Alpha-thrombin stimulation of heparin secretion by the peritoneal mast cells in rats

The interaction of alpha-thrombin with connective tissue-type mast cells (CTMC) purified by Ficoll density gradient centrifugation has been examined. It was demonstrated that exposure of CTMC to polymixin (widely used histamine liberator) (3 mg/ml) induced the release of heparin and histamine. Exposure of CTMC to 10(-11) M alpha-thrombin resulted in increase of heparin secretion by 75.5% in relation to basal level. CTMC which were stimulated by very low concentrations of alpha-thrombin (10(-11)-10(-8) M) can release high level of heparin, but not histamine. We have a suggestion that the thrombin specificity is connected with the additional recognition binding site for high molecular substrates (HMS) distinct from the active centre. Unlike alpha-thrombin which has both the active centre and the recognition site for HMS, beta/gamma-thrombin with catalytic activity but with disrupted recognition site induced the heparin release from mast cells only at higher concentrations than alpha-thrombin. It was revealed that DIP-alpha-thrombin without proteolytic activity was unable to activate mast cells in contrast to alpha-thrombin. We consider that alpha-thrombin induced release of heparin by CTMC account for proteolytic and hormone-like activity enzyme by means of both the active centre and the additional recognition site for HMS.     

Heparin secretion by mast cells as an index of the status of the anticoagulant system

The status of the mast cell population was studied and compared after administration of trypsin or alpha-thrombin in similar molar concentrations. Morphometry disclosed a substantial shift of the mast cell population towards light, heparin-free cells within one minute after alpha-thrombin administration. The index of mast cell saturation with heparin dropped below 1. The maximal heparin secretion was observed at the 5th minute of experiment. The morphometric criteria of the mast cell population returned to basal level in 120 minutes. These data along with a significant increase in the level of complex heparin compounds and plasma thrombin time indicate heparin release as a result of the effector action of the anticoagulation system. No changes were observed in the activity of complex heparin compounds and in thrombin time after intravenous injection of trypsin. It is suggested that high heparin secretion by mast cells may serve as criterion of the active status of the anticoagulation system.     

Thymosin alpha(1)--an endogenous modulator of alpha-thrombin recognition site]

Thymosin alpha 1-inhibited fibrinogen clotting activity of alpha-thrombin, but not amidolysis of H-D-Phe-Pip-Arg-pNA. Modulation of thrombin interaction with rat peritoneal mast cells (RPMC) by suppressors of additional recognition binding site (thymosin and heparin) was studied. Thrombin-induced pHi changes of RPMC were controlled with pH-sensitive fluorescent dye, BCECF. Thrombin caused a biphasic changes in pHi: rapid cell acidification (0.02) followed by slow alkalinization (0.06 above baseline for 18 min). Thymosin suppressed thrombin-induced pHi increase above resting level. Similar changes in pHi were observed after modification of additional recognition binding site by heparin. Beta/gamma-thrombin with disrupted additional binding site was shown to induce only a decrease of pHi. It is concluded that thymosin alpha 1 is endogenous modulator of alpha-thrombin activity.     

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.     

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.     

Role of thrombin exosites in inhibition by heparin cofactor II.

We determined the role of specific thrombin "exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and gamma T-thrombin (produced by partial proteolysis of alpha-thrombin by elastase and trypsin, respectively). All of the thrombin derivatives were inhibited in a similar manner by AT, either in the absence or presence of heparin, which confirmed the integrity of both heparin binding abilities and serpin reactivities of epsilon- and gamma T-thrombin compared to alpha-thrombin. Antithrombin activities of HC in the absence of a glycosaminoglycan with alpha-, epsilon, and gamma T-thrombin were similar with rate constants of 3.5, 2.4, and 1.2 x 10(4) M-1 min-1, respectively. Interestingly, in the presence of glycosaminoglycans the maximal inhibition rate constants by HC with heparin and dermatan sulfate, respectively, were as follows: 30.0 x 10(7) and 60.5 x 10(7) for alpha-thrombin, 14.6 x 10(7) and 24.3 x 10(7) for epsilon-thrombin, and 0.017 x 10(7) and 0.034 x 10(7) M-1 min-1 for gamma T-thrombin. A hirudin carboxyl-terminal peptide, which binds to anion-binding exosite-I of alpha-thrombin, dramatically reduced alpha-thrombin inhibition by HC in the presence of heparin but not in its absence. We analyzed our results in relation to the recently determined x-ray structure of D-Phe-Pro-Arg-chloromethyl ketone-alpha-thrombin (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475). Our results suggest that the beta-loop region of anion-binding exosite-I in alpha-thrombin, which is not present in gamma T-thrombin, is essential for the rapid inhibition reaction by HC in the presence of a glycosaminoglycan. Therefore, alpha-thrombin and its derivatives would be recognized and inhibited differently by HC and AT in the presence of a glycosaminoglycan.     

