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PubMed1975

Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.

Animals↗
PubMed2003

Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation.

Thrombin substrate binding is mediated through fibrinogen recognition "exosite 1" in thrombin, resulting in fibrinopeptide cleavage to form fibrin. In addition, thrombin exhibits "non-substrate" binding to fibrin, an activity termed "Antithrombin I". Antithrombin I (AT-I) is characterized by two classes of thrombin binding sites, the first of "low affinity" in the fibrin E domain, and the other of high affinity, that is situated between C-terminal residues 414 and 427 of a variant gamma chain termed gamma'(1-427L), Plasma fibrinogen molecules containing gamma' chains ("fibrinogen 2") are virtually all heterodimers containing one gamma(A) chain (platelet-binding) and one gamma' chain. The remaining fibrinogen (approximately 85%) is homodimeric, lacks high affinity thrombin-binding potential, and is termed " fibrinogen 1" (gamma(A)/gamma(A)). Thrombin generation in recalcified fibrinogen-depleted or congenital afibrinogenemic plasma is increased. Repletion with fibrinogen 1 has a modest effect in normalizing thrombin generation, whereas repletion with fibrinogen 2 (gamma(A)/gamma') has a more marked effect. A post-translational gamma' chain derivative, gamma'(1-423P), accounts for 3%-34% of the gamma' chain population, lacks thrombin binding potential, and arises by proteolytic processing at the expense of gamma' (1-427L) chains. Little is known about its effect on plasma AT-I activity under normal or pathological circumstances. In summary, fibrin formation (Antithrombin I) inhibits thrombin generation in clotting blood by sequestering thrombin, and "high-affinity" thrombin-binding (i.e., via gamma' chains) plays a dominant role in this process. AT-1 should be considered when assessing the pathogenesis of thromboembolic disease.

Binding Sites↗
PubMed1992

Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo.

Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.

Animals↗
PubMed1991

Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific binding models.

Equilibrium binding of human alpha-thrombin to heparin was investigated at pH 7.4 as a function of thrombin and heparin concentrations, NaCl concentration, temperature, and heparin chain length with the extrinsic fluorescence probe, p-aminobenzamidine, or by quantitative affinity chromatography, in order to distinguish between sequence-specific and nonspecific electrostatic modes of binding. Analysis of binding data by a nonspecific binding model developed for protein-nucleic acid interactions, or by the discrete binding site model previously used to analyze the thrombin-heparin interaction, indicated that both models described the binding interaction equally well over the range of thrombin binding densities accessible to measurement. However, the strong dependence of the thrombin-heparin binding interaction on NaCl concentration, its minimal dependence on temperature, and the increase in apparent binding affinity with increasing heparin oligosaccharide chain length were best accounted for by a nonspecific electrostatic association of thrombin with 5 to 6 anionic residues contained in a 3-disaccharide binding site of heparin. This interaction was characterized by an intrinsic dissociation constant (KD,obs) of 6-10 microM at physiological ionic strength. Although the nonspecific binding model satisfactorily described the binding of thrombin to heparin chains ranging in size from 3 to approximately 13 disaccharides in terms of a single intrinsic KD,obs, deviations from this model were apparent with longer heparin chains (approximately 22 to approximately 35 disaccharides) from a progressive decrease in the intrinsic KD,obs of up to 4-fold. Sedimentation equilibrium analyses of thrombin-heparin complexes suggested a second weaker binding site on thrombin for heparin, which accounted for these deviations as well as the observed insolubility of thrombin-heparin complexes at high thrombin binding densities.

Binding Sites↗
PubMed1990

Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis.

The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program "Ligand" suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and [3H]thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak [3H] thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin.

Animals↗
PubMed1990

Thrombin generation in newborn plasma is critically dependent on the concentration of prothrombin.

The ability to generate thrombin is decreased and delayed in plasma from the healthy newborn infant compared to the adult. Only 30 to 50% of peak adult thrombin activity can be produced in neonatal plasma. To test whether this observation can be explained by the low neonatal levels of the contact or vitamin K dependent factors, we measured neonatal thrombin generation after raising the concentration of these factors to adult values. We also determined whether the addition of a variety of blood products to neonatal plasma improved thrombin generation. An amidolytic method was used to quantitate intrinsic (APTT) and extrinsic (PT) pathway thrombin generation in defibrinated pooled cord plasma from healthy term infants. Added individually, factors VII, IX, X or the contact factors (CF) failed to alter the rate or the total amount of thrombin generated in neonatal plasma. In contrast, the addition of prothrombin increased the total amount of thrombin generated to above adult values in both the APTT and the PT systems but did not alter the rate of thrombin generation. The rate of thrombin generation in cord plasma shortened after a combination of II, IX, X and CF was added to the APTT system or II, VII and X to the PT system. In both systems, the total amount of thrombin generated was linearly related to the initial prothrombin concentration. Each of fresh frozen plasma, cryoprecipitate, plasma from platelet concentrates, or factor IX concentrate (in amounts used therapeutically) caused an increase in the total amount of thrombin generated which was related to the increase in prothrombin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation Factors↗
PubMed1990

[Effects of thrombin produced in blood prior to mixing with anticoagulant on the platelet function and blood coagulation tests].

