Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “THROMBIN”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,387 records · Page 77Linked to original sources
PubMed1985

Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin-binding sites that are not directly related to heparan sulfate.

Animals↗
PubMed1993

The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).

Animals↗
PubMedDate not supplied

Ultrasound-guided thrombin injection is a safe and durable treatment for femoral pseudoaneurysms.

Ultrasound-guided percutaneous thrombin injection has recently been described as a treatment for postcatheterization femoral pseudoaneurysms. Although ultrasound guided compression offers another nonoperative treatment option, thrombin injection has shown superior initial success rates. Reports of follow-up for thrombin injection longer than 30 days are currently lacking. The authors reviewed their initial experience with thrombin injection and prospectively evaluated patients for occult late recurrences of pseudoaneurysm and for distal circulatory complications. Records and vascular laboratory data for all patients treated with ultrasound-guided thrombin injection were reviewed for an 18-month period. Tibial vessel Doppler waveforms and ankle/brachial indices were routinely obtained before and after thrombin injection. Follow-up duplex examinations were performed within 24 hours of initial treatment. In the prospective portion of the study, successfully treated patients underwent a repeat femoral duplex scan and lower extremity arterial examination for comparison with the pretreatment studies. Forty-nine of 52 femoral pseudoaneurysms (94%) were successfully treated with ultrasound guided thrombin injection. One immediate failure and 2 early recurrences were treated surgically. There was 1 thrombotic complication of the native circulation identified at the time of injection. Follow-up studies were obtained in 32 of 46 available patients with a mean length of follow-up of 9 months (range 3-17 months). No late recurrences of the pseudoaneurysms or arterial-venous fistulas were observed. No distal circulatory complications were detected by arterial waveform analysis. Three deaths occurred in the interim (cardiac related). Two patients were lost to follow-up. The remaining 12 patients reported no additional limb complications but declined to be restudied. Ultrasound-guided thrombin injection is a safe, effective, and durable treatment for iatrogenic pseudoaneurysms. Thrombin injection should be the therapy of choice for catheter-related femoral false aneurysms.

Adult↗
PubMed2003

Synergistic effect of sphingosine 1-phosphate on thrombin-induced tissue factor expression in endothelial cells.

Sphingosine 1-phosphate (S1P), a bioactive lipid, is produced and stored in platelets and is released from activated platelets during blood coagulation activation. Thrombin, which is also generated during blood coagulation, has been shown to induce tissue factor (TF), the initiator of blood coagulation, in endothelial cells (ECs); however, the effect of S1P on this process is not evaluated. Here we demonstrated that S1P strongly potentiated thrombin-induced TF expression in ECs and that S1P itself did not induce TF expression. Among signaling lipids, platelet-activating factor slightly enhanced thrombin-induced TF expression; other lipids, including lysophosphatidic acid, lysophosphatidylcholine, sphingosine, and C2-ceramide exert no effect on TF expression. S1P enhanced TF expression at the transcriptional level, possibly via promoting the activation of transcription factors nuclear factor-kappaB (NF-kappaB) and Egr-1. Thrombin weakly and S1P strongly activated extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase and, in the presence of both stimulants, enhanced and sustained activation of this kinase was observed. The ERK1/2-specific inhibitor PD98059 significantly inhibited enhanced TF expression induced by both stimulants but only weakly inhibited thrombin-induced TF expression, thus indicating the requirement of the ERK1/2 pathway in synergistic induction of TF expression. In addition, we found that thrombin and S1P rapidly up-regulated the expression of S1P receptors, endothelial differentiation gene-1 (EDG-1) and EDG-3, thereby suggesting that the effect of S1P on TF expression and other EC functions may be enhanced by thrombin and S1P itself. The present data reveal the synergistic effect of S1P on thrombin-induced TF expression in ECs, which may promote further thrombin and S1P generation, thus propagating a positive feedback reaction.

Cells, Cultured↗
PubMed2002

Thrombin functions during tissue factor-induced blood coagulation.

