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PubMed1980

Evidence that the effects of thrombin on arachidonate metabolism in cultured human endothelial cells are not mediated by a high affinity receptor.

The effect of thrombin and its derivative, diisopropylphosphoryl-thrombin on [3H]arachidonic acid metabolism is studied in cultured umbilical vein endothelial cell monolayers. Thrombin causes a dose-dependent release of radioactivity from endothelial cells fed [3H]arachidonate. Thin layer radiochromatography of acidified supernatants reveals that most of the radio-activity is [3H]arachidonate and its metabolites, 6-ketoprostaglandin F1 alpha and prostaglandin E2. Diisopropylphosphoryl-thrombin, which is enzymatically inactive, does not cause release of arachidonic acid or metabolites. A 50-fold excess of diisopropylphosphoryl-thrombin, despite causing 98% inhibition of binding of 125I-thrombin to its high affinity binding sites, does not inhibit thrombin-induced release. We conclude that the high affinity, active site-independent thrombin binding sites are not involved in thrombin-induced mobilization of esterified arachidonic acid.

Arachidonic Acids↗
PubMed1994

Glu192-->Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor.

Modeling studies have ascribed the remarkable resistance of thrombin to inhibition by the Kunitz type inhibitors, bovine pancreatic trypsin inhibitor (BPTI), and tissue factor pathway inhibitor (TFPI), to steric inhibition by the 60-loop insertion, especially Trp60D (in the chymotrypsin numbering system). Indeed, deletion of Pro60B, Pro60C, and Trp60D from this loop (des-PPW) enhances BPTI inhibition (Ki = 16 nM) (Le Bonniec, B. F., Guinto, E. R., MacGillivray, R. T. A., Stone, S. R., and Esmon, C. T. (1993) J. Biol. Chem. 268, 19055-19061). Activated protein C, however, lacks an equivalent insertion loop but is nevertheless resistant to inhibition by these Kunitz inhibitors. A unique feature of thrombin and activated protein C is the presence of Glu at position 192. Substitution of Glu192 with Gln in activated protein C dramatically enhances inhibition by BPTI and TFPI (Rezaie, A. and Esmon, C. T. (1993) J. Biol. Chem. 268, 19943-19948). We now demonstrate that thrombin E192Q (the Glu192-->Gln mutant) is inhibited by BPTI (Ki = 24 nM) or TFPI (Ki = 14 nM) much more effectively than wild type thrombin (Ki > 1 microM for both inhibitors). A thrombin mutant having both the des-PPW deletion and E192Q substitution binds BPTI (Ki = 35 pM) and TFPI (Ki = 25 pM) even tighter. BPTI can displace dansylarginine N-(-3-ethyl-1,5-pentanediyl)-amide from the active site of thrombin E192Q (Ki = 19 nM), indicating that BPTI interacts directly with the S1 binding site in thrombin. The E192Q mutation and PPW deletion contribute comparably and additively to the binding energy of thrombin with the Kunitz inhibitors. We suggest that access to the active center of thrombin is less restricted than predicted from previous studies.

Amino Acid Sequence↗
PubMed1994

Thrombin regulation in children differs from adults in the absence and presence of heparin.

The physiologic mechanisms that protect children from thromboembolic complications are not known. We investigated the regulation of thrombin in children because of its central importance to thrombosis. The capacity to generate thrombin in vitro (chromogenic assay) was decreased by 26% in plasmas from children (1-16 yrs; n = 102) compared to adults ([20-45 yrs; n = 20; p < 0.001]). The addition of purified prothrombin to plasmas from children increased thrombin generation to adult values. The capacity of plasmas to inhibit 125I-alpha-thrombin was increased by 21% in children compared to adults (p = 0.020), with significantly more thrombin complexed to alpha 2-macroglobulin (alpha 2M) in children. When DVT occur in children, adult guidelines for heparin therapy are used. At low heparin concentrations (0.1 and 0.2 U/ml), thrombin generation was decreased by 30% in children compared to adults (p < 0.001). At high heparin levels (0.4 U/ml), thrombin generation was negligible in all plasmas. ATIII inhibited over 95% of thrombin in all plasmas in the presence of heparin. In summary, thrombin regulation differs in children from adults and may protect children from thromboembolic complications. When DVT do occur, heparin requirements may differ in children compared to adults.

