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.     

Antithrombin reactions with alpha- and gamma-thrombins.

Human alpha-thrombin with high clotting activity and its proteolyzed derivative gamma-thrombin with virtually no clotting activity reacted in an essentially identical manner with antithrombin. The two enzyme forms bound proflavin with similar constants and showed identical behavior with small substrates. No significant differences were found for the antithrombin reactions (measured by proflavin displacement or active site titration) with respect to kinetics, extent of reaction, or effect of added heparin. The enzyme--antithrombin complexes could not be dissociated with sodium dodecyl sulfate (NaDodSO4) but the NaDodSO4-denatured complexes were dissociated by hydroxylamine treatment. The gamma-thrombin-antithrombin complex has an approximate molecular weight of 75 000 by disc gel electrophoresis as compared with 100 000 for the alpha-complex, consistent with the polypeptide structures of the two proteins. The gamma-thrombin--antithrombin complex did not inhibit clotting catalyzed by alpha-thrombin. In addition, fibrinogen did not affect the reaction of gamma-thrombin with antithrombin or antithrombin--heparin. Thus, the antithrombin and antithrombin--heparin reactions do not involve the fibrinogen recognition sites which are destroyed by proteolytic conversion of alpha-thrombin to the noncoagulant gamma form.     

4,4'-Bis[8-(phenylamino)naphthalene-1-sulfonate] binding to human thrombins: a sensitive exo site fluorescent affinity probe

The binding of the fluorescent probe 4,4'-bis[8-(phenylamino)naphthalene-1-sulfonate] (bis-ANS) to human alpha- and gamma-thrombins was investigated. Bis-ANS binds in a 1:1 complex to both forms of the enzyme, with Kd = 14.8 +/- 2.2 microM and 5.8 +/- 1.0 microM for alpha- and gamma-thrombin, respectively, at pH 7.0 [25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaC1]. Fluorescence changes upon complexation included a considerable (approximately 30-nm) blue shift in the fluorescence emission maximum as well as a dramatic increase in the fluorescence emission intensity: a 70-fold enhancement was observed with alpha-thrombin vs. a approximately 220-fold enhancement with gamma-thrombin. Proflavin was not displaced upon bis-ANS binding. The unknown thrombin effectors ATP, Ca(II)ATP, Co(III)ATP, phosphate, and pyrophosphate bound with enhancement of the fluorescence of the bis-ANS-alpha-thrombin complex. The two inhibitors benzamidine and p-chlorobenzylamine as well as heparin caused decreases in bis-ANS-thrombin fluorescence: valerylamidine had no effect on the fluorescence of the bis-ANS-thrombin complex. Kinetic measurements with two chromogenic substrates, S-2238 and S-2160, indicated that bis-ANS acts as a partial noncompetitive inhibitor of thrombin amidase activity. The kinetic evidence combined with the ligand binding results suggests that bis-ANS does not overlap the catalytic site. The fluorophore ANS complexed with equal affinity to both alpha- and gamma-thrombins (Kd = 24 +/- 4 microM); however, the gamma-thrombin-ANS complex emission at 470 nm was enhanced 26% more than that for the alpha form.     

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.     

Structural and functional properties of human alpha-thrombin, phosphopyridoxylated alpha-thrombin, and gamma T-thrombin. Identification of lysyl residues in alpha-thrombin that are critical for heparin and fibrin(ogen) interactions

alpha-Thrombin derivatives obtained either by site-specific modification at lysyl residues (phosphopyridoxylated) or by limited trypsinolysis (gamma T-thrombin) were compared to correlate structural modifications with the functional reactivity toward fibrin(ogen) and heparin. alpha-Thrombin phosphopyridoxylated in the absence of heparin (unprotected) showed approximately 2 mol of label incorporated/mol of thrombin, but only 1 mol of label incorporated/mol of proteinase when modified in the presence of added heparin (protected). In contrast to native alpha-thrombin, both phosphopyridoxylated alpha-thrombin derivatives failed to interact with a fibrin monomer-agarose column and had reduced fibrinogen clotting activity, which is very similar to gamma T-thrombin. Heparin accelerated the rate of antithrombin III inhibition of alpha-thrombin, heparin-protected modified-alpha-thrombin, and gamma T-thrombin in a manner consistent with a template mechanism but was without effect on unprotected modified alpha-thrombin. In a heparin-catalyzed antithrombin III inhibition assay of alpha-thrombin, we found that D-Phe-Pro-Arg chloromethyl ketone-active site-inactivated gamma T-thrombin competed for heparin binding. It has been shown that limited proteolysis/autolysis of the B-chain of alpha-thrombin in the area around Arg-B73 (in beta T/beta- and gamma T/gamma-thrombin), but not that around Lys-B154 (in gamma T/gamma-thrombin), diminishes specific interactions with fibrinogen (Hofsteenge, J., Braun, P. J., and Stone , S. R. (1988) Biochemistry 27, 2144-2151). In unprotected modified alpha-thrombin, lysyl residues B21, B65, B174, and B252 were phosphopyridoxylated. In heparin-protected modified alpha-thrombin, only lysyl residues B21 and B65 were phosphopyridoxylated. These observations suggest that lysyl residues 21/65 of the B-chain of alpha-thrombin are involved in fibrin(ogen) interactions, and lysyl residues 174/252 of the B-chain are important in heparin interactions.     

