Inhibition of calcium oxalate crystal growth and aggregation by prothrombin and its fragments in vitro: relationship between protein structure and inhibitory activity.
(25/1307)
During blood coagulation, prothrombin (PT) is ultimately degraded to three fragments, thrombin, fragment 1 (F1) and fragment 2 (F2), which, collectively, contain all of the structural features of PT. One of these fragments, F1, is excreted in human urine and is the principal protein occluded into calcium oxalate (CaOx) crystals precipitated from it. This urinary form of F1, which we have named urinary prothrombin fragment 1 is present in calcium stones and is a potent inhibitor of CaOx crystallization in urine in vitro. The aim of this study was to determine whether PT itself and its other activation products, namely, thrombin, F1 and F2 also inhibit CaOx crystallization, by comparing their effects in a seeded, inorganic crystallization system. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. PT was isolated from a human blood concentrate rich in vitamin K-dependent proteins. Following initial cleavage by thrombin, the resulting fragments, F1 and F2, were purified by a combination of reversed phase HPLC and low pressure column chromatography. The purity of the proteins was confirmed by SDS/PAGE and their individual effects on CaOx crystallization were determined at the same concentration (16.13 nM) in a seeded, metastable solution of CaOx using a Coulter Counter. [14C]Oxalate was used to assess deposition of CaOx and crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that the proteins reduced the size of precipitated crystals in the order F1 > PT > F2 > thrombin. These findings were confirmed by scanning electron microscopy which showed that the reduction in particle size resulted from a decrease in the degree of crystal aggregation. [14C]Oxalate analysis demonstrated that all proteins inhibited mineral deposition, in the order F1 (44%) > PT (27.4%) > thrombin (10.2%) > F2 (6.5%). It was concluded that the gamma-carboxyglutamic acid domain of PT and F1, which is absent from thrombin and F2, is the region of the molecules which determines their potent inhibitory effects. The superior potency of F1, in comparison with PT, probably results from the molecule's greater charge to mass ratio. (+info)
Changes of hemostasis, endogenous fibrinolysis, platelet activation and endothelins after percutaneous transluminal coronary angioplasty in patients with stable angina.
(26/1307)
OBJECTIVES: This study investigated parameters of endogenous fibrinolysis, activation of coagulation and platelets, and endothelin levels before and after elective percutaneous transluminal coronary angioplasty (PTCA) in patients with stable coronary artery disease (CAD). BACKGROUND: Abrupt vessel closure is a serious short-term complication after PTCA and is often unforeseeable. Detailed insight into the effect of PTCA on hemostasis, platelets and the release of vasoconstrictive substances, which are among the mainly discussed mechanisms of abrupt vessel closure, is needed to enhance the safety of coronary intervention. METHODS: Plasma levels of markers of platelet activity, coagulation, endogenous fibrinolysis and endothelins were determined in 20 patients with stable CAD undergoing elective PTCA. The blood specimens were drawn before, immediately after, 1 h after intervention and on the next morning. RESULTS: All patients showed an initially uncomplicated PTCA. Regarding the efficacy of anticoagulation after receiving 15.000 IU heparin during PTCA, two groups were compared. In eight patients with ineffective anticoagulation production of thrombin and platelet activation directly after and 1 h after PTCA was significantly higher compared with 12 patients with effective anticoagulation. Despite the strong activation of coagulation, only a low fibrinolytic response could be observed. Endothelins rose significantly after PTCA in both groups but stayed longer on higher levels in patients with distinct thrombin generation. Three of the eight patients without sufficient heparin treatment suffered abrupt vessel closure. CONCLUSIONS: Initially uncomplicated dilation of coronary arteries leads to systemically measurable activation of coagulation and platelets in patients with ineffective doses of heparin and release of endothelins in all patients. Therefore, individual adjustment of anticoagulation and platelet inhibition in combination with effective antivasospastic substances are needed in every patient before, during and after initially uncomplicated PTCA to prevent this serious complication. (+info)
Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.
(27/1307)
We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin. (+info)
IgG reactivity to phospholipid-bound beta(2)-glycoprotein I is the main determinant of the fraction of lupus anticoagulant activity quenched by addition of hexagonal (II) phase phospholipid in patients with the clinical suspicion of antiphospholipid-antibody syndrome.