Adolescent↗
PubMed1995

Solid tumor cells express functional "tethered ligand" thrombin receptor.

Previous work demonstrated that alpha-thrombin promoted tumor cell adhesion to endothelium and extracellular matrix as well as enhanced the metastatic capacity of tumor cells. This study was initiated to investigate whether the thrombin effect on tumor cells is mediated through the "tethered ligand" thrombin receptor. RT-PCR analysis using primers based on the human thrombin receptors detected mRNA in human colon adenocarcinoma cells (clone A), whose authenticity was confirmed by Southern hybridization. The presence of thrombin receptor mRNA in rat (W256 carcinosarcoma) and mouse (melanoma) tumor cells was demonstrated by RT-PCR/Southern blotting using species-specific PCR primers. Sequencing of the PCR fragment of clone A cells revealed complete homology with the reported human cDNA sequence. Subsequently, tumor cells derived from three species, i.e., human, rat, and mouse, were found to express the thrombin receptor protein as revealed by immunoblotting using ligand peptide-derived mAb ATAP138, whose reactivity towards the M(r) approximately 66,000, potential thrombin receptor was blocked by preincubating the antibody with the immunogen peptide SFLLRNPNDKYEPF (TRP 14). Finally, peptides TRP 14 and TRP 7 (SFLLRNP), but not TRP 5 (FLLRN), were found to mimic alpha-thrombin in stimulating tumor cell adhesion to fibronectin, suggesting that the thrombin receptors expressed on solid tumor cells are biologically functional.

Amino Acid Sequence↗
PubMed1994

A model for the tissue factor pathway to thrombin. I. An empirical study.

The activation of prothrombin, factor V, factor VIII, factor IX, and factor X by the tissue factor-factor VIIa complex, in vitro, in a system in which each precursor protein was present at plasma concentration, was evaluated using a combination of activity assays, immunoblots, active-site blots, and autoradiography. The thrombin generation curves observed were distinctly nonlinear and typically displayed a time lag in which little or no thrombin was observed. This was followed by an almost linear propagation phase of thrombin formation. The lag was a function of tissue factor/factor VIIa concentration and represented primarily the interval of factor V and factor VIII activation. The postlag propagation phase of thrombin generation was nearly independent of the initial activator (factor VIIa or tissue factor) concentration over a 10(3)-fold range in factor VIIa-tissue factor concentration. Maximum thrombin generation rates were observed when less than 1% of the factor IX and X present was activated but when nearly 100% activation of the cofactors, factor V and factor VIII, was achieved. Analyses of the activation pattern of factor V indicated that the cofactor is activated by both factor Xa and thrombin which are formed at low levels during the lag phase of the reaction. When the initial reaction mixture contained factor Va instead of factor V, the lag was substantially reduced. When factor V was deleted from the reaction mixture, no thrombin formation was observed. When either factor VII or factor IX was deleted from the reaction system, the propagation phase of thrombin formation (at 5 pM tissue factor-factor VIIa complex) was only one-third that observed for reactions which contained factor VIII and factor IX. The addition of factor XI to the experimental system increased the rate of thrombin formation by 15% during the propagation phase but had no effect upon the lag phase of the reaction. Our data suggest that normal hemostasis may be initiated by the factor VIIa-tissue factor complex and support the concept of multiple feedback reactions which amplify and propagate the hemostatic response.

Blood Coagulation Factors↗
PubMed1993

Glucocorticoid inhibits thrombin-induced expression of platelet-derived growth factor A-chain and heparin-binding epidermal growth factor-like growth factor in human aortic smooth muscle cells.

