Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TF mediated initiation of coagulation appears to play a critical role in normal hemostasis and probably pathologic thrombosis as well. Although teleological considerations would seem to suggest that a specific regulator of this process should exist, and although the presence in plasma of such an inhibitor was documented many years ago, it was not until the past five years that the inhibitor was characterized and its mechanism of action defined. LACI produces factor Xa-dependent feedback initiation of the VIIa/TF catalytic complex. The mechanism of this feedback inhibition is novel. First, LACI, a multi-headed protease inhibitor, binds factor Xa, a product of VIIa/TF catalysis, at one of its inhibitory domains. The Xa-LACI complex, possibly acting as a pseudosubstrate, then is able to bind to VIIa/TF in an appropriate conformation such that a second inhibitory domain of LACI is positioned to interact with factor VIIa in the VIIa/TF complex. Whether such a unique means of eliciting feedback inhibition in a protease cascade is repeated in nature is unknown. The existence of LACI appears to help explain the clinical need for both "extrinsic" and "intrinsic" coagulation pathways. In addition, data to the present are consistent with the notion that, in normal hemostasis at least, TF is responsible for an initial burst of factor Xa generation which provides sufficient thrombin to induce the aggregation of platelets and the activation of the critical coagulation cofactors factor V and factor VIII. Ultimate and persistent hemostasis, however, appears to require the continued production of additional factor Xa through the action of factor IXa and factor VIII. The fact that patients with factor XI deficiency suffers a variable but usually mild bleeding diathesis suggests that under certain conditions the initial burst of factor IXa formed through the action of VIIa/TF is insufficient and supplemental factor IXa generated by factor XIa is needed for normal hemostasis. The mechanism by which this factor XIa is generated in vivo, however, has not been determined. We stress that the predicted in vivo role of LACI is simply that--a prediction based on its known in vitro properties. Documentation of its physiologic importance remains to be provided and is an area of active research. Further, although significant progress has been made over the past few years in the characterization of LACI, many questions remain unanswered. For example: What is the mechanism for LACI's association with lipoproteins in plasma? What function, if any, does the third Kunitz-type protease inhibitor domain in LACI serve? (ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The lipoprotein-associated coagulation inhibitor. 200 33

The aim of this study was to investigate the mechanism involved in beta-thromboglobulin (BTG) release induced by platelet activating factor (PAF) in human platelet-rich plasma (PRP) and washed platelets (WP) during aggregation. PAF was used in PRP at increasing concentrations starting at its threshold concentration for irreversible aggregation (TAC: 90-150 nM). In citrated PRP, PAF induced release of BTG (80-95% of total content) and thromboxane B2 (TXB2) formation (30-40 pmol/ml). At low PAF concentrations aggregation and BTG release were blocked by apyrase (a scavenger of ADP), by ASA (an inhibitor of cyclooxygenase) and by BM 13177 (a thromboxane receptor antagonist). Higher concentrations of PAF overcame the effect of apyrase, but only induced reversible aggregation and minor release in the presence of ASA or BM 13177. In heparinized PRP, PAF induced full irreversible aggregation, but only very low BTG release (about 25% of total content) and thromboxane synthesis (2-3 pmol/ml). WP resuspended in the presence of 2 mmol/l Ca2+ seldom responded to PAF alone, as previously shown by others, but full aggregation could be induced by concomitant addition of subthreshold concentrations of PAF (25-50 nM) and epinephrine (1 microM). In these conditions average BTG release from WP was less than 20% of the amount releasable by thrombin. In contrast, when WP were resuspended in the absence of Ca2+, stimulation by PAF + EPI induced sustained BTG release (40-50% of total content) and TXB2 synthesis (15-20 pmol/ml). We conclude that at low Ca2+ concentration PAF induces BTG release mainly through thromboxane-endoperoxides formation. In contrast, when [Ca2+] is normal, PAF does not or weakly induces thromboxane formation and BTG release.
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PMID:At low extracellular calcium concentration platelet activating factor induces beta-thromboglobulin release from human platelets through thromboxane-endoperoxides formation. 296 Dec 18

Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.
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PMID:Recombinant E. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock. 760 Jun 36

Using a fast kinetic centrifugal analyzer, the inhibitory effects of glycosylated and unglycosylated full-length and truncated forms of TFPI on protease generation were studied in fibrinogen-deficient human plasma after extrinsic (EA) or intrinsic (IA) activation of coagulation. When the assay system was supplemented with increasing amounts of the TFPI variants the generation of both thrombin and factor Xa was inhibited in a concentration-dependent manner. Clear differences in the effectiveness of the TFPI variants were found. After EA, the unglycosylated full-length TFPI was most effective followed by the glycosylated full-length form. The C-terminal truncated TFPI showed the lowest inhibitory activity in this system. However, its efficiency increased several fold when coagulation was activated via the intrinsic pathway. Comparing the IC50 values after IA, the truncated TFPI was more effective than the unglycosylated full-length form and nearly as effective as the glycosylated full-length TFPI. After both EA and IA the thrombin generation inhibition by TFPI variants was more pronounced than the inhibition of factor Xa generation. The results show that chemical modifications of the TFPI structure can result in changes of TFPI's inhibitory properties to activated clotting factors leading to differences in protease generation inhibition.
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PMID:Inhibitory effects of TFPI variants on thrombin and factor Xa generation in fibrinogen-deficient human plasma. 790 Jan 3

