Alpha2-antiplasmin regulates the development of dermal fibrosis in mice by prostaglandin F(2α) synthesis through adipose triglyceride lipase/calcium-independent phospholipase A(2).

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin-α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor ß (TGFß) production. This study was undertaken to clarify how α2AP induces TGFß production and the development of fibrosis. METHODS: To clarify the detailed mechanism by which α2AP induces TGFß production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A(2) (iPLA(2)) and examined whether ATGL/ iPLA(2) is associated with α2AP-induced TGFß production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFß and prostaglandin F(2α) (PGF(2α)). RESULTS: We found that α2AP binding to ATGL promoted PGF(2α) synthesis through iPLA(2) in fibroblasts, and the PGF(2α) synthesis that was promoted by α2AP induced TGFß production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFß and PGF(2α) in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF(2α) synthesis, while the administration of PGF(2α) to α2AP-deficient mice facilitated α2AP deficiency-attenuated fibrosis. CONCLUSION: These findings suggest that α2AP regulates the development of fibrosis by PGF(2α) synthesis through ATGL/iPLA(2). The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.

Involvement of α2-antiplasmin in dendritic growth of hippocampal neurons.

The α2-Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule-associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP⁻/⁻ mice than in the neurons from α2AP⁺/⁺ mice. Exogenous treatment with α2AP enhanced the microtubule-associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP-induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen⁻/⁻ mice. The activation of p38 MAPK was involved in the α2AP-induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function.

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

alpha2-antiplasmin is associated with the progression of fibrosis.

Systemic sclerosis results in tissue fibrosis due to the activation of fibroblasts and the ensuing overproduction of the extracellular matrix. We previously reported that the absence of alpha2-antiplasmin (alpha2AP) attenuated the process of dermal fibrosis; however, the detailed mechanism of how alpha2AP affects the progression of fibrosis remained unclear. The goal of the present study was to examine the role of alpha2AP in fibrotic change. We observed significantly higher levels of alpha2AP expression in the skin of bleomycin-injected systemic sclerosis model mice in comparison with the levels seen in control mice. We also demonstrated that alpha2AP induced myofibroblast differentiation, and the absence of alpha2AP attenuated the induction of myofibroblast differentiation. Moreover, we found that connective tissue growth factor induced the expression of alpha2AP through both the extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways in fibroblasts. Interestingly, alpha2AP also induced transforming growth factor-beta expression through the same pathways, and the inhibition of ERK1/2 and JNK slowed the progression of bleomycin-induced fibrosis. Our findings suggest that alpha2AP is associated with the progression of fibrosis, and regulation of alpha2AP expression by the ERK1/2 and JNK pathways may be an effective antifibrotic therapy for the treatment of systemic sclerosis.

The absence of urokinase-type plasminogen activator receptor plays a role in the insulin-independent glucose metabolism.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions. In the present study, we examined whether the uPAR plays any role in the regulation of glucose metabolism. The experiments were performed using male wild-type (uPAR) and uPAR knockout (uPAR) C57BL/6J mice. The blood glucose levels after the intraperitoneal injection of glucose were significantly decreased in uPAR mice compared with uPAR mice. On the other hand, there were no differences in the insulin secretion induced by glucose injection and the reactivity of insulin between uPAR and uPAR mice. The expression levels of glucose transporter 2 (GLUT2) in the liver and GLUT4 in the skeletal muscles from the uPAR mice were significantly increased compared with those of the uPAR mice. In addition, we found that the level of phosphorylation of AMP-activated protein kinase in skeletal muscles and myoblasts from the uPAR mice increased compared with those in uPAR mice. These data suggest that the increase in the GLUT2 and GLUT4 expression and the activation of AMP-activated protein kinase by uPAR deficiency enhances the glucose intake. These findings therefore provide new insights into the role of uPAR in the glucose metabolism.

Abnormal production of macrophage inflammatory protein-1alpha by microglial cell lines derived from neonatal brains of Sandhoff disease model mice.

