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.

SummaryMegakaryocytopoiesis 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 αIIbβ3-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 phosphatidyl-inositol-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.

Summary. 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 aIIbb3 on platelets and av-integrins on different megakaryoblastic cell lines, even though they also expressed aIIbb3. 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 aIIbb3 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.

Max-Planck-lnstitut, Bad Nauheim, Germany !ntroduction contacts between the basolateral face of endothelial cells and the underlying basal lamina as well as to w,urent tight anchorage of smooth muscle cells and fibroblasts with their ECM. While integrins initiate cell attachment and spreading to extracellular matrix molecules, heparinoid-type proteoglycans are critical for the reorganization of actin microfilaments into stress-fibers and for the formation of focal adhesions. In particular, vascular cell syndecan-4 was fonnd associated as coreceptor with 0l-and B3-integrins in these stable adhesion points (4). Although integrin ligation appears to be necessary for triggering phosphorylation of specific intracellular focal adhesion components, cell surface proteoglycans are required to transduce signals for adhesion receptor clustering through activation of protein kinase C (5). Modulation of cell-cell and cell-ECM contacts by changes in the expression, affinity or specificity of adhesion molecules or by pericellular proteolysis results in variable and dynamic short-term adhesion or detachment events and is pivotal for the control of cell migration and invasion (6-8). The strength and duration of adhesive interactions not only depend on the expression pattern or the extent of adhesion receptor clustering (9) but also on the availability

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-SO) 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 m/33 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. In the present study, bioactive fragments of the multifunctional adhesion protein vitronectin were identified in hemofiltrate and characterized. Vitronectin belongs to a group of RGD-containing adhesion proteins including fibronectin, fibrinogen and von Willebrand factor, which are found distributed between the circulation and extracellular matrices [9]. Due to the ability of vitronectin to bind to macromolecular ligands or cellsurface receptors via multiple domains, it has been iniplicated as molecular link between cellular adhesion, pericellular proteolysis and immune defense [ 10, 111. In particular, the RGD-attachCorrespondence to K. T. Preissner, Haemostasis Research Unit, Max-Planck-Institut, Kerckhoff-Klinik, Sprudelhof 11, D-61231 Bad Nauheim, Germany Fux: +49 60 32 996 707. Abbreviarions. AcNCH,, carboxamido-methyl ; PAI-1, plasminogenactivator inhibitor-I. ment site is flanked by an N-terminal 44-amino-acid segment that serves as the primary high-affinity-binding site for plasminogen-activator inhibitor-1 (PAI-1) [12]. In the fibrinolytic system, SO-kDa PAI-1 appears to be the main physiological inhibitor of tissue plasminogen activator. Active PAI-1 was shown to be associated with vitronectin in plasma [I31 and at vascular matrix sites [14, 151. A search for acidic bioactive peptides from hemofiltrate led to the purification and characterization of peptides that corresponded to N-terminal fragments of the vitronectin molecule. These pepti...

No abstract

No abstract

Source/Description: XLIB18-88 is a 2.1 kb EcoRI fragment isolated from a Charon 21A human chromosome 3 library (1). Polymorphism: MspI digestion of genomic DNA and hybridization with the probe detects a two-allele polymorphism: 14.0 kb (Al) and 9.6 kb (A2). Frequency: Estimated from 120 unrelated Caucasians. A1: 0.41 A2: 0.59 Frequency of Heterozygosity: 0.48.