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PubMed1987

Uptake and inactivation of thrombin on the subendothelium: comparisons with endothelium.

One aspect of the non-thrombogenicity of endothelium is the property to bind and inhibit thrombin. Loss of the endothelial layer gives rise to a thrombogenic surface. The present study concerns uptake and inactivation of thrombin on endothelium as well as subendothelium. Both the immediate subendothelial layer and the media were investigated. Binding and inhibition of thrombin was assayed using both a synthetic chromogenic substrate and fibrinogen. In the latter case thrombin enzymatic activity was assessed by measuring liberation of fibrinopeptide A. There was a linear relationship between thrombin in the solution and thrombin enzymatic activity recovered on the surface on both endothelium and media. 30-50 per cent of the surface bound thrombin measured with the chromogenic substrate was also active towards fibrinogen. Preincubation of endothelium with plasma gave rise to an increased loss of thrombin from the solution at the same time as less thrombin was recovered on the surface. This indicates increased inactivation. Increased inactivation could also be obtained by pretreatment with antithrombin (AT) but not with AT-depleted plasma. The same general pattern was seen on the subendothelium but not on the media. It is concluded that inhibition of thrombin occurs on the endothelium and can be augmented by prior binding of AT to the surface. This effect although less pronounced can also be obtained on the subendothelium. The deeply injured vessel is much less capable of inhibiting thrombin.

Animals↗

PubMed1991

Interaction of the sulfated lactobionic acid amide LW 10082 with thrombin and its endogenous inhibitors.

The synthetic polyanion LW 10082 prolonged thrombin clotting times when human or bovine thrombin were used to induce clotting. In the clotting time prolongation LW 10082 was 15 times more potent in the presence of human thrombin than of bovine thrombin. In an amidolytic thrombin inhibition assay, LW 10082 inhibited bovine and human thrombin when plasma was present. Here, too, thrombin inhibition was much more pronounced when human thrombin was used. When purified antithrombin III was used, neither human nor bovine thrombin were inhibited by LW 10082. LW 10082 proved to be a potent inhibitor of human thrombin in the presence of heparin cofactor II in an amidolytic assay. The time course of the inhibition was slow and at least 10 minutes of incubation were required to obtain complete inhibition of thrombin by an excess of HC II and LW 10082. The anticoagulant activity of LW 10082 in the APTT was unchanged in the presence of anti-antithrombin III antibodies while the potency of heparin was clearly reduced. This indicates that the overall anticoagulant effect of LW 10082 was as well independent of antithrombin III.

Animals↗

PubMed1995

A screening procedure to evaluate the anticoagulant activity and the kinetic behaviour of direct thrombin inhibitors.

The development of a fibrin clot microassay to define both the kinetic behaviour and the anticoagulant activity of direct thrombin inhibitors targeting various domains of thrombin (catalytic site, anion binding exosite or both) is described. Since classical kinetics studies are difficult to perform in a fibrin-clot assay, methodological conditions were selected in order to obtain a linear relationship between fibrin formation and the thrombin concentration i.e. 0.67 nM thrombin, 6 microM fibrinogen, 5 minutes reaction. Under those conditions, the concentration of the complex thrombin-inhibitor can easily be calculated from a standard curve performed with increasing concentrations of thrombin and fitted versus the total inhibitor concentration using adapted equations. To detect the slow establishment of the thrombin inhibition, results obtained with a protocol in which the inhibitor is pre-incubated with thrombin before the addition of fibrinogen is compared to a protocol in which the inhibitor is pre-incubated with fibrinogen before thrombin is added. Our assay which is validated using different types of thrombin inhibitors (classical competitive: NAPAP and hirudin 55-65; tight binding: r-hirudin; slow tight binding: DUP-714), provides a rapid screening protocol allowing to evaluate the biochemical and anticoagulant properties of any direct thrombin inhibitor.

Amino Acid Sequence↗

PubMed1995

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases.

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca2+]i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca2+]i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca2+]i studies. The kinetic profile for [Ca2+]i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca2+]i in guinea pigs, had no effect in rat, and increased [Ca2+]i in all other species. Staurosporine inhibited SFLLRN-induced [Ca2+]i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca2+]i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.

Animals↗

PubMed1990

Thrombin and its inhibitors regulate morphological and biochemical differentiation of astrocytes in vitro.