Sandhoff disease (SD) is a lysosomal beta-hexosaminidase deficiency involving excessive accumulation of undegraded substrates, including terminal N-acetylglucosamine-oligosaccharides and GM2 ganglioside, and progressive neurodegeneration. Our previous study demonstrated remarkable induction of macrophage inflammatory factor-1alpha (MIP-1alpha) in microglia in the brains of SD model mice as a putative pathogenic factor for SD via microglia-mediated neuroinflammation. In this study, we established microglial cell lines (WT- and SD-Mg) from wild-type and SD mice, and first demonstrated the enhanced production of MIP-1alpha in SD-Mg. Inhibitors of protein kinase C (PKC) and Akt reduced the production of MIP-1alpha by SD-Mg. Elevated activation of Akt and partial translocation of PKC isozymes (alpha, betaI, betaII, and delta) from the cytoplasm to the membrane in SD-Mg were also revealed by means of immunoblotting. Furthermore, it was demonstrated that intracellular extracellular signal-regulated kinase, c-Jun N-terminal kinase, and phospholipase C (PLC), but not phosphoinositide 3-kinase, should contribute to the induction of MIP-1alpha in SD-Mg, and that PLC could independently regulate the activation of both PKC and Akt. We proposed here that the deregulated activation of PLC should cause the enhanced MIP-1alpha production via plural signaling pathways mediated by PKC and Akt, followed by extracellular signal-regulated kinase and c-Jun N-terminal kinase, in SD-Mg.

Effect of staphylokinase-derived nonadecapeptide on the activation of plasminogen.

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin. The interaction between the plasmin-SAK complex and plasminogen was investigated using synthesized peptides, which were constructed according to the amino acid sequence of the SAK molecule. A synthetic nonadecapeptide (SAK22-40) corresponding to Glu22-Leu40 by the SAK molecule enhanced the activation of Glu-plasminogen by the plasmin-SAK complex. Analysis of IAsys resonant mirror biosensor showed that SAK22-40 bound to Glu-plasminogen. This binding was completely inhibited by IgG against the B-chain in the plasminogen molecule. But, this binding was not inhibited by IgG against lysine-binding sites (LBS) of the A-chain in the plasminogen molecule. The substitution of Lys35 with Ala in SAK22-40 did not enhance the activation of Glu-plasminogen by the plasmin-SAK complex. When SAK22-40 was administrated in a mouse thrombosis model, earlier recanalization was observed than in mice with vehicle administration. Thus, a newly synthesized peptide, SAK22-40 enhanced Glu-plasminogen activation and induced effective thrombolysis.

Alpha2-antiplasmin on cardiovascular diseases.

Circumstantial evidence has been provided of a role of the plasminogen/plasmin system in a variety of biological phenomena, including thrombolysis, vascular stenosis, reproduction, embryogenesis, cell invasion, angiogenesis, brain function and chronic lung or kidney inflammatory disorders. Inhibition of the system occurs either at the levels of plasminogen activator, regulated by specific plasminogen activator inhibitors (PAIs) or at the levels of plasmin, mainly regulated by alpha2-antiplasmin (alpha2-AP). alpha2-AP is a specific plasmin inhibitor. We investigated the role of alpha2-AP on arterial or venous thrombus formation using mice deficient alpha2-AP and the interactions among lack of alpha2-AP, antiplatelet, anticoagulant and thrombolytic compounds were evaluated using murine thrombus model. These results clearly indicate that alpha2-AP plays a different role in acute arterial thrombosis or venous thrombosis. Additionally, lack of alpha2-AP significantly affected anti-coagulant and thrombolytic action, but not anti-platelet compounds, on the development of thrombus formation in vivo. Recent findings reported that plasmin cleaves vascular endothelial growth factor (VEGF) in extracellular matrix. Our findings newly indicate that lack of alpha2-AP enhances the secretion of VEGF in acute myocardial infarction and over secretion of VEGF promotes heart failure by pulmonary edema. Moreover, regulation of VEGF by alpha2-AP significantly affected reendothelialization after vascular injury. These findings indicate a potential new aspect in this field and could be a useful report for the development of novel antithrombotic compounds.

Novel ideas of gene therapy for atherosclerosis: modulation of cellular signal transduction of TGF-beta family.

Atherosclerosis is a disease characterized by accumulation of lipids and fibrous elements in the innermost layer of the arterial wall. An asymptomatic atherosclerotic plaque is characterized by a lipid core, composed of modified lipids, macrophages and T cells, which were separated from the lumen vessel by a thick fibrous cap, composed of vascular smooth muscle cell-secreted solid collagen matrix. Recently, it has been reported that expressions of TGF-beta family were up regulated in human atherosclerotic plaques. In addition TGF-beta family seems to plays pivotal roles for the development of atherosclerosis: TGF-beta and activin A were suggested to play protective roles against the development of atherosclerotic plaques. On the other hand bone morphogenetic protein seems to play pivotal roles for the calcification of the atherosclerotic plaques. Therefore, it is debatable whether gene therapy modulating cellular signal transductions of TGF-beta family is a useful tool for the inhibition of progression of atherosclerotic disease. In this review, our discussion is focused on the possibilities of gene therapies for atherosclerosis either by enhancement or suppression of cellular signaling of TGF-beta family in target cells, in atherosclerotic plaques.