Activation of heparin cofactor II by calcium spirulan

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit alpha-thrombin is accelerated by a variety of sulfated polysaccharides in addition to heparin and dermatan sulfate. Previous investigations have indicated that calcium spirulan (Ca-SP), a novel sulfated polysaccharide, enhanced the rate of inhibition of alpha-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by Ca-SP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Delta74) inhibited alpha-thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of alpha-thrombin by rHCII-Delta74 was 2.0 x 10(8) M(-1) min(-1) in the presence of 50 microgram/ml Ca-SP and 10, 000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-Delta74 for the inhibition of gamma-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of alpha-thrombin was essential for the rapid inhibition reaction by HCII in the presence of Ca-SP and that the N-terminal acidic domain of HCII was not required. Therefore, we proposed a mechanism by which HCII was activated allosterically by Ca-SP and could interact with the anion-binding exosite I of thrombin not through the N-terminal acidic domain of HCII. The Arg(103) --> Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited alpha-thrombin in a manner similar to native rHCII. These results indicate that Arg(103) in HCII molecule is not critical for the interaction with Ca-SP.     

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.     

Antithrombin III inhibition of the reaction to thrombin excitation of the anticoagulation system

It has been established that intravenous administration of alpha-thrombin-antithrombin III preparations (1 mkM) has practically no effect on anticoagulation parameters (thrombin time, additive fibrinolytic activity, nonenzymatic fibrinolysis and nonenzymatic fibrinolytic activity). Administration of 1 mkM of alpha-thrombin caused a statistically significant increase of all the parameters. The experiments on perfusion of the humorally isolated sinocarotid area of the rabbit with alpha-thrombin-antithrombin III preparations (1.25 mkM) showed no changes peculiar to the induction of anticoagulation response with thrombin. It is concluded that antithrombin III blocks the ability of thrombin to activate anticoagulation system function.     

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.     

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.     

Initiation of proliferative events by human alpha-thrombin requires both receptor binding and enzymic activity

To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.     

Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II.

Bothrojaracin, a 27-kDa C-type lectin from Bothrops jararaca venom, is a selective and potent thrombin inhibitor (K(d) = 0.6 nM) which interacts with the two thrombin anion-binding exosites (I and II) but not with its catalytic site. In the present study, we analyzed the allosteric effects produced in the catalytic site by bothrojaracin binding to thrombin exosites. Opposite effects were observed with alpha-thrombin, which possesses both exosites I and II, and with gamma-thrombin, which lacks exosite I. On the one hand, bothrojaracin altered both kinetic parameters K(m) and k(cat) of alpha-thrombin for small synthetic substrates, resulting in an increased efficiency of alpha-thrombin catalytic activity. This effect was similar to that produced by hirugen, a peptide based on the C-terminal hirudin sequence (residues 54-65) which interacts exclusively with exosite I. On the other hand, bothrojaracin decreased the amidolytic activity of gamma-thrombin toward chromogenic substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with alpha-thrombin. In agreement with these observaions, bothrojaracin produced opposite effects on the fluorescence intensity of alpha- and gamma-thrombin derivatives labeled at the active site with fluorescein-Phe-Pro-Arg-chloromethylketone. These observations support the conclusion that bothrojaracin binding to thrombin produces two different structural changes in its active site, depending on whether it interacts exclusively with exosite II, as seen with gamma-thrombin, or with exosite I (or both I and II) as observed with alpha-thrombin. The ability of bothrojaracin to evoke distinct modifications in the thrombin catalytic site environment when interacting with exosites I and II make this molecule an interesting tool for the study of allosteric changes in the thrombin molecule.     

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.     

Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor.

Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a "tethered-ligand" to activate the receptor. A thrombin receptor activating peptide (SFLLRNPNDKYEPF) homologous to the new N-terminus was shown to activate platelets. We synthesized this peptide and demonstrated that it desensitized platelets to activation by low concentrations of alpha-thrombin but not gamma-thrombin. We also synthesized a thrombin exosite inhibitor (BMS 180742) that inhibited platelet aggregation induced by low, but not high, concentrations of alpha-thrombin. In contrast, a thrombin active site inhibitor, N alpha-(2-naphthylsulfonyl-glycyl)-D,L-amidinophenylalanylpiperi dide, competitively inhibited thrombin-induced platelet aggregation. We conclude that thrombin-induced platelet activation is mediated by at least two pathways: one activated by low concentrations of alpha-thrombin and blocked by a thrombin exosite inhibitor that appears to be coupled to the "tethered-ligand" thrombin receptor, and another that is stimulated by higher concentrations of alpha-thrombin and by gamma-thrombin and does not require the thrombin exosite for activation. Both pathways are blocked by a thrombin active site inhibitor.     

