Experimental Hematology - Articles in Press

Comparison between an artificial neural network and logistic regression in predicting acute graft-versus-host disease following unrelated donor hematopoietic stem cell transplantation in thalassemia patients - Accepted Manuscript

2010-03-08

Abstract: Objective: There is growing interest in the development of prognostic models for predicting the occurrence of acute graft-versus-host disease (aGVHD) following unrelated donor hematopoietic stem cell transplantation (UD-HSCT). A high number of variables have been shown to play a role in aGVHD, but the search for a predictive algorithm is still ongoing. Artificial neural networks (ANNs) represent an attractive alternative to multivariate analysis for clinical prognosis. So far, no reports have investigated the ability of ANNs in predicting HSCT outcome.Methods: We compared the prognostic performance of ANNs with that of logistic regression (LR) in 78 beta-thalassemia major patients given UD-HSCT. Twenty-four independent variables were analyzed for their potential impact on outcome.Results: Twenty-six patients (33.3%) developed grade II-IV aGVHD. In multivariate analysis, homozygosity for donor KIR haplotype A (p=0.03), donor age (p=0.05) and donor homozygosity for the deletion of the HLA-G 14-bp polymorphism (p=0.05) were independently significantly correlated to aGVHD. The mean sensitivity of LR and ANNs (capability of predicting aGVHD in patients who developed aGVHD) in test data sets was 21.7% and 83.3% respectively (p<0.001); the mean specificity (capability of predicting absence of aGVHD in patients who did not develop aGVHD) was 80.5% and 90.1% respectively (p=NS).Conclusion: Although ANNs are unable to calculate the weight of single variables on outcome, they were found to have a better performance than LR. A combination of these two methods could be more efficient in predicting outcomes and help tailor GVHD prophylaxis regimens according to the predicted risk of each patient. Whether ANN technology will provide better predictive performance when applied to other data sets remains to be confirmed.

Runx1 Isoforms Show Differential Expression Patterns During Hematopoietic Development But Have Similar Functional Effects in Adult Hematopoietic Stem Cells - Accepted Manuscript

Grant A. Challen, Margaret A. Goodell — 2010-03-04

Abstract: Objective: RUNX1/AML1 is an essential regulator of hematopoiesis and has multiple isoforms arising from differential splicing and utilization of two promoters. We hypothesized that the rare Runx1c isoform has a distinct role in hematopoietic stem cells (HSCs).Methods: We have characterized the expression pattern of Runx1c in mouse embryos and human embryonic stem cell (hESC)-derived embryoid bodies using in situ hybridization, and expression levels in mouse and human HSCs by real-time PCR. We then determined the functional effects of Runx1c using enforced retroviral over-expression in mouse HSCs.Results: We observed differential expression profiles of RUNX1 isoforms during hematopoietic differentiation of hESCs. The RUNX1a and RUNX1b isoforms were expressed consistently throughout hematopoietic differentiation whereas the RUNX1c isoform was only expressed at the time of emergence of definitive HSCs. RUNX1c was also expressed in the AGM region of E10.5-11.5 mouse embryos, the region where definitive HSCs arise. These observations suggested that the RUNX1c isoform may be important for the specification or function of definitive HSCs. However, using retroviral over-expression to study the effect of RUNX1 isoforms on HSCs in a gain-of-function system, no discernable functional difference could be identified between RUNX1 isoforms in mouse HSCs. Over-expression of both RUNX1b and RUNX1c induced quiescence in mouse HSCs in vitro and in vivo.Conclusions: Although the divergent expression profiles of Runx1 isoforms during development suggest specific roles for these proteins at different stages of HSC maturation, we could not detect an important functional distinction in adult mouse HSCs using our assay systems.