(28/1307)
BACKGROUND AND OBJECTIVE: Autoantibodies to beta(2)-glycoprotein I (beta(2)-GPI) and/or prothrombin (FII) have been involved in the expression of lupus anticoagulant (LA) activity, an in vitro phenomenon associated with an increased risk of arterial and/or venous thromboembolic events. However, LA activity sustained by anti-FII antibodies has a much weaker association with thrombosis than LA activity sustained by anti-beta(2)-GPI antibodies. Because assays aimed at detecting LA activity are now commercially available, we evaluated the relative sensitivity to anti-FII and anti-beta(2)-GPI antibodies of a commercial LA assay in a consecutive series of patients with the clinical suspicion of anti-phospholipid antibody (APA) syndrome. DESIGN AND METHODS: One hundred and ten consecutive patients with the clinical suspicion of APA syndrome (primary in 39) and 36 healthy controls were evaluated for the presence of LA activity (LA, Staclot, Stago), anticardiolipin antibodies (Quanta Lite aCL IgG, IgM, Inova Diagnostics), and IgG binding to solid-phase and/or phospholipid (PL)-bound beta(2)-GPI and FII by ELISA assays developed an optimized in our laboratory. Odds ratios for the association of IgG binding activity with LA and the aCL IgG status were calculated. In LA patients, dependency of LA potency (as assessed by clotting time prolongation in absence or presence of hexagonal phospholipid) on autoantibody titers was analyzed by the generalized linear model. Total IgG fractions were purified from selected patients to evaluate their ability to inhibit prothrombin activation at low FII concentration. RESULTS: Anticardiolipin antibodies (aCL) of the IgG or IgM type were found in 64 and 23 patients and LA activity in 49 patients. Anti-beta(2)-GPI and anti-FII (solid-phase and PL-bound) IgG titers exceeding by more than 3 standard deviations the mean values observed in control subjects were found in 46 and 47 patients and in 56 and 30 patients respectively, with the highest titers detected in the subgroup of patients with both LA and aCL IgG. The relative risk of LA for patients free of anti-FII and/or anti-beta(2)-GPI IgG was 0.03 after stratification for the aCL IgG status. Anti-beta(2)-GPI (solid-phase and PL-bound) IgG (RR 34.4 and 12.6) and anti-FII (solid-phase) IgG (RR 6.33) were all associated with LA activity. However, when taking into account co-existence of anti-FII and anti-beta(2)-GPI IgG in the same patients, the relative risk of LA for patients with isolated anti-FII IgG (solid-phase and/or PL-bound) was 0.50, whereas it ranged from 4.24 to 8.70 for all the antibody combinations including anti-beta(2)-GPI IgG. Anti-beta(2)-GPI (PL-bound) and aCL IgG titers were the only significant predictors of LA potency determined in absence phospholipid (anti-beta(2)-GPI IgG) or in presence of hexagonal phospholipid (aCL IgG). Total IgG fractions purified from 12 patients (6 with anti-FII IgG) did not significantly inhibit factor II activity up to a 150-fold molar excess. INTERPRETATION AND CONCLUSIONS: These results highlight the high prevalence of anti-FII and anti-beta(2)-GPI IgG in patients with the clinical suspicion of APA syndrome and particularly in the subgroup of patients with LA activity. The fraction of LA activity which can be quenched by addition of hexagonal phospholipid is, however, only dependent on IgG directed to PL-bound beta(2)-GPI. Other antibodies associated with anticardiolipin IgG may explain residual clotting time prolongation observed in the presence of hexagonal phospholipid. (+info)
The risk of recurrent deep venous thrombosis among heterozygous carriers of both factor V Leiden and the G20210A prothrombin mutation.
(29/1307)
BACKGROUND: Point mutations in the factor V gene (factor V Leiden) and the prothrombin gene (the substitution of A for G at position 20210) are the most common causes of inherited thrombophilia. Whether or not factor V Leiden increases the risk of recurrent deep venous thrombosis is controversial, and there is no information on the risk of recurrence among carriers of both mutations. METHODS: We studied a retrospective cohort of 624 patients who were referred for a first episode of deep venous thrombosis. After excluding 212 patients with other inherited or acquired causes of thrombophilia, we compared 112 patients who were heterozygous carriers of factor V Leiden with 17 patients who were heterozygous for both factor V Leiden and the prothrombin mutation and 283 patients who had neither mutation. The relative risk of recurrent deep venous thrombosis was calculated with use of a proportional-hazards model. RESULTS: Patients who were heterozygous for factor V Leiden alone had a risk of recurrent deep venous thrombosis that was similar to that among patients who had neither mutation (relative risk, 1.1; 95 percent confidence interval, 0.7 to 1.6; P=0.76). In contrast, patients who were heterozygous for both factor V Leiden and the prothrombin mutation had a higher risk of recurrent thrombosis than did carriers of factor V Leiden alone (relative risk, 2.6; 95 percent confidence interval, 1.3 to 5.1; P=0.002). When the analysis was restricted to patients with spontaneous recurrences (i.e., ones that occurred in the absence of transient risk factors for venous thrombosis), the risk among carriers of both mutations, as compared with carriers of factor V Leiden alone, remained high (relative risk, 3.7; 95 percent confidence interval, 1.7 to 7.7; P<0.001), particularly if the first event had also been spontaneous (relative risk, 5.4; 95 percent confidence interval, 2.0 to 14.1; P<0.001). In contrast, the risk of recurrence in the presence of transient risk factors was similar among carriers of both mutations and carriers of factor V Leiden alone. CONCLUSIONS: The risk of recurrent deep venous thrombosis is similar among carriers of factor V Leiden and patients without this mutation. Carriers of both factor V Leiden and the G20210A prothrombin mutation have an increased risk of recurrent deep venous thrombosis after a first episode and are candidates for lifelong anticoagulation. (+info)
"Normal" thrombin generation.
(30/1307)
We have investigated the influence of alterations in plasma coagulation factor levels between 50% and 150% of their mean values for prothrombin, factor X, factor XI, factor IX, factor VII, factor VIII, factor V, protein C, protein S, antithrombin III (AT-III), and tissue factor pathway inhibitor (TFPI) as well as combinations of extremes, eg, 50% anticoagulants and 150% procoagulants or 50% procoagulants and 150% anticoagulants in a synthetic "plasma" system. The reaction systems were constructed in vitro using purified, natural, and recombinant proteins and synthetic phospholipid vesicles or platelets with the reactions initiated by recombinant tissue factor (TF)-factor VIIa complex (5 pmol/L). To investigate the influence of the protein C system, soluble thrombomodulin (Tm) was also added to the reaction mixture. For the most extreme situations in which the essential plasma procoagulants (prothrombin, and factors X, IX, V, and VIII) and the stoichiometric anticoagulants (AT-III and TFPI) were collectively and inversely altered by 50%, a 28-fold difference in the total available thrombin generated was observed. Variations of most of these proteins 50% above and below the "normal" range, with the remainder at 100%, had only modest influences on the peak and total levels of thrombin generated. The dominant factors influencing thrombin generation were prothrombin and AT-III. When these 2 components were held at 100% and all other plasma procoagulants were reduced to 50%, there was a 60% reduction in the available thrombin generated. No increase in the thrombin generated was observed when the 150% level of all plasma procoagulants other than prothrombin was evaluated. When only prothrombin was raised to 150%, and all other factors were maintained at 100%, the thrombin generated increased by 71% to 121%. When AT-III was at 50% and all other constituents were at 100%, thrombin production was increased by 104% to 196%. The additions of protein C and protein S over the 50% to 150% ranges with Tm at 0.1 nmol/L concentration had limited influence on thrombin generation. Individual variations in factors VII, XI, and X concentrations had little effect on the duration of the initiation phase, the peak thrombin level achieved, or the available thrombin generated. Paradoxically, increases in factor IX concentration to 150% led to lowered thrombin generation, while decreases to 50% led to enhanced thrombin generation, most likely a consequence of factor IX as a competitive substrate with factor X for factor VIIa-TF. Reductions in factor V or factor VIII concentration led to prolongations of the initiation phase, while the reduction of TFPI to 50% led to shortening of this phase. However, none of these alterations led to significant changes in the available thrombin generated. Based on these data, one might surmise that increases in prothrombin and reductions in AT-III, within the normal range, would be potential risk factors for thrombosis and that algorithms that combine normal factor levels may be required to develop predictive tests for thrombosis. (+info)
Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.
(31/1307)
During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml. (+info)
Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.
(32/1307)
A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation. (+info)