reduced shear-induced hemolysis compared to untreated blood samples (35.2 % vs. 44.6% control, p =0.033). Conclusion: Vepoloxamer can significantly reduce firm cell adhesion to both VCAM-1 and HUVECS under physiologic flow conditions. Shear-induced hemolysis is also reduced in a statistically significant manner, consistent with the proposed membrane protecting effects of vepoloxamer. Interestingly, P-selectin mediated rolling adhesion was not significantly affected. This may suggest that vepoloxamer may have distinct inhibitory effects to VCAM-1 vs. P-selectin. The current findings suggest that combinatory assessment of the response of adhesive properties and membrane fragility to vepoloxamer treatment may facilitate selection of patients most likely to benefit from vepoloxamer therapy. The microfluidic flow-based platform presented in this study may have the potential to predict and monitor individual patient response to anti-adhesive therapies such as vepoloxamer. There are few reliable biomarkers that objectively predict and assess vaso-occlusive potential, thus further clinical study of this microfluidic flow-based platform are warranted. Disclosures White: Functional Fluidics: Employment, Equity Ownership. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment, Equity Ownership. Benjamin:Mast Therapeutics: Consultancy. Gao:Functional Fluidics: Employment, Equity Ownership. Emanuele:Mast Therapeutics: Employment.

Description: Sickle cell disease (SCD) is caused by a point mutation in the beta-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Among other abnormalities, sickle red blood cells (RBCs) are more adhesive than normal RBCs. Sickle RBC adhesion is an important pathway leading to both VOC and hemolysis: adhesive interactions promote the formation of blood flow-obstructing heterocellular aggregates that induce ischemic tissue damage and slow the transit of RBCs through the vasculature, promoting sickle hemoglobin polymerization and hemolysis. The capacity of sickle RBCs to adhere to endothelium positively correlates with disease severity. Interventions that reduce sickle RBC adhesion, in particular that of reticulocytes, by blocking specific molecular targets may limit or prevent disease sequelae. Very Late Antigen – 4 (VLA-4 or α4β1 integrin) is an important integrin on reticulocytes that mediates adhesive interactions to endothelial and plasma vascular cell adhesion molecule - 1 (VCAM-1), plasma fibrinogen, and other ligands. VLA-4 is expressed on the surface of sickle reticulocytes, with levels decreasing during maturation such that mature RBCs do not express surface VLA-4. Natalizumab is a recombinant humanized antibody that binds to the α4 subunit of VLA-4 and is used to treat multiple sclerosis (MS) and Crohn’s Disease (CD) by preventing leukocyte trafficking into tissues at sites of inflammation. We investigated the ability of natalizumab to block VLA-4 in the context of sickle whole blood, and evaluated the effect on sickle reticulocyte, mature erythrocyte, and leukocyte adhesion during physiologic flow conditions. Whole blood samples obtained from SCD donors were analyzed for saturation binding of natalizumab to surface VLA-4 on leukocytes and reticulocytes using flow cytometry. Up to 20% of reticulocytes from SCD donors (n=13) were positive for VLA-4 surface staining, whereas VLA-4 was undetectable on the surface of reticulocytes from healthy donors (n=4). VLA-4 on SCD reticulocytes and mononuclear leukocytes was saturated by natalizumab concentrations lower than known plasma trough concentrations achieved in MS and CD patients after natalizumab treatment, with SCD reticulocyte EC50 = 0.11 ± 0.01 μg/mL and SCD leukocyte EC50 = 0.16 ± 0.01 μg/mL (n=6). This translates to a binding affinity of 0.7 to 1.3 nM, similar to that found for healthy donor leukocytes. Both whole blood cells and isolated leukocytes from SCD donors adhered to immobilized VCAM-1 during physiologic flow conditions simulating post-capillary venules (shear stress =1 dynes/cm2) using a microfluidic flow-based adhesion system. Leukocytes from SCD donors adhered more to VCAM-1 (Mean = 20.7 + 10.0 from n=7) than leukocytes from healthy donors (Mean = 5.0 + 1.4 from n=5). The adhesion of leukocytes and reticulocytes to VCAM-1 was blocked by natalizumab in a dose-dependent manner and as a function of natalizumab saturation of cell surface VLA-4. Therapeutic IgG4 antibodies, including natalizumab, undergo chain shuffling in vivo with endogenous IgG4, leading to mono-specific IgG4 molecules, with one Fab arm specific to the antigen it was raised against. Compared to divalent natalizumab, monovalent chain-shuffled natalizumab bound to SCD reticulocytes at a7-fold higher EC50 and inhibited SCD reticulocyte adhesion to VCAM-1 at higher antibody concentrations (10 and 1 µg/mL). While increased, these values are still below trough natalizumab levels achieved in natalizumab-treated patients. In summary, natalizumab bound VLA-4 on the surface of SCD reticulocytes and leukocytes similarly to healthy donor leukocytes and blocked adhesion of SCD reticulocytes and leukocytes to immobilized VCAM-1 under shear conditions. Natalizumab binding and adhesion inhibition occurred at plasma concentrations similar to those seen in natalizumab treated MS and CD patients. Based on these findings, natalizumab may have potential as an anti-adhesive therapy for SCD. Further clinical studies are needed to evaluate the safety and efficacy of natalizumab in SCD. Disclosures Hines: Biogen Idec: Research Funding. Krishnamoorthy:Biogen Idec: Employment, Equity Ownership. White:Biogen Idec: Research Funding. Gupta:Biogen Idec: Employment, Equity Ownership. van der Flier:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership. Hobbs:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership.

