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At least 19 recordsLinked to original sources
PubMed1992

PPACK-thrombin is a noncompetitive inhibitor of alpha-thrombin binding to human platelets.

Recent studies from our laboratory indicate that purified kininogens are noncompetitive inhibitors of human alpha-thrombin but not PPACK-thrombin, binding to human washed platelets. In order to understand the mechanism by which the kininogens inhibit alpha-thrombin binding, investigations were initiated to determine if alpha-thrombin and PPACK-thrombin bound to the same site on human platelets. Initial investigations reveal that alpha-thrombin is a more potent inhibitor of 125I-PPACK-thrombin binding than PPACK-thrombin. Further studies show that PPACK-thrombin is a noncompetitive inhibitor of 125I-alpha-thrombin binding to platelets. These studies suggest that human alpha-thrombin binds on the platelet surface to a different site or binds differently to the same site from PPACK-thrombin. These data indicate that the ability of the kininogens to block alpha-thrombin binding to platelets but not PPACK-thrombin binding results from these thrombins having either two different binding sites or one binding site on the platelet surface which they interact with differently.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity.

We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.

Amino Acid Sequence↗
PubMed1992

Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor.

We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor's extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin's assorted actions on non-platelet cells? We now report that the novel thrombin receptor agonist peptide reproduces thrombin-induced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin-responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA. Our data show that important thrombin-induced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors.

Amino Acid Sequence↗
PubMed1992

"Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.

Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were found to be specific antagonists of receptor activation by thrombin. The effectiveness of these designed antagonists in blocking thrombin-induced platelet activation suggests a model for thrombin-receptor interaction and possible strategies for the development of novel antithrombotic agents.

Amino Acid Sequence↗
PubMed1992

Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.

To better define thrombin-receptor interactions, we synthesized human thrombin peptides and identified binding-domain peptides that bind thrombin receptors and activate mitogenic signals (Glenn, K.C., G.H. Frost, J.S. Bergmann, and D.H. Carney. 1988. Pept. Res. 1:65-73). Treatment of full dermal dorsal incisions with a single topical application of thrombin receptor-activating peptide (TRAP-508) or human alpha-thrombin in saline enhances 7-d incisional breaking strength in normal rats up to 82% or 55% over saline-treated controls, respectively. Control wounds require approximately 11.5 d to achieve breaking strength equivalent to TRAP-treated wounds at day 7. Thus, a single application of TRAP accelerates healing, shifting the time course forward by up to 4.5 d. Histological comparisons at day 7 show more type I collagen, less evidence of prolonged inflammation, and an increase in number and maturity of capillaries in TRAP- and thrombin-treated incisions. Angiograms also show 50-65% more functional vascularization going across thrombin- and TRAP-treated surgical incisions. Thus, alpha-thrombin and thrombin peptides, such as those released following injury, appear to initiate or enhance signals required for neovascularization and wound healing. The ability to accelerate normal wound healing events with synthetic peptides representing receptor binding domains of human thrombin may offer new options for management of wound healing in man.

Amino Acid Sequence↗
PubMed1992

Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin.

Radioactivity from I125-labeled human platelets was measured to estimate the extent of binding of platelet surface proteins to immobilized thrombin. 1-3% of the radioactivity was bound with 10-20% of this amount apparently irreversibly bound to the thrombin matrix. Site-specific chemical modification of thrombin with pyridoxal-5'-phosphate, N-bromosuccinimide or tetranitromethane resulted in a variable reduction of the amount of radiolabel bound. When thrombin modified with H-D-PheProArg-chloromethyl ketone (PPACK) was coupled to the matrix, there was no difference in the binding of platelet membrane proteins when compared to a control thrombin preparation while thrombin modified with tosyl-Lys-chloromethyl ketone (TLCK) coupled to the matrix did not bind radiolabel any more effectively than albumin which served as the control. However, when thrombin was modified with PPACK after coupling to the agarose matrix, ability to bind radiolabel was lost. Thrombin bound to platelets remained catalytically active when assayed with a peptide nitroanilide substrate. These results suggest tight binding between thrombin and platelets that is not only not dependent on active site integrity but leaves the bound thrombin catalytically competent.