The possibility of novel antiplatelet peptides: the physiological effects of low molecular weight HSPs on platelets.

Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported on a physiological role of HSP20, HSP27 and alphaB-crystallin on platelet function in vitro and ex vivo. HSP20 and alphaB-crystallin inhibited platelet aggregation using human platelets dose-dependently induced by thrombin or botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When HSP20 or alphaB-crystallin was injected intravenously as a bolus in hamsters, the development of thrombus after endothelial injury was prevented. Moreover, 9 amino-acid sequences isolated from HSP20 or alphaB-crystallin significantly reduced platelet aggregation induced by TRAP, but not a PAR-4 agonist. These findings strongly suggest that HSP20 or alphaB-crystallin can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defensive system to thrombus formation in vivo.

Differential roles of MAP kinases in atorvastatin-induced VEGF release in cardiac myocytes.

Statins, specific inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia. In addition to the reduction of cholesterol biosynthesis, accumulating evidence indicates that statins have several pleiotropic effects especially on cardiovascular system. However, the exact role of statin in cardiac myocytes remains unclear. In the present study, we investigated whether atorvastatin induces vascular endothelial growth factor (VEGF) release in cardiac myocytes, and the underlying mechanism. We observed that atorvastatin significantly stimulated VEGF release in a dose-dependent manner. It induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The atorvastatin-induced VEGF release was enhanced by PD98059, which is a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Further, it was significantly reduced by SB203580, a specific inhibitor of p38 MAP kinase. Furthermore, the atorvastatin-induced phosphorylation of p38 MAP kinase was attenuated by SB203580, whereas it was enhanced by PD98059. Taken together, these results suggest that the atorvastatin-induced VEGF release in cardiac myocytes is positively regulated by p38 MAP kinase and negatively regulated byp44/p42 MAP kinase and that the atorvastatin-induced phosphorylation of p38 MAP kinase is regulated by p44/p42 MAP kinase in these cells.

CD62-mediated activation of platelets in cerebral white matter lesions in patients with cognitive decline.

BACKGROUND: Vascular dementia is related to intracranial arteriosclerosis associated with deep white matter lesions (DWMLs). DWMLs have been linked to thrombogenesis due to sustained platelet activation; therefore, an accurate hematological marker is needed. This study was done to evaluate the usefulness of a new method to examine the function of activated platelets in order to assess DWMLs associated with cognitive decline. METHODS: A total of 143 individuals (70.4 ± 6.1 years old) who underwent hospital-based health screening using head MRI were evaluated. DWLs were evaluated on T2-weighted and FLAIR images by semi-quantitatively grading them from Grade 0 (none) to Grade 3 (severe) using the Fazekas classification. Cognitive function was evaluated using the MMSE and the word fluency test. Platelet activation was assessed using fluorescence-labeled anti-human platelet monoclonal antibodies and semi-quantitatively determining PAC-1- and CD62P-positive rates by flow cytometry. RESULTS: Significant increases in hypertension and CD62P levels were observed with increasing DWML grade (2.6% in Group 0, 3.1% in Group 1, 4.1% in Group 2, and 5.0% in Group 3). CD62P levels were defined as elevated when they were above the mean+2SD of the Grade 0 group, and the odds ratio of the Grade 2+3 group was 3.03. A significant negative correlation was observed between CD62P levels and word fluency tests or the MMSE score. CONCLUSION: Elevations in CD62P levels, which reflect platelet function activation, were associated with white matter lesions accompanied by a decline in cognitive function. CD62P levels may be useful as a sensitive clinical marker for the early detection of DWMLs with cognitive decline.

Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases.

We investigated whether transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-beta increased the level of HSP27 but had no effect on the HSP70 level. TGF-beta stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-beta induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-beta was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-beta-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-induced increase of mRNA for HSP27. The amplification of TGF-beta-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-beta-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-beta-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.

P2Y12 receptors play a significant role in the development of platelet microaggregation in patients with diabetes.