Heterogeneous Expression And Function Of Il21r And Suceptibility To Il21-Mediated Apoptosis In Follicular Lymphoma Cells - Accepted Manuscript

2010-03-02

Abstract: Objectives: IL21, a member of the IL2 family, has anti-tumor activity and is now being tested in non-Hodgkin's lymphoma in combination with anti-CD20 antibodies. IL21 may either induce apoptosis or promote growth in different lymphoid malignancies. We therefore investigated the IL21/IL21R system in follicular lymphoma (FL) cells.Methods: IL21R expression was studied by RT-PCR, immunofluorescence and Western blot analyses. Apoptosis was measured by Annexin V-propidium iodide staining. Signaling via IL21R was studied using antibodies specific for phosphorylated JAK and STAT proteins by Western Blot.Results: IL21R was found on primary FL cells in 15/15 cases at diagnosis and IL21 increased apoptosis in 10/10 FL samples. However, cells from areas of diffuse growth in FL and from two diffuse lymphomas evolved from previous FL, showed low IL21R expression. The latter were also resistant to IL21-mediated apoptosis. Among lymphoma cell lines bearing the t(14;18) translocation, only 1 out of 7 showed increased apoptosis in response to IL21 stimulation. This cell line was IL21R-positive, whereas 5/6 of the non-responsive cell lines showed very low IL21R expression. Intriguingly, one of the IL21-resistant cell lines (DOHH2) expressed high levels of IL21R. Treatment with IL21 or IL4 up-regulated SOCS3 gene expression in the IL21-responsive cell line but not in DOHH2 cells, which showed defective JAK/STAT signaling in response to IL21, in relationship to the lack of JAK3 gene expression.Conclusion: These data indicate that low IL21R expression or defective signal transduction downstream IL21R may cause refractoriness to IL21-mediated effects in some FL cells.

Structural And Biological Properties Of Erythropoietin In Xenopus Laevis - Accepted Manuscript

2010-03-02

Abstract: Objective: Erythropoietin (EPO) and its receptor (EPOR) are key regulators of red blood cell production in mammals and fish. We aimed to investigate the structural and functional conservation of the EPO-EPOR system in amphibian erythropoiesis, using Xenopus laevis as a model.Methods: X. laevis epo (xlepo) cDNA was identified by referring to the X. tropicalis genome database. Biological activity of recombinant xlEPO expressed in COS-1 cells was evaluated using xlEPOR-expressing murine FDC/P2 cells and human EPO-dependent UT-7/EPO cells. Expression of xlepo mRNA in adult X. laevis tissues in the normal state and under the condition of phenylhydrazine-induced anemia was evaluated by real-time reverse-transcription polymerase chain reaction.Results: In the encoded protein, the positions of four cysteine residues were conserved; however, xlepo had only 38% identity with human EPO. N-glycosylation sites were absent. Recombinant xlEPO induced proliferation of cell lines expressing xlEPOR and UT-7/EPO, confirming biological activity and cross-species reactivity. Despite little primary amino acid sequence similarity, the evolutionary highly conserved sequence NFLRGK was identified in the EPOR-binding site 1 region as in the human EPO protein. Strong expression of xlepo mRNA was detected in the lung and liver, especially in fractionated hepatocytes. No marked increase in xlepo expression was seen in the lung and liver of PHZ-induced anemic X. laevis.Conclusion: We confirmed that xlEPO is the ligand to the previously reported xlEPOR in X. laevis. xlEPO shares structural and functional similarities and differences with mammalian counterparts, and regulation of xlepo expression and its influence on the erythropoietic system appears to be unique.

F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: Proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents - Accepted Manuscript

Norma E. Fox, Jihyang Lim, Rose Chen, Amy E. Geddis — 2010-02-25

Abstract: Objective: To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists.Methods: We created cell line models of type II c-Mpl mutations identified in CAMT. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing WT or F104S c-Mpl in response to thrombopoetin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD 4665) domains of the receptor by MTT assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species.Results: Cells expressing F104S c-Mpl proliferated in response to LGD 4665 but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD 4665 stimulates signaling with delayed kinetics in both WT and F104S c-Mpl expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen bonding interactions that are critical for binding to thrombopoietin.Conclusions: These findings suggest that a small subset of patients with CAMT might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin.

