Combined Treatment With Exenatide and Cyclosporine A or Parstatin 1-26 Results in Enhanced Reduction of Infarct Size in a Rabbit Model.

Exenatide and cyclosporine A have been shown to moderately protect against myocardial reperfusion injury leading to reduction of infarct size in patients. Our objective was to investigate whether the combined treatment with exenatide (glucagon-like peptide 1 receptor agonist) and cyclosporine A or parstatin 1-26 (inhibitors of mitochondrial permeability transition pore and/or inflammation) is more beneficial than either agent alone. Rabbits underwent 40 minutes of ischemia and 120 minutes of reperfusion. Intravenous bolus administration of exenatide or cyclosporine A, 10 minutes before reperfusion, reduced infarct size by 38% (P < 0.05) and 40% (P < 0.05), and cardiac troponin I (cTnI) plasma levels by 48% (P < 0.05) and 36% (P < 0.05), respectively, compared with control. The combined administration of both agents resulted in an additive decrease of infarct size by 55% (P < 0.05) and cTnI release by 61% (P < 0.05). Also, combined treatment of exenatide and parstatin 1-26 enhanced infarct size reduction (62%, P < 0.05), compared with monotherapies (41% for parstatin 1-26, P < 0.05; 43% for exenatide, P < 0.05). In contrast, the combined administration of parstatin 1-26 and cyclosporine A canceled out the cardioprotective effects observed by monotherapies. These results suggest that, for the therapy of myocardial reperfusion injury the combined administration of exenatide and cyclosporine A or parstatin 1-26 is more effective than monotherapies and may provide advantageous clinical outcome.

NGF promotes hemodynamic recovery in a rabbit hindlimb ischemic model through trkA- and VEGFR2-dependent pathways.

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 µm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.

Parstatin prevents renal injury following ischemia/reperfusion and radiocontrast administration.

BACKGROUND/AIMS: Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the protease-activated receptor 1. Parstatin was recently found to be cardioprotective against myocardial ischemia/reperfusion (IR) injury. In the present study, it was hypothesized that parstatin would protect the kidneys in acute renal failure. METHODS: We investigated the effects of parstatin on the renal dysfunction and injury caused either by renal IR injury or contrast-induced nephropathy (CIN) in two animal models. Renal IR injury was induced in rats by bilateral occlusion of renal arteries and veins for 45 min followed by 4 h of reperfusion, while CIN was induced in rabbits by intravenous injection of the radiocontrast medium Iopromide. RESULTS: Treatment with parstatin 15 min before or immediately after renal ischemia attenuated the resulting renal dysfunction as demonstrated by the improved biochemical indicators (serum creatinine and fractional excretion of Na(+)) and scintigraphic analysis. The effect was dose depended and provided evidence for a more prominent protection of tubular than glomerulal function. Histopathological examination of the kidneys revealed severe renal damage, which was significantly suppressed by the parstatin. Similarly, administration of a single dose of parstatin before the induction of CIN significantly protected against the resulting renal dysfunction and histologically evidenced renal tubular injury. CONCLUSION: These results suggest that parstatin is able to act as nephroprotective agent and may be useful in enhancing the tolerance of the kidney against renal injury associated with clinical conditions of acute renal failure. Further investigation on the mechanism underlying the nephroprotective properties of parstatin is deemed necessary.

Parstatin(1-26): the putative signal peptide of protease-activated receptor 1 confers potent protection from myocardial ischemia-reperfusion injury.

