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PubMed2000

Most clot-bound thrombin activity is inhibited by plasma antithrombin during clot aging, but a very small fraction survives.

Inhibitory effects of thrombin inhibitors against clot-bound thrombin have been evaluated using clots prepared from human plasma as the source of clot-immobilized active enzyme, and the clot-bound thrombin has been reported to be protected from its inhibition by antithrombin. However, we found that the clot-bound thrombin was not intrinsically protected from inhibition by antithrombin, i.e., a large fraction of initially active clot-bound thrombin was inhibited by antithrombin present in human plasma time dependently, and only very small fractions (0.04--0.08%) of the thrombin retained their enzymatic activity after clot-aging. These results suggest that the extent of clot-aging determines the sensitivity of clot-bound thrombin to antithrombin, and that inhibitory effects of drugs against clot-bound thrombin in vitro must be interpreted with caution to estimate their effects in vivo.

Antithrombins↗
PubMed1998

Stimulation of human smooth muscle cell proliferation by thrombin involves increased synthesis of platelet-derived growth factor.

OBJECTIVES: Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can also signal other events related to cell mitogenesis and migration. In this report, we investigated the effects of thrombin on the proliferation of human arterial smooth muscle cells (SMCs) in culture and its interaction with platelet-derived growth factor (PDGF). MATERIAL AND METHODS: Human arterial SMCs originated from a renal artery were grown in cell culture. The effect of thrombin on DNA synthesis was evaluated by 3H-thymidine incorporation. The effect of thrombin on inositol-phosphate formation by SMCs was also analyzed as well as the binding of PDGF AA and BB to these cells. PDGF secretion was analyzed by radioimmunoassay (RIA). RESULTS: Exposure of cultured human SMCs to thrombin caused an increased rate of DNA synthesis in a dose-response manner, with a maximal stimulatory effect at a concentration of 2.0 U/mL. Thrombin was found to increase the accumulation of inositol phosphates and to increase the production of PDGF as measured by RIA. Exposure of cells to 2.0 U/mL thrombin resulted in a strong decrease in PDGF AA binding to PDGF receptors and did not change PDGF BB binding, probably indicating that PDGF alpha-receptors could be occupied by endogenously produced PDGF A. CONCLUSION: Thrombin stimulates human vascular SMC proliferation in a dose-response way, in part by the formation of inositol phosphates. The mechanism responsible for this effect involves, at least in part, an increased endogenous synthesis of PDGF.

Anticoagulants↗
PubMed2005

Thrombin-induced oxidative stress contributes to the death of hippocampal neurons in vivo: role of microglial NADPH oxidase.

The present study investigated whether thrombin, a potent microglial activator, can induce reactive oxygen species (ROS) generation through activation of microglial NADPH oxidase and if this may contribute to oxidative damage and consequent neurodegeneration. Seven days after intrahippocampal injection of thrombin, Nissl staining and immunohistochemistry using the neuronal-specific nuclear protein NeuN revealed a significant loss in hippocampal CA1 neurons. In parallel, thrombin-activated microglia, assessed by OX-42 and OX-6 immunohistochemistry, and ROS production, assessed by hydroethidine histochemistry, were observed in the hippocampal CA1 area in which degeneration of hippocampal neurons occurred. Reverse transcription-PCR at various time points after thrombin administration demonstrated an early and transient expression of inducible nitric oxide synthase (iNOS) and several proinflammatory cytokines. Western blot analysis and double-label immunohistochemistry showed an increase in the expression of and the localization of iNOS within microglia. Additional studies demonstrated that thrombin induced the upregulation of membrane (gp91(phox)) and cytosolic (p47(phox) and p67(phox)) components, translocation of cytosolic proteins (p47(phox), p67(phox), and Rac1) to the membrane, and p67(phox) expression of the NADPH oxidase in microglia in the hippocampus in vivo, indicating the activation of NADPH oxidase. The thrombin-induced oxidation of proteins and loss of hippocampal CA1 neurons were partially inhibited by an NADPH oxidase inhibitor and by an antioxidant. To our knowledge, the present study is the first to demonstrate that thrombin-induced neurotoxicity in the hippocampus in vivo is caused by microglial NADPH oxidase-mediated oxidative stress. This suggests that thrombin inhibition or enhancing antioxidants may be beneficial for the treatment of neurodegenerative diseases, such as Alzheimer's disease, that are associated with microglial-derived oxidative damage.