Proliferation of smooth muscle cells (SMCs) in atherosclerosis may be modulated by several growth regulatory molecules. At least two mitogens for SMCs, platelet-derived growth factor (PDGF) A-chain and heparin-binding epidermal growth factor-like growth factor (HB-EGF), can be produced by SMCs themselves and may stimulate smooth muscle proliferation in an autocrine or paracrine fashion. We examined the effects of thrombin, which may be generated at the site of vascular injury during atherogenesis, and the potent anti-inflammatory glucocorticoid, dexamethasone (DEX), on the expression of the genes encoding these two growth factors. Since both PDGF A-chain and HB-EGF have affinity for heparin, we also examined the effect of thrombin and DEX on the release of heparin binding mitogenic activity from SMCs. Treatment of SMCs with thrombin resulted in increases both in the level of the PDGF-A and HB-EGF transcripts in the cells, as well as in released heparin-binding growth factor activity. DEX inhibits the thrombin-stimulated release of mitogenic activity in a dose-dependent manner. An enzyme-linked immunoadsorbent assay showed that DEX inhibits both constitutive and thrombin-stimulated release of PDGF-AA. DEX also decreases both constitutive and thrombin-stimulated mRNA levels for PDGF A-chain and HB-EGF and destabilizes the transcripts for both growth factors. A nuclear run-on assay revealed that DEX acts, in addition, to inhibit constitutive and thrombin-stimulated transcription of the PDGF A-chain and HB-EGF genes. Thus, these findings indicate that expression of PDGF A-chain and HB-EGF may be regulated by thrombin and glucocorticoid at the transcription level. Our results are consistent with the involvement of thrombin-induced growth factor expression in neointimal SMC proliferation and suggest the possibility that intimal proliferation may be attenuated by glucocorticoids.

Aorta, Thoracic↗
PubMed1993

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin.

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

Ancrod↗
PubMed1994

Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases.

The thrombin receptor, a member of the seven membrane-spanning superfamily of G-protein coupled receptors, is activated by an irreversible proteolytic mechanism, but signaling by activated thrombin receptors shuts off soon after receptor activation. This shut-off mechanism is thought to be required for concentration-dependent responses to thrombin and an important determinant of the cell's sensitivity to thrombin. We report that the thrombin receptor is rapidly phosphorylated upon activation, consistent with the action of a G-protein-coupled receptor kinase. Moreover, the G-protein coupled receptor kinase BARK2 (beta-adrenergic receptor kinase 2) blocked signaling by thrombin receptors coexpressed in Xenopus oocytes. In this system, rhodopsin kinase was inactive and BARK1 was markedly less effective than BARK2. Thrombin receptor mutants which lacked potential serine and threonine phosphorylation sites in the receptor's cytoplasmic tail were insensitive to inhibition by exogenous BARK2 but did confer concentration-dependent responses to thrombin. Our studies demonstrate that a G-protein coupled receptor kinase can shut off thrombin receptor signaling but that additional mechanism(s) for terminating signaling exist. These studies also reveal functional specificity among G-protein coupled receptor kinases in a novel in vivo reconstitution system and show that heterologous expression of these kinases can be used to manipulate cellular responsiveness.

Amino Acid Sequence↗
PubMed1993

Understanding thrombin and hemostasis.

Unlike other factors in blood coagulation and fibrinolysis, thrombin has several functions in hemostasis from injury to recovery. Because of continual consumption, thrombin generation controls prethrombotic thrombin functions and may be prevented by inactivation of its precursors or by inhibition of thrombin-mediated amplification steps. The direct activation product of prothrombin, alpha-thrombin, not only converts fibrinogen into clottable fibrin but also is actively incorporated into the forming thrombus, where it is protected and transformed into other or inactive forms with thrombus maturation. Larger protein inhibitors, such as antithrombin III, cannot penetrate the thrombus, whereas hirudin and small thrombin inhibitors can. Unique structural features of thrombin allow the design and synthesis of a variety of small inhibitors. Such small inhibitors may prevent rethrombosis upon lysis of immature thrombi. On the other hand, such intervention must be used with caution, because low levels of thrombin appear to promote wound healing. In this regard, the scars of healing are but manifestations of the many functions of thrombin.

Amino Acid Sequence↗
PubMed1993

Reocclusion after thrombolytic therapy: strategies for inhibiting thrombin-induced platelet aggregation.

