Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function.

Recent studies on G-protein-coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The gamma-amino-n-butyric acid type B (GABA(B)) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane alpha-helices, loops and the C-terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G-protein signalling. However, the HD of GB1 improves coupling efficacy. Conversely, although GB1 ECD is sufficient to bind GABA(B) ligands, the ECD of GB2 increases the agonist affinity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild-type functioning of the GABA(B) receptor and highlight further the importance of the dimerization process in GPCR activation.

C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABA(b) receptors.

Assembly of fully functional GABA(B) receptors requires heteromerization of the GABA(B(1)) and GABA(B(2)) subunits. It is thought that GABA(B(1)) and GABA(B(2)) undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABA(B(1)) retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABA(B(1)) trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABA(B(1)) that when deleted or mutagenized allows for surface delivery in the absence of GABA(B(2)). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in K(ATP) channels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABA(B(2)) is able to mask the RSRR signal and to direct the GABA(B(1)) monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABA(B(2)) participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABA(B(1)) and GABA(B(2)) supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABA(B) receptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

A novel site on the Galpha -protein that recognizes heptahelical receptors.

Specific domains of the G-protein alpha subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between alpha4 and alpha5 helices of the G-protein alpha subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Galpha(15) and its human homologue Galpha(16), whereas metabotropic glutamate receptor 8 activated Galpha(15) only. The extreme C-terminal 20 amino acid residues are identical between the Galpha(15) and Galpha(16) and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Galpha(16) that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of Galpha(16). One module comprises alpha4 helix, alpha4-beta6 loop (L9 Loop), beta6 sheet, and alpha5 helix. The other, not described previously, is located within the loop that links the N-terminal alpha helix to the beta1 strand of the Ras-like domain of the alpha subunit. Coupling of Galpha(16) protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully.

Linear and non-linear 24 h heart rate variability in chronic heart failure.

It has recently been demonstrated that SDNN of heart rate variability (HRV) is a useful independent prognostic tool in chronic heart failure (CHF). The purpose of the present study was to evaluate if spectral and non-linear analysis of 24-h HRV, considered markers of autonomic cardiac modulation, contain independent prognostic information in CHF patients. Twenty normal subjects and thirty consecutive outpatients with clinically stable CHF were studied for 2 years. Periods of 300 R-R intervals were analyzed from Holter recordings. The power spectral analysis, the slope of the linear relationship between log-power versus log-frequency (1/f), and the complexity content (using corrected conditional entropy; CCE) of the R-R series were calculated. The normalized power of the low frequency spectral component (LF) and the 1/f slope were significantly lower in patients compared to controls (respectively 30.1 +/- 3.0 vs. 48.6 +/- 3.4 and -1.27 +/- 0.04 vs. -1.08 +/- 0.05; P < 0.05). Moreover, the patients who died during the study presented a reduced LF (20.9 +/- 4.1 vs. 35.5 +/- 3.5 nu; P < 0.05) and a steeper 1/f slope (-1.40 +/- 0.09 vs. -1.21 +/- 0.04 nuts, P < 0.05) compared to survivors. These results remained significant in a logistic model including heart rate and SDNN. The information content present in spectral and non-linear analysis of HRV in CHF patients has prognostic relevance independently from the time domain measures of HRV. In particular, the reduction of LF power seems the best indicator among those considered.

Neurotrophin dependence domain: a domain required for the mediation of apoptosis by the p75 neurotrophin receptor.

The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.

Protein isoaspartyl methyltransferase protects from Bax-induced apoptosis.

Protein L-isoaspartyl methyltransferase (Pimt) is a highly conserved enzyme utilising S-adenosylmethionine (AdoMet) to methylate aspartate residues of proteins damaged by age-related isomerisation and deamidation. We have been particularly interested in this enzyme since addition of the compound CGP3466 to primary rat astroglia cell cultures resulted in an upregulation of Pimt at the mRNA level, as shown here by semi-quantitative RT-PCR. CGP3466 is a compound related to the anti-Parkinson's drug R-(-)-deprenyl, which has been shown to protect from neural apoptosis induced by trophic factor withdrawal [Tatton et al., 1994. J. Neurochem. 63, 1572]. The pro-apoptotic gene Bax is required in the cascade of events following withdrawal [Deckwerth et al., 1996. Neuron 17, 401]. We therefore investigated whether Pimt overexpression was able to affect Bax-induced apoptosis in primary mouse cortical neurons. Our results show that Pimt is indeed able to protect from Bax-induced apoptosis. Furthermore, this activity is not restricted to brain-specific cell types, since the same effect is also demonstrated in COS1 cells. In addition, mutational analysis suggests that the protective effect is dependent on the adenosine methionine-binding motif, which is well conserved in protein methyltransferases, and that a mutation destroying this motif crucially affects cytoskeletal structures of the cell.

