Platelets store laminins 411/421 and 511/521 in compartments distinct from α- or dense granules and secrete these proteins via microvesicles.

BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. OBJECTIVES: Our aim was to compare the subcellular localization of laminins 411/421 and 511/521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. METHODS: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. RESULTS AND CONCLUSIONS: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express α-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum.

Laminin-8 (alpha4beta1gamma1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation.

Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin alpha4, beta1 and gamma1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin beta1 and gamma1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(alpha5beta1gamma1/alpha5beta2gamma1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using alpha6beta1 integrin, but minimally to laminin-1 (alpha1beta1gamma1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and -10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.

Monocytic cells synthesize, adhere to, and migrate on laminin-8 (alpha 4 beta 1 gamma 1).

Laminins, a growing family of large heterotrimeric proteins with cell adhesive and signaling properties, are major components of vascular and other basement membranes. Expression, recognition, and use of laminin isoforms by leukocytes are poorly understood. In monoblastic THP-1 cells, transcripts for laminin gamma(1)-, beta(1)-, and alpha(4)-chains were detected by RT-PCR. Following immunoaffinity purification on a laminin beta(1) Ab-Sepharose column, laminin beta(1)- (220 kDa), gamma(1)- (200 kDa), and alpha(4)- (180/200 kDa) chains were detected by Western blotting in THP-1 cells and in two other monoblastic cell lines, U-937 and Mono Mac 6. After cell permeabilization, a mAb to laminin gamma(1)-chain reacted with practically all blood monocytes by immunofluorescence flow cytometry, and laminin-8 (alpha(4)beta(1)gamma(1)) could be isolated also from these cells. Monoblastic JOSK-I cells adhered constitutively to immobilized recombinant laminin-8, less than to laminin-10/11 (alpha(5)beta(1)gamma(1)/alpha(5)beta(2)gamma(1)) but to a higher level than to laminin-1 (alpha(1)beta(1)gamma(1)). Compared with the other laminin isoforms, adhesion to laminin-8 was preferentially mediated by alpha(6)beta(1) and beta(2) integrins. Laminin-8 and, to a lower extent, laminin-1 promoted spontaneous and chemokine-induced migration of blood monocytes, whereas laminin-10/11 was inhibitory. Altogether, the results indicate that leukocytes, as other cell types, are able to synthesize complete laminin molecules. Expression, recognition, and use of laminin-8 by leukocytes suggest a major role of this laminin isoform in leukocyte physiology.

Erythromegakaryocytic cells synthesize laminin-8 (alpha4beta1gamma1).

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.

Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin.

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.

[Monoclonal antibodies IGR to surface antigens of neutrophilic granulocytes in human blood]. / Monoklonal'nye antitela IGR k poverkhnostym antigenam neitrofil'nykh granulotsitov krovi cheloveka.

Four monoclonal antibodies (MAbs) from series IGR to human peripheral blood neutrophilic granulocyte cell surface antigens were obtained by the conventional hybridoma technique. Specificity of MAbs AGR was determined to various leukemic cell lines and human peripheral blood cells. Overlapping in characteristics of antigens (molecular weight, localization, expression on induced leukemic cell line HL-60) to MAbs IGR-1 4C7, IGR-1 5B6 and IGR-2 IA6 suggests their identity. These, apparently, cannot be analogous to the well known granulocyte cell surface glycoproteins LFA-1, CR-3, p150, 95 or GP 130. The characteristics of MAbs IGR-1 and IGR-2 permit concluding that the antibodies should be useful in normal and leukemic myelomonocytic cell linear differentiation studies.

[Monoclonal antibodies IGR to nuclear antigens of neutrophilic granulocytes of human blood]. / Monoklonal'nye antitela IGR k iadernym antigenam neitrofil'nykh granulotsitov krovi cheloveka.

Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic granulocytes of human peripheral blood were obtained. Mabs IGR-1 2B8 and IGR-1 6B5 are bound to their specific antigens in the nuclei of all the investigated human cell lines. These Mabs were also specific for metaphase chromosomes of cell lines HL-60 and U-937. Investigations on the ultrastructural level showed that Mabs IGR-1 6B5 reacted with the HL-60 nuclear heterochromatin region. Mabs IGR-1 3D3 and IGR-2 2F1 manifested high specificity only for the nuclei of mature neutrophils and of plasma cells.