Analysis of Variance↗
PubMed1999

Thrombin stimulates synthesis of type IV collagen and tissue inhibitor of metalloproteinases-1 by cultured human mesangial cells.

Glomerular accumulation of extracellular matrix (ECM) is the common pathologic feature following glomerular injury, and the alteration in the synthesis and degradation of ECM may be involved in the glomerular accumulation of ECM. Glomerular fibrin formation occurs in various forms of human and experimental glomerulonephritis, and it may play an important role in progressive glomerular injury. Thrombin, a multifunctional serine proteinase that is generated at the site of vascular injury, has central functions in hemostasis and it also shows various biologic effects. In this study, it is hypothesized that thrombin may alter the production and the degradation of type IV collagen, which is an important component of ECM in the glomeruli. Human mesangial cells (HMC) were cultured, and the levels of type IV collagen, tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-2 (MMP-2) in the culture supernatants were measured by enzyme immunoassay using specific antibodies. MMP-2 activity was also evaluated by zymography using polyacrylamide/ sodium dodecyl sulfate gel-containing gelatin. Thrombin increased the production of type IV collagen and TIMP-1 in a dose-and time-dependent manner, but it did not increase MMP-2. Thrombin also stimulated the gene expressions of the type IV collagen and TIMP-1 in HMC in a dose- and time-dependent manner. Thrombin treated with diisopropylfluorophosphate, a serine proteinase inhibitor, did not show any of these effects. Hirudin, a natural thrombin inhibitor, and anti-transforming growth factor-beta-neutralizing antibody inhibited the stimulating effect of thrombin. These findings suggest that thrombin may contribute to the excessive accumulation of ECM and progression of glomerulosclerosis through an increase of type IV collagen production and a decreased matrix degradation presumably via a transforming growth factor-beta-dependent mechanism.

Cells, Cultured↗
PubMed1997

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells.

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.

Animals↗
PubMed2003

Iatrogenic femoral pseudoaneurysms that are unresponsive to percutaneous thrombin injection: potential causes.

OBJECTIVE: We sought to determine whether any quantitative selection criteria can be established to predict which pseudoaneurysms of the common femoral artery will fail to respond to thrombin. SUBJECTS AND METHODS. Under sonographic guidance, we injected thrombin into 54 consecutive iatrogenic pseudoaneurysms of the common femoral artery. We statistically compared the volume, maximum dimension, and neck diameter of the pseudoaneurysms and thrombin doses injected into the successfully treated (persistent thrombosis on a follow-up duplex sonogram obtained 7-10 days after thrombin injection) and unresponsive (sonographically documented recurrence within 24 hr) pseudoaneuryms to determine whether any of these factors could help to predict a failure to respond to thrombin. For all patients in whom treatment failed, findings at surgical repair were noted and sonograms were reviewed to confirm the dimensions and appearance of the pseudoaneurysm and the absence of an associated arteriovenous fistula. RESULTS: Complete and persistent thrombosis was achieved in 49 (91%) of the 54 common femoral artery pseudoaneurysms. In comparing the 49 successfully treated patients and the five patients in whom treatment failed, we identified no significant differences in the volume, maximum dimension, or neck diameter of the pseudoaneurysm (two-sample t test) or in the thrombin dose (Wilcoxon's rank sum test). In all five patients with failed treatment, an arteriotomy site laceration measuring at least 8.0 mm (n = 4) or infection (n = 1) was identified at surgical repair, none of which were identifiable even in retrospect on duplex and color Doppler sonography. CONCLUSION: Failure of an iatrogenic pseudoaneurysm of the common femoral artery to respond to thrombin may indicate an occult vascular injury. In these patients, surgical repair, rather than reinjection of thrombin, should be considered.

Adult↗
PubMed1992

Inhibition of human alpha-, beta- and gamma-thrombin by mono-, bis-, tris- and tetra-benzamidine structures: thermodynamic study.