Flat, amorphous astroblasts in culture differentiate into rounded process-bearing cells after removal of serum from the media or following addition of dibutyryl cyclic-AMP (dbcAMP). We report here that addition of thrombin (10 nM) to rat primary astroglial cultures reversed both the spontaneous morphological differentiation of astroblasts caused by serum removal, and the more extensive morphological differentiation caused by pre-treatment with dbcAMP. The astroblasts retained the ability to differentiate upon removal of thrombin from the medium. Proteolytic activity of thrombin was required for the reversal of differentiation. Moreover, addition of serine protease inhibitors active against thrombin elicited a prolonged morphological differentiation rivaling that induced by dbcAMP, suggesting that inactivation of cell-associated thrombin might be sufficient for morphological differentiation to occur. Two other serine proteases with a cleavage specificity similar to thrombin were ineffective in reversing differentiation. Both the induction of morphological differentiation by dbcAMP and its reversal by thrombin were rapid, being essentially complete by 1 h. With more prolonged treatments, thrombin also reduced the dbcAMP-mediated increase in glutamine synthetase, a biochemical marker for astroglial differentiation. Thrombin also inhibited morphological differentiation in C6 glioma and altered the morphology of microglial cells; however, thrombin did not prevent neurite outgrowth in primary central neuronal cultures in contrast to its previously reported effects on the neuroblastoma 2a cell line. These findings indicate that a proteolytic mechanism mediated by thrombin and its inhibitors may underlie the regulation of astroglial differentiation.

Animals↗

PubMed1986

Multiple complexes of thrombin and heparin.

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.

Carbohydrate Conformation↗

PubMed1988

Blood coagulation and bone metabolism: some characteristics of the bone resorptive effect of thrombin in mouse calvarial bones in vitro.

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.

Adrenal Cortex Hormones↗

PubMed2006

Induction of interleukin-8 secretion and activation of ERK1/2, p38 MAPK signaling pathways by thrombin in dermal fibroblasts.

It was reported that thrombin could induce IL-8 secretion from human dermal fibroblasts (HDFs) through activation of proteinase activated receptor (PAR)-1. However, little is known of intracellular signaling pathways involved in the event. In the present study, expression of PARs in primarily cultured HDFs was determined by flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR), levels of IL-8 were determined by using ELISA and signaling pathways were examined by using Western blot. It was found that HDFs express PAR-1 and PAR-3, and thrombin induces approximately 7.4-fold increase in IL-8 secretion from HDFs. Hirudin and a PAR-1 blocking antibody completely abolish the action of thrombin. It was also found that PD98059, a mitogen-activated protein kinase (MAPK) pathway inhibitor and U0126, an inhibitor of extracellular signal-regulated kinase (ERK) blocks thrombin-induced phosphorylation of ERK1/2 and IL-8 secretion, indicating the involvement of MAPK/ERK signaling pathway in thrombin-induced IL-8 secretion. p38 MAPK pathway appears also being involved as SB203580, a selective inhibitor of p38 MAPK inhibit phosphorylation of p38 MAPK and thrombin-induced IL-8 secretion. Furthermore, Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway, but not phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway may also be activated by thrombin. In conclusion, thrombin potently induce IL-8 release via PAR-1 from HDFs. Thrombin elicited IL-8 release is predominantly conducted through MAPK/ERK and p38 MAPK signaling pathways. Discovery of the signaling pathways of thrombin in HDFs may help to understand the role of thrombin in inflammation and tissue remodeling.

Adolescent↗

PubMed2004

Modes and consequences of thrombin's interaction with fibrin.

Thrombin mediates the balance between coagulant and fibrinolytic forces and has numerous cellular effects. This intricate balance is maintained by biochemical mechanisms that regulate thrombin activity. Disruption of this balance could lead to bleeding or thrombosis. Once thrombin is generated, two major mechanisms regulate its activity. By binding fibrin, thrombin's activity is localized to the thrombus, a process that limits its systemic procoagulant effects. Thrombin that escapes into the circulation is efficiently inactivated by plasma inhibitors, such as antithrombin, or is sequestered by thrombomodulin on the endothelium. Although thrombin's interaction with fibrin limits its systemic effects, fibrin-bound thrombin resists inactivation and can produce a local procoagulant stimulus that triggers thrombus growth. Direct thrombin inhibitors were developed, at least in part, to target fibrin-bound thrombin. These agents are finding their niche for the prevention and treatment of venous and arterial thrombosis. The mechanisms by which thrombin binds fibrin are reviewed in this paper. As well, the potential pathological consequences of thrombin's interaction with fibrin are discussed.