The coagulation enzyme thrombin, a serine protease like all other coagulation factors, plays a central role in the hemostatic processes engaged after injurious events. It induces, with particular efficacy, the aggregation of blood platelets (primary hemostasis) and accounts, via splitting of fibrinogen to fibrin, for the event actually responsible for the coagulation of blood (secondary hemostasis). As is well-known, thrombin itself is generated by a cascade of activation events involving various coagulation factors (F). In this respect the "tissue factor" (TF, formerly known as thromboplastin), in combination with F VIIa, attains decisive significance, not only in the extrinsic pathway of coagulation (activation of F X-->Xa), but also in the intrinsic pathway (activation of F IX-->IXa). Under physiological circumstances, platelet aggregation and coagulation are restricted to the area of the vascular lesion, since the surrounding intact endothelium inhibits an intraluminal spreading of both processes. These "antithrombotic" features of the endothelium encompass antiaggregatory mechanisms (formation and release of prostacyclin [PGI2], adenosine, EDRF [NO], degradation of ADP and other nucleotides mediated by ecto-nucleotidases) as well as anti-coagulatory properties (formation and release of "tissue factor pathway inhibitor" [TFPI], which blocks the coagulation cascade by joining F Xa, TF and F VIIa into an inactive complex, thrombomodulin--thrombin induced activation of protein C, which, together with protein S, inactivates F Va and F VIIIa, thereby attenuating further generation of thrombin, and the heparan sulfate-enhanced activation of antithrombin III and heparin-cofactor II).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Interaction of blood and the vascular wall: hemostatic aspects]. 815 53

Tissue factor pathway inhibitor is a multivalent, Kunitz-type proteinase inhibitor. It directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex which is responsible for the initiation of coagulation. Human recombinant TFPI (rTFPI) produced in Escherichia coli was used to define the kinetic constants describing the human factor Xa:TFPI interaction. The inactivation of factor Xa by E. coli-rTFPI is indistinguishable from that of rTFPI produced in mammalian SK-hepatoma cells, suggesting that post-translational modifications such as glycosylation and phosphorylation do not play a major role in the inhibitory process. The slow, tight-binding inhibition of factor Xa follows the scheme: [formula: see text] Where the enzyme (E) and inhibitor (I) form an initial, immediate collision complex (EI) that then isomerizes slowly to a tightened final EI* complex. In the absence of other additions, the initial Ki (=k2/k1) and final Ki* for the inhibition of factor Xa by E. coli-rTFPI are 1.24 nM and 26.4 pM, respectively. In the presence of calcium ions (5 mM) the interaction between factor Xa and rTFPI is substantially weaker, with a Ki of 42.7 nM and Ki* of 85.2 pM. The addition of other components of the prothrombinase complex produces enhanced factor Xa inhibition predominantly through an effect on the initial Ki. In the presence of calcium ions and saturating concentrations of phospholipids and factor Va, the Ki and Ki* for factor Xa inactivation are 2.04 nM and 52.3 pM. The enhancing effect of heparin on the inhibitory process is concentration dependent and exhibits an optimum, reminiscent of the "template" model for heparin's acceleration of thrombin and factor IXa inhibition by antithrombin III. At optimal concentrations, the major mechanism of heparin action is also a reduction in the Ki of the initial encounter complex between factor Xa and rTFPI.
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PMID:Kinetics of factor Xa inhibition by tissue factor pathway inhibitor. 826 29

As discussed in the accompanying paper [Markland, W., Ley, A. C., & Ladner, R. C. (1996) Biochemistry 35, 8045-8057], we generated libraries from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) using multivalent M13 III display and derived potent inhibitors of human plasmin (PLA) by iterative variegation and selection. Here, we show that high-affinity, high-specificity binders to human plasma kallikrein (pKAL) and human thrombin (THBN) can be obtained starting from the identical library and employing the same iterative variegation procedures used to obtain PLA inhibitors. Lib#1 (allowing 31 200 variants involving five positions near the P1 residue of LACI-D1) and its pKAL-biased derivative, Lib#4 (allowing an additional 1600 variants at residues 31, 32, 34, and 39), were screened against pKAL, yielding potent inhibitors. One of these, EPI-K401, has Ki = 284 pM, very high specificity, and excellent stability. We used information from Lib#4 selectants to design Lib#5 (allowing 1.5 x 10(6) amino-acid sequences involving nine varied positions) from which we obtained an inhibitor (EPI-K503) having high affinity for pKAL (Ki = 40 pM) and retaining the high specificity of EPI-K401. When we screened Lib#1 and its THBN-tailored derivative, Lib#6, against THBN, we obtained a different and very homogeneous population of selected molecules. The purified proteins derived from Lib#6 selectants bound to THBN-agarose beads but did not inhibit proteolytic activity of THBN, suggesting that these selectants bind to a site on THBN other than the catalytic site. Thus, a single large combinatorial library can serve as a source to obtain highly specific, high-affinity binding molecules for each of several targets. Furthermore, the results with THBN show that the binding of Kunitz domains to other proteins is not limited to the catalytic sites of trypsin-homologous proteases.
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PMID:Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin. 867 10