Ninety-eight diabetic patients (type 2) were studied together with 24 healthy normotensive controls. Microaggregates (particle scale, <25 microm) of platelets were detected by a laser scattering system. Microaggregates in the control group showed a time-dependent reversible change; however, they existed continuously in 82 of 98 diabetic patients. When platelets of diabetics were stimulated by a shear stress alone without ADP, 74 also showed spontaneous and irreversible microaggregates even though they were not observed in all control subjects. In control subjects, microaggregates were inhibited by MRS2279 (a P2Y1 antagonist), but not AR-C69931MX (a P2Y12 antagonist). However, AR-C69931MX prevented irreversible microaggregates in diabetic patients. When either aspirin or ticlopidine was administered to diabetic patients with irreversible microaggregates, both drugs significantly decreased microaggregates induced by a low dose of ADP. Ticlopidine additionally reduced the microaggregates induced by shear stress alone. In conclusion, microaggregates of platelets via P2Y12 receptors could play a key role in the hypersensitivity of platelets in diabetic patients, and the measurement of microaggregation could be a useful marker to estimate of thrombogenesis. These findings present a possible new means for patients with diabetes to prevent ischemic events.

Tissue type plasminogen activator facilitates NMDA-receptor-mediated retinal apoptosis through an independent fibrinolytic cascade.

PURPOSE: To investigate the association between apoptosis and the fibrinolytic system in retinal cell damage. METHODS: Tissue type plasminogen activator-deficient (tPA(-/-)), urokinase type plasminogen activator-deficient (uPA(-/-)), plasminogen activator inhibitor-1-deficient (PAI-1(-/-)), alpha2 antiplasmin-deficient (alpha2 AP(-/-)) mice, and their wild-type counterparts were used. Retinal cell damage was induced by intravitreal injection of the excitotoxin N-methyl-d-aspartate (NMDA). The TdT-dUTP terminal nick-end labeling (TUNEL) method was used to examine retinal cell damage. RESULTS: tPA(-/-) mice were resistant to retinal cell damage caused by administration of NMDA, and PAI-1(-/-) mice were more injured than their wild-type. No significant difference was observed between uPA(-/-) or alpha2 AP(-/-) and their wild-type mice. CONCLUSIONS: The results strongly suggest that endogenous tPA, but not uPA acts as a facilitator in NMDA-induced retinal cell damage, and that its mechanism may not be associated with cleavage of plasminogen into plasmin in the fibrinolytic cascade.

Sphingomyelinase amplifies BMP-4-induced osteocalcin synthesis in osteoblasts: role of ceramide.

We previously reported that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblast-like MC3T3-E1 cells and that mitogen-activated protein (MAP) kinases are involved in bone morphogenetic protein (BMP)-4-stimulated osteocalcin synthesis in these cells. In the present study, we investigated whether sphingomyelinase affects BMP-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. Sphingomyelinase significantly enhanced the BMP-4-stimulated osteocalcin synthesis. Among sphingomyelin metabolites, C(2)-ceramide enhanced the BMP-4-stimulated osteocalcin synthesis while sphingosine and sphingosine 1-phosphate had little effect on the synthesis. D-erythro-MAPP, an inhibitor of ceramidase, amplified the sphingomyelinase-effect on the osteocalcin synthesis. C(2)-ceramide suppressed the BMP-4-induced phosphorylation of p44/p42 MAP kinase, while having little effect on the phosphorylation of Smad1 and p38 MAP kinase. Taken together, our results strongly suggest that extracellular sphingomyelinase enhances the BMP-stimulated osteocalcin synthesis via ceramide in osteoblasts and that the effect of ceramide is exerted at a point upstream from p44/p42 MAP kinase.

Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase.

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.

Plasmin generation plays different roles in the formation and removal of arterial and venous thrombus in mice.

The role of plasminogen (Plg) and alpha2-antiplasmin (alpha2-AP) in vascular thrombolysis in vivo was investigated in mice deficient in plasminogen (Plg-/-) or a2-AP (alpha2-AP-/-) or their wild type (PAI-1+/+, alpha2-AP+/+). A thrombus was induced in the murine carotid artery or the internal jugular vein by endothelial injury. Blood flow was continuously monitored for 90 min and for 6 h 30 min after the initiation of endothelial injury. The times to occlusion by the developing thrombus in the carotid artery and the jugular vein of wild type mice were 12+/-1.8 and 7.2+/-1.9 min, respectively. The arterial thrombus formation in alpha2-AP-/- mice was indistinguishable from the one in wild type mice, whereas the time to occlusion in Plg-/- was significantly shortened to 5.9+/-1.7 min. Vascular patency after spontaneous reperfusion was markedly improved in alpha2-AP-/- mice. On the contrary, arteriarpatency in Plg-/- mice was aggravated. In venous thrombus formation, the time to occlusion in alpha2-AP-/- mice was significantly prolonged (27.1+/-5.2 min), whereas in Plg-/- it was slightly shortened to 6.5+/-2.5 min. Vascular patency after spontaneous reperfusion was also improved in alpha2-AP-/- mice, but not in Plg-/- mice. Histological observations using SEM indicated that fibrin nets were firmly fixed on the injured area in Plg-/- mice, but not in alpha2-AP-/- mice. The tail bleeding time was not different in any type of mice. However, re-bleeding time using a template bleeding device was significantly prolonged in alpha2-AP-/- as compared with that of wild type mice. In conclusion, lack of plasminogen markedly reduces the antithrombotic activities in vivo, whereas alpha2-AP plays a more important role in the formation and removal of venous thrombus in mice. Consequently, the inhibition of alpha2-AP could be a useful tool for the therapy of venous thrombosis and the prevention of re-thrombus formation.

