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PubMed1995

Monocyte chemoattractant activity of Ser195-->Ala active site mutant recombinant alpha-thrombin.

alpha-Thrombin is chemotactic for human monocytes with optimal activity between 10-100 nM. The mechanism by which this response is mediated remains a point of controversy. The purpose of this study was to compare the chemotactic activity of proteolytically inactive thrombin (active site Ser195-->Ala mutant or Phe-Pro-Arg-chloromethyl ketone-inactivated thrombin) to thrombin and the "tethered ligand" thrombin receptor agonist peptide SFLLRN (single-letter amino acid code). Monocyte chemotaxis was compared to an optimal concentration (10 nM, considered to be 100%) of formyl-Met-Leu-Phe (fMLP). Proteolytically inactive thrombin (38% of fMLP) had similar chemotactic activity to active thrombin (46% of fMLP) at a concentration of 100 nM. Chemotaxis to SFLLRN was comparable to that of a control hexapeptide (FSLNLR) which is not an agonist for the tethered ligand thrombin receptor. Cross-desensitization experiments showed that pretreatment of monocytes with either mutant or active thrombin reduced subsequent chemotaxis to both thrombin chemotaxins. Pretreatment with SFLLRN did not decrease subsequent chemotaxis to either form of thrombin. Calcium flux measurements showed that both active thrombin and SFLLRN induced a rapid increase in monocyte and platelet intracellular calcium concentration. However, there was no intracellular calcium change in response to mutant thrombin or FSLNLR. Likewise, active thrombin and SFLLRN induced a rapid net increase in polymerized actin, but mutant thrombin and FSLNLR did not. By contrast, both active and mutant thrombin induced a polarization of monocyte morphology and actin distribution. This polarization has been associated with directed migration in many cell types. SFLLRN, however, induced a symmetrical increase in polymerized actin. These results suggest that measurements of intracellular calcium and polymerized actin are not perfect surrogate tests for true chemotactic activity. These results show that thrombin proteolysis is not required for monocyte chemotaxis and may be mediated by interaction with a binding site other than the tethered ligand thrombin receptor.

Actins↗
PubMed1992

Thrombin enhances degradation of heparan sulfate in the extracellular matrix by tumor cell heparanase.

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.

Animals↗
PubMed1999

Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.

The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of alpha-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by approximately 2 orders of magnitude, TM protects alpha-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of alpha-thrombin, the exosite 1 variant gamma-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k(2) = (7.9 +/- 0.5) x 10(2) M(-)(1) s(-)(1)] of alpha-thrombin with PAI-1 approximately 200- and approximately 240-fold, respectively. The k(2) value for gamma-thrombin [(7.9 +/- 0.7) x 10(1) M(-)(1) s(-)(1)] was impaired 10-fold, but was enhanced by heparin and VN approximately 280- and approximately 75-fold, respectively. Similar to inactivation of gamma-thrombin, PAI-1 inactivation of alpha-thrombin in complex with the epidermal growth factor-like domains 4-6 of TM (TM4-6) was impaired approximately 10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k(2) = (1.3-1.5) x 10(3) M(-)(1) s(-)(1) for R93,97,101A thrombin and k(2) = (3.6-5.1) x 10(2) M(-)(1) s(-)(1) for meizothrombin]. Unlike heparin, however, VN enhanced the k(2) of R93,97,101A thrombin and meizothrombin inactivation approximately 80- and approximately 30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (K(d)) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of alpha-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of alpha-thrombin by lowering the K(d) for initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of alpha-thrombin, (3) alpha-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.

Binding Sites↗
PubMed1990

Thrombin regulates components of the fibrinolytic system in human mesangial cells.

Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis. Northern blot analysis indicated that thrombin induced an increase in the mRNA levels of t-PA and of PAI-1. We conclude that thrombin increases DNA synthesis in human mesangial cells and enhances the synthesis of both t-PA and PAI-1. The latter is released in a large excess as compared to t-PA. Hence, thrombin may have a role in provoking a localized hypofibrinolytic state and may contribute to the persistence of glomerular fibrin deposits during proliferative glomerulonephritis.