Fibrin↗

PubMed2004

Inhibition of thrombin-induced signaling by resveratrol and quercetin: effects on adenosine nucleotide metabolism in endothelial cells and platelet-neutrophil interactions.

BACKGROUND: Thrombin downregulates endothelial ectonucleotidase activity resulting in high levels of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) which lead to platelet, leukocyte and endothelial activation. Depending on adenosine nucleotide levels, resting platelets inhibit and thrombin-activated platelets increase respiratory burst of neutrophils. Whether the red wine polyphenols quercetin and resveratrol affect thrombin-dependent adenosine nucleotide, metabolism and thrombin-induced signaling is unknown. MATERIALS AND METHODS: ATP and ADP secretion by platelets, the impact on subsequent oxidative burst activity in neutrophils and CD39/ATPdase function in endothelial cells (ECs)was studied. Cell migration was measured in modified Boyden chambers; adenosine metabolites were quantified by high-performance liquid chromatography (HPLC). Signal transduction was studied by Western blotting. RESULTS: Quercetin and resveratrol inhibited thrombin-induced ADP and ATP secretion from platelets in a concentration-dependent manner. Augmented respiratory burst of neutrophils in response to thrombin-activated platelets was also inhibited by the two polyphenols as was neutrophil migration toward thrombin-induced supernatants of platelets. Quercetin and resveratrol restored the decreased CD39/ATPdase activity in human umbilical vein endothelial cells, in response to thrombin as demonstrated by adenosine monophosphate (AMP) and adenosine increases in endothelial culture supernatants. Both polyphenols inhibited thrombin-induced MAPK, JNK and focal adhesion kinase activities in endothelial cells. CONCLUSION: Quercetin and resveratrol interfere with the proinflammatory signaling of thrombin resulting in the inhibition of adenosine nucleotide secretion from activated platelets and decreased neutrophil function. Moreover, the polyphenols protect endothelial adenosine nucleotide metabolism when downregulated by thrombin. These observations may explain cardioprotective effects of grape products.

Adenine Nucleotides↗

PubMed2004

Reactive oxygen species-sensitive p38 MAPK controls thrombin-induced migration of vascular smooth muscle cells.

Thrombin has been implicated in the development of atherosclerosis and restenosis, in which migration of vascular smooth muscle cells (VSMC) is a crucial event. Thrombin-stimulated VSMC migration is associated with increased generation of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPKs), and production of growth factors and chemoattractants. In this study, we examined the interrelation of these signals to determine the pathway controlling thrombin-directed migration of human VSMC. Our results show that thrombin stimulated the production of ROS and activation of p38 MAPK. ROS were required for thrombin-induced VSMC migration since both generation of ROS and cell migration were significantly attenuated by inhibitors of NAD(P)H oxidase, diphenyleneiodonium (DPI) and apocynin (Apo.), and by the hydrogen peroxide scavenger, catalase (Cat.). Activation of p38 MAPK by thrombin was inhibited by DPI, Apo. and Cat., indicating ROS are used as messengers for activating this kinase. p38 MAPK is an important step since SB 203580, a selective inhibitor of p38 MAPK, suppressed the cell migration induced by thrombin. Furthermore, thrombin increased the expression of vascular endothelial growth factor (VEGF), a chemoattractant for VSMC, and this expression was inhibited by DPI, Apo., Cat. and SB 203580. Addition of anti-VEGF antibody significantly attenuated thrombin-induced migration. Collectively, the data presented here show that thrombin has stimulated VSMC migration and VEGF expression through an ROS-sensitive p38 MAPK pathway. VEGF synthesized and released by the cell served as a secondary mediator in thrombin-directed migration.

Cell Movement↗

PubMed2000

The effects of thrombin preconditioning on focal cerebral ischemia in rats.