Amino Acid Chloromethyl Ketones↗
PubMed1976

Effects of source and concentration of thrombin, and divalent cations, on thrombin time of heparinized plasma.

The effects of the source and concentration of thrombin, and those of divalent cations, on the thrombin time (TT) of heparinized plasma were investigated. A correlation between TT and the heparin concentration was obtained only when the thrombin was of human origin and when it was reconstituted in divalent cation solutions. Relatively small variations in thrombin concentration resulted in marked differences in TT of heparinized plasma. Bovine thrombin gave a very prolonged TT of heparinized plasma compared with human thrombin, though the two thrombins gave identical TT's for non-heparinized control plasma. Divalent cation solution, in which thrombin was reconstituted, had a profound influence on TT of heparin plasma. When thrombin was reconstituted in 0.1 M MnCl2 solution, the TT of a plasma containing 0.5 unit heparin per ml. was the same as that of a plasma containing no heparin. The reliability of the thrombin time test as a means of monitoring heparin anticoagulation must be established by individual laboratories via extensive testing of clinical samples.

Animals↗
PubMed1992

Actions of thrombin and thrombin receptor peptide analogues in gastric and aortic smooth muscle: development of bioassays for structure-activity studies.

We have examined the biological activities of thrombin and the thrombin-receptor-related polypeptides, S42FLLRNPNDKYEPF55(TRP42-55), S42FLLRNPND50(TRP42-50), and A42FLLRNPND50(A42-TRP42-50) as well as an arginine-containing basic peptide beginning with the SF motif (SFRGHITR), in rat aortic (RA) rings and in a gastric guinea pig longitudinal (LM) smooth muscle preparation. In the RA preparation, thrombin, as well as the three receptor-related peptides caused a relaxation in tissue that was precontracted with noradrenaline; the basic peptide, SFRGHITR, was inactive either as an agonist or as an antagonist to TRP42-55. In the LM bioassay, which unlike the RA preparation did not persistently desensitize in response to thrombin, all three receptor-related peptides, like thrombin, caused a prompt phasic reproducible contraction. The basic peptide, SFRGHITR, was inactive. In the LM assay, TRP42-55, TRP42-50 and A42-TRP42-55 all caused comparable contractile responses. We conclude that the gastric LM smooth muscle possesses a thrombin receptor and provides a convenient and reliable assay for the activities of thrombin receptor-related peptides. Our data also demonstrated that neither the C-terminal hirudin-related pentapeptide nor the N-terminal serine hydroxyl group are required for the biological activity of the thrombin receptor-derived peptide previously described (TRP42-55). Based on our findings we suggest that only a small portion of the N-terminal sequence of TRP42-55 may be required for thrombin-like biological activity.

Amino Acid Sequence↗
PubMed1979

Thrombin receptors of human platelets: thrombin binding and antithrombin properties of glycoprotein I.

Washed human platelets were solubilized and the proteins were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulphate. The gel was cut into slices and the effect of the eluted proteins on the clotting of fibrinogen by thrombin was evaluated. The isolate from only one gel slice strongly inhibited the clotting of fibrinogen. The prolongation of the clotting time was dependent on the concentration of the protein and reached a plateau around 5 microgram. Gel electrophoresis of this isolate showed a prominent glycoprotein with an apparent Mr=150 000. Gel filtration studies with [125I]thrombin showed that the protein isolate bound a significant amount of thrombin which could be displaced with unlabelled thrombin. Another preparation from the same gel or purified gamma-globulin did not bind thrombin or prolong the clotting time of fibrinogen. Glycoprotein I was isolated from human platelets by affinity chromatography on lectin-Sepharose columns. The isolated glycoprotein prolonged the clotting of fibrinogen and bound [125I]thrombin which could be displaced by unlabelled thrombin. It is proposed that the high affinity receptor of thrombin on human platelets is glycoprotein I. In addition, the antithrombin activity of intact platelets is due to binding of thrombin to this glycoprotein.