Natural Killer cell amplification for adoptive leukemia relapse immunotherapy: Comparison of 3 cytokines IL-2, IL-15 or IL-7 and impact on NKG2D, KIR2DL1 and KIR2DL2 expression - Accepted Manuscript

2010-02-22

Abstract: Objective: Natural Killer cells are a lymphocyte subset which, in an hematopoietic stem cell transplantation setting, mediates a graft versus leukemia effect without any graft versus host disease. We aimed to evaluate an isolation method which can be used with GMP grade reagents and to compare 3 cytokines for expansion in order to design future clinical protocols based on donor NK cell infusions to cure relapse after allograft.Methods: NK cells were enriched using a CD3 / CD19 depletion method and expanded for 13 days in presence of 2, 10 and 50 ng/ml IL2, IL15 or IL7. NK cell cytotoxicity was evaluated after isolation and culture. Expression of NKG2D and KIR2DL2 and KIR2DL1 was monitored during expansion.Results: Highly T and B cell depleted NK cells were obtained and enriched 2.6 fold. The optimal cytokine concentration for expansion was 10 ng/ml for IL2 or 50 ng/ml for IL15. Natural Killer cell cytotoxicity was significantly improved after an overnight incubation with 10 or 50 ng/ml IL2 or with 2, 10 or 50 ng/ml IL15, and after 13 days with 50 ng/ml IL15. The use of a combination of IL2 and IL15 showed no additional benefit and negative results were obtained with IL7. The 3 NK cell receptors were significantly upregulated after culture, mainly with IL2 or IL15.Conclusion: In our study, 10 ng/ml IL2 or 50 ng/ml IL15 were the optimal concentrations for expansion and were equivalent in significantly enhancing cytotoxicity and modifying NK cell receptor expression patterns.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells - Uncorrected Proof

2010-02-18

Objective: To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero.Materials and Methods: A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow.Results: Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well.Conclusion: We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow−derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas. Published by Elsevier Inc. on behalf of the Society for Hematology and Stem Cells

Persistence of donor-derived protein in host myeloid cells after induced rejection of engrafted allogeneic bone marrow cells - Uncorrected Proof

Toshiki I. Saito, Joji Fujisaki, Alicia L. Carlson, Charles P. Lin, Megan Sykes — 2010-02-17

Objective: In recipients of allogeneic hematopoietic stem cell transplantation to treat hematologic malignancies, we have unexpectedly observed anti-tumor effects in association with donor cell rejection in both mice and humans. Host-type CD8 T cells were shown to be required for these anti-tumor effects in the murine model. Because sustained host CD8 T-cell activation was observed in the murine bone marrow following the disappearance of donor chimerism in the peripheral blood, we hypothesized that donor antigen presentation in the bone marrow might be prolonged.Materials and Methods: To assess this hypothesis, we established mixed chimerism with green fluorescent protein (GFP)−positive allogeneic bone marrow cells, induced rejection of the donor cells by giving recipient leukocyte infusions, and utilized in vivo microscopy to follow GFP-positive cells.Results: After peripheral donor leukocytes disappeared, GFP persisted within host myeloid cells surrounding the blood vessels in the bone marrow, suggesting that the host myeloid cells captured donor-derived GFP protein.Conclusions: Because the host-vs-graft reaction promotes induction of anti-tumor responses in this model, this retention of donor-derived protein may play a role in the efficacy of recipient leukocyte infusions as an anti-tumor therapy.

Therapeutic efficacy of the pan-cdk inhibitor PHA-793887 in vitro and in vivo in engraftment and high-burden leukemia models - Uncorrected Proof

2010-02-17

Objective: The aim of the work was to determine and characterize, in vitro and in vivo, the therapeutic activity of PHA-793887, a new potent pan-cdk inhibitor, in the context of hematopoietic neoplasms.Materials and Methods: Thirteen leukemic cell lines bearing different cytogenetic abnormalities and normal hematopoietic cells were used in cytotoxicity and colony assays. The drug activity at the molecular level was analyzed by Western blotting. PHA-793887 was also tested in vivo in several leukemia xenograft models.Results: PHA-793887 was cytotoxic for leukemic cell lines in vitro, with IC50 ranging from 0.3 to 7 μM (mean: 2.9 μM), regardless of any specific chromosomal aberration. At these doses, the drug was not cytotoxic for normal unstimulated peripheral blood mononuclear cells or CD34+ hematopoietic stem cells. Interestingly, in colony assays PHA-793887 showed very high activity against leukemia cell lines, with an IC50 <0.1 μM (mean: 0.08 μM), indicating that it has efficient and prolonged antiproliferative activity. PHA-793887 induced cell-cycle arrest, inhibited Rb and nucleophosmin phosphorylation, and modulated cyclin E and cdc6 expression at low doses (0.2−1 μM) and induced apoptosis at the highest dose (5 μM). It was also effective in vivo in both subcutaneous xenograft and primary leukemic disseminated models that better mimic naturally occurring human disease. Interestingly, in one disseminated model derived from a relapsed Philadelphia-positive acute lymphoid leukemia patient, PHA-793887 showed strong therapeutic activity also when treatment was started after establishment of high disease burden.Conclusions: We conclude that PHA-793887 has promising therapeutic activity against acute leukemias in vitro and in vivo.