Description: The possible role of physiologic stress hormones in enhancing adhesion of sickle erythrocytes (SS RBCs) to endothelial cells (ECs) in sickle cell disease (SCD) has not been previously explored. We have now found that up-regulation of intracellular cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA) by epinephrine significantly increased sickle but not normal erythrocyte adhesion to both primary and immortalized ECs. Inhibition of serine/threonine phosphatases also enhanced sickle erythrocyte adhesion at least partially through a PKA-dependent mechanism. Adhesion was mediated through LW (intercellular adhesion molecule-4 [ICAM-4], CD242) blood group glycoprotein, and immunoprecipitation studies showed that LW on sickle but not on normal erythrocytes undergoes increased PKA-dependent serine phosphorylation as a result of activation. The major counter receptor for LW was identified as the αvβ3 integrin on ECs. These data suggest that adrenergic hormones such as epinephrine may initiate or exacerbate vaso-occlusion and thus contribute to the association of vaso-occlusive events with physiologic stress.

Description: Background: The cause and continuation of vaso-occlusion in sickle cell disease (SCD) are fueled by the sickle-Red Blood Cells interactions with multiple other cell populations, promoting inflammation, obstructing the vasculature, and injuring the endothelium, leading to broad manifestations that affect most vital organs. Recent studies have identified multiple cellular components and molecular factors that contribute to the pathophysiology of SCD as reviewed by Zhang et al 2016 in Blood. It is likely that a multi-targeted approach for addressing SCD vaso-occlusion will be required to achieve the best clinical outcome. Sevuparin (DF02), a novel drug in Phase 2 for acute treatment of vaso-occlusive crisis in SCD (NCT02515838), is a polysaccharide blocking abnormal adhesion and thereby normalizing obstructed blood flow. In vitro and in vivo studies have shown potent anti-adhesive effects with a multimodal mechanism of action. In this study, we evaluate the effects of sevuparin on the adhesion of sickle whole blood from individual patients to endothelial cells (HUVECs) and vascular cell adhesion molecule-1 (VCAM-1) (Flow Firm Adhesion) and sickle-leukocyte rolling adhesion on L-selectin (Flow Dynamic Adhesion) using a standardized microfluidic flow-based adhesion assay. Methods: Blood was obtained from homozygous SCD patients (n = 12, age range 15-25yrs) in sodium citrate after obtaining informed consent. A comprehensive assessment of the effect of sevuparin on whole blood adhesive properties during simulated blood flow was assessed using standardized Flow Firm Adhesion and Flow Dynamic Adhesion assays (Functional Fluidics, Detroit MI). Flow Firm Adhesion: Whole blood firm adhesion was measured during physiologic flow in microfluidic channels (Fluxion-Bioflux 1000, San Francisco, CA) coated with either VCAM-1 or cultured HUVECs. HUVECs were activated by TNF-alpha (25ng/mL x 24 hrs.) and Histamine (100mM x 10min) prior to the assay. Whole blood was treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of whole blood adhesion index (cells/mm2) to sevuparin was measured. Flow Dynamic Adhesion: Rolling adhesion of isolated sickle-leukocytes on an L-selectin coated microfluidic channel was measured during physiologic flow. Isolated sickle-leukocytes were treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of rolling cell density (cells/mm2), rolling cell percentage (%), and average rolling velocity (µm/s) to sevuparin was assessed. Cell identification and tracking of rolling were digitally analyzed. Results:Statistically significant inhibition of sickle whole blood adhesion to HUVECs was observed at 3.0 µg/mL of sevuparin (p

Description: The vasoocclusive crisis is the major clinical feature of sickle cell anemia, which is believed to be initiated or sustained by sickle (SS) red blood cell (RBC) adhesion to the vascular wall. SS RBCs, but not unaffected (AA) RBCs, adhere avidly to multiple components of the vascular wall, including laminin. Here we report a novel role for epinephrine and cyclic adenosine monophosphate (cAMP) in the regulation of human SS RBC adhesiveness via the laminin receptor, basal cell adhesion molecule/Lutheran (BCAM/Lu). Our data demonstrate that peripheral SS RBCs contain greater than 4-fold more cAMP than AA RBCs under basal conditions. Forskolin or the stress mediator epinephrine further elevates cAMP in SS RBCs and increases adhesion of SS RBCs to laminin in a protein kinase A (PKA)–dependent manner, with the low-density population being the most responsive. Epinephrine-stimulated adhesion to laminin, mediated primarily via the β2-adrenergic receptor, occurred in SS RBC samples from 46% of patients and was blocked by recombinant, soluble BCAM/Lu, implicating this receptor as a target of cAMP signaling. Thus, these studies demonstrate a novel, rapid regulation of SS RBC adhesion by a cAMP-dependent pathway and suggest that components of this pathway, particularly PKA, the β2-adrenergic receptor, and BCAM/Lu, should be further explored as potential therapeutic targets to inhibit SS RBC adhesion.

