Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.
We have examined the morphological and secretory behavior of rat and guinea pig megakaryocytes exposed for up to 24 hours to extracellular matrix produced by cultured bovine endothelial cells. By phase-contrast microscopy of living cells and in more detail by scanning electron microscopy, the megakaryocytes showed a nonreversible adherence, an extensive formation of filopodia around the periphery like the rays of the sun, and a tendency toward flattening. These filopodia were generally linear with attenuated tips and were larger than, but resembled the filopodia of, rat or guinea pig platelets exposed to this extracellular matrix. In contrast, isolated megakaryocytes on glass or on uncoated plastic surfaces did not show these responses; adherence, in the face of gentle agitation before fixation, was minimal, with rare filopodia and no flattening. Megakaryocytes that interacted with the extracellular matrix produced significant amounts of thromboxane A2, but this did not occur on uncoated surfaces and could not be attributed to other contaminating cells in the megakaryocyte suspensions. The appearance in megakaryocytes of these typical platelet responses indicates that megakaryocytes acquire the functional capabilities of platelets by the synthesis and assembly of platelet substances and organelles. Thromboxane production by megakaryocytes stimulated by the extracellular matrix is a readily quantifiable measure of this capacity.
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