Parstatin, the N-terminal 41-amino-acid peptide cleaved by thrombin from the protease-activated receptor 1, protects against rat myocardial ischemia and reperfusion injury. In this study, we determined that the parstatin fragment 1-26, the putative signal peptide of protease-activated receptor 1, contains the functional domain of parstatin. We assessed a synthesized parstatin(1-26) peptide in an in vivo rat model of myocardial regional ischemia-reperfusion injury (n = 6/group). Infarct size in control rat hearts was 58 +/- 1% area at risk. Parstatin(1-26) was able to reduce infarct size to 13 +/- 1% (P < 0.001) and 22 +/- 1% area at risk (P < 0.01) when given before or after reperfusion. The infarct-sparing effects of parstatin(1-26) were abolished by inhibition of G(i) proteins (pertussis toxin), phosphoinositide 3-kinase/Akt (wortmannin), nitric-oxide synthase (NOS; N(G)-monomethyl-l-arginine), soluble guanylyl cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)], and sarcolemmal and mitochondrial K(ATP) channels [glibenclamide, 5-hydroxydecanoic acid, and sodium (5-(2-(5-chloro-2-methoxybenzamido)ethyl)-2-methoxyphenylsulfonyl) (methylcarbamothioyl)amide (HMR 1098)]. Parstatin(1-26) cardioprotection was also abolished by atractyloside, a mitochondrial permeability transition pore (mPTP) opener. The inhibitors and opener alone had no effect on infarct size. Furthermore, preischemic treatment with parstatin(1-26) increased Akt and endothelial NOS phosphorylation at the time of reperfusion. After a 120-min reperfusion, parstatin(1-26) increased nitric oxide levels (12 +/- 0.4 to 17 +/- 0.9 mmol/g tissue) and cyclic GMP levels (87 +/- 21 to 395 +/- 36 pmol/g tissue). Parstatin(1-26) treatment either before or after ischemia results in an extremely efficacious protection against ischemia-reperfusion injury that depends on a G(i) protein-mediated pathway involving mPTP, the end effector of the preconditioning pathway. This suggests that parstatin(1-26) has a potential therapeutic role in the treatment of ischemia and reperfusion injury.

Parstatin, the cleaved peptide on proteinase-activated receptor 1 activation, is a potent inhibitor of angiogenesis.

The proteolytic activation by thrombin of the proteinase-activated receptor 1 unveils the tethered peptide ligand and cleaves a 41-amino acid peptide. In this report, we show that this peptide, which we have designated as "parstatin," is a potent inhibitor of angiogenesis. Synthesized parstatin suppressed both the basic angiogenesis and that stimulated by basic fibroblast growth factor and vascular endothelial growth factor in the chick embryo model in vivo and in the rat aortic ring assay. Parstatin also abrogated endothelial cell migration and capillary-like network formation on the Matrigel and fibrin angiogenesis models in vitro. Treatment of endothelial cells with parstatin resulted in inhibition of cell growth by inhibiting the phosphorylation of extracellular signal-regulated kinases in a specific and reversible fashion and by promoting cell cycle arrest and apoptosis through a mechanism involving activation of caspases. We have shown that parstatin acts as a cell-penetrating peptide, exerting its biological effects intracellularly. The uptake into cells and the inhibitory activity were dependent on parstatin hydrophobic region. These results support the notion that parstatin may represent an important negative regulator of angiogenesis with possible therapeutic applications.

Thrombin promotes arteriogenesis and hemodynamic recovery in a rabbit hindlimb ischemia model.

BACKGROUND: Compared with angiogenesis, arteriogenesis is a distinct process based on the remodeling and maturation of pre-existing arterioles into large conductance arteries. Therapeutic angiogenesis has been proposed as a potential treatment for ischemic atherosclerotic diseases. Since a variety of angiogenic factors have been tested with inconsistent so far clinical results, the challenge remains in identifying the factor(s) that will stimulate functional neovascularization. Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by regulating and organizing a network of angiogenic mediators. Also, it was recently demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, providing evidence on a potential role of thrombin in vascular protection and maintenance of vessel integrity. Based on these observations, we hypothesized that thrombin may promote the development of mature functional blood vessels. METHODS: Seventy-four (n=74) rabbits underwent bilateral femoral artery surgical excision. On the 20th postsurgical day increasing doses of VEGF or bFGF or thrombin were injected in one ischemic limb per rabbit and an equal volume of normal saline to the contralateral control limbs. Quantification of newly developed collateral vessels (diameter >500 mum) was performed by transauricular intra-arterial subtraction angiography. Computerized quantitative analysis of collateral vessels in angiography images was based on the concept of multiscale structural tensor. Perfusion analysis of an in vivo dynamic computed tomography study was performed to investigate hemodynamic recovery of the distal ischemic limbs. Tissue perfusion analysis was performed with the semiquantitative slope methodology, which focuses on the first-pass arterial phase. RESULTS: A single administration of thrombin exhibited a dose-dependent increase of arteriogenic outcome. Thrombin at 5000 IU induced a 30.2 +/- 7.4% (P < 0.05) increase of total collateral area and length. Both VEGF and bFGF were without any significant effect at the concentrations used. Functional estimation of limb perfusion showed a statistically significant increase of blood flow recovery only for thrombin. The semiquantitative slope method perfusion score differed significantly in the 5000 IU thrombin treated limbs (5.7 +/- 0.3 vs 5.0 +/- 0.3 in control ischemic limbs; P < .05), and was not significantly inferior from the score of normal nonoperated limbs (6.5 +/- 0.3) suggesting a trend towards hemodynamic recovery of distal limb perfusion. CONCLUSIONS: In a rabbit hindlimb ischemia model, thrombin promoted the formation of large collateral vessels and improved the perfusion of distal ischemic tissue. These results provide new insights in understanding the involvement of thrombin in vascular formation and point to a novel role of thrombin in arteriogenesis.