Blotting, Northern↗
PubMed1997

Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin-III, and heparin cofactor-II.

The effects of the stoichiometric inhibitors tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III) and heparin cofactor-II (HC-II) on thrombin generation were evaluated in a reaction system composed of coagulation factors VIIa, X, IX, VIII, and V and prothrombin initiated by tissue factor (TF) and phospholipids. Initiation of the reaction in the absence of inhibitors resulted in explosive thrombin generation for factor VIIa.TF concentrations varying from 100 to 0.25 pM with the lag time or initiation phase of thrombin generation increasing from 0 to 180 s with decreasing factor VIIa.TF concentrations. During the propagation phase, prothrombin is quantitatively activated to 1.4 micro;M alpha-thrombin. At normal plasma concentration (2.5 nM) full-length recombinant TFPI prolonged the initiation phase of thrombin generation 2-fold, and the rate of thrombin generation in the propagation phase of the reaction was 25-50% that of the uninhibited reaction when the reaction was initiated with 1.25-20 pM factor VIIa.TF. Inhibition of the reaction by TFPI is associated with a delay in factor V activation. In the presence of TFPI no explosive thrombin generation was observed when factor VIII was omitted from reactions initiated by factor VIIa.TF concentrations </=20 pM. This indicates that in the presence of TFPI the factor IXa.factor VIIIa pathway becomes essential at low factor VIIa.TF concentrations. In the reconstituted system, AT-III (3.4 micro;M) did not prolong the initiation phase of thrombin generation when the reaction was initiated with 1.25 pM factor VIIa.TF, nor did AT-III delay factor V activation. The rate of thrombin formation in the presence of AT-III was reduced to 30% that of the uninhibited reaction, and the alpha-thrombin formed was rapidly inhibited subsequent to its generation. The addition of HC-II alone at its physiological concentration (1.38 micro;M) to the procoagulant mixture did not alter the rate or extent of thrombin generation. Subsequently, the thrombin formed was slowly inhibited by HC-II. The slow inactivation of thrombin by HC-II does not contribute to thrombin inhibition in the presence of AT-III. In contrast, the combination of physiological levels of AT-III and TFPI inhibited explosive thrombin generation initiated by 1.25 pM factor VIIa.TF completely. The absence of prothrombin consumption indicated that the combination of TFPI and AT-III is able to prevent the formation of prothrombinase activity at low factor VIIa.TF concentrations. The data indicate that TFPI potentiates the action of AT-III by decreasing the rate of formation and thus the amount of catalyst formed in the reaction, enabling AT-III to effectively scavenge the limited traces of factor IXa and factor Xa formed in the presence of TFPI. The initiation of thrombin generation by increasing factor VIIa.TF concentrations in the presence of physiological concentrations of TFPI and AT-III showed dramatic changes in the maximal rates of thrombin generation over small changes in initiator concentration. These data demonstrate that significant thrombin generation becomes a "threshold-limited" event with regard to the initiating factor VIIa.TF concentration in the presence of TFPI and AT-III.

Antithrombin III↗
PubMed2003

Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin.

Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.

Binding Sites↗
PubMed2006

The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII.

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

Amino Acid Sequence↗
PubMed1990

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers.

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1976

The binding of human and bovine thrombin to human platelets.

Human thrombin binds to specific receptors on the surface of human platelets in a manner analogous to bovine thrombin. Thus, two classes of binding are observed--high affinity with a dissociation constant (Kdiss) of 0.02 U/ml and low affinity with a Kdiss of 5 U/ml. Bovine and human thrombin bind to the same platelet receptors, although bovine thrombin binds with slightly greater affinity. When the amount of thrombin bound to platelets is related to the extent of 14C-serotonin release, bovine and human thrombin are equally effective. Antibodies to human and bovine thrombin were found to differ markedly in their ability to precipitate thrombin of the two species. Thus, antibovine thrombin precipitated eightfold more bovine thrombin than human thrombin, while antihuman thrombin precipitated tenfold more human thrombin than bovine thrombin. Similar differences were found in the ability of Fab fragments of these antibodies to block the interaction of thrombin of each species with human platelets. The finding that both species of thrombin, despite significant evolutionary differences in primary structure, retain essentially identical binding sites to platelets suggests that this part of the thrombin molecule is physiologically important and supports our hypothesis of a role for thrombin binding to platelets in platelet function and hemostasis.