Antithrombins↗
PubMed1979

Routes of thrombin action in the production of proteolytically modified, secondary forms of antithrombin-thrombin complex.

The reaction between thrombin and antithrombin results in the formation of an inactive, stable, equimolar complex between the two proteins. However, under most reaction conditions several secondary complex forms, which have lower apparent molecular weights in dodecyl sulfate/polyacrylamide gel electrophoresis, appear concomitantly with or immediately following the production of the primary form of the complex. Purification of nascent, intact complex and treatment of this complex form with thrombin demonstrated that these subsidiary forms of antithrombin-thrombin complex may arise by proteolysis of the nascent complex by excess thrombin. Dissociation of such proteolytically modified complex preparations by hydroxylamine, and examination of the dissociation products by dodecyl sulfate/polyacrylamide gel electrophoresis suggested that degradation occurs primarily in the thrombin part of the complex, and only after prolonged proteolysis in its antithrombin moiety also. Incubation of antithrombin with several autolytically modified thrombin preparations showed that formation of subsidiary complex forms can also occur by an alternative route, i.e. between premodified thrombin forms and the inhibitor. In contrast, complex formation between thrombin and active forms of antithrombin, which have been modified by thrombin before complex formation, is unlikely, since no such active forms of antithrombin could be demonstrated.

Animals↗
PubMed1992

Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.

Amino Acid Sequence↗
PubMed1977

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.

Blood Coagulation↗
PubMed1978

The influence of thrombin on the clotting activity of factor VIII. A study with insolubilized thrombin.

An investigation of the influence of thrombin on the clotting activity of factor VIII was made. Purified factor VIII and different amounts of thrombin complexed to Sepharose 4 B were mixed and incubated for various periods of time. The factor VIII activities of these incubation mixtures were determined by the one- and two-stage analytical procedures in the presence of the thrombin-sepharose and in its absence following the latter removal from the test sample by filtration. The results so obtained confirm the view that thrombin inactivates factor VIII. Evidences for a thrombin-induced potentiation of the factor VIII activity, seen only in the thrombin-sepharose containing test samples analyzed by the one-stage method, are here interpreted as thrombin-effects peculiar to this factor VIII test system and not as potentiation by thrombin of the factor itself.

Blood Coagulation↗
PubMed1992

The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.

Animals↗
PubMed1992

Determination of r DNA hirudin and A-human thrombin- hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs. chromogenic thrombin substrate assay.

rDNA hirudin plasma concentrations in man and rhesus monkeys were determined over a period of 15 and 24 h. The plasma concentration of alpha-human thrombin-hirudin complex was measured after administration of the complex to rhesus monkeys. The complex was also determined after administration of hirudin to man and rhesus monkeys to study a possible formation of a complex with alpha-human thrombin in blood. The determination of hirudin was performed by a sandwich ELISA, using polyclonal and monoclonal antibodies and the chromogenic thrombin substrate assay. The alpha-human thrombin-hirudin complex concentration in the plasma of rhesus monkeys was measured over a period of 48 hours. The results of a sandwich ELISA were compared with those of the chromogenic thrombin substrate assay. A good agreement between the total hirudin concentrations analyzed by the hirudin ELISA and the alpha-human thrombin-hirudin complex ELISA and those of the chromogenic thrombin substrate assay, measuring total hirudin, too, was observed.

Animals↗
PubMed1992

Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.

Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptor's extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thrombin receptor is necessary for platelet activation by thrombin, and provides a strategy for developing blocking monoclonal antibodies of potential therapeutic value.

Amino Acid Sequence↗
PubMed1976

Comparison of the actions of thrombin and the thrombin-like venom enzymes ancrod and batroxobin.

Thrombin acts on several coagulant proteins to produce products with physiologic, pharmacologic and pathologic potential. The most sensitive thrombin substrate seems to be factor VIII. Some thrombin dependent reactions studied in vitro and proposed as control reactions seem too insensitive to the action of thrombin to be of in vivo significance. The only enzymic reaction the thrombin-like venom enzymes, Ancrod and Batroxobin, have in common with thrombin is the removal of fibrinopeptide A.

Ancrod↗