Fifth complement cascade protein (C5) cleavage fragments disrupt the SDF-1/CXCR4 axis: Further evidence that innate immunity orchestrates the mobilization of hematopoietic stem/progenitor cells - Uncorrected Proof

2010-02-15

Objective: Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/desArgC5a) in human HSPC mobilization.Materials and Methods: C5 cleavage fragments in the plasma were evaluated by enzyme-linked immunosorbent assay using human anti-desArgC5a antibody, and expression of the C5a/desArgC5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and expression of stromal cell−derived factor-1 (SDF-1)−degrading proteases that perturb retention of HSPC in bone marrow, namely matrix metalloproteinase (MMP)-9, membrane type 1−MMP, and carboxypeptidase M.Results: We found that plasma levels of desArgC5a are significantly higher in patients who are good mobilizers and correlate with CD34+ cell and white blood cell counts in mobilized peripheral blood. C5 cleavage fragments did not chemoattract myeloid progenitors (colony-forming unit granulocyte-macrophage), but desArgC5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34+ cells, but appeared on more mature myeloid precursors, monocytes, and granulocytes. Moreover, granulocyte colony-stimulating factor−mobilized peripheral blood mononuclear cells and polymorphonuclear cells had a significantly higher percentage of cells expressing CD88 than nonmobilized peripheral blood. Furthermore, C5a stimulation of granulocytes and monocytes decreased CXCR4 expression and chemotaxis toward an SDF-1 gradient and increased secretion of MMP-9 and expression of membrane type 1−MMP and carboxypeptidase M.Conclusion: C5 cleavage fragments not only induce a highly proteolytic microenvironment in human bone marrow, which perturbs retention through the CXCR4/SDF-1 axis, but also strongly chemoattracts granulocytes, promoting their egress into mobilized peripheral blood, which is crucial for subsequent mobilization of HSPC.

Erythrophagocytosis by angiogenic endothelial cells is enhanced by loss of erythrocyte deformability - Uncorrected Proof

2010-02-10

Objective: Angiogenic endothelial cells can function as phagocytes, and phagocytosis is initiated via the opsonin lactadherin. In this study, we examined the interaction between lactadherin-opsonized erythrocytes with reduced deformability and angiogenic endothelium, as loss of deformability is characteristic for suicidal and aged erythrocytes.Materials and Methods: We used the RGD-modified erythrocyte model and investigated the deformability parameter by cross-linking erythrocyte membranes through treatment with glutaraldehyde. Association in vitro with primary endothelial cells was detected by flow cytometry and visualized by light, fluorescent, and electron microscopy. Involvement of two crucial factors in phagocytosis, αv-integrins and Rho guanosine triphosphatase family member Rac1, was studied using small interfering RNA technology. Modified erythrocytes were administered in vivo into tumor-bearing mice to detect phagocytosis by endothelial cells.Results: Glutaraldehyde-treated (rigid) RGD-modified erythrocytes showed a strongly enhanced endothelial cell association compared to flexible RGD-modified erythrocytes. Knockdown by small interfering RNA lipoplexes of αv-integrins and Rac1 confirmed classical tethering and internalization of rigid RGD-erythrocytes. Upon in vivo administration, tumor endothelium showed pronounced erythrophagocytosis.Conclusion: The pronounced phagocytosis of opsonized erythrocytes with reduced deformability by angiogenic growth factor−activated endothelial cells evokes new insights in endothelial cell function and suggests a role for these endothelial cells in (hematological) disorders because of their capacity to clear disordered erythrocytes.

Effects of high-dose chemotherapy on bone marrow multipotent mesenchymal stromal cells isolated from lymphoma patients - Uncorrected Proof

2010-02-08

Objective: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT.Materials and Methods: We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing α−minimum essential medium plus 15% fetal bovine serum.Results: The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkin's disease and non-Hodgkin's lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group.Conclusions: Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.