Function of tissue-type plasminogen activator releaser on vascular endothelial cells and thrombolysis in vivo.

The effect of monosodium[2-(6-hydroxynaphthalen-2-yl)-6-methylpyrimidin-4-yloxy]acetate dihydrate (JTV-926) on fibrinolysis was investigated in vitro and in vivo. JTV-926 released tissue-type plasminogen activator (t-PA) from human vascular endothelial cells in a dose-dependent manner. The thrombolytic effect of JTV-926 was studied using three animal thrombosis models; a photo-irradiation-induced mouse carotid artery thrombosis model, a photo-irradiation-induced rat femoral artery thrombosis model and a thrombin-induced rat venous thrombosis model. In the mouse thrombosis model, t-PA deficient mice (t-PA-/- mice) and their wild-type (t-PA+/+) were used. JTV-926 was injected as a bolus 30 min after the interruption of blood flow by an occlusion thrombi. Blood flow was continuously monitored for 180 min after intravenous administration of JTV-926 (1 mg/kg). Although the recanalization rate of the occluded artery was 37.5% in t-PA +/+ mice with the vehicle control, it increased to 75% in t-PA+/+ mice after JTV-926 administration. However, when JTV-926 was administrated in t-PA-/- mice, vascular recanalization was not observed in any arteries. In the photo-irradiation-induced rat femoral artery thrombosis model, intra-duodenal administration of JTV-926 induced thrombolysis. Moreover, in the thrombin-induced rat venous thrombosis model, the dose-dependent thrombolysis was also observed by oral administration of JTV-926. It was suggested that JTV-926 revealed a sufficient thrombolytic effect through the absorption from the intestine. Thus, a newly synthesized compound, JTV-926 induced t-PA release from vascular endothelial cells and effective thrombolysis in vivo.

Pharmacokinetic and pharmacodynamic properties of a new thromboxane receptor antagonist (Z-335) after single and multiple oral administrations to healthy volunteers.

The pharmacokinetics and pharmacodynamics of a new oral thromboxane (TX) A2 receptor antagonist, Z-335, were studied in healthy male volunteers following single doses (0.5-40 mg, PO) in a dose-escalating manner and multiple doses (40 mg, PO, once daily for 7 consecutive days) with a single-blind, placebo-controlled design. Serial blood and urine samples were analyzed for Z-335 and its metabolites to obtain key pharmacokinetic parameters. In the single-dose (10, 20, and 40 mg) study, the maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) increased in proportion to the dose when administered afterfasting, while the mean elimination half-life (t1/2beta) was essentially unchanged (7.79-7.93 h). Recovery of the unchanged and taurine-conjugated drugs in the urine within 24 hours was 6.5% to 8.4% and 11.9% to 14.2%, respectively. These parameters essentially remained unchanged when the effect of meal intake was evaluated at the dose of 20 mg with a crossover design. Ex vivo platelet aggregation in the plasma by a TXA2 analogue, U46619, was completely inhibited within 2 hours after all doses, and complete inhibition was maintained for 12 to 14 hours, depending on the dose. The aggregation induced by collagen was also inhibited to a lesser extent, whereas that by adenosine diphosphate was hardly influenced. In the multiple-dose study, Cmax and AUC0-24 were increased by 34% after the last dose compared with the first dose. Z-335 afforded extensive inhibition of platelet aggregation by U46619 throughout the administration period, which returned, however, almost to the control level 48 hours after the last dose. The agent was well tolerated without any abnormalities in subjective and objective symptoms, blood biochemistry, hematology, and urinalysis definitely attributable to the agent, except for the changes expected from its TXA2 receptor-antagonizing actions. Z-335 was concluded to be safe and to provide long-lasting blockade of TXA2 receptors on the basis of a once-daily regimen, promoting further clinical evaluation.