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PubMed1993

Thrombin generates monocyte chemotactic activity from complement factor H.

We have recently found that the complement factor H (H) was the precursor of the major macrophage chemotactic factor in the delayed-type hypersensitivity (DTH) reaction site in the skin and was converted to the factor by an unidentified trypsin-like protease in plasma. Thrombin and plasmin are also present in the site, and we, therefore, examined the possibility that these proteases converted H to be monocyte chemotactic. Intact H caused no monocyte migration, although it was able to do so after incubation with thrombin, but not with plasmin. The activity was chemotactic rather than chemokinetic and was absorbed by an anti-H IgG-conjugated column. The generation of monocyte chemotactic activity from H was dependent on incubation time with thrombin and also the protease activity of thrombin, and the activity was seen at concentrations of H lower than 10(-8) M. The inhibitory activity of H for C3b-Bb was not affected by incubation with thrombin or plasmin. Incubation of H with thrombin, but not with plasmin, generated a hydrophobic molecule, in a time-dependent manner, which had monocyte chemotactic activity. These results show that H becomes a monocyte chemotactic factor due to cleavage by thrombin, which converts H to a more hydrophobic molecule and also suggest that thrombin-treated H induces monocyte migration in the DTH reaction site.

Chemotaxis, Leukocyte↗
PubMed1993

Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets.

Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phospholipase A2 to liberate arachidonic acid for further conversion to thromboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets contain significant amounts (60 ng/10(9) platelets) of the recently identified Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic labeling of human platelets with 33Pi revealed that the extent of phosphorylation of cPLA2 was greatly increased after thrombin treatment. Upon stimulation of platelets with thrombin, cPLA2 exhibits enhanced catalytic activity, as well as a change in its electrophoretic and chromatographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demonstrating that they are the consequence of thrombin-stimulated phosphorylation. Thrombin-induced phosphorylation and activation of cPLA2 is rapid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-dependent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thrombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization and further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets, we found that while activated cPLA2 exhibits the same Ca2+ requirement and apparent substrate affinity (Km), its catalytic activity (Vmax) is increased compared with control cPLA2. We conclude that 1) cPLA2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.

Amino Acid Sequence↗
PubMed1993

Comparison of thrombin active site and exosite inhibitors and heparin in experimental models of arterial and venous thrombosis and bleeding.

Different pharmacological approaches to thrombin inhibition were compared for their effects on thrombosis and bleeding time in anesthetized rats. Thrombosis was induced in the carotid artery by transmural vessel injury and in the vena cava by partial blood flow stasis combined with mild endothelial disruption. Small mesenteric arteries were punctured with a hypodermic needle to measure the bleeding time. Dose-response relationships were determined with a thrombin active site inhibitor, N-methyl (GYKI 14,766); a thrombin exosite inhibitor, succinyl-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-Gln (BMS 180,742); and heparin. BMS 180,742 interferes with fibrinogen binding to the thrombin exosite but, unlike GYKI 14,766, it does not block thrombin's catalytic site. The effects on thrombosis and bleeding time were correlated with ex vivo clotting times using the activated partial thromboplastin time for heparin and the thrombin time for GYKI 14,766 and BMS 180,742. Venous thrombosis was inhibited more than 90% by all three inhibitors at doses that either produced threshold increases or had no effect on bleeding and clotting times. Arterial thrombosis was inhibited 82% by GYKI 14,766 and 63% by heparin but it was not inhibited by BMS 180,742. These antithrombotic activities were accompanied by a maximal activated partial thromboplastin time increase and doubling of the bleeding time with heparin and a maximal thrombin time prolongation and 35% increase in bleeding time with GYKI 14,766. These results suggest that thrombin inhibitors, which act at the active site or exosite or through antithrombin III, are equally efficacious against venous thrombosis but active site inhibitors are the most effective against arterial thrombosis.

Amino Acid Sequence↗
PubMed1994

Identification of basic amino acid residues in thrombin essential for heparin-catalyzed inactivation by antithrombin III.

The therapeutically important anticoagulant heparin catalyzes inactivation of thrombin by antithrombin III via formation of an intermediary ternary thrombin-heparin-antithrombin III complex that is subsequently converted to a stable thrombin-antithrombin III complex with the release of heparin. Point mutations at Arg-180, Arg-245, Lys-248, and Lys-252 in thrombin markedly reduced the efficiency of heparin catalysis by decreasing the stability of the ternary intermediate, whereas the inactivation of thrombin by antithrombin alone was not affected by these mutations. These results together with an analysis of the x-ray crystal structure of thrombin yielded a model for the thrombin-heparin interaction, wherein heparin forms salt linkages along a groove in thrombin defined by Lys-252, Lys-248, Arg-245, Arg-89, Arg-98, and Arg-180.