The inhibitory effect of mono-, bis-, tris- and tetra-benzamidine structures (benzamidine, DAPP, TAPB and TAPP, respectively) on the catalytic properties of human alpha-, beta- and gamma-thrombin (alpha-, beta- and gamma-thrombin, respectively) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C). The affinity of DAPP, TAPB and TAPP for alpha- and beta-thrombin is higher than that found for benzamidine association around neutrality, converging in the acidic pH limb; in contrast, benzamidine, DAPP, TAPB and TAPP show the same value of the association inhibition constant (Ki; M-1) for gamma-thrombin over the whole pH range explored. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to alpha-, beta- and gamma-thrombin, as well as for DAPP, TAPB and TAPP association to gamma-thrombin reflects the acidic-pK shift, upon inhibitor binding of a single ionizing group. On the other hand, values of Ki for DAPP, TAPB and TAPP binding to alpha- and beta-thrombin appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues over the same pH range. By considering molecular models of the serine proteinase:inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).

Amidines↗
PubMed1997

Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model.

Recently, the authors showed that thrombin contributes to the formation of brain edema following intracerebral hemorrhage. The current study examines whether the action of thrombin is due to an effect on cerebral blood flow (CBF), vasoreactivity, blood-brain barrier (BBB) function, or cell viability. In vivo solutions of thrombin were infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later; CBF and BBB permeability were measured. The actions of thrombin on vasoreactivity were examined in vitro by superfusing thrombin on cortical brain slices while monitoring microvessel diameter with videomicroscopy. In separate experiments C6 glioma cells were exposed to various concentrations of thrombin, and lactate dehydrogenase release, a marker of cell death, was measured. The results indicate that thrombin induces BBB disruption as well as death of parenchymal cells, whereas CBF and vasoreactivity are not altered. The authors conclude that cell toxicity and BBB disruption by thrombin are triggering mechanisms for the edema formation that follows intracerebral hemorrhage.

Animals↗
PubMed2001

The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.

Acute Disease↗
PubMed1999

Thrombin-induced growth cone collapse: involvement of phospholipase A(2) and eicosanoid generation.

The studies presented here explore intracellular signals resulting from the action of repellents on growth cones. Growth cone challenge with thrombin or thrombin receptor-activating peptide (TRAP) triggers collapse via a receptor-mediated process. The results indicate that this involves activation of cytosolic phospholipase A(2) (PLA(2)) and eicosanoid synthesis. The collapse response to repellents targets at least two functional units of the growth cone, the actin cytoskeleton and substratum adhesion sites. We show in a cell-free assay that thrombin and TRAP cause the detachment of isolated growth cones from laminin. Biochemical analyses of isolated growth cones reveal that thrombin and TRAP stimulate cytosolic PLA(2) but not phospholipase C. In addition, thrombin stimulates synthesis of 12- and 15-hydroxyeicosatetraenoic acid (HETE) from the released arachidonic acid via a lipoxygenase (LO) pathway. A selective LO inhibitor blocks 12/15-HETE synthesis in growth cones and inhibits thrombin-induced growth cone collapse. Exogenously applied 12(S)-HETE mimics the thrombin effect and induces growth cone collapse in culture. These observations indicate that thrombin-induced growth cone collapse occurs by a mechanism that involves the activation of cytosolic PLA(2) and the generation of 12/15-HETE.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2000

N-Ethylmaleimide induces disaggregation of platelets preaggregated by ADP and thrombin and decreases cytoplasmic calcium level.

The effect of N-ethylmaleimide (NEM, 1-200 microM) on ADP- and thrombin-induced platelet aggregation and thrombin-induced increase in intracellular Ca2+ concentration was studied. Addition of NEM to platelets preaggregated with ADP or thrombin induces platelet disaggregation. The anti-aggregant activity of NEM was different for ADP- and thrombin-induced aggregations. At 200 microM concentration, NEM completely disaggregated ADP-induced aggregates and only partially disaggregated thrombin-aggregated platelets. NEM did not influence the thrombin-induced increase in cytoplasmic Ca2+ and had no effect on the basal level of Ca2+ in the cytosol of non-activated platelets. However, NEM decreased the level of thrombin-mobilized Ca2+ in the cytosol of activated platelets. Thus, NEM can induce disaggregation of ADP- and thrombin-preaggregated platelets by activating a system which removes Ca2+ from the platelet cytosol.

Adenosine Diphosphate↗
PubMedDate not supplied

Hypercoagulable state in hypercholesterolemic subjects assessed by platelet-dependent thrombin generation: in vitro effect of cerivastatin.