The elusive link between coronary lesion morphology and dobutamine stress echocardiography results. The EDIC (Echo Dobutamine International Cooperative) Study Group.

BACKGROUND: Coronary lesion angiographic morphology of the complex type is associated to enhanced susceptibility to ischemia during vasodilator adenosinergic stress testing and attributed to the reduced vasodilatory capacity of the damaged endothelium. Whether coronary lesion morphology can also influence the results of adrenergic pharmacologic stress test remains unknown. The aim of our study was to assess the relationship between coronary plaque morphology and dobutamine-atropine stress echocardiography (DASE) results. METHODS AND RESULTS: We analyzed DASE (up to 40 mcg/kg/min plus atropine) and coronary angiography data of 42 patients with single vessel disease and no totally occluded vessel at angiography. 7 patients had angina, 35 had previous infarction. A diagnostic DASE was performed in all patients within 1-10 (mean 4.7 +/- 3.4) days before coronary angiography. An angiographic lesion was considered complex when irregular borders and/or intraluminal lucencies, suggestive of ulcer and/or thrombus were present. According to the angiographic lesion morphology (Ambrose classification), 2 groups were identified: Group I, with simple lesion; Group II with complex lesion. The two groups were similar for number of patients (n = 21), age (I = 55 +/- 11 vs II = 53 +/- 7 years, p = ns), coronary stenosis severity expressed as % diameter reduction (I = 77 +/- 14 vs II = 78 +/- 15%, p = ns), presence of previous infarction (I = 17 vs II = 18 pts, p = ns). No difference was found in the prevalence of positivity between the two groups (I = 72 vs II = 62%, p = ns). The two groups achieved a similar peak dobutamine dose (I = 32 +/- 9 vs II = 33 +/- 9 mcg/kg/min, p = ns) and peak Wall Motion Score Index (I = 1.5 +/- 0.26 vs II = 1.45 +/- 0.28, p = ns). CONCLUSIONS: In patients with non occlusive single vessel disease, coronary morphology of complex type is not associated with greater vulnerability to dobutamine induced ischemia.

Factors affecting the therapeutic choice in patients with multivessel coronary artery disease. The Studio Lombardo Angiografia Multivasali (SLAM) Study Group.

OBJECTIVE: To assess how clinical and angiographic findings are related to the decision to carry out coronary angioplasty (PTCA) or coronary bypass grafting in patients with multivessel coronary artery disease. DESIGN: Prospective survey carried out in 14 centres in the Lombardia region of Italy. PATIENTS: 1468 consecutive patients under going coronary arteriography for known or suspected ischaemic heart disease between May and October 1994, who were found to have multivessel coronary artery disease. MAIN OUTCOME MEASURES: Multivariate analysis was undertaken using stepwise logistic regression to identify the clinical and angiographic variables correlated with revascularisation (v medical treatment) in all of patients, and with surgery (v angioplasty) in the subset of revascularised patients. RESULTS: In all patients the clinical decision after coronary arteriography was made by physicians of each participating centre on the basis of their experience and clinical judgment: 53% of patients had bypass surgery, 28% had PTCA, and 19% continued medical treatment. The choice of a revascularisation procedure was directly related to a clinical diagnosis of unstable angina (P < < 0.001), the presence of left anterior descending artery disease (P < < 0.001), and to an ejection fraction > or = 40% (P < < 0.001), and inversely related to history of previous coronary bypass surgery (P < < 0.001). In revascularised patients, bypass surgery was the preferred treatment in patients with left anterior descending artery disease (P < < 0.001), three-vessel disease (P < < 0.001), and in those with at least one occluded vessel (P = 0.008). The choice of PTCA was significantly related to history of previous PTCA (P < < 0.001) or coronary bypass surgery (P < < 0.001), to a clinical diagnosis of non-Q wave myocardial infarction (P = 0.002), and to the possibility of implanting an intracoronary stent (P = 0.01). CONCLUSIONS: Bypass surgery is still the most widely used treatment for patients with multivessel coronary artery disease. This analysis provides a basis for comparison with future developments in the treatment of such patients. Further advancements in PTCA technology are needed to tilt the balance in favour of this less invasive procedure.