Amino Acid Sequence↗
PubMed1993

[Study on the mechanisms of thrombin to inhibit neurite outgrowth].

In the present study, we investigated the effects and signal transduction mechanisms of thrombin on neuronal differentiation of human primitive neuroectodermal tumor cell line CHP-100. Thrombin inhibited neurite outgrowth of CHP-100 cells in culture induced by serum removal. The inhibitory effects were found to be associated with activation of phosphatidylinositol turnover/calcium mobilization signal transduction pathway by thrombin, since stimulation with thrombin induced significant inositol-1, 4, 5 trisphosphate accumulation and intracellular free calcium transient in CHP-100 cells. In addition, the most potent inhibitor of thrombin hirudin, which reversed the inhibitory effect of thrombin on neurite outgrowth of CHP-100 cells, also inhibited thrombin-stimulated calcium transient. These results suggest a specific role of thrombin signal transduction pathway in the regulation of neuronal differentiation.

Brain Neoplasms↗
PubMed1993

Protein-tyrosine kinase p72syk is activated by thrombin and is negatively regulated through Ca2+ mobilization in platelets.

Activation of platelets by thrombin results in a dramatic increase in tyrosine phosphorylation on multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3603-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). However, none of the responsible protein-tyrosine kinase has been reported so far. We report here that p72syk, one of the non-receptor-type protein-tyrosine kinases, is activated following thrombin stimulation in blood platelets. Washed porcine platelets were stimulated by thrombin, and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 5 s, reached a maximum at 10 s, and decreased to a basal level within 60 s after 0.5 unit/ml thrombin stimulation. The amount of immunoprecipitated p72syk was not altered throughout the time course. This activation was greatly enhanced in a dose-dependent manner and was completely canceled by the pretreatment of platelet suspension with hirudin, a specific antagonist of thrombin. In the Ca(2+)-depleted condition both extra- and intracellularly, the activation of p72syk was still persistent; in contrast, the deactivation process was completely abrogated even at 120 s after thrombin stimulation. In addition, the replenishment of Ca2+ resulted in a similar deactivation pattern as seen in the Ca(2+)-rich condition. Furthermore, this deactivation was also canceled by the pretreatment of platelets with W7, a calmodulin antagonist, as well as ML9, a myosin-light-chain kinase inhibitor. These results indicate that p72syk can be a responsible enzyme to the protein-tyrosine phosphorylation events following the platelet activation by thrombin and may be negatively regulated by Ca2+ in a calmodulin-dependent manner, inter alia myosin light-chain kinase, in thrombin-stimulated platelets.

Animals↗
PubMed1993

The effect of antithrombin III-independent thrombin inhibitors and heparin on fibrin accretion onto fibrin-coated polyethylene.

Prosthetic vascular grafts become coated with a layer of fibrin that contributes to graft thrombosis and occlusion. We compared the effect of antithrombin III-independent inhibitors of thrombin with heparin for their ability to prevent fibrin accretion onto a model of a vascular graft formed in vitro by coating polyethylene tubing with thrombin bound to a layer of polymerized fibrin. Equivalent antithrombin concentrations of heparin, D-Phe-Pro-Arg CH2Cl (PPACK), recombinant hirudin (r-hirudin), and Hirulog-1 were added to barium chloride-absorbed plasma containing radiolabelled fibrinogen. Whereas, PPACK and r-hirudin persistently inhibited fibrin accretion, the inhibition by heparin was transient. Hirulog-1 had no effect on early fibrin accretion and was actually associated with enhanced accretion at 30 min (control 11.7 +/- 2.0 micrograms fibrin/cm2; Hirulog-1, 18.4 +/- 3.5 micrograms fibrin/cm2, p < 0.001). Both Hirulog-1 and r-hirudin displaced radiolabelled thrombin from the fibrin surface. Whereas hirudin-thrombin complexes are stable, Hirulog-1 produces only transient inhibition of the displaced thrombin thereby accounting for the enhanced fibrin accretion with this anticoagulant. These studies show that the antithrombin III-independent inhibitors, r-hirudin and PPACK, are more effective inhibitors of fibrin accretion onto fibrin-coated polyethylene than heparin or Hirulog-1. In addition, they emphasize the importance of determining the ability of anticoagulants to displace thrombin from fibrin and to form stable thrombin-inhibitor complexes; lack of stability of thrombin-inhibitor complexes must be countered by levels of anticoagulant that are adequate to maintain its effectiveness.

