Antiangiogenic Activity of a Domain Deletion Mutant of Tissue Plasminogen Activator Containing Kringle 2

Abstract

Objective-The thrombolytic therapy drug, Reteplase, is a domain deletion mutant of tissue plasminogen activator (tPA), comprising the kringle 2 and protease (K2P) domains. Some kringle domains of hemostatic proteins are antiangiogenic and promote apoptosis. The objective of this study was to investigate whether K2P is an angiogenesis inhibitor because of the presence of kringle 2. Methods and Results-K2P inhibited basic fibroblast growth factor-induced human endothelial cell proliferation and migration. Inhibition was not dependent on the protease activity of K2P because similar results were obtained with catalytically inactivated K2P. Purification of the kringle 2 domain derived from elastase cleavage of K2P at the Arg 275 -Ile 276 bond revealed that inhibition was mediated by this domain. In addition, K2P inhibited angiogenesis in vivo and increased endothelial cell apoptosis. Conclusions-Wound healing and angiogenesis are severely compromised by K2P. These data provide new mechanistic insights into the bleeding complications observed in some patients while undergoing thrombolytic therapy with this drug. In addition, we identify the kringle 2 domain of tPA as a novel target for antiangiogenic therapy. Key Words: antiangiogenesis Ⅲ reteplase Ⅲ kringle 2 Ⅲ endothelial cells Ⅲ wound healing T issue plasminogen activator (tPA) is a serine protease that activates fibrinolysis through the conversion of plasminogen to plasmin. Plasmin, in turn, degrades fibrin in the vasculature during clot dissolution and wound healing. 1 tPA is expressed in a variety of cell types including endothelial cells 2 and tumor cells. 3,4 It is a multidomain protein that consists of a finger domain, an epidermal growth factor-like domain, 2 kringle modules, and a protease domain. Its widespread use for treating thrombosis during acute myocardial infarction has led to the development of tPA derivatives with improved pharmacokinetic properties. 5 One of these is the domain deletion mutant comprising just the kringle 2 and protease domains (K2P) of tPA. 6 Kringles are triple-loop structures of Ϸ80 amino acids. 7 Recent studies have shown that isolated kringle domains of proteins involved in the hemostatic system are antiangiogenic. These include plasminogen, which has 5 kringle domains, 8 hepatocyte growth factor, which has 4, 9 prothrombin, which has 2 kringles, 10 and urokinase plasminogen activator (uPA), which has just 1. 11 Angiostatin, consisting of kringles 1 to 4 of plasminogen, was first shown to inhibit basic fibroblast growth factor (bFGF)-mediated endothelial cell proliferation and tumor angiogenesis. 12 Further studies have shown that the individual plasminogen kringles have varying efficacy on endothelial cells. Kringles 1 to 3 and kringle 5 inhibit endothelial cell growth, 13,14 whereas the kringle 4 domain is a weaker inhibitor of cell growth but significantly blocks endothelial cell migration. 13,15 Angiostatin-specific binding sites have been identified on endothelial cells, including ATP-synthase and angiomotin, 16,17 and i...

Antimigratory effect of TK1-2 is mediated in part by interfering with integrin α2β1

“…DGEA peptide inhibits collagen-induced platelet reactivity as an antagonist through a 2 h 1 integrin (45) and also inhibits endothelial progenitor cell differentiation on collagen matrix (46). Interestingly, such similar sequence, DGDA, exists only in kringle 2 of tissue-type plasminogen activator among kringle molecules, which is consistent with the result that the kringle domain 2 alone is a novel molecule for antiangiogenic therapy (8). Therefore, a possibility of DGDA sequence associating with antimigratory effect of TK1-2 has been raised, and we are studying the question whether TK1-2 perturbs a 2 h 1 integrin through DGDA sequence and whether DGDA is able to function as an antiangiogenic peptide.…”

“…It has also been shown to inhibit in vivo angiogenesis in chick chorioallantoic membrane and tumor growth in lung or colon cancer xenograft murine models (5 -7). Later, a domain deletion mutant of tissue-type plasminogen activator containing kringle 2, a thrombolytic agent named Reteplase, has been found to elicit antiangiogenic activity by other researchers, providing new mechanistic insights into the bleeding complications of this drug (8). They also identified the kringle domain 2 alone as a novel molecule for antiangiogenic therapy.…”

Inhibition of Pathological Retinal Neovascularization by a Small Peptide Derived from Human Tissue-Type Plasminogen Kringle 2

“…The recombinant protein TK1-2 (kringle 1-2 from human t-PA) inhibited angiogenesis partly by the interaction with integrin a2b1 (Kim et al, 2003;Kim et al, 2008). TKII-12 was derived from the extended antiparallel b-sheet within the t-PA kringle 2 domain which also demonstrated antiangiogenic activity (Byeon et al, 1991;Carroll et al, 2005). Another peptide, TP-7, derived from t-PA kringle 2 inhibited VEGF or FGF-induced phosphorylation of ERK1/2 and FAK (Kim et al, 2017).…”

The Kringle-2 domain of tissue plasminogen activator significantly reduces mortality and brain infarction in middle cerebral artery occlusion rats

“…Kringle-2 and P were purified as described previously (23), digesting reteplase by plasmin at Arg275-Ile276 (24). Reteplase (2 mg/ml) was incubated with 2 μg/ml rat plasminogen in the digest buffer (0.7 mol/l Arg in PBS, pH 8.0) at 37˚C overnight.…”

“…1A, Lanes 3 and 4). The full biological activity of purified Kringle-2 and P were recovered, as the denatured protein did not bind to lysine-or ETI-Sepharose 4B (23).…”