Description: Vaso-occlusive episodes (VOEs) in sickle cell disease (SCD) are characterized by severe pain and microvascular obstruction precipitated by adhesive interactions among endothelial cells (ECs), red blood cells, white blood cells (WBCs), and platelets. P-selectin contributes to microvascular occlusion by supporting abnormal interactions between blood cells and the vascular endothelium. Inhibition of p-selectin mediated adhesion reverses acute, VOEs in SCD mice. Additionally, SCD mice, deficient in either P- or E-selectin, develop fewer vascular occlusions. These reports led to the development of anti-adhesive therapies targeting p-selectin. For example, Crizanlizumab, an anti-P-selectin antibody, reduced the annual rate of VOEs prospectively and Rivipansel, a pan-selectin inhibitor, decreased the length of active VOEs. Objective measures of VOEs are nonexistent. We developed a standardized, flow-based adhesion assay to measure p-selectin mediated adhesion in SCD. Whole blood (Wb) and isolated WBC samples were perfused (1.0 dynes/cm2, 1.67Hz) through micro-fluidic channels. Adhered cells were enumerated and an adhesion index was calculated (total # of adhered cells/mm2). Blood samples were collected from SCD subjects (n = 35) every 3 weeks for 6 months at baseline and within 24 and 48hrs of a patient-reported VOE. Baseline and VOE states were confirmed by an electronic patient reported outcomes (ePRO) device, as previously described. VOEs were separated into 2 groups: VOEs managed at-home (Home-VOE) or during ER visit and/or hospitalization (Contact-VOE). WB (n = 288; mean=42 ± 48 cells/mm2, median=31 cells/mm2) and WBC (n = 282; mean=163 ± 149 cells/mm2; median= 116 cells/mm2) adhesion to p-selectin varied in SCD subjects (n=35) at baseline. WBC adhesion to P-selectin was significantly (p=0.36) higher during VOE (n=59; 205 ± 179) when compared to baseline (n = 282; mean=163±149 cells/mm2, median=116 cells/mm2). For individual subjects, the geometric mean for WBC adhesion to P-selectin was significantly (p = 0.023) higher for VOEs vs. baseline (VOE= 109, n= 23, 95% confidence interval 94-126 vs. baseline= 148, n = 35, 95% confidence interval 114-192). Additionally, WB adhesion to p-selectin correlated with VOE frequency (time to 2ndVOE; r = -0.71, n = 10, p = 0.02 or time between 1stand 2ndVOEs; r = -0.68, n = 10, p = 0.032). WB adhesion to p-selectin positively correlated with C-reactive protein (CRP; r = 0.12, p = 0.047), reticulocyte % (r = 0.15, p = 0.017), and WBC counts (r = 0.26, p 〈 0.0001) at baseline. WBC adhesion to p-selectin positively correlated with hematocrit (Hct; r = -0.37, p = 0.027) and reticulocyte % (r = 0.44, p = 0.0058) during VOEs managed at home; WB adhesion to p-selectin positively correlates with CRP (r = 0.44, p = 0.011) and WBC count (r = 0.63, p 〈 0.0001); WB adhesion to p-selectin positively correlated with WBC count (r = 0.51, p = 0.0021) during contact-VOE. These data confirm the normal range and longitudinal variability of SCD adhesion to p-selectin at baseline and during VOEs. WB adhesion to p-selectin was inversely related to VOE frequency although there was no difference between baseline and VOE samples. These data suggest that WB adhesion to p-selectin may be a better predictor of disease severity and not SCD symptomology. In contrast, WBC adhesion to p-selectin differentiated baseline from VOE samples suggesting this assay may be a more suitable reflection of SCD clinical state. Both WB and WBC adhesion to VCAM-1 correlates with biomarkers of hemolysis and inflammation. P-selectin may be selective for the identification of pro-adhesive phenotypes directly related to hemolytic and inflammatory influencers in SCD. Disclosures Liu: Functional Fluidics: Equity Ownership. Gao:Functional Fluidics: Equity Ownership. White:functional fluidics: Equity Ownership. Callaghan:Alnylam Pharmaceuticals: Equity Ownership; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Employment, Membership on an entity's Board of Directors or advisory committees; Hema Pharmaceuticals: Honoraria; Grifols: Honoraria; Sancilio Pharmaceuticals Company: Employment; Global Blood Therepeutics: Employment; Roche/Genentech: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Honoraria; Octapharma: Honoraria; Pfizer: Employment, Honoraria, Research Funding; Amgen: Employment. Hines:functional fluidics: Equity Ownership.

Description: Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the 1, 2, β1, and γ1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin 5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin 5 chain. © 1998 by The American Society of Hematology.