Erythromegakaryocytic Cells Synthesize Laminin-8 (α4β1γ1)

Abstract

Blood platelets contain laminin-8 (α4β1γ1), 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 α4, β1, and γ1 laminin chains were readily detected by RT-PCR. Immunofluorescence flow cytometry demonstrated reactivity of mAbs to laminin β1 and γ1 chains with permeabilized cells. Metabolic labeling of HEL cells using [35S]methionine and [35S]cysteine followed by immunoprecipitation with monoclonal antibodies to β1 and γ1 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 β1 and γ1 chains, respectively, whereas immunoaffinity-purified rabbit antibodies to laminin α4 chain gave inconclusive results. However, following immunoaffinity purification on a laminin β1 antibody–Sepharose column, a 200-kDa band was readily detected by the antibodies to laminin α4 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 α4 chain was noncovalently associated to disulfide-bonded β1γ1 heterodimer, as in platelets. We conclude that erythromegakaryocytic cells synthesize laminin-8.