Hypoxia mediates low cell-cycle activity and increases the proportion of long-term−reconstituting hematopoietic stem cells during in vitro culture - Corrected Proof

2010-02-04

Objective: Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)−1α.Materials and Methods: HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O2, and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1α was studied using methods of protein stabilization and gene silencing.Results: When long-term FLT3−CD34− Lin−Sca-1+c-Kit+ (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G0. Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21cip1, p27Kip1, and p57Kip2 increased in LSK cells by hypoxia, only p21cip1 was upregulated in FLT3−CD34−LSK cells. We could demonstrate that expression of p27Kip1 and p57Kip2 was dependent of HIF-1α. Surprisingly, overexpression of constitutively active HIF-1α or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability.Conclusions: Our results imply that hypoxia, in part via HIF-1α, maintains HSCs by decreasing proliferation and favoring quiescence.

Timing of captopril administration determines radiation protection or radiation sensitization in a murine model of total body irradiation - Uncorrected Proof

2010-01-29

Objective: Angiotensin II (Ang II), a potent vasoconstrictor, affects the growth and development of hematopoietic cells. Mixed findings have been reported for the effects of angiotensin-converting enzyme (ACE) inhibitors on radiation-induced injury to the hematopoietic system. We investigated the consequences of different regimens of the ACE inhibitor captopril on radiation-induced hematopoietic injury.Materials and Methods: C57BL/6 mice were either sham-irradiated or exposed to 60Co total body irradiation (0.6 Gy/min). Captopril was provided in the water for different time periods relative to irradiation.Results: In untreated mice, the survival rate from 7.5 Gy was 50% at 30 days postirradiation. Captopril treatment for 7 days prior to irradiation resulted in radiosensitization with 100% lethality and a rapid decline in mature blood cells. In contrast, captopril treatment beginning 1 hour postirradiation and continuing for 30 days resulted in 100% survival, with improved recovery of mature blood cells and multilineage hematopoietic progenitors. In nonirradiated control mice, captopril biphasically modulated Lin− marrow progenitor cell cycling. After 2 days, captopril suppressed G0−G1 transition and a greater number of cells entered a quiescent state. However, after 7 days of captopril treatment Lin− progenitor cell cycling increased compared to untreated control mice.Conclusion: These findings suggest that ACE inhibition affects hematopoietic recovery following radiation by modulating the hematopoietic progenitor cell cycle. The timing of captopril treatment relative to radiation exposure differentially affects the viability and repopulation capacity of spared hematopoietic stem cells and, therefore, can result in either radiation protection or radiation sensitization.

Antiplatelet antibodies in WASP(−) mice correlate with evidence of increased in vivo platelet consumption - Uncorrected Proof

2009-09-04

Objective: To study the role of antiplatelet antibodies in the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS).Materials and Methods: A flow cytometric method was developed for detection of serum antiplatelet antibodies via their binding to intact target platelets lacking surface antibodies. Platelets were labeled with 5-chloromethylfluorescein diacetate in order to track their clearance from the circulation. WASP(−)μMT(−/−) mice were generated by standard breeding methods.Results: Serum antiplatelet antibodies were detected in approximately 40% of WASP(−) males. Mean level of reticulated platelets is significantly increased in these antibody(+) males. While WASP(−) males show an approximately 50% reduction in platelet counts, 5% to 10% show a more severe thrombocytopenia associated with increased reticulated platelets, suggesting the presence of clearance-inducing antiplatelet antibodies. In support of that inference, 90% of the latter mice show detectable serum antiplatelet antibodies. The antibodies are primarily immunoglobulin G, and are also detected in >30% of CD47(−/−) males. WASP(−)μMT(−/−) males, which demonstrate no serum- or platelet-associated antibodies, show a degree of thrombocytopenia similar to that of WASP(−) males. Their platelet clearance rates remain accelerated—more so in WASP(−)μMT(−/−) than WASP(+)μMT(−/−) recipients.Conclusions: These findings suggest that platelet WASP deficiency results in an increase in platelet clearance rates by two mechanisms, i.e., an antibody-independent mechanism that largely requires WASP deficiency in trans, and an antibody-dependent mechanism that does not. Both an increased incidence of antiplatelet antibodies and an increased susceptibility to their effects contribute to antibody-dependent clearance of WASP(−) platelets.