Platelet adhesion to cyanogen-bromide fragments of collagen alpha 1(I) under flow conditions

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.     

Polyclonal hematopoietic reconstitution in leukemia patients at remission after suppression of specific gene rearrangements [see comments]

Clonality studies of hematopoietic reconstitution after remission were performed in 24 female patients (pts) with leukemias characterized by specific molecular markers. At diagnosis, 13 pts had promyelocytic leukemia (PML) retinoic acid receptor-alpha (RAR-alpha)-rearranged acute promyelocytic leukemia (APL), 8 Philadelphia positive (Ph'+) break-point cluster region (BCR+) chronic myeloid leukemia (CML), and 3 Ph'+ (BCR+) acute lymphoblastic leukemia (ALL). All pts were analyzed at presentation and after Southern blot suppression of specific rearrangements after various treatments, including conventional chemotherapy, autologous or allogeneic bone marrow transplant (BMT), all-trans retinoic acid, and alpha-2b interferon. DNA from BM samples collected at diagnosis and, during remission phases, were subjected to Southern blot analysis with the M27 beta probe to detect X chromosome methylation differences, and with BCR, in CML and ALL cases, or PML/RAR- a probes for gene rearrangements, in APL cases. Twenty-one of the 24 pts had polyclonal methylation patterns at remission, together with disappearance of the specific rearrangement, whereas 3 pts retained the same single unmethylated DXS255 allele detected at diagnosis despite no evidence of gene rearrangement. Concerning these 3 pts, such an apparently clonal pattern was also observed in one case in T lymphocytes and skin-derived DNA; in a second case in BM fibroblasts and T lymphocytes; and, in the third case, in blood mononuclear cells obtained from her healthy female BM donor. All these 3 pts are in unmaintained clinical and cytogenetic remission after more than 20 months off therapy. These data suggest that (1) polyclonal and presumably normal hematopoiesis occurs in APL, CML, and Ph'+ ALL pts once the major burden of leukemic cells carrying a specific rearrangement is suppressed by treatment; and (2) unbalanced X chromosome methylation patterns, or aberrant methylation of X chromosome regions may be observed in some cases, most likely reflecting constitutional features simulating a clonal picture.