Amino Acid Chloromethyl Ketones↗
PubMed1996

The effects of dermatan sulfate at submicrogram/ml concentrations on in vitro thrombin generation.

Dermatan sulfate is an antithrombotic glycosaminoglycan which has been shown to be effective in preventing deep venous thrombosis in general surgery patients when present at concentrations less than 1 microgram/ml. It has also been found to circulate physiologically in similar concentrations in pregnant women at term and in cord blood. We investigated the ability of dermatan sulfate added to plasma at 0.2, 0.5 and 1.0 microgram/ml to inhibit thrombin generation initiated by low concentrations of recombinant human tissue factor in defibrinated plasma. A dose dependent decrease in thrombin potential was demonstrated at therapeutically relevant concentrations of dermatan sulfate (0.5 and 1.0 microgram/ml) but there was no induction of a lag phase in thrombin generation. We were unable to demonstrate a significant effect on thrombin potential of dermatan sulfate at a concentration similar to that found in pregnancy plasma (0.2 microgram/ml). This indicates that either the dermatan sulfate concentration found in pregnancy plasma is not physiologically relevant or that our experimental system (which lacks platelets and fibrin) does not accurately reflect physiologic conditions. The effect on the thrombin potential was somewhat greater at the lowest concentration of tissue factor and amounted to a maximum inhibition of approximately 50% at 1 microgram/ml dermatan sulfate. A dose dependent increase in formation of thrombin-heparin cofactor II complexes and a decrease in thrombin-antithrombin complex formation with increasing dermatan sulfate concentration were observed at all dermatan sulfate concentrations. Prothrombin consumption was not changed by any dose of dermatan sulfate. We conclude that dermatan sulfate, at the concentrations tested, catalyses inhibition of free thrombin by heparin cofactor II but not efficiently enough to inhibit prothrombinase formation.

Dermatan Sulfate↗
PubMed1996

Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster.

BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes. MATERIALS AND METHODS: The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes. RESULTS: Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating these two genes is less than 100 kb. CONCLUSIONS: The fact that the thrombin receptor and PAR2 genes share an identical structure and are located within approximately 100 kb of each other in the genome demonstrates that these genes arose from a gene duplication event. These results define a new protease-activated receptor gene cluster in which new family members may be found.

Amino Acid Sequence↗
PubMed1997

A novel degradation pathway of tissue factor pathway inhibitor: incorporation into fibrin clot and degradation by thrombin.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from anti-thrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).

Animals↗
PubMed1998

Aging and venous thromboembolism influence the pharmacodynamics of the anti-factor Xa and anti-thrombin activities of a low molecular weight heparin (nadroparin).

Venous thromboembolism may be efficiently treated by one single daily administration of a high dose of low molecular weight heparin (LMWH). The present study investigates if the physiological deterioration of renal function associated with normal aging or the presence of an acute venous thromboembolism influences the pharmacodynamic pattern of the anti-factor Xa and anti-thrombin activities. Three groups of 12 subjects were investigated. The first 2 groups were composed of healthy volunteers differing by age (25 +/- 4 and 65 +/- 3 yrs) and creatinine clearance (114 +/- 15 and 62 +/- 6 ml x min(-1)). The third group was composed of patients hospitalized for deep vein thrombosis, having a mean age of 65 +/- 11 yrs and creatinine clearance of 76 +/- 8 ml x min(-1). Nadroparin was administered subcutaneously once daily at the dose of 180 anti-factor Xa IU.kg(-1) for 6 to 10 days. Serial sampling on day 1 and on the last day of administration (day n) allowed the pharmacodynamic parameters of the anti-factor Xa and anti-thrombin activities to be compared at the beginning and at the end of the treatment. The main findings were the following: (1) After repeated administration, a significant accumulation of the anti-factor Xa activity was observed in the healthy elderly and in the patients but not in the healthy young subjects (accumulation factor: 1.3). There was no evidence of accumulation of anti-thrombin activity; (2) There were significant correlations between the clearance of creatinine and the clearance of the anti-factor Xa activity but not with that of the anti-thrombin activity; (3) In the patients, the clearance of the anti-factor Xa and of the anti-thrombin activities were 1.4 and 2 times higher respectively than those calculated in the healthy elderly; (4) The mean ratio of the of anti-factor Xa and anti-thrombin clearances was close to 2 in the healthy subjects but equal to 5.4 in the patients. These results suggest that the mechanisms involved in the clearance of polysaccharide chains which support the anti-thrombin activity are different from those of the anti-factor Xa activity and that the enhanced binding properties of plasma proteins to unfractionated heparin reported in patients presenting an acute venous thromboembolism also exists for LMWH, predominantly for the anti-thrombin activity.