The amount of thrombin produced in the withdrawn blood of healthy donors prior to mixing with anticoagulants was estimated with thrombin-antithrombin III complex (TAT) methods. No samples showed production of thrombin in the blood mixed with anticoagulants within 2 min. After 3 min or more incubation, four donors out of 14 showed elevation of TAT, and a technician could suspect trouble only in a case during taking blood. Although two samples with low thrombin production (5.8, 12 ng/ml, normal: less than 5 ng/ml) showed no changes of APTT or prothrombin time, samples with high thrombin production (74, 98 ng/ml) showed 1.4-2 sec shortening of APTT and 0.8-1 sec shortening of prothrombin time. Platelet counts and platelet volume showed no remarkable changes even when samples with large amount of thrombin were suspended in tubes containing EDTA-2 K. Plasma beta-thromboglobulin concentration was elevated in the blood containing high amount of thrombin and close correlation was observed amount of thrombin in the blood and the amount of plasma beta-thromboglobulin (r = 0.90, P value less than 0.001). Platelet aggregation induced by collagen was not influenced by amount of thrombin produced. ADP-induced aggregation was suppressed in 3 out of 4 cases with thrombin production although no close correlation was observed between maximal aggregation rate and the concentration of thrombin. As improper handling of blood samples including withdrawal of blood leads to misunderstanding of test results. So, blood samples only for these sensitive tests should be taken and mix with anticoagulants within 2 min.

Adolescent↗
PubMed1989

Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization.

Thrombin stimulates polyphosphoinositide hydrolysis in embryonic chick heart cells and in 1321N1 astrocytoma cells and increases intracellular Ca2+ in the 1321N1 cells. The serine protease trypsin mimics these actions in a dose-dependent fashion, whereas the proteolytically inactive thrombin derivatives diisopropyl fluorophosphate-thrombin (DIP-thrombin) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin (PPACK-thrombin) are ineffective in this regard. The phosphoinositide responses to thrombin or trypsin and the muscarinic agonist carbachol are additive, but no additivity is observed between the responses to thrombin and trypsin. Unlike the response to carbachol, the phosphoinositide and Ca2+ responses to thrombin and trypsin desensitize, with no recovery of the calcium response even when Ca2+ stores are replenished. Cross-desensitization of phospholipase C activation and calcium mobilization between these proteases is also observed. In addition, PPACK-thrombin, which elicits no response itself, effectively inhibits trypsin-stimulated phosphoinositide hydrolysis. It is proposed that thrombin and trypsin act through the same receptor. Proteolysis appears to be important in the mechanism by which these agonists elicit phosphoinositide hydrolysis, calcium mobilization, and, perhaps, subsequent receptor desensitization.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Human neutrophil elastase alters human alpha-thrombin function: limited proteolysis near the gamma-cleavage site results in decreased fibrinogen clotting and platelet-stimulatory activity.

During blood coagulation, polymorphonuclear leukocytes release elastase in amounts that can exceed 100 nmol/L. We therefore studied the interaction between human leukocyte elastase and human alpha-thrombin. Elastase cleaved the thrombin B chain (Ala 150-Asn 151) near the gamma-cleavage site, resulting in two fragments held together by noncovalent interactions. The NH2-terminal fragment (FI), mol wt approximately 18,000, was disulfide-linked to the thrombin A chain. The COOH-terminal fragment (FII), mol wt approximately 13,000, contained the active-site serine and formed a covalent bond with antithrombin III. Heparin accelerated proteolysis of alpha-thrombin by elastase. Proteolyzed alpha-thrombin (T theta) retained full amidolytic activity; however, the concentration of T theta causing 50% maximal platelet aggregation and adenosine triphosphate (ATP) release was 7.9 nmol/L (1.1 nmol/L for alpha-thrombin and 220 nmol/L for gamma-thrombin). Fibrinogen clotting activity of T theta and gamma-thrombin was 32% and 1% that of alpha-thrombin, respectively. Elastase released during the coagulation process may modulate thrombin activity. In addition, elastase-modified thrombin may be a useful probe of the structure and function of the gamma-cleavage region.

