The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences.

Summary. Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamatebased metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.Keywords: soluble interleukin-6 receptor, shedding, metalloproteinase, multiple myeloma, tissue inhibitor of metalloproteinase-3.

Careful control of surface chemistry results in strong surface enhanced resonance Raman scattering from dye-labelled oligonucleotides assembled on nanostructured gold surfaces, releasing their potential as reliable enhancing surfaces.

Collagen fibers or a glycoprotein VI-specific collagenrelated peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125 fak (FAK), in human platelets. An integrin ␣ 2 ␤ 1 -specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O ‫؍‬ Hyp) was without effect. Antibodies to the ␣ 2 and ␤ 1 integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin ␣ IIb ␤ 3 . The intracellular Ca 2؉ chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin ␣ 2 ␤ 1 is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor ␣ IIb ␤ 3 is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca 2؉ and protein kinase C activity are essential intermediaries of FAK activation.Hemostasis, prevention of blood loss from damaged blood vessels, is dependent upon the activation of platelets by subendothelial collagens (types I and III) of the vessel wall. Platelet activation involves shape change, adhesion, aggregation, and secretion of granule contents. These events lead to the formation of a clot at the site of injury (1).Platelets possess several receptors for collagen, recently reviewed (2-4), including the integrin ␣ 2 ␤ 1 (glycoproteins IaIIa), CD36 1 (glycoprotein IV), and glycoprotein VI (GpVI). Platelet activation by collagen is a two-stage process involving sites in collagen, which support platelet adhesion, and others, which support both platelet adhesion and activation (5, 6). At present, ␣ 2 ␤ 1 is considered primarily an adhesive co-receptor (7), whereas other collagen receptors, notably glycoprotein VI, activate platelets (8). This study was designed to clarify which collagen receptors transmit signals to the platelet interior, information which is crucial for the development of anti-platelet therapy based on collagen receptor antagonism (9).Recent evidence suggests that CD36, ␣ 2 ␤ 1 , and GpVI each contribute to both signaling and adhesion to collagen (10). GpVI, recently cloned (11), acts with the Fc receptor ␥-chain (12, 13) as a crucial signaling receptor complex, and platelets deficient in GpVI fail to aggregate in response to collagen, although the tyrosine kinase c-Src, but not p72 syk , is activated (14). The role of ␣ 2 ␤ 1 in platelet signaling is unclear: ␣ 2 ␤ 1 -reactive snake venoms fuel the debate on the integrin's role in platelet signaling (15, 16), and overexpression of ␣ 2 ␤ 1 has recently been advanced as a risk factor in myocardial infarction and stroke (17,18).We have synthesized a collagen-related peptide (CRP) recognized by GpVI, which shares both the triple-helica...

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