The use of first generation plasminogen activators, urokinase, streptokinase and tissue plasminogen activator has revolutionized thrombolytic therapy for myocardial infarction and ischaemia, and potentially stroke. However, thrombolytic therapy employing these activators is limited by reocclusion of the very arteries being opened, which follows in a small but significant number of patients. The development of second generation plasminogen activators, e.g. staphylokinase and anisoylated plasminogen streptokinase activator complex, has not alleviated the problems encountered with classical plasminogen activators. It is now widely recognized that aberrant platelet aggregation induced primarily by thrombin, rather than plasmin, is one of the major causes of recurrent thrombosis following pharmacologic thrombolysis. Agents that (a) inhibit enzymatic and/or coagulant activity of thrombin, (b) block binding of thrombin to its receptor, and (c) interfere with the generation of thrombin by the prothrombinase complex may compromise haemostasis resulting in haemorrhage. We recently demonstrated that thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a putative ADP-receptor on the platelet surface, and that these events are indirectly mediated by intracellularly activated calpain expressed on the surface. In this review, we discuss the known mechanisms of thrombin-induced platelet aggregation and suggest relative advantages of potential pharmacological agents, being developed in our laboratory, over those that have been previously developed and tested. These inhibitors selectively prevent aggregation of platelets induced by thrombin by inhibiting calpain expressed on the surface. Moreover, one of these inhibitors which blocks thrombin-induced platelet aggregation does not interfere with other platelet responses mediated by thrombin or platelet aggregation induced by other agonists, such as, ADP, collagen, phorbol myristate acetate and thromboxane A2 mimetics. This selectivity could reduce the chances of perturbing the formation of a haemostatic plug.

Amino Acid Sequence↗
PubMed1996

Inogatran, a novel direct low molecular weight thrombin inhibitor, given with, but not after, tissue-plasminogen activator, improves thrombolysis.

Coronary artery often reoccludes after therapy of acute myocardial infarction with recombinant tissue plasminogen activator rt-PA, most likely due to in situ thrombin generation during thrombolysis. Previous studies with high molecular weight thrombin inhibitors, such as hirudin, have shown variable improvement in the frequency of sustained thrombolysis. This study was conducted to examine the modulation of thrombolysis, indices of thrombin generation and activated partial thromboplastin time (APTT) by a novel low molecular weight direct thrombin inhibitor, inogatran. A stable occlusive intracoronary thrombus was created in 19 dogs. Nine dogs were given an intravenous bolus of saline (group A), and 5 dogs were given inogatran (group B) followed by rapid infusion of rt-PA (1 mg/kg over 20 min), whereas saline or inogatran was continuously infused for 2 hr. Five other dogs were given inogatran (bolus and continuous infusion) only after thrombolysis by rt-PA was obtained (group C). Time to reflow was similar in all dogs. None of the reperfused coronary arteries reoccluded in group B dogs (vs. 75% and 40% reocclusion rates in groups A and C, respectively, P < .02). Accordingly, the mean duration of reflow was > 120 min in group B dogs (vs. 39 +/- 7 and 44 +/- 14 min in group A and C dogs, respectively, P < .05). After infusion of inogatran, APTT was increased to 1.6 to 1.9 times the base-line value, and the changes in APTT were similar in group B and C dogs. Thrombin generation and activity, assessed by thrombin-antithrombin complex and fibrinopeptide A levels, increased in all dogs during thrombus formation. The increase in thrombin-antithrombin complex and fibrinopeptide A levels during thrombolysis was not evident in group B dogs. These data show that direct thrombin inhibition with inogatran, when initiated before rt-PA, results in sustained thrombolysis and only a modest increase in APTT. However, inogatran given after thrombolysis only partially prevents reocclusion because large amounts of thrombin generation occur during the early stages of thrombolysis.

Animals↗
PubMed1996

Thrombin in the synovial fluid of patients with rheumatoid arthritis mediates proliferation of synovial fibroblast-like cells by induction of platelet derived growth factor.

OBJECTIVE: To investigate the clotting and fibrinolytic activities in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to examine the role of thrombin in synovial hyperplasia. METHODS: We measured the amounts of thrombin-antithrombin-III complex (TAT), antithrombin-III (AT-III), thrombin, plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI), and plasmin-alpha 2-antiplasmin complex (PAP) in SF of 20 patients with RA and 16 patients with osteoarthritis (OA). The proliferative response of synovial fibroblast-like cells to thrombin was measured using [3H] thymidine incorporation. Expression of platelet derived growth factors (PDGF) in conditioned medium was analyzed using a Western blot method, and expression of the mRNA of PDGF and their receptors was analyzed by reverse transcription polymerase chain reaction. RESULTS: The amounts of clotting factors (TAT, AT-III) and fibrinolytic factors (plasminogen, alpha 2-PI, and PAP) were significantly higher in the patients with RA than in patients with OA (p < 0.01). Moreover, SF thrombin concentrations of patients with RA correlated significantly with erythrocyte sedimentation rates (rs = 0.751, p < 0.01) and serum C-reactive protein concentrations (rs = 0.531, p < 0.05). Thrombin exhibits mitogenic activity toward synovial fibroblast-like cells in vitro, and this mitogenic activity is associated with an increase in the expression of mRNA of both PDGF-alpha receptor and PDGF-beta-receptor. CONCLUSION: The high levels of thrombin activity in the SF of patients with RA and strong mitogenic activity of thrombin toward the synovial fibroblast-like cells suggest that thrombin plays an important role in the pathogenesis of RA.

