Blood platelets contain laminin-8 (alpha4beta1gamma1), a recently described laminin isoform, but the origin of platelet laminin is at present unknown. Laminin of platelets could be synthesized by megakaryocytes or, alternatively, endocytosed from plasma or other sources. In the present study, the synthesis and presence of laminin-8 in erythromegakaryocytic HEL and DAMI cells were explored. In HEL cells, transcripts for alpha4, beta1, and gamma1 laminin chains were readily detected by RT-PCR. Immunofluorescence flow cytometry demonstrated reactivity of mAbs to laminin beta1 and gamma1 chains with permeabilized cells. Metabolic labeling of HEL cells using [(35)S]methionine and [(35)S]cysteine followed by immunoprecipitation with monoclonal antibodies to beta1 and gamma1 chains revealed bands of approximately 220 and 200 kDa. In the HEL cell lysate, polypeptides of 220 and 200 kDa were recognized by monoclonal antibodies to laminin beta1 and gamma1 chains, respectively, whereas immunoaffinity-purified rabbit antibodies to laminin alpha4 chain gave inconclusive results. However, following immunoaffinity purification on a laminin beta1 antibody-Sepharose column, a 200-kDa band was readily detected by the antibodies to laminin alpha4 chain. Similar results were obtained with DAMI cells. The size of laminin chains of HEL/DAMI cells was similar, though not identical, to the one of platelets, and the alpha4 chain was noncovalently associated to disulfide-bonded beta1gamma1 heterodimer, as in platelets. We conclude that erythromegakaryocytic cells synthesize laminin-8.
Copyright 2000 Academic Press.