BACKGROUND: Hypercholesterolemia is an important risk factor to develop acute thrombotic complications of atherosclerosis like to myocardial infarction and ischemic stroke. Platelets and coagulation factors are strictly involved in the genesis of such thrombotic events and their hyperactivity in hypercholesterolemic patients has been previously reported. Moreover some cholesterol-lowering molecules (statins) seem to be able of reducing platelet activity. METHODS: We performed platelet-dependent thrombin generation (colorimetric method) to assess the coagulative potential of 40 caucasian hypercholesterolemic subjects with respect to normal controls and to the grade of hypercholesterolemia. Moreover we observed the effect of platelets from hypercholesterolemics on thrombin generation in plasma from normal subjects. The effect of Cerivastatin on thrombin generation was evaluated too. RESULTS: Our data show an increased thrombin generation both in mild and high hypercholesterolemic subjects with respect to controls (424.6+/-30.5 vs. 197.1+/-27.4 mIU/ml). No significant difference in the amount of thrombin generation was found between mild and high hypercholesterolemics (399.6+/-20.7 vs. 440.2+/-21.4 mIU/ml). Platelets directly influence thrombin generation and they present an intrinsic hyperactivity that can be modulated by Cerivastatin (223.6+/-24.8 vs. 424.6+/-30.5 mIU/ml). CONCLUSIONS: Mild hypercholesterolemia is associated with an increased thrombinic potential that may be considered an added risk factor to develop thrombotic events. Platelets directly influences this hypercoagulative state and Cerivastatin is able to reduce thrombin generation by way of a direct interaction with platelets.

Blood Coagulation Disorders↗
PubMed2001

Ultrasound-guided thrombin injection for the treatment of iatrogenic pseudoaneurysm of the femoral artery.

BACKGROUND: Pseudoaneurysm occurring after catheterization of the femoral artery is associated with significant morbidity. Percutaneous ultrasound-guided thrombin injection has recently emerged as a potential first-line therapy. OBJECTIVES: To evaluate the efficacy of this treatment in eight patients with iatrogenic femoral artery pseudoaneurysm. METHODS: After attempted treatment with external compression had failed, eight patients with iatrogenic femoral artery pseudoaneurysm were treated with thrombin injection. Treatment performed between 2 and 9 days following arterial puncture. The study group comprised seven males and one female ranging in age from 23 to 89 years (median 70). Seven had undergone cardiac catheterization with or without intervention, and five were receiving antiplatelet and/or anticoagulant drugs. Arterial pseudoaneurysm resulted from femoral vein catheterization in one patient. Using a sterile technique and real-time Doppler ultrasound guidance, a dilute solution of bovine thrombin (average dose 250 units, range 100-600), was slowly injected directly into the pseudoaneurysm until cessation of flow was seen. Patients were allowed to walk within 2 hours of the procedure and were followed up clinically and by color Doppler ultrasound during the admission. RESULTS: Cardiac catheterization had been inadvertently performed via the superficial or profunda femoris arteries in four of the eight patients. Thrombin injection was initially successful in all eight patients without complication. Thrombosis occurred immediately in every case. Early recanalization of pseudoaneurysm occurred in one patient despite repeat thrombin injection and attempted ultrasound-guided compression. He eventually required surgical repair. The final success rate was 87.5% (7/8). CONCLUSION: Faulty puncture technique is an important risk factor for the development of post-catheterization femoral artery pseudoaneurysm. Ultrasound-guided thrombin injection is a safe, rapid, well-tolerated, inexpensive and successful therapy. If initial external compression with a sandbag fails to result in thrombosis of the pseudoaneurysm then thrombin injection should be considered as first-line therapy. If unsuccessful, it does not preclude the use of alternative treatment modalities. Further study is necessary to assess the long-term effects of thrombin injection.

Adult↗
PubMed2001

Selective and sustained inhibition of surface-bound thrombin activity by intimatan/heparin cofactor II and its relevance to assessing systemic anticoagulation in vivo, ex vivo and in vitro.

We compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001. We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.

Animals↗
PubMed1999

[The mediation effects of endothelin-1 on human mesangial cell proliferation stimulated by thrombin].

