Steingrimur Stefansson

Neuroserpin, a Brain-associated Inhibitor of Tissue Plasminogen Activator Is Localized Primarily in Neurons IMPLICATIONS FOR THE REGULATION OF MOTOR LEARNING AND NEURONAL SURVIVAL

A cDNA clone for the serine proteinase inhibitor (serpin), neuroserpin, was isolated from a human whole brain cDNA library, and recombinant protein was expressed in insect cells. The purified protein is an efficient inhibitor of tissue type plasminogen activator (tPA), having an apparent second-order rate constant of 6.2 × 105 m −1 s−1 for the two-chain form. However, unlike other known plasminogen activator inhibitors, neuroserpin is a more effective inactivator of tPA than of urokinase-type plasminogen activator. Neuroserpin also effectively inhibited trypsin and nerve growth factor-γ but reacted only slowly with plasmin and thrombin. Northern blot analysis showed a 1.8 kilobase messenger RNA expressed predominantly in adult human brain and spinal cord, and immunohistochemical studies of normal mouse tissue detected strong staining primarily in neuronal cells with occasionally positive microglial cells. Staining was most prominent in the ependymal cells of the choroid plexus, Purkinje cells of the cerebellum, select neurons of the hypothalamus and hippocampus, and in the myelinated axons of the commissura. Expression of tPA within these regions is reported to be high and has previously been correlated with both motor learning and neuronal survival. Taken together, these data suggest that neuroserpin is likely to be a critical regulator of tPA activity in the central nervous system, and as such may play an important role in neuronal plasticity and/or maintenance.

Inhibition of Angiogenesis in Vivo by Plasminogen Activator Inhibitor-1

The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1s anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.

Plasminogen Activator Inhibitor-1 in Tumor Growth, Angiogenesis and Vascular Remodeling

Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of urokinase type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), and as such is thought to play an important role in the regulation of extracellular matrix remodeling. In blood, PAI-1 is bound to the adhesion protein vitronectin and is associated with vitronectin in fibrin clots and the provisional matrix. Elevated levels of PAI-1 are associated with atherosclerosis and an increased thrombotic tendency, while PAI-1 deficiency leads to increased fibrinolysis and bleeding. PAI-1 is also elevated in many solid tumors and is associated with a poor prognosis in cancer. PAI-1 has been shown to be a potent regulator of both vascular cell migration in vitro and of angiogenesis and tumor growth in vivo. PAI-1 can both promote and inhibit tumor growth and angiogenesis. Low concentrations of PAI-1 can stimulate tumor angiogenesis while treatment of animals with high doses of PAI-1 inhibits angiogenesis and tumor growth. Hence, PAI-1 appears to have a multifunctional role in regulating the migratory and fibrinolytic activity of vascular cells, and this, in turn, may help to explain the many varied actions of PAI-1.

Plasminogen Activator Inhibitor-1 Contains a Cryptic High Affinity Binding Site for the Low Density Lipoprotein Receptor-related Protein

Much of the controversy surrounding the binding of plasminogen activator inhibitor-1 (PAI-1) to the low density lipoprotein receptor-related protein (LRP) may be due to the labile structure of PAI-1 and the distinct conformations that it can adopt. To examine this possibility and to test the hypothesis that PAI-1 contains a specific high affinity binding site for LRP, a sensitive and quantitative assay for PAI-1 binding to LRP was developed. This assay utilizes a unique PAI-1 mutant that was constructed with a hexapeptide tag at the NH2 terminus, which is recognized by the protein kinase, heart muscle kinase and can be specifically labeled with 32P. Our results show that only 32P-PAI-1 in complex with a proteinase binds LRP with high affinity and is efficiently endocytosed by cells, indicating that a high affinity site for LRP is generated on PAI-1 only when in complex with a proteinase. In addition, PAI-1 in complex with different proteinases is shown to cross-compete for LRP binding, demonstrating that the binding site is independent of the proteinase and therefore must reside on the PAI-1 portion of the complex. Finally, mutagenesis of PAI-1 results in loss of LRP binding, confirming that the high affinity binding site is located on PAI-1 and suggesting that the LRP binding site lays within a region of PAI-1 previously shown to contain the heparin binding domain.

Characterization of the Binding of Different Conformational Forms of Plasminogen Activator Inhibitor-1 to Vitronectin IMPLICATIONS FOR THE REGULATION OF PERICELLULAR PROTEOLYSIS

Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into β-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with β-anhydrotrypsin, which does not undergo rearrangement of β-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to β-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1·plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.

Old dogs and new tricks: proteases, inhibitors, and cell migration.

A new model for the actions of plasminogen activator inhibitors (PAIs) on cell migration may resolve the conflicting research data on these proteins in metastasis and angiogenesis. Results from two groups reveal a role for PAI-1 in promoting cycles of attachment and detachment of the cell from the extracellular matrix that is independent of its role as an enzymatic inhibitor of urokinase-type plasminogen activator (uPA). Through the formation of a complex of integrins, uPA and its receptor, and the clearance receptors of the low-density lipoprotein family, PAI-1 may promote endocytosis and recycling of these adhesion-controlling proteins, allowing cycling of cellular attachment and detachment.

The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with ∼10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins αvβ3 and αvβ5 at the cell-matrix interface.

Avian urokinase-type plasminogen activator (u-PA) lacks the putative binding site for plasminogen activator inhibitor (PAI) and is resistant to inhibition by human PAI-1 and PAI-2

Summary In mammalian cultures, the activation and catalytic activity of both urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) is regulated in part by naturally occurring plasminogen activator inhibitors PAI-1 and PAI-2, members of the serpin family of serine protease inhibitors. Interaction of human PAIs with human u-PA (h-u-PA) and t-PA appears to be mediated by a putative PAI binding sequence in the enzymes. Comparison of the amino acid sequences of human, murine, porcine, bovine, simian and avian u-PA revealed specific sequences in the mammalian enzymes that are homologous to the recently discovered PAI-binding site in human u-PA and t-PA which is characterized by conserved basic amino acids. However, no homology was seen in the same region in chicken u-PA (c-u-PA), which is devoid of the consensus arginine and/or lysine residues. The chicken enzyme is a close structural and catalytic homolog of mammalian u-PA. The lack of the putative PAI binding sequence would predict that c-u-PA cannot interact with and be inhibited by human PAIs. To test this hypothesis, the effects of human PAI-1 and PAI-2 on avian u-PA were examined, using purified enzyme and inhibitors. Under conditions where h-u-PA activity is inhibited by human recombinant PAI-1 (rPAI-1), c-u-PA retains 95% activity. c-u-PA was also resistant to inhibition by human PAI-2. High molecular weight, SDS-stable complexes do not form readily when c-u-PA is incubated with PAI-1 or PAI-2, while human as well as murine u-PA formed complexes with both PAIs under the identical conditions. The c-u-PA molecule, thus, is a naturally occurring u-PA variant which lacks a PAI-binding region. The resistance of this enzyme to inhibition by human PAI-1 and PAI-2 supports the functional relevance of this sequence in mammalian u-PA and t-PA.