Adult↗
PubMed1998

Risk and clinical significance of developing antibodies induced by topical thrombin preparations.

OBJECTIVE: TO determine the clinical relevance, prevalence, and risk of antibody development in patients exposed to topical bovine thrombin preparations. DESIGN: A prevalence study of individuals previously exposed to topical bovine thrombin was done by screening using Western blot assay to detect antibodies against bovine thrombin preparations. SETTING: A large tertiary care center. PATIENTS: A convenience sample of 120 stored blood specimens from patients previously exposed to topical bovine thrombin was identified from hospital records. A control sample of 114 stored blood specimens from nonexposed patients was used. Case reviews for 2 exposed patients with severe bleeding complications and difficult clinical management are presented. RESULTS: Twelve of the bovine thrombin-exposed patients were found to have antibodies directed against bovine thrombin and other coagulation factors (95% CI, 4.6%-15.4%). Patients receiving multiple exposures were 8 times more likely to develop antibodies than were patients with a single exposure (P < .001). CONCLUSIONS: (1) Topical bovine thrombin is associated with formation of antibodies to coagulation factors; (2) Patients receiving multiple exposures are more likely to develop antibodies to coagulation factors; and (3) Topical bovine thrombin use may cause severe bleeding problems and should be avoided if there has been previous exposure.

Administration, Topical↗
PubMed1998

Antibody to thrombin receptor inhibits neointimal smooth muscle cell accumulation without causing inhibition of platelet aggregation or altering hemostatic parameters after angioplasty in rat.

An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH2-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (P<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (P<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.

Angioplasty↗
PubMed1995

Effect of Thrombin Inhibition on Patients with Peripheral Arterial Obstructive Disease: A Multicenter Clinical Trial of Argatroban.

Background: Enhanced thrombin generation has been found in patients with peripheral arterial obstructive disease (PAOD). The objective of this study is to investigate the effect of thrombin inhibition in PAOD patients. Methods: Argatroban (20 mg/day) was infused intravenously over 2 hours for 14-68 days in 27 patients with chronic PAOD of the lower extremities. Plasma thrombin-antithrombin III complex (TAT) levels and clinical signs were assessed. Results: TAT levels before argatroban therapy were significantly higher in the PAOD patients than in age- and gender-matched controls. In the PAOD patients, TAT levels increased stepwise in the presence of rest pain classified as Fontain III and IV. To assess the effect of thrombin inhibition, we divided the patients into a high-TAT group (pretreatment TAT level >/=5 ng/ml, n = 12) and a low-TAT group (pretreatment TAT level <5 ng/ml, n = 15). In the high-TAT group, TAT levels were suppressed in 8 of 12 patients after the administration of argatroban, along with improvement of their clinical symptoms and a decrease in the size of ischemic skin ulcers, indicating that argatroban clearly inhibited thrombin generation in vivo. Even in the low-TAT group, argatroban improved the clinical signs and symptoms, and also reduced the size of ischemic skin ulcers although TAT levels remained within the low range (<5 ng/ml) in 13 of the 15 patients, indicating that PAOD signs and/or symptoms may be related to small amounts of thrombin generated locally at the sites of atherothrombotic stenotic lesion. Conclusions: These results suggest that thrombin generation was enhanced in PAOD and that the amount was related to disease severity. Thrombin inhibition by argatroban may break this vicious cycle and lead to clinical improvement in PAOD.

Journal Article↗
PubMed1991

Effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE) on thrombin induced platelet adherence to endothelial cells in vitro.