On the mode of action of thrombin-induced angiogenesis: thrombin peptide, TP508, mediates effects in endothelial cells via alphavbeta3 integrin.

In a previous report we have presented evidence that thrombin interacts with alpha(v)beta(3) integrin in endothelial cells at the molecular and cellular level. This interaction was shown to be of functional significance in vitro and in vivo and contributed to activation of angiogenesis by thrombin. In the present study, we have used a synthetic thrombin peptide, TP508, which represents residues 183 to 200 of human thrombin. This peptide lacks the catalytic site of thrombin but contains the thrombin RGD sequence. Immobilized (surface-coated) TP508 peptide, like thrombin, supported alpha(v)beta(3) integrin-dependent endothelial cell attachment and haptotactic migration. These effects were specific (a scrambled TP508 peptide was without effect), and dosedependent. The RGD sequence was essential since a modified TP508 peptide, which contained RAD sequence instead of RGD, was inactive. Immobilized TP508 peptide stimulated phosphorylation of mitogen-activated protein kinases and focal adhesion kinase, the signal transduction pathways characteristic for integrin activation. On the other hand, TP508 peptide, when in solution, did not mimic other thrombin-promoted angiogenic effects, such as that of activation gelatinase A, upregulation of expression of vascular endothelial growth factor receptor mRNA or prostacyclin PGI(2) release in endothelial cells. On the contrary, soluble TP508 acted as an antagonist for the aforementioned effects of thrombin. TP508 peptide inhibited these thrombin-induced effects through a RGD and alpha(v)beta(3)-related mechanism. The antagonism with thrombin or thrombin receptor activating peptide was specific and involved at least in part mitogen-activated protein kinases activation. These results point to the importance of RGD sequence of thrombin in mediating effects on endothelial cells and angiogenesis.

Highly functionalized 2-oxopiperazine-based peptidomimetics: an approach to PAR1 antagonists.

A series of pseudodipeptide-based chiral 1,3,4,5-tetrasubstituted-2-oxopiperazines has been designed and synthesized as potential PAR1 antagonists. These highly functionalized piperazines were synthesized from aromatic and basic amino acid derived Ψ[CH(CN)NH]pseudodipeptides through a four step pathway that involves reduction of the cyano group to build the 2-oxopiperazine ring, followed by selective functionalization at the N4-, N1-positions, and at the exocyclic moiety at position C5. This regioselective functionalization required the fine tuning of reaction conditions. All new compounds were screened as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cell lines. Some of the compounds displayed moderate PAR1 antagonist activity, while, others were cytotoxic at µM concentration. No correlation was observed between both types of activities.

Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1.

By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and protecting group removal. All new compounds have been evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed significant antagonist activity.

The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression.

The protease-activated receptor 1 (PAR1) is activated by thrombin cleavage releasing the physiologically-relevant parstatin peptide (residues 1-41). However, the actual length of parstatin was unclear since the receptor may also possess a cleavable signal peptide (residues 1-21) according to prediction programs. Here, we show that this putative signal peptide is indeed functional and removed from the PAR1 resolving the question of parstatin length. Moreover, we show that the sequence encoding the signal peptide may surprisingly play a role in stabilization of the PAR1 mRNA, a function which would be novel for a G protein-coupled receptor.

Parstatin suppresses ocular neovascularization and inflammation.