The inhibition of thrombin generation (TG) was studied in plasma from human volunteers after single subcutaneous administrations of 4000, 8000 or 12,000 anti-Xa units (i.e., 6, 12 or 18 mg) of the synthetic pentasaccharide (SR 90107/ORG 31540) (SP). SP impaired TG in plasma for up to 18 h after injection, and the time-courses of TG and factor Xa inhibitions were similar. In untreated plasma supplemented in vitro with SP to obtain the same anti-Xa activity as in ex vivo samples, equivalent TG inhibitions were observed thus showing that no transformed SP molecules were involved in the TG inhibition ex vivo. Functional as well as immunological assay of TFPI indicated that subcutaneous injection of 12,000 anti-Xa units of SP did not induce any TFPI release, whereas under the same conditions, 13,000 IU of Fraxiparine produced a significant rise of TFPI in plasma. The plotting of TG inhibition versus SP concentration could be fitted with a good correlation (r = 0.94) to the graphical representation linking [ATIII-SP] to [SP]. These results demonstrate that following subcutaneous administration to man, SP inhibits TG ex vivo and likely in vivo exclusively through the same selective ATIII-mediated inhibition of factor Xa as the one elicited in vitro.
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PMID:The effect of the synthetic pentasaccharide SR 90107/ORG 31540 on thrombin generation ex vivo is uniquely due to ATIII-mediated neutralization of factor Xa. 877 23

The effects of the components of the protein C pathway on thrombin generation were studied in a reconstituted model in which thrombin is generated by factor VIIa and relipidated tissue factor (TF) via the activation of the purified coagulation factors X, IX, VIII, V, and prothrombin. The influence of protein C and soluble thrombomodulin on thrombin generation was correlated with factor Xa generation, factor V(a) and factor VIII(a) formation/inactivation, and protein C activation. Thrombin generation initiated by low concentrations of factor VIIa.TF (1.25 pM) occurs in an explosive fashion during a propagation phase which occurs after an initiation phase of approximately 1 min in which only traces of thrombin are formed. In the absence of other inhibitors, protein C (65 nM) in combination with high concentrations of soluble thrombomodulin (10 nM) resulted in a reduced rate of thrombin generation during the propagation phase without affecting the initiation phase; the activated protein C generated failed to neutralize prothrombinase activity and did not prevent prothrombin consumption. In the presence of plasma levels of the tissue factor pathway inhibitor (2. 5 nM recombinant TFPI), the protein C pathway reduced the rate of thrombin generation, initiated by 1.25 pM factor VIIa.TF, and completely eliminated prothrombinase activity at soluble thrombomodulin concentrations of >/=1 nM. The neutralization of prothrombinase activity coincided with cleavages at Arg-506 and subsequent cleavage at Arg-306 of the factor Va heavy chain by activated protein C. Thus, the protein C pathway combined with TFPI creates a minimal inhibitory potential required to shut down TF-initiated thrombin generation. The protein C pathway constituents did not influence factor Xa generation or factor VIIIa degradation over the interval in which prothrombinase activity was neutralized. Our data thus suggest that the protein C pathway regulates thrombin generation solely by the inactivation of factor Va. At low initiating factor VIIa.TF (1.25 pM) and high thrombomodulin concentrations (10 nM), the factor Va heavy chain is cleaved before significant amounts of light chain are generated. The ability of the protein C pathway to inhibit thrombin generation was greatly reduced when the reaction was initiated in the presence of factor Va, supporting the hypothesis that effective down-regulation of thrombin generation by the protein C pathway, in reactions initiated with the procofactor, occurs by prevention of the coexistence of the factor Va heavy and light chains.
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PMID:Inhibitory mechanism of the protein C pathway on tissue factor-induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor. 906 69

The effect of the Arg506 --> Gln mutation in factor VLEIDEN on thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pM factor VIIa.TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 microM thrombin. The factor VLEIDEN mutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nM TFPI, 65 nM protein C, and 10 nM recombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nM thrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDEN increased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nM, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDEN suggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI. Markedly increased thrombin generation in the presence of both 1.25 nM TFPI and factor VLEIDEN was also observed when antithrombin-III was added to the system to complete the natural set of coagulation inhibitors. Protein S (300 nM) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with either normal factor V or factor VLEIDEN. Protein S also failed to significantly potentiate the action of the protein C pathway in the presence of antithrombin-III in reactions employing normal factor V or factor VLEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in the reaction. Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis when combined with the Arg506 --> Gln mutation in factor VLEIDEN.
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PMID:Increased tissue factor-initiated prothrombin activation as a result of the Arg506 --> Gln mutation in factor VLEIDEN. 925 93


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