Animals↗
PubMed2001

Aptamer inhibits degradation of platelet proteolytically activatable receptor, PAR-1, by thrombin.

We investigated the in vitro effects of the site-directed thrombin inhibitor-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTTGG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and platelet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin-dependent degradation of proteolytically activatable receptor, PAR-1, exposed on platelet surface membrane at micromolar concentrations. The incubation of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up to fourfold, P<.0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46+/-20% inhibition at 0.15 U/ml thrombin, P<.001), as well as thrombin-mediated platelet aggregation in PRP (7+/-10% inhibition at 1 U/ml thrombin, P<.05). Furthermore, aptamer inhibited the thrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17%, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 nM (P<.025 by randomised block analysis; P(regression slope)<.0001). We conclude that aptamer is able to considerably attenuate thrombin proteolytic activity regardless of the molecular size of thrombin substrates. Our observations directly proved that aptamer may be successfully used for the inhibition of thrombin activity towards various physiological targets: one related to fibrin generation in the final stage of coagulation cascade, and another concerning the interaction of thrombin with its surface membrane receptor, PAR-1, in blood platelets.

Adult↗
PubMed1981

Preparation and application of a photoreactive thrombin analogue: binding to human platelets.

alpha-Thrombin has previously been shown to bind to specific, saturable glycoproteins on the platelet surface. Modification of the thrombin active site with tosyllysyl chloromethyl ketone (TosLysCH2Cl) does not alter thrombin's binding characteristics. Interaction of alpha-thrombin with high-affinity binding sites (KD = 10(-9) M) initiates the platelet response which involves proteolytic hydrolysis of this glycoprotein. Although TosLysCH2Cl--thrombin binds to and competes for the same sites as alpha-thrombin, it cannot induce platelet stimulation because it is enzymatically inactive. In this study, we describe the preparation and application of photoreactive tritium-labeled thrombin analogues. The alpha-thrombin derivative retains its platelet-stimulating and enzymatic activities and, upon photoactivation, covalently binds to specific platelet membrane components. When freshly washed human platelets are exposed to less than saturation doses (less than or equal to 2 nM) of the thrombin derivatives in the dark and photoactivated, a single labeled complex is detected. The same experiment with greater than saturating doses (greater than or equal to 20 nM) of the thrombin derivative yields a similar complex as well as two additional ones. Molecular weight estimates of these thrombin-bound complexes were obtained by gel filtration and NaDodSO4--polyacrylamide gel electrophoresis. The low dose (high affinity) complex with TosLysCH2Cl--thrombin has an approximate molecular weight of 200 000, while that with active alpha-thrombin is smaller, approximately 120 000, due to enzymatic cleavage. The additional complexes detected with the high thrombin dose had estimated molecular weights of 400 000 and 46 000, respectively, and appeared to be the same for TosLysCH2Cl--thrombin and for the alpha-thrombin coupled platelets. These isolated complexes appear to correspond to the two previously detected populations of thrombin binding sites on the platelet.

Azides↗
PubMed1994

Thrombin binding to platelets and their activation in plasma.

The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.

Blood Platelets↗
PubMed1992

The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.

The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.

Amino Acid Sequence↗
PubMed1994

Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling.