PURPOSE: Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor. The authors recently showed that parstatin is a potent inhibitor of angiogenesis. The purpose of the present study was to evaluate the therapeutic effect of parstatin on ocular neovascularization. METHODS: Choroidal neovascularization was generated in mice using laser-induced rupture of Bruch's membrane and was assessed after 14 days after perfusion of FITC-dextran. Oxygen-induced retinal neovascularization was established in neonatal mice by exposing them to 75% O(2) at postnatal day (P)7 for 5 days and then placing them in room air for 5 days. Evaluation was performed on P17 after staining with anti-mouse PECAM-1. The effect of parstatin was tested after intravitreal administration. The effects of subconjunctival-injected parstatin on corneal neovascularization and inflammation in rats were assessed 7 days after chemical burn-induced corneal neovascularization. Retinal leukostasis in mice was assessed after perfusion with FITC-conjugated concanavalin A. RESULTS: Parstatin potently inhibited choroidal neovascularization with an IC(50) of approximately 3 µg and a maximum inhibition of 59% at 10 µg. Parstatin suppressed retinal neovascularization with maximum inhibition of 60% at 3 µg. Ten-microgram and 30-µg doses appeared to be toxic to the neonatal retina. Subconjunctival parstatin inhibited corneal neovascularization, with 200 µg the most effective dose (59% inhibition). In addition, parstatin significantly inhibited corneal inflammation and VEGF-induced retinal leukostasis. In all models tested, scrambled parstatin was without any significant effect. CONCLUSIONS: Parstatin is a potent antiangiogenic agent of ocular neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.

Thrombin's central role in angiogenesis and pathophysiological processes.

A plethora of endogenous modulators of angiogenesis have been identified and their roles in the molecular and cellular events that mediate and regulate angiogenesis have been proposed. In this review, we summarize the recent findings on the role of thrombin/thrombosis on angiogenesis and other related pathophysiological processes. The mechanisms by which thrombin itself and its receptor PAR1 orchestrate many cellular events through interaction with a variety of other factors and cell types are discussed. These new data point to the complexity of the regulatory processes involved in the angiogenic cascade, which may be tissue specific, and dependent upon the pathology involved. The understanding of these events may provide targets for therapeutic intervention in disease states where angiogenesis is disturbed.

Parstatin: a cryptic peptide involved in cardioprotection after ischaemia and reperfusion injury.

AIMS: Thrombin activates protease-activated receptor 1 by proteolytic cleavage of the N-terminus. Although much research has focused on the activated receptor, little is known about the 41-amino acid N-terminal peptide (parstatin). We hypothesized that parstatin would protect the heart against ischaemia-reperfusion injury. METHODS AND RESULTS: We assessed the protective role of parstatin in an in vivo and in vitro rat model of myocardial ischaemia-reperfusion injury. Parstatin treatment before, during, and after ischaemia decreased infarct size by 26%, 23%, and 18%, respectively, in an in vivo model of ischaemia-reperfusion injury. Parstatin treatment immediately before ischaemia decreased infarct size by 65% and increased recovery in ventricular function by 23% in an in vitro model. We then assessed whether parstatin induced cardioprotection by activation of a Gi-protein-dependent pathway. Gi-protein inactivation by pertussis toxin completely abolished the cardioprotective effects. The cardioprotective effects were also abolished by inhibition of nitric oxide synthase (NOS), extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and K(ATP) channels in vitro. Furthermore, parstatin increased coronary flow and decreased perfusion pressure in the isolated heart. The vasodilatory properties of parstatin were confirmed in rat coronary arterioles. CONCLUSION: A single treatment of parstatin administered prior to ischaemia confers immediate cardioprotection by recruiting the Gi-protein activation pathway including p38 MAPK, ERK1/2, NOS, and K(ATP) channels. Parstatin exerts effects on both the cardiomyocytes and the coronary circulation to induce cardioprotection. This suggests a potential therapeutic role of parstatin in the treatment of cardiac injury resulting from ischaemia and reperfusion.

Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms.

Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by indirectly regulating and organizing a network of angiogenic molecules. In addition, it has been proposed that thrombin can directly activate endothelial cell proliferation. However, in this report it was shown that thrombin is a poor growth factor for human endothelial cells, and its modest mitogenic activity is mediated indirectly by the release of heparin-binding epidermal growth factor, subsequent to proteinase-activated receptor 1 (PAR1) activation. On the other hand, it was demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, pointing to a novel role of thrombin in vascular protection. Analysis by annexin V-propidium iodide double staining revealed that thrombin, specifically, promoted survival of serum-starved endothelial cells in a concentration-dependent manner. In contrast to its mitogenic effect, the anti-apoptotic effect of thrombin was largely independent of its catalytic activity and was mediated through interaction with alphanubeta3 and alpha5beta1 integrins, whereas the involvement of PAR1 was limited. These results provide new insights in understanding the role of thrombin in endothelial cell signaling and vascular biology.

Inhibition of angiogenesis by small-molecule antagonists of protease-activated receptor-1.

