Laminin of platelets and leukocytes : molecular characterization, integrin receptors and functional roles

Abstract

Laminins are multifunctional, multidomain glycoproteins found predominantly in basement membranes. They are [alpha]/[beta]/[gamma] heterotrimers assembled from known chains. To date, twelve cell- and tissue-specific laminin isoforms have been reported laminins play at least two essential roles. First, they are major structural elements of all basement membranes. Second, they are signaling molecules that interact with cellular receptors mediating processes such as cell adhesion, migration, differentiation and tumor progression. In addition, five laminin genes have been associated with inherited human diseases and three more targeted laminin gene deletions resulted either in developmental lethality or potential disease processes. Though previous studies have Implicated laminins In various platelet and leukocyte functions expression, recognition and use of laminin isoforms by these cells are poorly understood. This thesis describes the synthesis, expression, integrin receptors and functional roles of laminin-8 in haematopoietic cells.

As a first step, chain specificity of commonly used monoclonal antibodies (mAb) to human laminins was defined by using recombinant laminin [beta]1 and [gamma]1 chains. Thereafter, western blotting of platelet cell lysate and immunoaffinity purified platelet laminin revealed polypeptides of 180/230/220 KDa, corresponding to laminin [alpha]4, [beta]1 and [gamma]1 chains, respectively. Subsequent microsequencing confirmed the identity of the chains, hence constituting laminin-8 ([alpha]4[beta]1[gamma]1), a laminin isoform first described in 1997 and expressed, together with laminin-10 ([alpha]5,[beta]1[gamma]1), by vascular endothelial cells. Results from RT-PCR, metabolic labeling, immunoaffinity purification and Western blotting showed that erythromegakaryocytic cells as well as monocytic and lymphoid (T, B and NK) cells were able to synthesize laminin-8. In blood platelets and leukocytes (lymphocytes, monocytes and PMNs), laminin-8 had mainly an intracellular localization as detected by immunofluorescence, and was partially secreted following stimulation of the cells.

Cellular receptors for laminin-8 are not known and no evidence for the functional role of laminin-8 has hitherto been described. Immobilized recombinant laminin-8 promoted adhesion of platelets, monocytic cells and stimulated blood lymphocytes This adhesive Interaction was largely inhibited by mAbs to integrin [alpha]6 and [beta]1 chains, indicating that [alpha]6[beta]1 integrin is a receptor for laminin-8 The adhesion of monocytic cells was also partially inhibited by mAbs to [beta]2 integrins, laminin-10, which was more adhesive than laminin-8, was similarly recognized by [alpha]6[beta]1 integrin in blood lymphocytes. Recombinant laminin-8 largely promoted chemokine-induced migration of blood monocytes and, to a lower extent, of lymphocytes and PMNs. In contrast, laminin-10 was either Inactive or inhibitory. Laminin-8 was more effective than the prototype laminin-1 ([alpha]1[beta]1[gamma]1). In further studies, immobilized recombinant laminin-8 together with mAb to CD3 strongly stimulated proliferation of Isolated blood T cells via a 6,61 integrin, whereas laminin-8 or the CD3 mAb on their own failed to induce any significant cell proliferation. Laminin-10, but not laminin-1, also costimulated T cell proliferation. Finally, platelet laminin-8 was found to inhibit agonist-induced platelet aggregation and P-selectin surface expression. The inhibitor effect on the platelet responses was independent of the type of agonist used. Moreover, the inhibition of platelet aggregation was reversed by mAbs to integrin [beta]1 and, to a lower extent, [alpha]6 chain. Thus, platelet laminin-8, through [beta]1 integrin receptors may limit platelet activation.

For the first time, the presence of a molecularly defined laminin in blood platelets and leukocytes is described and the synthesis of a complete laminin molecule by haematopoietic cells Is shown. In addition, integrin receptors for the novel laminin-8 are identified and the role of laminin-8 in leukocyte migration, lymphocyte proliferation and platelet activation are described. Differential recognition and use of laminin-8 and -10 by leukocytes is also reported. Altogether, the results Indicate a major role of laminin-8 In platelet and leukocyte physiology. This laminin isoform may participate in hemostasis and thrombosis, wound healing, immune and inflammatory responses and the pathophysiology of hematolymphoid malignancies.