Arie Reijerkerk

Tissue-Type Plasminogen Activator Is a Multiligand Cross-β Structure Receptor

Abstract Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin [1]. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA [1], but the structural basis for this interaction is unknown. Amyloid β (Aβ) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimers disease [2]. Like fibrin, it stimulates tPA-mediated plasmin formation [3–5]. Intermolecular stacking of peptide backbones in β sheet conformation underlies cross-β structure in amyloid peptides [6]. We show here that fibrin-derived peptides adopt cross-β structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Aβ and islet amyloid polypeptide (IAPP) (associated with pancreatic β cell toxicity in type II diabetes [7]), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-β structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-β structure is the common denominator in tPA binding ligands.

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor.

Summary.  Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin‐mediated fibrin degradation by removing the C‐terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAFI is an important determinant in the control of tissue repair, we investigated the effect of TAFI deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAFI knockout mice, including an altered pattern of epithelial migration. The time required to completley heal the cutaneous wounds was slightly delayed in TAFI‐deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in 3 of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI‐deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re‐epithelialization and scar formation, occurs in TAFI‐deficient mice, TAFI appears to be important for appropriate organization of the healing process.

Recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro

Endostatin is a fragment of collagen XVIII that acts as an endogenous inhibitor of tumor angiogenesis and tumor growth. Anti‐tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study we have investigated the structure and cellular effects of insoluble endostatin. We found that insoluble endostatin shows all the hallmarks of amyloid aggregates. Firstly, it binds Congo red and shows the characteristic apple‐green birefringe when examined under polarized light. Secondly, electron microscopy shows that endostatin forms short unbranched fibrils. Thirdly, X‐ray analysis shows the abundant presence of cross‐β sheets, the tertiary structure that underlies fibrillogenesis. None of these properties was observed when examining soluble endostatin. Soluble endostatin can be triggered to form cross‐β sheets following denaturation, indicating that endostatin is a protein fragment with an inherent propensity to form amyloid deposits. Like β‐amyloid, found in the brains of patients with Alzheimers disease, amyloid endostatin binds to and is toxic to neuronal cells, whereas soluble endostatin has no effect on cell viability. Our results demonstrate a previously unrecognized functional difference between soluble and insoluble endostatin, only the latter acting as a cytotoxic amyloid substance.

No grip, no growth: the conceptual basis of excessive proteolysis in the treatment of cancer

The formation of new bloodvessels, called angiogenesis, is critical for a tumour to grow beyond a few mm(3) in size. A provisional matrix promotes endothelial cell adhesion, migration, proliferation and survival. Synthesis and degradation of this matrix closely resemble processes that occur during coagulation and fibrinolysis. Degradation of the matrix and fibrinolysis are tightly controlled and balanced by stimulators and inhibitors of the plasminogen activation system. Here we give an overview of these processes during tumour progression. We postulate a novel way to inhibit angiogenesis by removal of the matrix through specific and localised overstimulation of the plasminogen activation system.

Early endostatin treatment inhibits metastatic seeding of murine colorectal cancer cells in the liver and their adhesion to endothelial cells

Endostatin, a carboxy-terminal fragment of collagen XVIII, potently inhibits angiogenesis and tumour growth, presumably through induction of apoptosis in endothelial cells and/or inhibition of their migration. Here we have tested how the timing of recombinant human endostatin (rh-E) administration affects its antitumour activity in a liver metastasis model of mouse C26 colorectal carcinoma cells. The effects of rh-E treatment on hepatic tumour load and on early tumour cell seeding were evaluated. Recombinant human endostatin was most effective in reducing intrahepatic tumour growth when administered prior to tumour cell inoculation. Analysis of early tumour cell seeding by using [125I]iododeoxyuridine-labelled C26 cells or by in vivo microscopy showed that rh-E reduced tumour cell seeding in the liver sinusoids. Recombinant human endostatin did not inhibit tumour growth when administered later than 4 days after tumour injection. Pretreatment of human umbilical vein endothelial cells with rh-E in vitro reduced C26 tumour cell adhesion under flow conditions two-fold as assessed by video microscopy and multiphoton laser scanning microscopy. Our results show that rh-E, in addition to antiangiogenic effects, reduces tumour cell adhesion in the liver sinusoids during the very early phases of metastasis formation. These data point towards a previously unknown mode of action of endostatin, that is, its ability to interfere with tumour cell seeding. Such insights may be helpful in the design of trials to improve (surgical) treatment of colorectal carcinoma and liver metastases.

The role of the fibrinolytic system in corneal angiogenesis

The plasminogen activation system has been implicated in angiogenesis and angiogenesis-dependent diseases such as cancer, atherosclerosis and ocular diseases. The identification and development of inhibitors of angiogenesis offer new possibilities for the treatment of these diseases. To clarify the role of proteins involved in the regulation of fibrinolysis during corneal angiogenesis, we have studied corneal vessel formation in mice deficient for urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen, plasminogen activator inhibitor-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI). Our results corroborate earlier findings that angiogenesis in the mouse cornea is dependent on PAI-1 and plasminogen. The absence of tPA, uPA or TAFI did not affect the formation of new vessels in the cornea.