Adult↗
PubMed1997

Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

Amino Acid Sequence↗
PubMed1997

Human alveolar macrophages synthesize endothelins by thrombin.

Alveolar macrophages (AM) play pivotal roles in the defense mechanism and the regulation of inflammatory processes in the airways. Macrophages express receptors for thrombin on their surfaces, and thrombin induces the chemotaxis and the proliferation of macrophages. Thrombin acts on vascular endothelial cells to synthesize endothelin (ET)-1. AM have been known to express prepro ET-1 mRNA. Thus, we hypothesized that thrombin stimulates AM so as to synthesize ET. Surgically resected human lungs were irrigated by saline to remove intravascular blood, then saline was instilled into the bronchus, and the fluids were recovered. AM were separated by Percoll density centrifugation (density, 1.060 g/ml). AM were resuspended in culture medium without FCS in the presence or absence of thrombin. ET was synthesized by thrombin in a concentration-dependent manner, and the amounts of ET synthesized by thrombin (10 U/ml) were equivalent to those by LPS (10 microg/ml). Dexamethasone (10(-6)-10(-10) M), IL-4 (100 U/ml), and TGF-beta (10 ng/ml) significantly suppressed the ET synthesis by thrombin (p < 0.05). In contrast, 1,25-dihydroxyvitamin D3 (10(-8)-10(-10) M) enhanced the ET synthesis up to approximately 300%. The analysis using high pressure liquid chromatography revealed that AM-derived ET consists of ET-1, ET-2, and ET-3. Major constituents were ET-2 and ET-1, and the ratio of ET-2/ET-1 was 1.7 +/- 0.4 (mean +/- SE). These results indicate that thrombin is a potent agonist for AM to synthesize ET.

Cells, Cultured↗
PubMed1998

Rapid activation of protein C by factor Xa and thrombin in the presence of polyanionic compounds.

A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (approximately 50 to 100 micromol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations ( approximately 80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.

Blood Coagulation↗
PubMed1998

Effects of radiographic contrast agents on thrombin formation and activity.

Clinical trials suggest that the risk of thrombosis during coronary angioplasty is lower with ionic contrast agents than with nonionic contrast agents. However, the molecular mechanisms underlying this effect are unknown. This study examined the effects of contrast agents on thrombin formation and its interaction with substrates, inhibitors, and ligands to define potential mechanisms by which contrast agents affect thrombus formation. Two ionic agents, diatrizoate and ioxaglate, and one nonionic agent, ioversol, were studied. Ionic agents inhibited factor X activation by the tissue factor-factor VIIa complex more potently than ioversol (53 +/- 3.7, 43.0 +/- 1.9, and 26.5 +/- 2.4% inhibition by diatrizoate, ioxaglate, and ioversol, respectively, at concentrations of 5%). Ionic contrast agents were potent inhibitors of prothrombinase function, inhibiting thrombin formation by >75% at contrast concentrations of 0.6% (p <0.005). Ioversol inhibited prothrombinase to a significantly lesser extent than ionic agents. Clotting assays suggested that ioxaglate was the most potent inhibitor of thrombin generation in plasma despite having the least effect on fibrin polymerization. Contrast agents inhibited binding of thrombin to fibrin, with ionic agents producing a more potent effect than ioversol (p <0.02). However, contrast agents did not inhibit thrombin-mediated platelet activation, had only a minor effect on inhibition of thrombin by antithrombin III, and did not affect thrombin-hirudin interactions. In summary, these studies identify specific mechanisms by which radiographic contrast agents inhibit thrombin formation and function -- i.e. inhibition of tissue factor-dependent factor Xa generation, inhibition of the prothrombinase complex, and inhibition of thrombin binding to fibrin. These findings may help to explain the reduced risk of thrombosis during coronary angioplasty associated with ionic contrast agents.