Tissue factor-induced blood coagulation was studied in 20 individuals, for varying periods of time during 54 months, in contact pathway-inhibited whole blood at 37 degrees C and evaluated in terms of the activation of various substrates. After quenching over time with inhibitors, the soluble phases were analyzed for thrombin-antithrombin III (TAT) complex formation, prothrombin fragments, platelet activation (osteonectin release), factor Va generation, fibrinopeptide (FP) A and FPB release, and factor XIII activation. TAT complex formation, for 35 experiments, showed an initiation phase (up to 4.6 +/- 0.6 minutes) in which thrombin was generated at an average rate of 0.93 +/- 0.3 nM/min catalyzed by about 1.3 pM prothrombinase yielding approximately 26 nM thrombin. During a subsequent propagation phase, thrombin was generated at a rate of 83.9 +/- 3.8 nM/min by about 120 pM prothrombinase, reaching ultimate levels of 851 +/- 53 nM. Clot time, determined subjectively, occurred at 4.7 +/- 0.2 minutes and correlated with the inception of the propagation phase. The thrombin concentrations associated with the transitions to rapid product formation are 510 +/- 180 pM for platelet activation (1.9 +/- 0.2 minutes), 840 +/- 280 pM for factor XIII activation and factor Va generation (2.2 +/- 0.6 minutes), 1.3 +/- 0.4 nM for FPA release (2.5 +/- 0.7 minutes), 1.7 +/- 0.5 nM for FPB release and prethrombin 2 (2.8 +/- 0.8 minutes), 7.0 +/- 2.2 nM for thrombin B chain (3.6 +/- 0.2 minutes), and 26 +/- 6.2 nM for the propagation phase of TAT formation (4.6 +/- 0.6 minutes). These results illustrate that the initial activation of thrombin substrates occurs during the initiation phase at less than 2 nM thrombin (0.2%). Most thrombin (96%) is formed well after clotting occurs.

Adult↗
PubMed2001

The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment.

Thrombin, the terminal serine protease in the coagulation cascade, is a proinflammatory molecule in vivo and induces endothelial activation in vitro. The cellular signaling mechanisms involved in this function are unknown. The role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in thrombin-induced chemokine production was studied. Phosphorylation of both p38 MAPK and its substrate, ATF-2, was observed in human umbilical vein endothelial cells (HUVECs) stimulated with thrombin, with a maximum after 5 minutes of stimulation. Using the selective p38 MAPK inhibitor SB203580, there was a significant decrease in thrombin-induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) protein production and messenger RNA steady-state levels. In addition, SB203580 decreased IL-8 and MCP-1 production induced by the thrombin receptor-1 agonist peptide (TRAP), suggesting functional links between the thrombin G protein-coupled receptor and the p38 MAPK pathway. Furthermore, endothelial activation in the presence of SB203580 decreased the chemotactic activity of thrombin-stimulated HUVEC supernatant on neutrophils and monocytic cells. In contrast, the p42/p44 MAPK pathway did not appear to be involved in thrombin- or TRAP-induced endothelial chemokine production, because there was no reduction in the presence of the p42/p44-specific inhibitor PD98059. These results demonstrate that the p38 rather than p42/44 MAPK signaling pathway plays an important role in thrombin-induced endothelial proinflammatory activation and suggest that inhibition of p38 MAPK may be an interesting target for anti-inflammatory strategies in vascular diseases combining thrombosis and inflammation. (Blood. 2001;98:667-673)

Cells, Cultured↗
PubMed2005

Thrombin-mediated IL-10 up-regulation involves protease-activated receptor (PAR)-1 expression in human mononuclear leukocytes.

Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease-activated receptors (PARs). Interleukin (IL)-10, besides its anti-inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL-12 production in human mononuclear cells and that such inhibition is accompanied by IL-10 up-regulation. To our knowledge, there are no data available to show that thrombin mediates IL-10 production by its interactions with PAR-1. We here report that human alpha-thrombin enhances IL-10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up-regulation requires PAR-1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR-1 agonist peptides, such as SFLLRN, results in a significant increase of IL-10 production. PAR-1 expression is required for thrombin-induced IL-10 production, as shown by experiments performed with antisense or sense PAR-1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac-6, results in an increased IL-10 production. This suggests that the observed IL-10 up-regulation may be the result of a direct interaction with monocytes. The observation that thrombin-mediated up-regulation of IL-10 may require the expression of the PAR-1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses.

Cell Line↗
PubMed2002

Differential effects of thrombin and hypoxia on endometrial stromal and glandular epithelial cell vascular endothelial growth factor expression.