OBJECTIVE: To investigate the effects of endothelin-1 (ET-1) on mesangial cell proliferation stimulated by thrombin. METHODS: With cultured human mesangial cells (HMC), the following tests were performed. (1) Stimulative tests: After thrombin stimulation, HMC proliferation was determined by MTT method, concentration of ET-1 in supernatant was measured with radioimmunoassay (RIA) and preproendothelin-1 (ppET-1) mRNA expression of HMC was analysed by Northern blot hybridization. (2) Inhibitory test: The influence of ET-1 inhibitors on the HMC proliferation following thrombin stimulation at three different levels, i.e. ET-1 antiserum, selective ET receptor-type A (ET(A)) antagonist and antisense oligodeoxynucleotide for ppET-1, was measured by MTT test respectively. RESULTS: (1) HMC proliferation was stimulated by thrombin in dose dependent manner and in relation to the time of stimulation. The peak growth level was attained in thrombin concentration of 16 U/ml and incubation time of 16 hours. ET-1 secretion by HMC was increased in parallel with the dose curve and time curve of HMC proliferation; HMC ppET-1 mRNA expression was up-regulated by thrombin 16 U/ml with incubation time of 8 hours and 16 hours respectively (7.9 fold, 3.4 fold versus control group). (2) ET-1 inhibitors, ET-1 antiserum, selective ET(A) receptor antagonist and antisense oligodeoxynucleotide for ppET-1 all significantly inhibit HMC proliferation stimulated by thrombin. CONCLUSION: Thrombin stimulates HMC to produce ET-1 and ET-1 mediates the HMC proliferation.

Cell Proliferation↗
PubMed2000

[The difference of thrombin-induced TF/uPAR mRNAs expressions in cultured endothelial cells and U937 cell line].

OBJECTIVE: To compare the effects of thrombin on the expression of the tissue factor (TF) and urokinase-type plasminogen activator receptor (uPAR) mRNA in the cultured vascular endothelial cells (VEC) and U937 cell line. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of TF and uPAR genes in the cultured cells under stimulation of thrombin, inactivated thrombin (DFP-T), or at the presence of the inducers and anti-thrombin receptor antibody (ATR-Ab). RESULTS: In U937 cells, the TF and uPAR mRNA expression induced by thrombin could be fully quenched with the ATR-Ab, but in VEC, even at the presence of ATR-Ab, there still was a little stimulative effect of thrombin revealed. DFP-T had no effect on the expression of TF and uPAR mRNA in U937 cells, but could elicit a faint reaction in VEC. CONCLUSION: The induction effects on TF and uPAR mRNA expression in both of the cultured cells by thrombin were mainly dependent on the TR activation, which may not be the unique pathway for thrombin to induce TF and uPAR mRNA expression in VEC.

Cells, Cultured↗
PubMed2002

The role of thrombin in the neo-vascularization of malignant gliomas: an intrinsic modulator for the up-regulation of vascular endothelial growth factor.

Thrombin is a key enzyme in the blood coagulation system where it converts fibrinogen to fibrin. It participates in a variety of biological processes such as the induction of mitogenesis and of morphological changes, the production of cytokines and growth factors, and apoptosis. To clarify the role of thrombin in the proliferation of human malignant gliomas, we investigated its effect on the expression of vascular endothelial growth factor (VEGF) in vitro and determined its intrinsic expression in human glioma tissues. In 3 human glioma cell lines tested, U-87 MG, U-251 MG, and U-105 MG, thrombin induced the VEGF mRNA expression and protein in a dose- and time-dependent manner. The thrombin receptor expression was detectable by RT-PCR and immunoblot. The secretion of VEGF protein in glioma cells was stimulated by the thrombin receptor agonist peptide and the induction of VEGF was significantly blocked by the thrombin inhibitor hirudin, indicating that the up-regulation of VEGF was mediated by the thrombin/thrombin receptor pathway. Immunoblot analysis demonstrated that prothrombin, the precursor of thrombin, was distributed in all 10 glioma tissues examined. In situ hybridization and immunohistochemical analysis revealed the co-localization of prothrombin mRNA-positive and GFAP-positive cells in the glioma tissues. Although various factors may be involved in the up-regulation of VEGF, our results suggest that human gliomas per se express prothrombin, and that thrombin, converted from prothrombin in glioma tissues, substantially stimulates angiogenesis in an autocrine fashion.

Blotting, Northern↗
PubMed2002

Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin.

Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 micrograms/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through "localized" TAFIa generation.

Blood Coagulation↗