According to current models, human thrombin receptors are activated when thrombin cleaves the receptor's N terminus, exposing the tethered ligand domain, SFLLRN. In the megakaryoblastic CHRF-288 cell line, thrombin receptor activation is followed by the rapid internalization of > 90% of the receptors. In the present studies, antibodies directed at the site of cleavage by thrombin were used to examine changes in receptor structure during activation, internalization, and recovery. As would be expected, the initial rate of receptor cleavage was directly related to the thrombin concentration. However, even after prolonged incubation, receptor cleavage was incomplete until the thrombin concentration exceeded the receptor concentration. Only cleaved receptors were internalized in response to thrombin and only catalytically active thrombin and active variants of SFLLRN-containing peptides caused receptor internalization. Over a 3-h period following receptor activation by thrombin, there was a gradual recovery of approximately one-quarter of the receptors on the cell surface. These receptors were detectable with antibodies directed at retained portions of the receptor N terminus, but not with antibodies directed at the proposed site of cleavage, confirming that they are recycled, rather than new, receptors. At 4 degrees C two-thirds of the receptors cleaved by thrombin were retained on the cell surface. Like recycled receptors these "cold-cleaved" receptors failed to self-activate when warmed to 37 degrees C, but could be activated by SFLLRN. Unlike recycled receptors, however, the cold-cleaved receptors were also internalized and appeared to be activated by a second addition of thrombin. These results 1) provide strong evidence at the protein level that thrombin cleaves its receptors at the predicted site, 2) show that receptor activation is necessary for internalization, 3) suggest that each thrombin molecule may not activate large numbers of receptors, 4) demonstrate that a substantial fraction of internalized thrombin receptors can be recycled, and 5) suggest that the failure of recycled receptors to be reactivated by thrombin may involve a change in the receptor that does not occur at 4 degrees C. Finally, the inability of cold-cleaved receptors to self activate in the absence of thrombin, suggests that in addition to cleaving the receptor, thrombin may also play an important role in guiding the tethered ligand domain to regions on the remainder of the receptor that mediate activation.

Amino Acid Sequence↗
PubMed1994

Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin.

Thrombin, in addition to being a key enzyme in hemostasis, affects a series of endothelial and leukocyte functions and thus may be involved in the regulation of inflammatory reactions. Because leukocyte recruitment and activation are important events in inflammatory and thrombotic processes, in this study we have examined the possibility that thrombin induces the production of a cytokine chemotactic for mononuclear phagocytes. Human peripheral blood mononuclear cells (PBMC) exposed in vitro to thrombin expressed transcripts of monocyte chemotactic protein-1 (MCP-1; alternative acronyms: JE, monocyte chemotactic and activating factor, tumor-derived chemotactic factor). Thrombin was two- to threefold less effective than endotoxin in inducing MCP-1 transcripts in PBMC. Among circulating mononuclear cells, monocytes were identified as the cells expressing MCP-1 in response to thrombin. Monocytes expressed thrombin receptor transcripts. Boiling, hirudin, antithrombin III, and mutation of the catalytic site serine 205 into alanine) blocked the capacity of thrombin to induce MCP-1 expression. The thrombin receptor-activating peptide mimicked the effect of thrombin in inducing MCP-1 expression. Induction of MCP-1 transcript by thrombin was not reduced by blocking interleukin-1 and tumor necrosis factor, suggesting that these mediators are not involved in thrombin-induced expression of MCP-1. In addition to monocytes, endothelial cells (EC) also expressed MCP-1 in response to thrombin, although at lower levels compared with monocytes. Actinomycin D experiments indicated that induction of MCP-1 by thrombin in PBMC and EC was gene transcription dependent. The inhibition of protein synthesis blocked thrombin-induced MCP-1 expression in PBMC, whereas it superinduced both constitutive and thrombin-inducible expression of MCP-1 in EC, indicating different mechanisms of regulation of this gene in mononuclear phagocytes versus endothelial cells. Thrombin stimulated mononuclear cells and EC to release chemotactic activity for monocytes that could be inhibited by absorption with anti-MCP-1 antibodies. Induction of a chemotactic cytokine for monocytes by thrombin points to the importance of this enzyme in regulating inflammatory processes and further indicates that hemostasis, inflammation, and immunity are strictly interconnected processes.

Base Sequence↗
PubMed1978

Cell surface action of thrombin is sufficient to initiate division of chick cells.