Ovarian steroids and/or premenstrual endometrial hypoxia are thought to restore the endometrial vasculature shed during menstruation by elevating endometrial vascular endothelial growth factor (VEGF) levels. During the luteal phase, VEGF levels peak, progesterone induces estradiol (E(2))-primed human endometrial stromal cells (HESCs) to decidualize and express tissue factor (TF), and endometrial vascular permeability is enhanced. The latter would present circulating clotting factors to decidual cell-expressed TF to form local thrombin. HESCs were incubated in serum-supplemented medium containing vehicle (control) or 10(-8) M E(2) or 10(-7) M medroxyprogesterone acetate (MPA) or E(2) + MPA for 7 d to induce decidualization, while monolayers of human endometrial glandular epithelial cells (HEGECs) formed during 4-d incubation of glands. The medium was exchanged for a defined medium containing corresponding vehicle or steroids +/- thrombin under normoxia or hypoxia (0-1% O(2)). Hypoxia enhanced secreted immunoreactive VEGF levels by severalfold in HESCs and HEGECs, but the steroids did not affect VEGF output in either cell type under normoxia or hypoxia. In E(2) + MPA-decidualized HESCs, VEGF levels were elevated by 0.1 U/ml of thrombin, and 0.5-2.5 U/ml of thrombin elicited maximum effects. The addition of 0.5 U/ml of thrombin evoked a time-dependent enhancement of VEGF levels and about an 8-fold increase at 48 h (P < 0.02; n = 6). Northern blotting indicated that E(2) + MPA-decidualized HESCs expressed VEGF(121), VEGF(165), and VEGF(189) mRNA, which were enhanced severalfold during 5- to 20-h incubation with thrombin. Moreover, TRAP, a synthetic peptide activator of the constitutively expressed protease activated receptor-1 thrombin receptor in decidualized HESCs, also elevated secreted VEGF levels. By contrast, HEGECs were unresponsive to thrombin added alone or with ovarian steroids. These results suggest that thrombin formed by progestin-augmented TF levels acts as an autocrine enhancer of VEGF expression in decidualized HESCs. Because angiogenesis occurs in a matrix of decidualized HESCs, these in vitro results provide a novel mechanism to account for both the peak in VEGF and angiogenesis in luteal phase human endometrium.

Cell Hypoxia↗
PubMed2001

Low-dose thrombin injection to treat iatrogenic femoral artery pseudoaneurysms.

OBJECTIVE: Treatment of iatrogenic femoral artery pseudoaneurysms with thrombin injection has been reported as an efficacious and safe procedure. The major risk of this procedure is distal limb ischemia from thrombosis, resulting from thrombin escape. The cumulative average dose of thrombin reported in the literature is approximately 1100 U per patient. Minimizing the thrombin dose may reduce the risks of the procedure. This study reports our experience with low-dose thrombin injection for the treatment of pseudoaneurysms. MATERIALS AND METHODS: Twenty-three patients with 26 postcatheterization femoral pseudoaneurysms were administered thrombin injection with color-flow Doppler sonographic guidance. Pseudoaneurysm volume ranged from 1 to 41 cm(3) with an average of 6.7 cm(3) and a median of 4 cm(3). Two patients received therapeutic doses of IV heparin for anticoagulation. When possible, the neck of the pseudoaneurysm was occluded during the injection to promote stagnation and prevent thrombin leakage. Sonographic follow-up was routinely performed after 24 hr. RESULTS: An average dose of 192 U of thrombin was used (range, 50-450 U), and time to coagulation ranged from 10 to 60 sec. All 26 pseudoaneurysms were successfully thrombosed, although one required repeated treatment because of recanalization noted at 1-day follow-up. There were no complications. CONCLUSION: Doses of thrombin at an average of fivefold lower than previously reported were effective in the treatment of 26 iatrogenic femoral pseudoaneurysms, even in the presence of anticoagulation. This experience shows that a much smaller dose of a potentially dangerous medication can achieve the same efficacy as previously used higher doses.

Aged↗
PubMed1989

Kinetics of pulmonary platelet deposition and clearance during thrombin-induced microembolism in rabbits.

