Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the ␣IIb3 receptor. In this study we identified regions on integrin 3 that control 7E3 binding. Murine͞ human amino acid substitutions were created in two regions of the A domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human 3 in complex with ␣IIb. The introduction of both the human C177-C184 region and human W129 into murine 3 was necessary and sufficient to permit 7E3 binding to the human ␣IIb͞murine 3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 Å of each other in the crystal structure and close to the 3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that ␣IIb3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an ␣IIb3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with ␣IIb3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in ␣IIb3 adopting a more extended conformation.T he integrin ␣IIb3 and ␣V3 receptors are important in a number of physiologic and pathologic phenomena, including hemostasis, thrombosis, tumor angiogenesis, and bone resorption (1, 2). The murine mAb 7E3 (3) blocks ligand binding to both ␣IIb3 and ␣V3 receptors (4, 5). Abciximab is a mouse͞ human chimeric Fab fragment of the mAb 7E3 that inhibits ␣IIb3-mediated platelet aggregation and is approved for human use to prevent the ischemic complications associated with percutaneous coronary interventions (6).Previous studies of 7E3 binding to cells expressing recombinant ␣IIb3 receptors demonstrated that: (i) swapping select murine for human ␣IIb sequences does not decrease 7E3 binding (7), (ii) removing the specificity determining loop (SDL) (K156-G189 sequence) from 3 results in loss of 7E3 binding (8), (iii) swapping the murine for human C177-C184 sequence within the SDL region in 3 results in loss of 7E3 binding (7), and (iv) swapping the murine S129-T133 sequence for the human W129-N133 sequence results in partial loss of 7E3 binding (7).The above human͞mouse swapping studies identified regions within 3 that affect 7E3 binding, but the biochemical and functional properties of these chimeric receptors were not characterized. In addition, the W129-N133 region contains two amino acid differences between human 3 (3Hu) and mouse 3 (3M) and the C177-C184 region contains three amino acid differences (Table 1). To define further the regions on ␣IIb3 th...
The molecular basis of Glanzmann thrombasthenia (GT) was studied in 40 families from southern India. Of 23 identified mutations (13 in the alphaIIb (ITGA2B) gene and 10 in the beta3 (ITGB3) gene), 20 were novel and three were described previously. Three mutations in the beta3 gene-p.Leu143Trp (Leu117Trp), p.Tyr307Stop (Tyr281Stop), and p.Arg119Gln (Arg93Gln)-were detected in 12, three, and two families, respectively, with definite founder effects observed for the first two mutations. Alternative splicing was predicted in silico for the normal variant and a missense variant of the beta3 gene, and for 10/11 frameshift or nonsense mutations in alphaIIb or beta3. The prediction was confirmed experimentally for a c.2898_2902dupCCCCT mutation in exon 28 of the alphaIIb gene that induced exon skipping. Seven out of nine missense mutations substituted highly conserved amino acids buried in the proteins' cores, predicting structural abnormalities. Among these, a beta3 substitution, p.Cys39Gly (Cys13Gly) was found to cause intracellular degradation of the beta3 subunit, in contrast to previous findings that mutations at Cys435, the partner of Cys13 in a disulfide bond, cause constitutive activation of alphaIIbbeta3. The two patients with a beta3 Arg93Gln mutation had normal clot retraction, consistent with a recent finding that this substitution is associated with normal surface expression of alphaIIbbeta3. In conclusion, this study demonstrates that a variety of mutations account for GT in southern Indian patients, provides new insights into mRNA splicing, and highlights the role of specific amino acids in structure-function correlations of alphaIIbbeta3.
While isolated factor VII (FVII) deficiency is being more frequently diagnosed owing to improved preoperative screening procedures, there is no specific guideline for perioperative management of such patients. To complicate the issue, FVII activity levels seem to correlate less well with the risk of hemorrhage than the patient's past and family bleeding history do. We have devised expert consensus recommendations for managing such patients perioperatively, taking into consideration the personal and family bleeding history, the FVII activity level and the inherent bleeding risk of the procedure itself. We hope that clinicians will find this a useful tool in the decision-making process, thereby limiting the use of recombinant factor VIIa to those who need it most, and preventing possible thrombotic complications in those without a strong indication for its use.