Transphosphorylation of the neurotrophin Trk receptors.

The potential for the activation of one Trk receptor by ligand binding to another Trk receptor was explored by determining if transphosphorylation on tyrosine residues can occur between receptors. For most of these experiments, functional chimeric receptors were used that contained the extracellular domain of the human type 2 tumor necrosis factor receptor and the transmembrane and cytoplasmic domains of rat TrkA, TrkB, or TrkC and that, when activated by the tumor necrosis factor, mediated the nerve growth factor-like biological activities in PC12 cells. Cotransfection experiments in COS-7 cells and fibroblasts showed that despite the presence of different extracellular regions, intermolecular transphosphorylation of homologous cytoplasmic domains occurred between TrkA or TrkB and their cognate chimeras. Heterologous transphosphorylation between TrkB and TrkC kinase domains was also observed when one partner was a chimeric receptor; however, TrkA did not transphosphorylate the TrkB or TrkC kinase domains of chimeric receptors or act as a transphosphorylation substrate for these two receptors. The failure of TrkA to take part in transphosphorylation reactions with TrkB and TrkC was confirmed using the natural receptors. Trk receptor transphosphorylation occurs in the two non-neuronal cell types, but TrkA is excluded from these reactions.

[Identification of multivessel coronary disease using myocardial Thallium-201 and dipyridamole scintigraphy in acute myocardial infarction in the thrombolytic era]. / L'identificazione della coronaropatia plurivasale mediante tomoscintigrafia miocardica con tallio-201 e dipiridamolo nell'infarto miocardico acuto nell'era trombolitica.

Recently some Authors observed a low sensitivity of submaximal exercise thallium-201 myocardial scintigraphy for predicting multivessel coronary disease in patients with recent acute myocardial infarction (AMI) treated with thrombolytic therapy. The aim of our study was to evaluate the accuracy of dipyridamole thallium-201 single photon emission computerized tomography (DIP-SPECT) for predicting the location and the extent of coronary artery disease in patients with recent uncomplicated AMI and to compare the results obtained in patients treated with thrombolytic therapy (Group T) to those obtained in patients treated with non-thrombolytic therapy (Group NT). We examined 61 consecutive patients with recent uncomplicated AMI by predischarge DIP-SPECT as well as by coronary angiography. In the Group T the total number of reversible perfusion defects per patient was 2.6 +/- 1.9 of which 2.1 +/- 1.6 at infarct site and 0.5 +/- 0.7 in other coronary territories; similarly, in the Group NT the total number of reversible perfusion defects per patient was 2.5 +/- 1.6 (NS compared to Group T) of which 1.9 +/- 1.2 (NS compared to Group T) at infarct site and 0.5 +/- 0.8 (NS compared to Group T) in other coronary territories. In Group T, DIP-SPECT demonstrated a sensitivity of 84% with specificity of 60% for predicting critical (> or = 70%) stenoses at infarct site as well as a sensitivity of 54% with specificity of 54% in the other coronary territories (data are expressed in number of patients per cent). The sensitivity and specificity observed in Group NT did not differ significantly from those of Group T.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the heparin-binding site of glia-derived nexin/protease nexin-1 by site-directed mutagenesis.

Recombinant rat glia-derived nexin was expressed in insect cells using the baculovirus system. The kinetics for the inhibition of thrombin by this recombinant material were indistinguishable from those observed with natural glia-derived nexin and recombinant nexin expressed in yeast. In addition, the dependence of the rate of inactivation on the concentration of heparin was similar for the three preparations. At the optimal heparin concentration, the association rate constant was 330-fold higher than that observed in the absence of heparin. A putative heparin-binding site is found in glia-derived nexin between residues 71 and 86; heparin-binding sites are found in homologous regions of antithrombin III and heparin cofactor II. Lysines in this region were mutated to glutamates, and the kinetics for the inhibition of thrombin by mutant proteins were determined. Concurrent mutation of all seven lysines in this region (residues 71, 74, 75, 78, 83, 84, and 86) did not affect the rate constant for the association of glia-derived nexin with thrombin in the absence of heparin, but it resulted in complete loss of the heparin acceleration of the rate of association. Mutations of residues 83, 84, and 86 together also caused a marked decrease in the acceleration by heparin of the reaction between glia-derived nexin and thrombin. These results support the hypothesis that the heparin-binding sites of glia-derived nexin, antithrombin III, and heparin cofactor II are found in homologous regions of the molecules. Heparin was also found to potentiate the ability of wild-type glia-derived nexin to inhibit the thrombin-induced retraction of neurites from neuroblastoma NB2a cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Chimeric tumor necrosis factor-TrkA receptors reveal that ligand-dependent activation of the TrkA tyrosine kinase is sufficient for differentiation and survival of PC12 cells.