Using 111In-labeled autologous platelets, we studied the kinetics of pulmonary platelet deposition and clearance in relation to hemodynamic and structural events during thrombin-induced pulmonary microembolism in rabbits. Autologous platelets were radiolabeled and returned to animals prior to infusion of thrombin (100 units/kg over 15 min) (n = 20) or saline (n = 6). All animals were pretreated with tranexamic acid, an inhibitor of fibrinolysis. Thrombin-treated animals manifested progressive increases in mean pulmonary platelet activity, reaching a maximum of 38% above baseline (p less than .0001), whereas no change was observed in saline-treated controls. Animals that died during, or immediately following, thrombin infusion manifested significantly greater increases in pulmonary platelet uptake (mean 1.55 +/- 0.47 times baseline), compared to surviving animals (1.14 +/- 0.16; p less than .05 survivors vs. nonsurvivors). In surviving animals, following cessation of thrombin, pulmonary platelet activity cleared gradually, with a half-time of approximately 12 min. Thrombin reduced circulating platelet counts (p less than .001), increased mean pulmonary artery pressure (13 +/- 3 mm Hg to 18 +/- 6 mm Hg; p less than .0001), and reduced mean systemic arterial pressure (55 +/- 10 mm Hg to 44 +/- 7 mm Hg; p less than .001). The time courses of these events approximated that of thrombin-induced pulmonary platelet uptake. Furthermore, the increase in pulmonary artery pressure occurred predominantly in the group of animals in which the increase in pulmonary radiolabeled platelet activity exceeded the median value of 20%. Postmortem histology showed extensive pulmonary thrombus extending from small arterial to capillary levels in animals that died during, or immediately following, thrombin infusion, but not in surviving animals. Our findings suggest that platelet aggregation plays an important role in the pathogenesis of hemodynamic change following thrombin-induced pulmonary embolization.

Animals↗
PubMed1995

Effect of indomethacin on thrombin-induced pulmonary edema in the rat.

The preventive effect of indomethacin on thrombin-induced pulmonary edema was studied in rats. Administration of thrombin caused a significant increase in lung weight, wet weight to dry weight ratio (WW/DW), and relative lung water content. During infusion of thrombin, mean pulmonary artery pressure rose and mean systemic artery pressure fell, PaO2 decreased progressively and there was a continuous rise in pH and PaCO2. An inhibitor of cyclooxygenase, indomethacin, at a dose of 1 mg/kg body weight, induced a significant further increase in lung weight (p < 0.05), and a tendency towards an increase in WW/DW and water content compared with animals given thrombin alone. Treatment with indomethacin, however, counteracted the elevated pulmonary artery pressure occurring in the early phase after thrombin infusion, but not that in the late phase. Systemic artery pressure was not affected by indomethacin. The increases in pH and PaCO2 after thrombin infusion were attenuated and remained stable almost at baseline level after indomethacin administration. Indomethacin did not prevent the hypoxemia induced by thrombin infusion. In conclusion, although indomethacin prevented the early increase in pulmonary artery pressure due to thrombin and the decrease in pH and the increase in PaCO2, it caused lung vascular permeability to protein to increase more than with thrombin alone.

Animals↗
PubMed1997

Seizures induced by intracerebral injection of thrombin: a model of intracerebral hemorrhage.

The coagulation cascade plays an important role in brain edema formation caused by intracerebral blood. In particular, thrombin produces brain injury via direct brain cell toxicity. Seizures and increased cerebral electrical activity are commonly associated with intracerebral blood and are possible effects of thrombin leading to cell injury in the brain. In this study, artificial clots containing concentrations of thrombin found in hematomas were infused intracerebrally in rats. The animals were observed clinically for seizure activity, behavior, and neurological deficits. Several animals underwent video electroencephalographic (EEG) monitoring during intracerebral infusion and for 30 minutes postinfusion. All animals were killed 24 hours after injection, and brain water and ion contents were measured to determine the amount of brain edema. Clinically, thrombin produced focal motor seizures in all animals. None of the control animals or those receiving N[alpha]-(2-Naphthalenesulfonyl-glycyl)-4-amidino-DL-phenylalanine -piperidide (alpha-NAPAP), a thrombin inhibitor added to the thrombin, showed clinical evidence of seizures. Of the rats undergoing EEG monitoring, all animals receiving thrombin showed electrical evidence of seizure activity, whereas none of the control animals exhibited seizure activity. There was no evidence of seizure activity on EEG monitoring when alpha-NAPAP was injected along with the thrombin. In addition, the artificial clots containing thrombin produced agitation and a circling tendency in the rats, along with brain edema. These results indicate that the coagulation cascade is involved in seizure production and increased brain electrical activity, which contribute to the neurological deficits and brain edema formation that are seen with intracerebral hemorrhage.