Thrombin covalently linked to carboxylate-modified polystyrene beads initiated division of quiescent chick embryo (CE) cells either in medium containing low levels of serum or in serum-free medium. Release of thrombin was monitored by measuring acid-precipitable radioactivity released from 125I-thrombin beads into the medium during incubation with cells. Even if all of the acid-precipitable material released from the beads were active thrombin, it was not sufficient to account for any of the observed cell division, and was 10-30 fold less than the amount necessary to produce the increase in cell number caused by the thrombin beads. Two other kinds of experiments also showed that material released into the medium did not account for the observed initiation of cell division. First, medium taken from cultures incubated with thrombin beads did not initiate cell division when added to new quiescent cultures. Second, in coverslip experiments where populations of cells with an without thrombin feads shared the same medium, only bead-contacted cells divided. Several results suggested that the material which was released from the thrombin beads resulted from cell-associated proteolysis rather than from "leakage" of intact thrombin from the beads. For example, after incubating 125I-thrombin beads with or without CE cells, we were unable to detect any intact thrombin released into the medium. In addition, most of the material released from the beads was acid-soluble and was only released in the presence of CE cells. A few thrombin beads were endocytosed by CE cells, but they were surrounded by an intact plasma membrane. Thus they did not directly interact with the cytoplasm. The close association of many of the beads with the cell surface and the presence of a few beads in endocytic vesicles made it important to consider the possibility that thrombin might be released from the beads directly into the cells. This possibility was explored using ultrastructural (EM) autoradiography. With this technique (where one grain represented 700--900 thrombin molecules), we found that beads inside the cells had approximately the same number of grains as beads not in contact with cells. This suggested that little, if any, additional radioactive material had been released from the beads which were in contact with the cells. In addition, we were unable to detect any grains in the cytoplasm which could be attributed to released thrombin, even using an amount of 125I-thrombin beads which was 8 fold greater than the amount which produced maximal cell division. Taken together, these results provide direct evidence that thrombin action at the cell surface is sufficient to initiate division of CE cells.

Cell Count↗
PubMed2001

Effect of different types of thrombin inhibitors on thrombin/thrombomodulin modulated activation of protein C in vitro.

The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human alpha-thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl(2). TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant (K(D)) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC(50) values+/-S.D. of 4.4+/-0.8, 20.0+/-1.1 and 6.4+/-0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC(50) value of 290+/-10 nmol/l, which was enhanced fourfold (IC(50) 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.

Antithrombins↗
PubMed1976

Evidence for a single class of thrombin-binding sites of human platelets.

We previously demonstrated that human platelets bind a small number of thrombin molecules with high affinity and a larger number with relatively lower affinity (Tollefsen, D. M., Feagler, J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). In the present report, equilibrium binding studies using [125I]DIP-thrombin (diisopropylphosphoryl-thrombin) over the range of 0.0002-10 U/ml yield a Hill coefficient of 0.775. Measurements of the rate of dissociation of [125I]DIP-thrombin bound to platelets at relatively high affinity (0.04 U/ml added) indicate a much faster dissociation in the presence of 14 U/ml unlabeled DIP-thrombin (T 1/2 = 1.0 min) than in its absence (T 1/2 - 140 min). [125I]DIP-thrombin bound at low affinity (1.0U/ml added) dissociates from platelets with a T 1/2 = 1.7 min in the absence of unlabeled DIP-thrombin. These results suggest a negative cooperative interaction among receptor sites for thrombin; i.e., as thrombin binds to unoccupied sites, high-affinity receptors are apparently converted to low-affinity receptors. In an attempt to detect whether there is heterogeneity of thrombin receptors, [125I]DIP-thrombin was covalently cross-linked to intact platelets using 1 mM glutaraldehyde. A single complex (apparent molecular weight, 200 000) containing [125I]DIP-thrombin was formed throughout a range of thrombin concentrations in which both high- and low-affinity binding was observed. Since incorporation of [125I]DIP-thrombin into this complex did not occur in the absence of platelets and was inhibited by unlabeled thrombin, the complex may represent thrombin cross-linked to its receptor. We conclude that a single class of receptor sites can account for both high- and low-affinity binding of thrombin to platelets, although interaction between nonidentical sites cannot be excluded.

Binding Sites↗