Our previous studies have shown that prior intracerebral infusion of a low dose of thrombin (thrombin preconditioning; TPC) reduces the brain edema that follows a subsequent intracerebral infusion of a high dose of thrombin or an intracerebral hemorrhage. In vitro studies have also demonstrated that low concentrations of thrombin protect neurons and astrocytes from hypoglycemia and oxidative stress-induced damage. This study, therefore, examines the hypothesis that TPC would offer protection from ischemic brain damage in vivo. This was a blinded design study. The rat brain was preconditioned with 1 U thrombin by direct infusion into the left caudate nucleus. Seven days after thrombin pretreatment, permanent middle cerebral artery occlusion (MCAO) was induced. Twenty-four hours post-ischemia, neurological deficit was evaluated and infarction volume, brain water and ion contents were measured. Compared to saline-treated rats, thrombin pretreatment significantly attenuated brain infarction in cortex (90+/-33 vs. 273+/-22 mm(3); P<0.05) and basal ganglia (56+/-17 vs. 119+/-12 mm(3); P<0.05) that followed 24 h of permanent MCAO. TPC also reduced the brain edema in cortex and basal ganglia by 50 and 53% (P<0.05). Neurological deficit was improved in thrombin pretreatment group (P<0.05). These effects of TPC were, in part, prevented by co-injection of hirudin, a thrombin inhibitor, indicating that the protection was indeed thrombin mediated. Cerebral TPC significantly reduces ischemic brain damage, perhaps by activation of the thrombin receptor. This finding provides a new mechanism by which to study ischemic tolerance.

Animals↗

PubMed2003

A novel approach to thrombin inhibition.

The inability of the heparin-antithrombin complex to inhibit fibrin-bound thrombin limits the utility of heparin for treatment of arterial thrombosis. In contrast with heparin, melagatran, a direct thrombin inhibitor (DTI), is equally effective at inhibiting fluid-phase thrombin and thrombin bound to fibrin. This reflects the ability of melagatran, a reversible, active site-directed DTI, to access the active site of thrombin even when it is fibrin-bound. Because bivalent DTIs, such as hirudin, compete with fibrin for access to the fibrin-binding site on thrombin, bivalent DTIs produce less inhibition of fibrin-bound thrombin than free thrombin when given in low doses. Consequently, higher doses of hirudin are needed for complete inhibition of fibrin-bound thrombin resulting in a steep dose-response curve. This phenomenon, combined with the fact that hirudin irreversibly inhibits thrombin, may account for hirudin's narrow therapeutic window. In a rabbit arterial thrombosis prevention and ear bleeding model, melagatran produced less bleeding than hirudin when the two agents were given at doses that produced nearly complete inhibition of thrombosis. The more favourable benefit-to-risk profile of melagatran in this model likely reflects better access to fibrin-bound thrombin and the reversible nature of the melagatran/thrombin complex. The theoretical advantages of melagatran may endow it with mechanistic advantages over heparin, and safety advantages over hirudin. Large-scale clinical trials are under way to evaluate the utility of an orally active prodrug form of melagatran for prevention and treatment of venous and arterial thrombosis.

Fibrinolytic Agents↗

PubMed2000

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells.

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.

Antibodies, Monoclonal↗

PubMed2003

The evolving role of direct thrombin inhibitors in acute coronary syndromes.

The central role of thrombin in the initiation and propagation of intravascular thrombus provides a strong rationale for direct thrombin inhibitors in acute coronary syndromes (ACS). Direct thrombin inhibitors are theoretically likely to be more effective than indirect thrombin inhibitors, such as unfractionated heparin or low-molecular-weight heparin, because the heparins block only circulating thrombin, whereas direct thrombin inhibitors block both circulating and clot-bound thrombin. Several initial phase 3 trials did not demonstrate a convincing benefit of direct thrombin inhibitors over unfractionated heparin. However, the Direct Thrombin Inhibitor Trialists' Collaboration meta-analysis confirms the superiority of direct thrombin inhibitors, particularly hirudin and bivalirudin, over unfractionated heparin for the prevention of death or myocardial infarction (MI) during treatment in patients with ACS, primarily due to a reduction in MI (odds ratio, 0.80; 95% confidence interval, 0.70 to 0.91) with little impact on death. The absolute risk reduction in the composite of death or MI at the end of treatment (0.8%) was similar at 30 days (0.7%), indicating no loss of benefit after cessation of therapy. Supportive evidence for the superiority of direct thrombin inhibitors over heparin derives from the recently reported Hirulog and Early Reperfusion or Occlusion (HERO)-2 randomized trial with ST-segment elevation ACS, which demonstrated a similar benefit of bivalirudin over heparin for the prevention of death or MI at 30 days (absolute risk reduction 1.0%), again primarily due to a reduction in MI during treatment (odds ratio, 0.70; 95% confidence interval, 0.56 to 0.87), with little impact on death. Further evaluation of hirudin and bivalirudin in the antithrombotic management of patients with ACS is warranted.

