The role of plasminogen activator inhibitor-1 as inhibitor of platelet and megakaryoblastic cell adhesion.

In the present study the ability of plasminogen activator inhibitor type-1 (PAI-1) to interfere with platelet and megakaryoblastic cell adhesion was investigated. Both cell types exhibited integrin-dependent adhesion in a static system, mediated by alphaIIb beta3 on platelets and alpha v-integrins on different megakaryoblastic cell lines, even though they also expressed alphaIIb beta3. In a concentration-dependent manner, active, but not latent or complexed, PAI-1 abrogated cell adhesion onto vitronectin but not onto fibrinogen or other matrix substrata. Urokinase as well as thrombin neutralized the anti-adhesive effect of active PAI-1. The direct binding of vitronectin, but not of other matrix proteins, to integrin alphaIIb beta3 was blocked by active PAI-1 in a purified system. Since activated platelets release active and latent PAI-1 as well as structurally and functionally distinct forms of vitronectin, the described interactions appear to be physiologically significant. Co-distribution of vitronectin and PAI-1 at sites of fibrin polymers within platelet thrombi was demonstrated by transmission electron microscopy, suggesting an extracellular functional relationship of both release products with regard to cell adhesion. Our data emphasize the regulatory role of active PAI-1 in platelet adhesion to provisional matrix proteins as found during wound healing independent of its anti-proteolytic activity. Furthermore, megakaryocyte maturation may depend on the intact vitronectin-integrin adhesion system that is influenced by PAI-1, thereby proposing a regulatory role for the inhibitor in cellular differentiation.

Molecular crosstalk between adhesion receptors and proteolytic cascades in vascular remodelling.

The multifunctionality of adhesion receptor ligands as well as the promiscuous nature of vascular integrins and nonintegrin-dependent adhesive interactions allow ligand-receptor binding of variable strength. The cooperation with pericellular proteolysis cascades is required for vascular remodelling during angiogenesis, atherogenesis or inflammatory processes. In particular, integrin-dependent cell contact, spreading and (trans-)migration can be modulated by ECM-associated PAI-1 and uPA-receptor driven reactions that are intimately linked to the invasive potential of cells. Recently, mechanisms of molecular crosstalk between these receptor systems have been recognized: (a) uPA-receptor may directly interact with beta 1- and beta 2-integrins on circulating blood cells; (b) av beta 3-integrin-directly binds to a matrix metalloproteinase; (c) uPA and PAI-1 balance the high affinity binding of vitronectin to uPA-receptor; (d) vitronectin-dependent cell adhesion and migration involving alpha v-integrins or uPA-receptor are blocked by active PAI-1 independent of its role as protease inhibitor. These results are compatible with vascular injury studies in uPA- and PAI-1 knock-out mice and provide new targets for the treatment of diseases associated with imbalanced vascular remodelling.

The urokinase-receptor (CD87) is expressed in cells of the megakaryoblastic lineage.

Megakaryocytopoiesis is governed in the bone marrow microenvironment by cellular interactions that include various adhesion receptor systems and pericellular proteolysis for proper regulation of cell motility and differentiation. In order to define the role of cell surface molecules required for these processes, we searched for protease receptors on these cells. In an in vitro system utilizing different cell lines of the megakaryoblastic lineage (MEG-01, Dami), low level surface expression of the urokinase (uPA) receptor was noted. Following stimulation with phorbolester (PMA), a 3-6 fold higher expression of uPA receptor over a period of up to 5 days could be observed by fluorescent activated cell-sorting as well as by direct ligand-binding of amino-terminal fragment of uPA or vitronectin. Together with elevated expression of alpha IIb beta 3-integrin (glycoprotein IIb/IIIa complex), double immuno-fluorescence staining of stimulated cells confirmed the increased cell surface localization of uPA receptor. Semi-quantitative RT-PCR, ligand blot analysis and measurement of cell-bound proteolytic activity revealed a differentiation-dependent upregulation of the uPA receptor expression in megakaryoblastic cell lines as in monocytic cells. Due to its glycolipid anchorage, incubation with phosphatidylinositol-specific phospholipase C reduced uPA receptor-mediated ligand binding by about 60%, uPA receptor mRNA was expressed in cultured megakaryocytes derived from bone marrow, whereas no uPA receptor mRNA was detectable in platelets. These results indicate a differentiation-dependent increase in the expression of uPA receptor in megakaryoblastic cells. The characteristics of surface expression and functionality of the receptor on megakaryocytic cells may influence their maturation by regulating cellular communication in the bone marrow micro-environment.

Structural and functional characterization of vitronectin-derived RGD-containing peptides from human hemofiltrate.

Bioactive peptides derived from the adhesive plasma protein vitronectin are present at submicromolar concentrations in human hemofiltrate of patients with renal diseases and were isolated by a combination of high-efficiency chromatographic steps. The structural and functional properties of these peptides were characterized. Sequencing and mass spectrometry revealed the existence of peptide isoforms (5-6 kDa) which corresponded to the N-terminus (residues 1 to 44-50) of vitronectin. The isolated peptides bound directly to plasminogen-activator inhibitor-1 (PAI-1) and were effective competitors of the interaction of PAI-1 with isolated intact vitronectin or extracellular matrix. These functional properties were indistinguishable from the binding properties of a recombinant fusion protein containing residues 1-52 of vitronectin linked to a portion of glutathione S-transferase, expressed in Escherichia coli. Peptides containing the RGD sequence of vitronectin competed for vitronectin binding to the alpha v beta 3 integrin. No indication for direct growth-factor binding was noted, whereas natural peptides were found associated with PAI-1 as the major binding protein in plasma. These data demonstrate that functionally active vitronectin-derived peptides are released by unknown protease(s) from the mature protein and that these peptides are identical, in terms of activity, to recombinant vitronectin fragments. These natural peptides may interact with active PAI-1 in plasma or at extravascular sites and thereby interfere with established biological functions of intact vitronectin.

Allelic dimorphism in the human tissue-type plasminogen activator (TPA) gene as a result of an Alu insertion/deletion event.

Polymerase chain reaction and direct sequencing were used to investigate an amplified DNA fragment containing the suspected polymorphic site of all known intragenic restriction fragment length polymorphisms (RFLPs) within the human tissue-type plasminogen activator (TPA) gene. Sequence data obtained showed that these RFLPs were all generated by the presence or absence of one of the two Alu sequences located in intron h of the human TPA gene. Furthermore, one of the direct repeats flanking this Alu sequence was absent in the minor allele. In addition to indicating the presence of an Alu insertion in an ancestral human TPA gene, these findings suggest a slip-replication mechanism for the deletion of this Alu repeat, once inserted into the gene. As both alleles have been observed in similar frequencies among different ethnic groups, the insertion or subsequent deletion of this Alu sequence in the human TPA gene must have occurred early in human evolution.