Cardiac Catheterization↗
PubMed1999

Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III.

The recent demonstration of a protein disulfide isomerase (PDI) on the surface of and secreted from blood platelets raises the possibility that proteins involved in hemostasis and wound healing are also substrates of this enzyme. In this study purified preparations of platelet PDI, thrombospondin-1 (TSP), alpha-thrombin, and antithrombin III (AT) were used to demonstrate that PDI catalyzes formation of a TSP-thrombin-AT complex consistent with previous results with supernatant platelet activation. Concentrations of 1.25 microg/ml of PDI were sufficient to convert almost 50% of thrombin to TSP-thrombin-AT complex. Complex formation requires low concentrations of a reduced thiol and the reaction can be prevented by N-ethymaleimide. The complex is dissociated by reducing agents such as mercaptoethanol. Absence of Ca2+ and the addition of EDTA increased the rate of complex formation, indicating that TSP in the Ca2+-free form is most effective. In the absence of AT a small amount of TSP-thrombin complex formed which was only 0-13% of maximal complex formation in the presence of AT. This result, in combination with kinetic studies showing rapid formation of thrombin-AT complex followed by conversion to ternary complex, suggests that the thrombin-AT complex is an obligatory intermediate in the reaction. Under optimal conditions over 70% of the thrombin is incorporated into the complex in 60 min. Heparin accelerated the reaction largely by enhancing formation of thrombin-AT complexes and had little effect on TSP. PDI coprecipitated with TSP from the supernatant solution of activated platelets, suggesting an association between PDI and its substrate. In summary, these data are consistent with a role for PDI-catalyzed formation of disulfide-linked complexes of TSP with other proteins.

Antithrombin III↗
PubMed1994

Surface 12-Lead Electrocardiographic Findings and Plasma Markers of Thrombin Activity and Generation in Patients with Myocardial Ischemia at Rest.

Background: Myocardial ischemia at rest is typically associated with atherosclerotic coronary artery disease, atherommous plaque rupture, and intracoronary thrombosis. In areas of advanced disease and vascular injury, the extent of thrombus is influenced largely by a delicate balance of procoagulant factors, favoring thrombus initiation, growth, and development, and anticoagulant factors, attempting to limit potentially flow-limiting coronary thrombosis. Thrombin, a 308 amino acid serine pretense, is considered the most patent procoagulant factor in the setting of acute vessel wall injury, playing an essential role in the conversion of fibrinogen to fibrin, accelerating the prothrombinase complex, activating platelets, and stabilizing fibrin polymers. The purpose of this study was to determine the relationship between electrocardiographic abnormalities and markers of thrombin activity and generation among patients with unstable angina and non-Q.wave myocardial infarction. Mehtods and Results: In a study of 36 patients (59.1+/- 11.0 years) with myocardial ischemia at rest participating in the Thrombolysis in Myocardial Ischemia (TIMI) IIIB trial, thrombin activity in plasma, as determined by fibrinopeptide A (FPA), prothrombin fragment 1.2 (F 1.2), and thrombin-antithrombin III complexes (TAT) concentrations, were found to be increased significantly when compared with healthy volunteers (p < 0.004). Thrombin generation was also increased modestly compared with age-matched patients with stable coronary artery disease undergoing elective cardiac catheterization. Given that,he surface 12-lead electrocardiogram (ECG) is frequently abnormal in patients with ischemic chest pain at rest and represents a readily available, first-line diagnostic test for assessing disease activity and treatment response, we investigated whether ECG abnormalities and thrombin activity/generation in plasma were correlated. Twenty-six patients (72%) had ECG changes compatible with myocardial ischemia at the time of study entry, including 18 (50%) with newly inverted T waves (or pseudonormalization), 14 (39%) with reversible ST-segment depression, and 4 (11%) with transient (<30 minutes) ST-segment elevation. Within the predefined ECG groups there were no differences in plasma thrombin activity between patients with and those without confirmed abnormalities. Similarly, there were no differences in either plasma thrombin activity or generation between the predefined ECG groups. Conclusion: Although ECG abnormalities supporting the presence of myocardial ischemia occur commonly in patients with chest pain at rest, they do not correlate closely with markers of thrombin activity and generation in plasma. The diagnostic and prognostic capabilities of these diagnostic tools, considered either alone or together, require further investigation.

Journal Article↗