Animals↗
PubMed1999

Factor XI dependent and independent activation of thrombin activatable fibrinolysis inhibitor (TAFI) in plasma associated with clot formation.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as plasma procarboxypeptidase B is activated by relatively high concentrations of thrombin in a reaction stimulated by thrombomodulin. In plasma an intact factor XI-dependent feed back loop via the intrinsic pathway is necessary to generate sufficient thrombin for TAFI activation. This thrombin generation takes place after clot formation with consequent down-regulation of fibrinolysis. We developed a specific and sensitive assay for activated TAFI (TAFIa) and studied its factor XI-dependent generation during clot formation. In the absence of thrombomodulin, addition of 20 nM thrombin to normal plasma generated 5-10% of the amount of TAFIa generated by 20 nM thrombin in the presence of 8 nM thrombomodulin. Minimal activation of TAFI was detected in factor II deficient plasma when clotting was initiated by 20 nM thrombin. Addition of 320-640 nM of thrombin to factor II deficient plasma resulted in the same amount of TAFIa as in normal plasma, suggesting that approximately 50% of factor II has to be converted to thrombin for extensive activation of TAFI. A Mab that neutralizes activated factor XII had no effect on TAFI activation indicating that an intact contact system is not necessary for the activation of TAFI. The dependency of TAFI activation of factor XI was tested using a Mab that neutralizes activated factor XI. When plasmas from 13 healthy individuals were tested, this Mab reduced TAFI activation by 65% (range 35-89%). Our results indicate that activation of TAFI in serum after clot formation can be quantitated and that it takes place in both factor XI-dependent and factor XI-independent mechanisms.

Blood Coagulation↗
PubMed2000

Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects.

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.

Amino Acid Sequence↗
PubMed2000

Measurement of antithrombin activity by thrombin-based and by factor Xa-based chromogenic substrate assays.

Functionally active antithrombin can be quantified by chromogenic substrate assays utilizing the heparin cofactor activity of antithrombin and the inhibition rates of thrombin or of activated factor X (FXa). Thrombin-based assays but not FXa-based assays may overestimate the antithrombin activity due to their sensitivity toward heparin cofactor II. We focused on the question whether an overestimation of antithrombin activity by thrombin-based assays involves the risk of misdiagnosing antithrombin-deficient individuals as being non-deficient. We determined antithrombin using two thrombin-based assays and one FXa-based assay in 27 plasma samples from patients with acquired antithrombin deficiency spiked with lepirudin, in antithrombin-deficient plasma and in mixtures of antithrombin-deficient plasma and normal plasma. We also measured antithrombin in healthy subjects, in patients with inherited and acquired antithrombin deficiency and in patients under high-dose heparin treatment. At therapeutic final concentrations of lepirudin, antithrombin activities were considerably overestimated by the thrombin-based assays but not by the FXa-based assay. The residual antithrombin activities in antithrombin-deficient plasma determined by the thrombin-based assays were markedly higher than the corresponding values obtained with the FXa-based assay. The thrombin-based assays also overestimated antithrombin activity in patients under high-dose heparin. However, the degree of overestimation in the range between 50 and 100 IU/dl was too low to misidentify individuals with inherited or acquired antithrombin deficiency as normal. We conclude that functionally active antithrombin can be reliably determined using FXa-based chromogenic substrate assays in all settings examined. Thrombin-based assays must not be used in patients under treatment with hirudin or other direct thrombin inhibitors.

Adult↗
PubMed2000

Anti-beta2-glycoprotein I autoantibodies, in vitro thrombin generation, and the antiphospholipid syndrome.