The effect of exogenous 13-HODE on alpha-thrombin induced adherence of platelets to monolayers of cultured pulmonary artery endothelial cells was determined using homologous sheep cells. In a separate series of experiments, endogenous 13-HODE was demonstrated in sheep endothelial cells by reverse phase high pressure liquid chromatography. Levels of endogenous 13-HODE were decreased by alpha-thrombin preincubation. Exogenous 13-HODE (10 microM) reduced the augmented platelet adherence produced by coincubation of alpha-thrombin with platelets and endothelial monolayers, and eliminated the enhancement of platelet adherence produced by preincubation of alpha-thrombin with endothelial monolayers. 13-HODE also reduced the alpha-thrombin induced adherence of platelets to monolayers pretreated with aspirin and to fixed monolayers indicating a direct effect of 13-HODE as opposed to secondary effects mediated by the release of prostacyclin (PGI2) or endothelial derived relaxing factor (EDRF). Platelet adherence to subendothelial matrix was also reduced by 13-HODE. 13-HODE inhibited platelet aggregation initiated by 0.2 U/ml alpha-thrombin but did not affect aggregation initiated by 2.0 U/ml alpha-thrombin. These data provide evidence for the ability of exogenous 13-HODE to attenuate the interaction of thrombin activated platelets with endothelial cells as well as with other platelets.

Animals↗
PubMed1999

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).

Amino Acid Sequence↗
PubMed1981

Detection of a new heparin-dependent inhibitor of thrombin in human plasma.

We have demonstrated that human plasma contains a heparin-dependent inhibitor of thrombin that is distinguishable from antithrombin III (AT III). When a 1:50 dilution of plasma was incubated with greater than or equal to 0.01 U/ml heparin and 1 U/ml 125I-thrombin, the labeled thrombin B-chains became incorporated into two complexes of Mr-96,000 and Mr-85,000 that were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Neither complex was detectable at heparin concentrations less than 0.01 U/ml. When a limiting amount of 125I-thrombin was present, the proportion of radioactivity incorporated into each of the two complexes varied with the heparin concentration. Thus, the Mr-85,000 complex predominated at 0.01-5 U/ml heparin, whereas the Mr-96,000 complex predominated at 5-100 U/ml heparin. The Mr-85,000 complex reacted with antibodies to human AT III and comigrated with the purified thrombin-AT III complex. The Mr-96,000 complex did not react with antibodies to AT III or to alpha 1-antitrypsin, and it was detected in normal quantities after incubating 125I-thrombin with plasma immunodepleted of AT III, alpha 2-antiplasmin, alpha 2-macroglobulin, C1 inactivator, alpha 1-antichymotrypsin, or inter-alpha-trypsin inhibitor. The protein that combines with thrombin to form the Mr-96,000 complex was estimated to be present at a minimum concentration of 90 +/- 26 micrograms/ml (mean +/- SD) in identical to any of the known plasma protease inhibitors and that at relatively high heparin concentrations in vitro it reacts with thrombin more rapidly than does AT III.

Antithrombin III↗
PubMed1994

Prothrombin fragment 1 + 2, thrombin-antithrombin III-complexes and fibrinopeptide A in spontaneously clotting whole blood in vitro. Effects of heparin addition and antithrombin III deficiency.

Previous in vitro studies using spontaneously clotting whole blood revealed thrombin formation and high fibrinopeptide A (FPA) concentrations measured during incubation time. This occurred in spite of normal concentrations of thrombin antagonists present in the blood of the healthy subjects examined. However, there are several reports showing that in vivo increased thrombin-antithrombin III-complex (TAT) concentrations and relatively low FPA concentrations may occur e.g. in patients with (pre)thrombotic disorders. These in vivo findings indicate more effective thrombin inhibition by antithrombin III, with almost no fibrin formation. To find an explanation for the differences observed in vitro and in vivo, we extended the in vitro studies by measuring concentrations of prothrombin fragment 1 + 2 (F1 + 2), TAT and FPA at several time points until 30 min. Our goal was to test whether thrombin at least initially is neutralized by antithrombin III, resulting in a lack of fibrin formation, either in the absence or in the presence of heparin (0.2 and 0.5 U/ml whole blood, respectively). In the absence of heparin a simultaneous increase in the concentrations of F1 + 2, TAT and FPA was observed. Thrombin was only partially neutralized by antithrombin III and large amounts of fibrin were formed. The addition of heparin virtually suppressed thrombin formation since the F1 + 2 concentration remained low. Moreover, the small amounts of thrombin formed were neutralized by antithrombin III to a greater extent than in the absence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