Angiogenesis, the growth of new blood vessels from preexisting ones, is a necessary component of embryogenesis, wound healing, and the proliferative phase of the female reproductive cycle. Angiogenesis also plays a critical role in important pathologic processes such as cancer and cardiovascular complications. In addition, clinical, laboratory, and pharmacologic evidence has shown a link between angiogenesis, coagulation, hemostasis, and thrombosis in the settings of these pathologies. Recent studies in our laboratory revealed that thrombin has a significant stimulatory effect on angiogenesis. This effect of thrombin is independent of fibrin formation and can be attributed mainly to the activation of protease-activated receptor-1 (PAR-1). PAR-1 is widely expressed in vascular cells and is involved in cardiovascular complications such as atherosclerosis, restenosis, and neointimal formation. It is also expressed in many cancer cells contributing to induction of tumor growth and metastasis. In this review, we will summarize our present-day understanding of the role of thrombin and PAR-1 in angiogenesis and the potential therapeutic utility of targeting PAR-1 in angiogenesis-related disease, such as atherosclerosis, restenosis, and cancer.

Blockade of angiogenesis by small molecule antagonists to protease-activated receptor-1: association with endothelial cell growth suppression and induction of apoptosis.

Many studies support the notion that protease-activated receptor (PAR)-1 plays a pivotal role in angiogenesis. However, direct evidence and understanding the molecular mechanisms involved were limited because PAR-1-specific antagonists have been developed only recently. In the present study, we evaluated the effects of two well characterized PAR-1 antagonists, SCH79797 ((N-3-cyclopropyl-7-{[4-(1-methylethyl)phenyl]-methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine)) and RWJ56110 [(alphaS)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide], in the angiogenic cascade. These antagonists suppressed both the basic angiogenesis and that stimulated by thrombin in the chick chorioallantoic membrane model in vivo. PAR-1 antagonists also abrogated tube formation in the in vitro Matrigel system. These inhibitory effects were dose-dependent and well correlated with the inhibitory effects of SCH79797 and RWJ56110 on primary endothelial cell proliferation and on the initiation of apoptosis. PAR-1 blockage resulted in inhibition of endothelial cell growth by increasing the sub-G0/G1 fraction and reducing the percentage of cells in the S phase. Consistent with this, PAR-1 antagonists reduced incorporation of [3H]thymidine in endothelial cells and blocked the phosphorylation of extracellular signal-regulated kinases in a fashion depending specifically on PAR-1 activation. Analysis by annexin V/propidium iodide staining and poly(ADP-ribose) polymerase cleavage revealed that PAR-1 blockage increased apoptotic cell death by a mechanism involving caspases. These results provide further evidence that PAR-1 is a key receptor that mediates angiogenesis and suggest PAR-1 as target for developing antiangiogenic agents with potential therapeutic application in cancer and other angiogenesis-related diseases.

Thrombin functions through its RGD sequence in a non-canonical conformation.

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with alpha(v)beta(3) and alpha(5)beta(1) integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Angstroms resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Angstroms away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.

Role of thrombin in angiogenesis and tumor progression.

Clinical, laboratory, histopathological, and pharmacological evidence support the notion that the coagulation system, which is activated in most cancer patients, plays an important role in tumor biology. Our laboratory has provided evidence that thrombin activates angiogenesis, a process which is essential in tumor growth and metastasis. This event is independent of fibrin formation. At the cellular level many actions of thrombin can contribute to activation of angiogenesis: (1). Thrombin decreases the ability of endothelial cells to attach to basement membrane proteins. (2). Thrombin greatly potentiates vascular endothelial growth factor- (VEGF-) induced endothelial cell proliferation. This potentiation is accompanied by up-regulation of the expression of VEGF receptors (kinase insert domain-containing receptor [KDR] and fms-like tyrosine kinase [Flt-1]). (3). Thrombin increases the mRNA and protein levels of alpha (v)beta (3) integrin and serves as a ligand to this receptor. Furthermore, thrombin increases the secretion of VEGF and enhances the expression and protein synthesis of matrix metalloprotease-9 and alpha (v)beta (3) integrin in human prostate cancer PC-3 cells. These results could explain the angiogenic and tumor-promoting effect of thrombin and provide the basis for development of thrombin receptor mimetics or antagonists for therapeutic application.

On the mechanism of thrombin-induced angiogenesis: involvement of alphavbeta3-integrin.

Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of alphavbeta3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with alphavbeta3 as demonstrated by direct binding of alphavbeta3 protein to immobilized thrombin. This interaction of thrombin with alphavbeta3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to alphavbeta3 or a specific peptide antagonist to alphavbeta3 can abolish all these alphavbeta3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to alphavbeta3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi.