To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. In this study, HSact precursor structures have been studied by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by 3'-phosphoadenosine 5'-phospho-35S and purified 3-O-sulfotransferase-1. This later in vitro labeling allows the regions of HS destined to become the antithrombin (AT)-binding sites to be tagged for subsequent structural studies. It was shown that six 3-O-sulfation sites exist per HSact precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HSact precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HSinact precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and HSinact precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HSact hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged HSact hexasaccharides exhibited the following structures, DeltaUA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS3(35)S+/-6S-++ +IdceA2S-[6-3H]Glc NS6S. The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the 3-O-sulfate moiety. The presence of five potential AT-binding precursor hexasaccharides in all HSact precursor chains demonstrates for the first time the processive assembly of specific sequence in HS. The difference in structures around potential 3-O-sulfate acceptor sites in HSact and HSinact precursors suggests that these precursors might be generated by different concerted assembly mechanisms in the same cell. This study permits us to understand better the nature of the HS biosynthetic pathway that leads to the generation of specific saccharide sequences. (+info)
Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II.
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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. (+info)
Thrombophilia as a multigenic disease.
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BACKGROUND AND OBJECTIVE: Venous thrombosis is a common disease annually affecting 1 in 1000 individuals. The multifactorial nature of the disease is illustrated by the frequent identification of one or more predisposing genetic and/or environmental risk factors in thrombosis patients. Most of the genetic defects known today affect the function of the natural anticoagulant pathways and in particular the protein C system. This presentation focuses on the importance of the genetic factors in the pathogenesis of inherited thrombophilia with particular emphasis on those defects which affect the protein C system. INFORMATION SOURCES: Published results in articles covered by the Medline database have been integrated with our original studies in the field of thrombophilia. STATE OF THE ART AND PERSPECTIVES: The risk of venous thrombosis is increased when the hemostatic balance between pro- and anti-coagulant forces is shifted in favor of coagulation. When this is caused by an inherited defect, the resulting hypercoagulable state is a lifelong risk factor for thrombosis. Resistance to activated protein C (APC resistance) is the most common inherited hypercoagulable state found to be associated with venous thrombosis. It is caused by a single point mutation in the factor V (FV) gene, which predicts the substitution of Arg506 with a Gln. Arg506 is one of three APC-cleavage sites and the mutation results in the loss of this APC-cleavage site. The mutation is only found in Caucasians but the prevalence of the mutant FV allele (FV:Q506) varies between countries. It is found to be highly prevalent (up to 15%) in Scandinavian populations, in areas with high incidence of thrombosis. FV:Q506 is associated with a 5-10-fold increased risk of thrombosis and is found in 20-60% of Caucasian patients with thrombosis. The second most common inherited risk factor for thrombosis is a point mutation (G20210A) in the 3' untranslated region of the prothrombin gene. This mutation is present in approximately 2% of healthy individuals and in 6-7% of thrombosis patients, suggesting it to be a mild risk factor of thrombosis. Other less common genetic risk factors for thrombosis are the deficiencies of natural anticoagulant proteins such as antithrombin, protein C or protein S. Such defects are present in less than 1% of healthy individuals and together they account for 5-10% of genetic defects found in patients with venous thrombosis. Owing to the high prevalence of inherited APC resistance (FV:Q506) and of the G20210A mutation in the prothrombin gene, combinations of genetic defects are relatively common in the general population. As each genetic defect is an independent risk factor for thrombosis, individuals with multiple defects have a highly increased risk of thrombosis. As a consequence, multiple defects are often found in patients with thrombosis. (+info)
Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets.
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We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization. (+info)
Antithrombin activity during the period of percutaneous coronary revascularization: relation to heparin use, thrombotic complications and restenosis.
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OBJECTIVES: This study evaluated changes in antithrombin (AT) activity around the time of percutaneous transluminal coronary revascularization (PTCR) with unfractionated heparin anticoagulation and the effects these changes had on major thrombotic complications of PTCR. BACKGROUND: Heparin is used during PTCR to prevent thrombosis. However, heparin, a cofactor for AT, causes AT activity to fall. AT activity <70% is associated with thrombosis. There is a prothrombotic state after heparin discontinuation that has not been well explained. METHODS: Antithrombin activity was sampled at the start and end of PTCR and the next two mornings in 250 consecutive patients. We recorded occurrence of major thrombotic events, defined as 1) major thrombotic complications of PTCR; 2) major in-lab thrombus formation; or 3) subacute occlusion. Discriminant analysis was employed to evaluate the relationship of AT activity to these events. Change in AT activity and its relationship to heparin was evaluated. Evidence of restenosis at six months was obtained. RESULTS: There were 14 major thrombotic events. Antithrombin activity <70% was strongly (p = 0.006) associated with these events. The AT activity fell significantly through the morning after PTCR when 21% of patients had AT activity <70%; AT activity did not normalize until >20 h after heparin discontinuation. Pre-PTCR use of heparin led to lower AT activity in proportion to duration of heparin use. There was no relationship between AT activity and restenosis. CONCLUSIONS: Low AT activity may contribute to major thrombotic complications of PTCR. The way heparin is used before and after PTCR is important to development of low AT activity. (+info)
Risk of venous thromboembolism and clinical manifestations in carriers of antithrombin, protein C, protein S deficiency, or activated protein C resistance: a multicenter collaborative family study.
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Deficiencies of antithrombin (AT), protein C (PC) or protein S (PS), and activated protein C resistance (APCR) are very well-established coagulation defects predisposing to venous thromboembolism (VTE). We performed a retrospective cohort family study to assess the risk for VTE in individuals with AT, PC, or PS deficiency, or APCR. Five hundred thirteen relatives from 9 Italian centers were selected from 233 families in which the proband had had at least 1 episode of VTE. We calculated the incidence of VTE in the whole cohort and in the subgroups after stratification by age, sex, and defect. The overall incidence of VTE (per 100 patient-years) in the group of relatives was 0.52. It was 1.07 for AT, 0.54 for PC, 0.50 for PS, 0.30 for APCR, and 0.67 in the group with a double defect. The incidence was associated with age, but not with sex. The mean age at onset was between 30 and 40 years for all the coagulation defects. Women had the peak of incidence in the age range of 21 to 40 years, earlier than men. The lifetime risk for VTE was 4.4 for AT versus APCR, 2.6 for AT versus PS, 2.2 for AT versus PC, 1.9 for PC versus APCR, and 1.6 for PS versus APCR. AT deficiency seems to have a higher risk for VTE than the other genetic defects. There is a relation between age and occurrence of thrombosis for both men and women. The latter had the peak of incidence earlier than the former. (+info)
Structure of heparin-derived tetrasaccharide complexed to the plasma protein antithrombin derived from NOEs, J-couplings and chemical shifts.
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A complex of the synthetic tetrasaccharide AGA*IM [GlcN, 6-SO3-alpha(1-4)-GlcA-beta(1-4)-GlcN,3, 6-SO3-alpha(1-4)-IdoA-alphaOMe] and the plasma protein antithrombin has been studied by NMR spectroscopy. 1H and 13C chemical shifts, three-bond proton-proton (3JH-H) and one-bond proton-carbon coupling constants (1JC-H) as well as transferred NOEs and rotating frame Overhauser effects (ROEs) were monitored as a function of the protein : ligand molar ratio and temperature. Considerable changes were observed at both 20 : 1 and 10 : 1 ratios (AGA*IM : antithrombin) in 1H as well as 13C chemical shifts. The largest changes in 1H chemical shifts, and the linewidths, were found for proton resonances (A1, A2, A6, A6', A1*, A2*, A3*, A4*) in GlcN, 6-SO3 and GlcN,3,6-SO3 units, indicating that both glucosamine residues are strongly involved in the binding process. The changes in the linewidths in the IdoA residue were considerably smaller than those in other residues, suggesting that the IdoA unit experienced different internal dynamics during the binding process. This observation was supported by measurements of 3JH-H and 1JC-H. The magnitude of the three-bond proton-proton couplings (3JH1-H2 = 2.51 Hz and 3JH4-H5 = 2.23 Hz) indicate that in the free state an equilibrium exists between 1C4 and 2S0 conformers in the ratio of approximately 75 : 25. The chair form appears the more favourable in the presence of antithrombin, as inferred from the magnitude of the coupling constants. In addition, two-dimensional NOESY and ROESY experiments in the free ligand, as well as transferred NOESY and ROESY spectra of the complex, were measured and interpreted using full relaxation and conformational exchange matrix analysis. The theoretical NOEs were computed using the geometry of the tetrasaccharide found in a Monte Carlo conformational search, and the three-dimensional structures of AGA*IM in both free and bound forms were derived. All monitored NMR variables, 1H and 13C chemical shifts, 1JC-H couplings and transferred NOEs, indicated that the changes in conformation at the glycosidic linkage GlcN, 6-SO3-alpha(1-4)-GlcA were induced by the presence of antithrombin and suggested that the receptor selected a conformer different from that in the free state. Such changes are compatible with the two-step model [Desai, U.R., Petitou, M., Bjork, I. & Olson, S. (1998) J. Biol. Chem. 273, 7478-7487] for the interaction of heparin-derived oligosaccharides with antithrombin, but with a minor extension: in the first step a low-affinity recognition complex between ligand and receptor is formed, accompanied by a conformational change in the tetrasaccharide, possibly creating a complementary three-dimensional structure to fit the protein-binding site. During the second step, as observed in a structurally similar pentasaccharide [Skinner, R., Abrahams, J.-P., Whisstock, J.C., Lesk, A.M., Carrell, R.W. & Wardell, M.R. (1997) J. Mol. Biol. 266, 601-609; Jin, L., Abrahams, J.-P., Skinner, R., Petitou, M., Pike, R. N. & Carrell, R.W. (1997) Proc. Natl Acad. Sci. USA 94, 14683-14688], conformational changes in the binding site of the protein result in a latent conformation. (+info)
Effect of thrombin inhibition in vascular dementia and silent cerebrovascular disease. An MR spectroscopy study.
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BACKGROUND AND PURPOSE: Silent cerebrovascular disease (CVD) has been proposed as a predisposing condition for clinically overt stroke and vascular dementia. Recently, we found increased thrombin generation in silent CVD patients. Here, we report the effect of thrombin inhibition using a potent selective thrombin inhibitor on the cerebral metabolism and function in peripheral arterial occlusive disease (PAOD) patients with or without silent CVD. METHODS: We examined 17 mild chronic PAOD patients, including 2 cases of vascular dementia. We divided the patients into 2 groups: 1 was the advanced CVD group with multiple lacunar infarction and/or advanced periventricular hyperintensity detected by brain MRI (n=12), and the other was the no CVD group that had none of these abnormalities (n=5). We assessed the cerebral biochemical compounds in the deep white matter area and cerebellar hemisphere (8 cm3) by proton MR spectroscopy before and after infusion of argatroban (10 mg/d IV) over 2 hours for 7 days. RESULTS: The ratio of N-acetylasparate (NAA) to total creatine (Cre) in the deep white matter area was significantly lower in the advanced CVD group than in the no CVD group, whereas there were no significant differences in this ratio in the cerebellar hemisphere between the 2 groups. In the former group, this decreased NAA/Cre ratio significantly increased after argatroban therapy, whereas there was no change in the latter group. The 2 patients with vascular dementia showed clinical improvement with marked increases in the NAA/Cre ratio and mini-mental score. CONCLUSIONS: These results suggest that increased thrombin generation may have some pathophysiological roles in developing vascular dementia and its chronic predisposing conditions. Thrombin inhibition may break this vicious cycle and lead to clinical improvement. (+info)