Acute Disease↗

PubMed1999

Thrombin injection versus compression of femoral artery pseudoaneurysms.

OBJECTIVE: The compression of femoral artery pseudoaneurysms is a time consuming, painful, and sometimes unsuccessful procedure. Thrombin injection has been advocated as a superior alternative. In this study, we compare our experiences with both techniques. METHODS: All the records of femoral artery false aneurysms that were treated in the vascular laboratory from January 1996 to April 1999 were retrospectively reviewed. Treatment with ultrasound scan-guided compression was compared with treatment with dilute thrombin injection (100 U/mL). RESULTS: Both groups had similar demographics and aneurysm sizes (P >.2). Of the pseudoaneursyms, 88% were caused by cardiac catheterization and the others were the results of femoral artery access for cardiac surgery (6%), arteriography (5%), and renal dialysis (1%). Compression was successful in 25 of 40 patients (63%). Nine persistent aneurysms necessitated operation, and six were treated successfully with thrombin injection. Primary thrombin injection successfully obliterated 21 pseudoaneurysms in 23 patients. Overall, 27 of 29 pseudoaneurysms were treated successfully with thrombin injection (93%). Thrombosis occurred within seconds of the thrombin injection and required, on average, 300 units of thrombin (100 to 600 units). The patients who underwent successful compression required an average of 37 minutes of compression (range, 5 to 70 minutes) and required analgesia on several occasions. No patients in the thrombin group required analgesia or sedation. Neither group had complications. A cost analysis shows that thrombin treatment results in considerable savings in vascular laboratory resource use but not in overall hospital expenditures. CONCLUSION: Ultrasound scan-guided thrombin injection is a safe, fast, and painless procedure that completely obliterates femoral artery pseudoaneurysms. The shift from compressive therapy to thrombin injection reduces vascular laboratory use and is less expensive, although it does not significantly impact hospital costs.

Adult↗

PubMed1995

Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin.

The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1-antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin.

Amino Acid Sequence↗

PubMed1994

Thrombin-bound structure of an EGF subdomain from human thrombomodulin determined by transferred nuclear Overhauser effects.

The EGF-like domains in human thrombomodulin interact with and change the specificity of thrombin from a procoagulant enzyme to an anticoagulant enzyme. Recent experiments identified the minimal thrombin-binding region of thrombomodulin as the most acidic loop of the fifth EGF-like domain with a sequence of E408CPEGYILDDGFI420CTDIDE. High-resolution NMR spectroscopy was employed to characterize the interaction of a des-Ile420 thrombomodulin peptide, Cys1(409)Pro2Glu3Gly4Tyr5Ile6- Leu7Asp8Asp9Gly10Phe11Cys12Thr13Asp14Ile15Asp16Glu17(426), with its target coagulation protein, thrombin. The disulfide-bonded peptide was found to be structured only upon binding, while neither the linear nor the cyclized peptide exhibited any structural preference free in solution. The thrombin-bound structure of the cyclic thrombomodulin peptide was determined by transferred nuclear Overhauser effects (transferred NOEs) and by distance geometry and Monte Carlo calculations. The thrombin-bound cyclic peptide assumes an overall conformation similar to those observed in the free but intact EGF molecules. There is a type II beta-turn involving residues Pro2-Tyr5, followed by an optimized antiparallel beta-sheet involving residues Gly4-Asp8 and residues Phe11-Ile15. The thrombomodulin peptide provides a potential thrombin-binding surface between residues Tyr5 and Phe11, which are brought close by a chain reversal within the central beta-sheet. Comparison of the thrombin-bound structure of the EGF-like subdomain with other thrombin-peptide complexes revealed that a common thrombin-binding surface can be organized by different secondary structure elements with entirely different peptide sequences. The thrombin-bound structure of the thrombomodulin peptide may serve as a basis to understand the regulatory functions of thrombomodulin and as a guide for the design of specific inhibitors for thrombin.

Amino Acid Sequence↗