To elucidate the function of the two nerve growth factor (NGF) receptors, p75NGFR and p140trk, chimeric molecules were constructed of tumor necrosis factor (TNF) and NGF receptors. Rat PC12 pheochromocytoma cells transiently transfected with TNF-p140trk chimeras, which contain the extracellular domain of TNF receptor and the transmembrane and cytoplasmic domains of p140trk, showed TNF-dependent neuronal differentiation and cell survival. The activity of TNF-p140trk chimeras was completely blocked by the tyrosine kinase inhibitor K252a, and TNF was unable to induce neurite elongation in PC12 cells transfected with a tyrosine kinase-defective chimeric receptor. The TNF-p75NGFR chimeras, which contain the cytoplasmic domain of p75NGFR, were nonfunctional. Our results suggest that p140trk may function as ligand-activated homodimers and that ligand-mediated activation of the cytoplasmic domain of p140trk alone is sufficient for inducing a neuronal phenotype.

Characterization of the heparin-binding site of glia-derived nexin/protease nexin-1.

The interaction of heparin with glia-derived nexin (GDN) has been characterized and compared to that observed between heparin and antithrombin III (ATIII). Heparin was fractionated according to its affinity for immobilized GDN, and the ability of various fractions to accelerate the inhibition rate of thrombin by either GDN or ATIII was examined. Fractions with different affinities for GDN accelerated the thrombin-GDN reaction to a similar extent; heparin with a high affinity for immobilized GDN stimulated the reaction only about 30% more than the fraction that did not bind to immobilized GDN. Slightly greater differences were observed for the effect of these fractions on the thrombin-ATIII reaction; heparin that did not bind to the GDN affinity column was about 60% more effective than heparin with a high affinity for GDN in accelerating the inhibition of thrombin by ATIII. The CNBr fragment of GDN between residues 63 and 144 was able to reduce the heparin-accelerated rate of inhibition of thrombin by GDN indicating that this region of GDN was able to bind the heparin molecules responsible for the acceleration. Shorter synthetic peptides within this sequence did not significantly reduce the rate, suggesting that the heparin-binding activity of fragment 63-144 depends on a specific conformation of the polypeptide chain. Fragment 63-144 was less effective in decreasing the heparin-accelerated rate of inhibition of thrombin by ATIII. The results are discussed in terms of the heparin species that are responsible for the acceleration of the GDN- and ATIII-thrombin reactions and the heparin-binding sites of GDN and ATIII.

Specific interaction of vitronectin with the cell-secreted protease inhibitor glia-derived nexin and its thrombin complex.

Interaction of vitronectin with glia-derived nexin (GDN), thrombin, and the complex GDN-thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN-thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin-III-thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50 approximately 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50 approximately 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN-thrombin complex to vitronectin. The interaction of the biotinylated GDN-thrombin complex with immobilized vitronectin (EC50 approximately 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN-trypsin complex were only weak competitors. Active-site-blocked urokinase and the complex GDN-urokinase also strongly competed for binding of the biotinylated GDN-thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN-thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN-thrombin complex.

Functional sites of glia-derived nexin (GDN): importance of the site reacting with the protease.

Glia-derived nexin (GDN) is a 43-kDa serine protease inhibitor with neurite promoting activity in mouse neuroblastoma cells (Guenther et al., 1985). In chick sympathetic neurons, GDN but not hirudin and synthetic peptide inhibitors promoted neurite outgrowth (Zurn et al., 1988). Thus, it was considered that the protease inhibitory activity cannot account for the total biological activity of GDN. We show here that synthetic peptide inhibitors with thrombin specificity mimic GDN at similar concentrations in neuroblastoma cells. Limited proteolysis of GDN with elastase causes a cleavage between sites P1 and P2, corresponding to residues Ala-344-Arg-345 of the molecule. The resulting fragments still copurify on heparin-Sepharose, but the protease inhibitor activity of GDN and the GDN neurite promoting activity are lost. The results confirm the necessity of an intact reactive site for the biological activity of GDN.

Synthesis of glia-derived nexin in yeast.