OBJECTIVE: Thrombin plays a pivotal role in the regulation of hemostasis. We examined the effect of antiphospholipid (aPL) antibodies, in particular those with specificity for beta2-glycoprotein I (beta2-GPI), on in vitro thrombin generation, and examined the association with clinical manifestations of the antiphospholipid syndrome (APS). METHODS: We studied plasma samples from 59 patients with aPL antibodies determined by the presence of either elevated anticardiolipin (aCL) antibodies or lupus anticoagulant (LAC). Direct antibody binding of IgG, IgM, and IgA to beta2-GPI and prothrombin (PT) was determined by ELISA. Affinity purification of total IgG and IgG anti-B2-GPI antibodies was performed using staphylococcal protein A and phospholipid liposomes. A chromogenic assay was used to determine the effect of plasma samples and purified autoantibodies on in vitro thrombin generation. RESULTS: Thirty-three of 59 (56%) plasma samples inhibited in vitro generation of thrombin and 7/59 (12%) accelerated thrombin formation. There was a strong negative correlation between thrombin generation and IgG aCL (r = -0.72, p < 0.001) and IgG anti-beta2-GPI (r = -0.71, p < 0.001) antibody levels, and a weaker correlation with LAC (r = -0.46, p = 0.001). This association was not found with anti-PT antibodies and could not be attributed to concurrent therapy with warfarin. Additional experiments with affinity purified IgG antibodies indicated a dose dependent inhibition of thrombin generation, which was restricted to anti-beta2-GPI antibodies. Patients with a history of core clinical manifestations of the APS [venous and arterial thrombosis, recurrent (> or = 2) fetal loss] had significantly greater inhibition of in vitro thrombin generation (mean +/- SEM Z score: -3.38 +/- 0.51 vs -1.42 +/- 0.56; p = 0.01) and higher levels of IgG aCL (mean +/- SEM Z score: 8.39 +/- 1.12 vs 5.39 +/- 0.88; p = 0.04) and IgG anti-beta2-GPI antibodies (mean +/- SEM Z score: 4.49 +/- 0.69 vs 2.26 +/- 0.54; p = 0.01). Odds ratios for these variables and clinical manifestations of the APS were 5.43, 4.17, and 3.28, respectively. CONCLUSION: aPL antibodies may accelerate or inhibit the rate of in vitro thrombin formation. The predominant effect is inhibition that is restricted to IgG anti-beta2-GPI antibodies and it is strongly associated with clinical manifestations of the APS.

Antiphospholipid Syndrome↗
PubMed2001

Identification of critical residues on thrombin mediating its interaction with fibrin.

Thrombin binding to fibrin may be important in localizing thrombin to the site of vascular injury. However, fibrin-bound thrombin retains its catalytic activity toward fibrinogen, and may be prothrombotic under certain conditions. A collection of 52 purified thrombin mutants was used to identify those residues mediating the thrombin-fibrin interaction. Comparison of fibrinogen clotting activity with fibrin binding activity identified twenty residues involved in fibrinogen recognition with four of these residues important in fibrin binding (Lys65, His66, Tyr71, Arg73). No mutant was identified with normal clotting activity and deficient fibrin binding, suggesting that these two properties are not readily dissociable. A DNA thrombin aptamer that binds to these residues was able to inhibit the thrombin-fibrin interaction, and displace thrombin that was already bound. Mapping of these fibrin-binding residues on thrombin revealed that they are localized within exosite I, and comprise a subset of the residues important in fibrinogen recognition.

Allosteric Regulation↗
PubMed1975

Adverse effect of heparin in thrombin-antithrombin III interaction.

Thrombin, while reacting in the presence of hepatin, impairs the inhibitory capacity of antithrombin III so that subsequent inhibition of thrombin or factor Xa is decreased or abolished. This adverse effect of hepatin has been observed directly with at least 1.5 Iowa units of thrombin per each unit of purified human antithrombin III participating in the reaction. The inhibitory capacity was then totally destroyed and some residual thrombin remained in the active form. With a lower enzyme/inhibitor ratio inactivation of thrombin in the presence of hepatin was fast and complete, however, a significant decrease of inhibitory capacity below that found in reaction without heparin, has been established by measuring the residual antithrombin III activity. In defibrinated human plasma at least 2 units of thrombin per each antithrombin III unit were required to demonstrate directly the adverse effect of heparin but a fast depletion of inhibitory capacity has been also observed after repeated additions of small thrombin portions into plasma heparinized in vitro or in vivo. Portions of enzyme initially added disappeared with great velocity; subsequent portions, however, accumulated building up a high thrombin level not seen in the absence of heparin. The accumulation of residual enzyme was more extensive in plasma containing about 1 heparin unit per ml than anticoagulant at lower concentrations and was particularly noticeable in antithrombin III deficient plasma. These results may have some bearings on the approach to heparin therapy in the event when thrombin continuously generates or when a marked deficiency of antithrombin III exists.

Antithrombins↗