Background: Increased baseline white blood cell count (WBC) has been associated with vaso-occlusive complications in sickle cell disease (SCD), raising concern for the use of mobilizing agents that increase WBC count for hematopoietic progenitor cell (HPC) collection for gene therapy. Indeed, G-CSF, which predominantly increases neutrophil counts, when administered to SCD patients can precipitate life-threatening vaso-occlusion. Such vaso-occlusion, however, does not always correlate with degree of white blood cell count elevation (Fitzhugh C et al, Cytotherapy 2009) and G-CSF is associated with increases in neutrophil activation and adhesion. This raises the question whether WBC might be a surrogate for other proposed contributors to vaso-occlusion, such as activation of white cells, platelets, and coagulation. We examined this issue in the context of a clinical study of HPC mobilization with a single dose of plerixafor alone in clinically stable SCD patients. Materials and Methods: 14 patients (13 SS, 1 SB0, median age 31 with range 21-46) have completed the study so far (6 patients at 80 ug/kg, 3 patients at 160 ug/kg, and 5 patients at 240 ug/kg). 10 patients were on hydroxyurea (HU) at a median dose of 25 mg/kg (range 16-28 mg/kg) with a mean HbF of 17% versus 7% for the non-HU treated patients. Only one patient (non-HU treated) had received recent red cell transfusion (for leg ulcers) and had HbA (54%) present. Whole blood was collected at means of 4 hr pre-dose and 12 hr post-dose for activation marker studies by flow cytometry (cell studies) or ELISA (coagulation studies) as listed in Table 1. Results: Increases in absolute total white cell, neutrophil, monocyte, and lymphocyte concentrations in whole blood with plerixafor treatment were all highly significant (Table 2, p < 0.002), without significant differences between HU- and non-HU treated patients or between dose levels. Twelve of 14 patients, however, did not experience any significant adverse events from plerixafor administration. Absolute neutrophil counts (ANC) were strongly correlated with absolute concentrations (but not percentages) of activated β2 integrin neutrophils (r= 1.0) and activated Mac-1 neutrophils (r= 0.8) but no other activation markers. We observed significant plerixafor-associated increases in activation markers (Figure 1) only for ANC subsets positive for activated β2 integrin (1.7± 0.44 fold, mean ± SD, p=0.002) and activated Mac-1 (1.7 ± 1.2 fold, p=0.05), which the correlations with ANC partly explain. The activated Mac-1 significance, however, disappeared with Bonferroni correction. There were no significant differences in activation markers between dose levels or between HU- and non-HU treated patients. There were moderate correlations between activated β2 integrin or activated Mac-1 and absolute reticulocyte count (r= 0.6 for both) and HbF (r= -0.5 for both) but not between these activation markers and LDH. Two patients had pain crises starting at 48 hr (80 ug/kg dose) and 81 hr (240 ug/kg dose) post-plerixafor administration; their pre and post-dose (12 hr) white counts are shown in Table 3. The fold-increase in activation markers for these 2 patients is summarized in Figure 2 (for each activation marker, color-coded arrows point to the two patients) Conclusion: As with G-CSF, elevation of WBC or their subsets with plerixafor is not necessarily associated with vaso-occlusion, as manifested by pain crises. In our study, however, ANC elevation correlated with absolute concentrations of neutrophils positive for activated β2 integrin and activated Mac-1, which were the only activation markers significantly increased with plerixafor. These activation markers in turn correlated with reticulocyte count and HbF, which influence SCD vaso-occlusive severity. Vaso-occlusion as manifested by pain crises, however, was not consistently associated with the highest fold increases in ANC or activation markers. Therefore it remains unclear which bioassays correlate with vaso-occlusion associated with plerixafor mobilization. Disclosures No relevant conflicts of interest to declare.
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