Glia-derived nexin (GDN) is a 43-kDa glycoprotein isolated from rat glioma cell cultures. It promotes neurite extension in cultures of neuroblastoma cells and chick sympathetic neurons. Moreover, GDN is a potent serine protease inhibitor (serpin), belonging to the family of protease nexins. We report here the expression of rat GDN in the Saccharomyces cerevisiae strain GRF18 under the control of the PHO5 promoter. We describe the purification of more than 6 mg total GDN from the cellular extract of 1 liter of yeast culture. The amino acid composition and the sequence of CNBr-fragments of the recombinant protein correlate with the values deduced from the rat GDN cDNA. We provide evidence that the recombinant GDN has exactly the same properties as the glioma-derived protein with respect to its protease-inhibitory activity and its ability to promote the extension of neurites from neuroblastoma cells. The large amounts of recombinant protein obtained from this expression system will allow further biochemical and physiological analysis of GDN and of the serpins in general.

Effect of heparin on the glia-derived-nexin-thrombin interaction.

In order to determine the specificity of the interaction between thrombin and glia-derived nexin (GdN), the inactivation of proteolytically modified human thrombin species by GdN has been studied. The second-order rate constants for the inactivation of alpha-, beta T-, gamma T- and epsilon-thrombin by GdN were 1.41, 0.63, 0.33 and 1.91 microM-1.s-1 respectively. The kinetic properties of gdN were also investigated in the presence of different types of heparin, fractionated according to antithrombin III-binding affinity. Association rate constants of both gdN and antithrombin III with alpha-thrombin were obtained using unfractionated, low- and high-affinity heparin types. The different heparin types gave optimal rates of inhibition at similar heparin concentrations for both inhibitors. At optimal heparin concentrations, the rate of inactivation of alpha-thrombin by GdN was 0.5-1.2 nM-1.s-1, which suggests that, under these conditions, the interaction is diffusion-controlled.

[Non-Q infarct. Clinical and prognostic aspects]. / L'infarto non Q. Aspetti clinici e prognostici.

Prognosis of on-Q wave myocardial infarction (nQMI) has been the subject of considerable controversy over the last few years and a systematically aggressive approach with PTCA or coronary by-pass surgery (CBS) has been advocated as a means to reduce subsequent coronary events rate. To investigate clinical outcome and possibly identify high-risk subgroups 131 consecutive patients (pts) meeting diagnostic criteria for nQMI, admitted to our CCU for their first myocardial infarction between January 1980 and June 1985, were followed-up for a mean period of 34 months (range 6-72). Mean age of pts was 59 +/- 7 yrs; 101 (76%) were males, 30 (24%) females. No pt was lost to follow-up. During the same period 684 pts were admitted for Q wave myocardial infarction. Major coronary events such as angina, reinfarction, CBS, cardiac death as well as overall early and late mortality were considered for statistical evaluation with uni-multivariate analysis taking into account multiple data from pts history and acute clinical presentation. Angina appeared or recurred in 71 pts (54%), reinfarction occurred in 14 (10.7%); 25 pts underwent CBC (19%). Early cardiac deaths were 7 (5.3%), late cardiac deaths 12 (9.2%); overall mortality rate 18.1%. Uni- and multivariate statistical analysis did not disclose significant criteria predicting major coronary events and no high-risk subgroup could be identified, confirming uncertainties and doubts from literature.

The use of sequence-specific antibodies to identify a secondary binding site in thrombin.

The peptide comprising residues 62-73 of the B-chain of human alpha-thrombin was synthesized and polyclonal antibodies raised against it. These antibodies were found to bind to the synthetic peptide, a CNBr fragment, and a proteolytic subfragment containing this sequence, as well as the entire thrombin molecule. The purified antibodies had no effect on the hydrolysis by thrombin of D-Phe-pipecolyl-Arg-p-nitroanilide and caused only a minimal decrease (20%) in the second-order rate constant for inactivation by antithrombin III. On the other hand, the antibodies competitively inhibited the binding of hirudin over the concentration range tested (0-43 nM), and a dissociation constant of 3.4 +/- 0.5 nM was found for the antibodies. The release of fibrinopeptide A from the A alpha-chain of fibrinogen by thrombin was competitively inhibited with an inhibition constant of 11.7 +/- 0.4 nM. The activation of protein C by thrombin in the presence of thrombomodulin was also inhibited by the antibodies, and an apparent inhibition constant of 10.7 +/- 1.5 nM was found. In contrast, the antibodies had no effect on the activation of protein C in the absence of thrombomodulin. These results are discussed in relation to data obtained recently on the interaction of well defined proteolytic derivatives of human alpha-thrombin with the ligands described above.