Amino Acid Sequence↗
PubMed1987

Correlation between thrombin-induced prostacyclin production and inositol trisphosphate and cytosolic free calcium levels in cultured human endothelial cells.

Cultured human umbilical vein endothelial cells (HUVEC) stimulated with thrombin are known to synthesize prostacyclin at least in part from arachidonate released by phospholipase A2, an enzyme directly activated by calcium. In this study, thrombin stimulation of Quin 2-loaded HUVEC caused rapid and dose-dependent rises in inositol trisphosphate (IP3) and cytosolic free calcium (Ca2+i) levels which preceded a similarly dose-dependent rise in prostacyclin production measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay (ED50 = 0.6-0.7 units/ml for all three effects). Thrombin induced these effects in the absence of extracellular calcium (EGTA) or in the presence of either 8-bromo-cAMP or the calmodulin inhibitor W7. Thrombin inactivated with either diisopropyl fluorophosphate or D-Phe-Pro-Arg-chloromethyl ketone was inactive. In contrast, Quin 2-loaded cultured bovine aortic endothelial cells failed to respond to thrombin, although stimulation with trypsin elevated IP3 and Ca2+i levels and increased 6-keto-PGF1 alpha production. Restimulation of HUVEC with thrombin or histamine 5 min after an initial stimulation with thrombin (2 units/ml for 5 min) failed to induce a second rise in either IP3 or Ca2+i levels or further production of 6-keto-PGF1 alpha, whereas restimulation with ionomycin in the presence or absence of extracellular calcium elevated Ca2+i levels and induced further 6-keto-PGF1 alpha production. However, if the initial stimulation with thrombin was terminated by addition of D-Phe-Pro-Arg-chloromethyl ketone within 10-60 s, restimulation with a second dose of thrombin induced second rises in both IP3 and Ca2+i levels and additional 6-keto-PGF1 alpha production that were greatest when the initial thrombin stimulus was briefest. These results are consistent with the conclusion that IP3 acts as a second messenger by which thrombin elevates Ca2+i levels and initiates prostacyclin synthesis in HUVEC and that in vivo endothelial cells may be stimulated multiple times to synthesize prostacyclin if each period of stimulation is brief.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Functional involvement of thrombospondin in platelet aggregation induced by low versus high concentrations of thrombin.

Thrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (less than or equal to 0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (greater than or equal to 60 micrograms/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (greater than or equal to 60 micrograms/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Characterization of thrombin binding to alpha 2-macroglobulin.

The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

The effect of thrombin on platelet accumulation on the vessel wall - influence of heparin and aspirin.

Rabbit aortae were removed from exsanguinated rabbits, washed, everted on probes, treated with thrombin, washed to remove unbound thrombin and used to measure the accumulation of 51Cr-labeled platelets in vitro. Thrombin pretreatment of normal rabbit aortae did not cause platelet accumulation on the endothelium; platelets appeared to accumulate only at sites where the subendothelium had been exposed. The quantitative data obtained with 51Cr-labelled platelets was reinforced by observations by scanning electron microscopy. 125I-labelled thrombin became associated with the endothelium and also with de-endothelialized vessels, and some of it could be displaced by high concentrations of heparin. Exposure of vessels to heparin after thrombin treatment eliminated the enhanced platelet accumulation caused by the thrombin treatment, probably because heparin displaced thrombin from the aortae, as demonstrated in experiments with 125I-thrombin. Inhibition of PGI2 production by aspirin treatment of the vessels did not enhance platelet accumulation on normal or thrombin-treated aortae. Thus, although thrombin treatment of the endothelium does not cause platelets to adhere to it, thrombin does cause increased platelet accumulation on the areas where the subendothelium is exposed or where endothelial cells are damaged.

Animals↗
PubMed1978

[Comparison of the catalytic properties of alpha- and beta-forms of thrombin].

18-25-fold purified alpha-thrombin, having high esterase activity and coagulating ability of 2500 NIH u per 1 mg of protein, was isolated using chromatography of commercial thrombin through SP-Sephadex C-50. Limited proteolysis of alpha-thrombin on the column with immobilized trypsin resulted in the appearance of beta-thrombin with alpha-thrombin-like esterase activity and tracing coagulating activity (2-5 NIH u per 1 mg of protein). Molecular weight analysis of alpha- and beta-thrombin forms suggests that a peptide (or peptides) with Mr of 1100 is splitted off under proteolysis. Some similarity is revealed in kinetic parameters (Km(app) and kkat) of TAME and BAME hydrolysis by alpha- and beta-thrombin, although Km(app) is somewhat low (approximately 2-fold) for alpha-thrombin. Investigation of TAME hydrolysis kinetics by both thrombin forms at a wide range of substrate concentrations has revealed the effect of substrate activation. Kinetic constants Ks and beta for high substrate concentrations are calculated. It is suggested that the similarity of alpha- and beta-thrombin action on arginine esters and sharp differences in their effect on fibrinogen may be a result of a disturbance of substrate-binding region of beta-thrombin active site.