The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin and chymotrypsin. Inhibition occurs when the protease attacks the reactive site peptide bond in HCII (Leu444-Ser445) and becomes trapped as a covalent 1:1 complex. Dermatan sulfate and heparin increase the rate of inhibition of thrombin, but not of chymotrypsin, greater than 1000-fold. The N-terminal portion of HCII contains two acidic repeats (Glu56-Asp-Asp-Asp-Tyr-Leu-Asp and Glu69-Asp-Asp-Asp-Tyr-Ile-Asp) that may bind to anion-binding exosite I of thrombin to facilitate covalent complex formation. To examine the importance of the acidic domain, we have constructed a series of 5' deletions in the HCII cDNA and expressed the recombinant HCII (rHCII) in Escherichia coli. Apparent second-order rate constants (k2) for inhibition of alpha-thrombin and chymotrypsin by each variant were determined. Deletion of amino acid residues 1-74 had no effect on the rate of inhibition of alpha-thrombin or chymotrypsin in the absence of a glycosaminoglycan. Similarly, the rate of inhibition of alpha-thrombin in the presence of a glycosaminoglycan was unaffected by deletion of residues 1-52. However, deletion of residues 1-67 (first acidic repeat) or 1-74 (first and second acidic repeats) greatly decreased the rate of inhibition of alpha-thrombin in the presence of heparin, dermatan sulfate, or a dermatan sulfate hexasaccharide that comprises the minimum high-affinity binding site for HCII. Deletion of one or both of the acidic repeats increased the apparent affinity of rHCII for heparin-Sepharose, suggesting that the acidic domain may interact with the glycosaminoglycan-binding site of native rHCII. The stimulatory effect of glycosaminoglycans on native rHCII was decreased by a C-terminal hirudin peptide which binds to anion-binding exosite I of alpha-thrombin. Furthermore, the ability of native rHCII to inhibit gamma-thrombin, which lacks the binding site for hirudin, was stimulated weakly by glycosaminoglycans. These results support a model in which the stimulatory effect of glycosaminoglycans on the inhibition of alpha-thrombin is mediated, in part, by the N-terminal acidic domain of HCII.     

Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.     

Activation of cyclic nucleotide formation in murine neuroblastoma N1E-115 cells by modified human thrombins

The activity of alpha-thrombin and chemically modified derivatives of this enzyme in stimulating cGMP formation in murine neuroblastoma clone N1E-115 cells is reported. The rank order potency of the compounds falls into three classes: 1) alpha-thrombin is the most potent and effective; 2) the catalytically active enzymes gamma-thrombin, trypsin, and nitro-alpha-thrombin are approximately 50-fold less potent than alpha-thrombin; and 3) active site blocked derivatives of alpha-thrombin are 100 to 1000-fold less potent than alpha-thrombin. Native alpha-thrombin consistently produces the most effective response, usually 1.5 to 3-fold greater than any of the other compounds tested. Preincubation of cells with quinacrine, an inhibitor of phospholipase A2, or with the lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid prior to thrombin challenge resulted in a concentration-dependent attenuation of the response. Indomethacin was without effect in modifying the response. These results suggest that thrombin stimulation of neuroblastoma cells involves the release of arachidonic acid and its metabolism by lipoxygenase. These results clearly demonstrate the activity of alpha-thrombin in stimulating a receptor-mediated response in cultured nerve cells. The results are discussed in relation to the interaction of endogenous alpha-thrombin with nerve cells following invasive trauma and to the possible presence of endogenous proteinases with a neurotransmitter-like function.     

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

Association of thrombin-antithrombin III complex with vitronectin in serum

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE. Upon prolonged boiling in SDS it dissociates into 56- and 32-kDa components which co-migrate in SDS-PAGE with purified antithrombin III and thrombin, respectively. The 82- and 56-kDa components react with an antiserum against antithrombin III, and an antiserum prepared against the 82-kDa complex reacts with purified antithrombin III. Thrombin-antithrombin III complex, from either serum or recalcified clotted plasma, bound to vitronectin immobilized on Sepharose or plastic. However, purified antithrombin III which had not reacted with thrombin lacked affinity for vitronectin as did antithrombin III from citrated plasma. Purified antithrombin III acquired affinity for immobilized vitronectin if it was complexed with thrombin or was modified by radioiodination. Binding of vitronectin to antithrombin III coated on plastic was demonstrated using enzyme-linked immunosorbent assay. These results demonstrate that vitronectin binds thrombin-antithrombin III complexes through a cryptic site in antithrombin III which can be exposed when antithrombin III is radioiodinated, bound to plastic, or complexed with thrombin. Since vitronectin can interact with cells, the binding of vitronectin to the thrombin-antithrombin III complex may serve to facilitate the interaction of this complex with cell surfaces.