Activation of Na/H exchange by thrombin in peritoneal mast cells]

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.     

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.     

Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.     

Binding of human thrombin to cultured human endothelial cells.

Binding of thrombin to monolayer cultures of human umbilical vein endothelium is studied. Binding is measured as inhibition by unlabeled ligand of the binding of 125I-thrombin to the cells. Radioactivity bound to cultures at equilibrium is measured after draining but not washing the cells. To correct for unremoved supernatant, 131I-albumin is included as a second label in the medium. Equilibrium between bound and free thrombin is attained within 1 min, and Scatchard analysis indicates a population of approximately 3 x 10(3) sites/cell with a dissociation constant of 10(-10) M, and a larger population with a dissociation constant greater than 10(-8) M. The two populations of sites are also indicated by a biphasic dissociation of bound label. Thrombin inactivated with diisopropyl fluorophosphate binds to the same receptor, with an affinity similar to that of active thrombin. Binding is unaffected by albumin (an acidic protein) and cytochrome c (a basic protein). Cultures of umbilical cord smooth muscle and fibroblasts bind thrombin at least 100 times more weakly than endothelium, and no binding to erythrocytes or a monolayer culture of mouse neuroblastoma is detected.     

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.     

Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin

Quiescent cultures of chick embryo fibroblasts incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal growth factors, insulin, and insulin-like growth factors did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at 22-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at 22 and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at 22 degrees C. Finally, confluent cultures of fibroblasts bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.     

Covalent binding of thrombin to specific sites on corneal endothelial cells

Binding of 125I-labeled human alpha-thrombin to endothelial cells derived from bovine corneas was studied in tissue culture. Specific and saturable binding to the cell surface occurred at 37 degrees C but to a much smaller extent at 4 degrees C. Binding of [125I]thrombin to a specific site on these cells with formation of a 77000-dalton complex was demonstrated by NaDodSO4 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I]thrombin was blocked by a 100-fold excess of unlabeled alpha-thrombin and by the thrombin inhibitor, hirudin. There are approximately 100000 of these thrombin binding sites on the cell surface. Formation of the complex could be detected as early as 15 s, increased rapidly over the next 20-30 min, and then continued at a slower rate for the next 2.5 h. The catalytically active site of the enzyme was required for formation of the NaDodSO4-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated in NaDodSO4 with 1.0 M hydroxylamine, suggesting an acyl linkage of the enzyme to the cellular binding site. The thrombin-endothelial cell complex was distinct from the thrombin-antithrombin III complex (Mr approximately 90000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes (Mr less than 77000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that alpha-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod-SO4-stable complex which requires the catalytically active enzyme.     

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.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin

Heparin (H) was previously shown to accelerate the inactivation of alpha-thrombin (T) by antithrombin III (AT) primarily by promoting the initial binding of thrombin and AT in a ternary T.AT.H complex intermediate without significantly influencing the subsequent product formation step (Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-14895). In the present study, the protein-heparin interactions which contribute to the assembly of the ternary complex intermediate and their linkage were quantitated by equilibrium binding and stopped-flow kinetic studies at pH 7.4, I 0.3, 25 degrees C, using p-aminobenzamidine (P) as a fluorescence probe. Equilibrium binding studies of the AT.H and T.H binary complex interactions monitored by the 40% enhancement in AT fluorescence or the 16-18% quenching of thrombin-bound p-aminobenzamidine fluorescence, respectively, indicated a 100-fold greater affinity of AT for heparin (KAT,H 0.23 microM) as compared to thrombin for heparin (KT,H 35-42 microM). Consistent with this large difference in affinities, rapid kinetic studies indicated that assembly of the ternary complex occurred predominantly as a bimolecular association between the AT.H binary complex and free thrombin. Thus, under pseudo-first order conditions ([AT]o, [H]o much greater than [T]o much less than [P]o), the observed thrombin inactivation rate constant (kobs) exhibited a saturable dependence on [AT]o or [H]o when [H]o much less than KT,H, reflecting a KAT,H (0.25 microM) similar to that directly determined by equilibrium binding. Moreover, competitive inhibition of the reaction by T.H binary complexes was indicated from the hyperbolic decrease in kobs produced by heparin with either high or low affinity for AT or active-site blocked thrombin at concentrations comparable to KT,H. This behavior was consistent with values of KT,H (27-51 microM) similar to those determined directly from equilibrium binding measurements. Comparison of the affinities of the binary protein-heparin interactions with the affinity of thrombin for AT.H complex in the ternary complex measured previously, indicated that the affinity of either protein for heparin was enhanced about 10-fold by the prior binding of the other protein to heparin. This linkage of the protein-heparin interactions implies that the ternary complex will be assembled at thrombin, AT, and heparin concentrations considerably lower than those predicted from previous reaction models which fail to account for this linkage.     

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.     

The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.     

Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions

We have developed a fluorescein isothiocyanate (FITC)-hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein-horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC-hapten immunoassay and a reference radioreceptor assay. The FITC-hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.     

Anophelin: kinetics and mechanism of thrombin inhibition

Anophelin is a 6.5-kDa peptide isolated from the salivary gland of Anopheles albimanus that behaves as an alpha-thrombin inhibitor. In this paper, kinetic analyses and the study of mechanism of alpha-thrombin inhibition by anophelin were performed. Anophelin was determined to be a reversible, slow, tight-binding inhibitor of alpha-thrombin, displaying a competitive type of inhibition. The binding of anophelin to alpha-thrombin is stoichiometric with a dissociation constant (K(i)) of 5.87 +/- 1.46 pM, a calculated association rate constant (k(1)) of 2.11 +/- 0.06 x 10(8) M(-1) s(-1), and a dissociation rate constant (k(-1)) of 4.05 +/- 0.97 x 10(-4) s(-1). In the presence of 0.15 and 0.4 M NaCl, a 17.6- and 207-fold increase in the K(i) of anophelin-alpha-thrombin complex was observed, respectively, indicating that ionic interactions are important in anophelin-alpha-thrombin complex formation. Incubation of alpha-thrombin with C-terminal hirudin fragment 54-65 that binds to alpha-thrombin anion binding exosite 1 (TABE1) attenuates alpha-thrombin inhibition by anophelin; anophelin also blocks TABE1-dependent trypsin-mediated proteolysis of alpha-thrombin. Using gamma-thrombin, an alpha-thrombin derivative where the anion binding exosite has been disrupted, anophelin behaves as a fast and classical competitive inhibitor of gamma-thrombin hydrolysis of small chromogenic substrate (K(i) = 0. 694 +/- 0.063 nM). In addition, anophelin-gamma-thrombin complex formation is prevented by treatment of the enzyme with D-Phe-Pro-Arg-chloromethyl ketone (PPACK), a reagent that irreversibly blocks the catalytic site of thrombin. It is concluded that anophelin is a potent dual inhibitor of alpha-thrombin because it binds both to TABE1 and to the catalytic site, optimal binding being dependent on the availability of both domains. Finally, anophelin inhibits clot-bound alpha-thrombin with an IC(50) of 45 nM and increases the lag phase that precedes explosive in vitro alpha-thrombin generation after activation of intrinsic pathway of blood coagulation. Because of its unique primary sequence, anophelin may be used as a novel reagent to study the structure and function of alpha-thrombin.