Arginine↗
PubMed1994

Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells.

Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRAP)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC). Here, we have compared the ability of these synthetic peptide ligands and thrombin to stimulate platelet-derived growth factor (PDGF) production by, and monocyte adhesion to, HUVEC. Thrombin (50 units/ml) and TRAP (25 microM) maximally stimulated monocyte adhesion. Furthermore, the stimulation of E-selectin cell surface expression and the steady-state E-selectin mRNA levels by thrombin and TRAP were comparable. Thrombin (50 units/ml) stimulated PDGF production 400% above the basal level in 24 h, whereas the 6-mer and 14-mer TRAP, even at 200 microM, did not significantly stimulate PDGF production. Northern analysis, however, revealed that TRAP at 100 microM stimulated PDGF-A and -B chain mRNA expression to a level similar to that induced by thrombin. These results suggest that activation of cell signaling by TRAP can mimic thrombin and is sufficient for the stimulation of monocyte adhesion to HUVEC; however, thrombin-stimulated PDGF production by HUVEC may require mechanisms in addition to the signaling events initiated by TRAP or may require the participation of a novel thrombin receptor.

Amino Acid Sequence↗
PubMed1995

Inhibition of thrombin by antithrombin III and heparin cofactor II in vivo.

The critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate.

Adult↗
PubMed1994

Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor.

The receptor for the protease thrombin is a member of the G protein-coupled receptor family, but is activated by a unique proteolytic mechanism. The irreversibility of this proteolytic mechanism and the fact that the ligand is tethered to its receptor raise special questions about inactivation of cleaved receptors and recovery of thrombin responsiveness. We compared the intracellular trafficking of the thrombin receptor to that of the beta 2-adrenergic receptor in transfected Rat1 fibroblasts. In unstimulated cells almost all beta 2 receptors were located on the plasma membrane; by contrast, part of a cell's thrombin receptors were found in an intracellular membrane compartment which co-localized with Golgi markers. Stimulation by agonist caused internalization and subsequent recycling of the beta 2-adrenergic receptor, but most activated thrombin receptors were internalized and targeted to lysosomes. The intracellular pool of thrombin receptors found in unstimulated cells was protected from activation by thrombin, but was translocated to the plasma membrane upon activation of cell surface thrombin receptors. Replenishment of plasma membrane thrombin receptors correlated with recovery of thrombin responsiveness. These observations reveal a novel trafficking mechanism for resensitizing the thrombin receptor as opposed to the internalization/recycling pathway of other G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1994

Low-affinity heparin stimulates the inactivation of plasminogen activator inhibitor-1 by thrombin.

The influence of heparin on the reaction between thrombin and plasminogen activator inhibitor-1 (PAI-1) has been examined. With a 50-fold excess of PAI-1, the rate constant for the inhibition of thrombin was 458 mol/L-1s-1, which increased to 5,000 mol/L-1s-1 in the presence of 25 micrograms/mL unfractionated heparin or heparin with low affinity for antithrombin. The effect of low affinity heparin was then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using close to equimolar concentrations of reactants. Thrombin and PAI-1 formed a stable stoichiometric complex in the absence of heparin, which did not dissociate after the addition of 25 micrograms/mL low-affinity heparin. In contrast, when low-affinity heparin was added at the beginning of the reaction, there was an initial increase in PAI-1-thrombin complex formation, but this was rapidly followed by substantial proteolytic cleavage of unreacted PAI-1 and of the thrombin-PAI-1 complex. The idea that the relative concentrations of thrombin and PAI-1, and the presence of low affinity heparin, could influence the products of the reaction was examined in detail. Quantitative zymographic analysis of tissue plasminogen activator and PAI-1 activities and chromogenic substrate assay of thrombin activity showed that low-affinity heparin stimulated the inactivation of PAI-1 by an equimolar amount of thrombin, but caused only a minimal stimulation of thrombin inhibition. It is concluded that low-affinity heparin stimulates thrombin inhibition when PAI-1 is in excess, but, unexpectedly, that low-affinity heparin enhances PAI-1 inactivation when thrombin is equimolar to PAI-1.

Amino Acid Sequence↗