Evidence suggesting the regulation of a coagulation factor levels in rabbits by a transferable plasma agent.
New Zealand white rabbits were given 30 ml of goat serum intravenously. This procedure resulted in an immediate decrease in platelet count, fibrinogen, and levels of coagulation factors II, V, VII, and X, due to consumption coagulopathy. These factors returned toward baseline levels approximately 12 hr after the injection. Plasma from rabbits who had received goat serum 48 hr previously (donor rabbits) was injected into recipient rabbits. This procedure resulted in a slight rise in the level of coagulation factor II (range, 20%-30%) and a significant rise in factors V (35%-75%), VII (35%-235%), and X (35%-75%) in the recipients. When plasma from control donor rabbits who had not received goat serum was injected into recipients, there was no change in these coagulation factors. It is postulated that the reduction in coagulation factor levels in donor rabbits induces a "coagulopoietin" for each factor or one "coagulopoietin" for all factors which stimulates increased synthesis and/or release of these factors in recipient rabbits. (+info)
Plasmin converts factor X from coagulation zymogen to fibrinolysis cofactor.
Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were approximately 28 and approximately 47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to approximately Lys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX. (+info)
Coagulation factors II, V, VII, and X, prothrombin gene 20210G-->A transition, and factor V Leiden in coronary artery disease: high factor V clotting activity is an independent risk factor for myocardial infarction.
Increased levels of hemostatic factors and genetic mutations of proteins involved in coagulation may play a role in the pathogenesis of coronary artery disease. We investigated clotting activity of factors II (FII:C), V (FV:C), VII (FVII:C), and X (FX:C), the prothrombin gene 20210G-->A transition, and the factor V Leiden mutation in 200 survivors of myocardial infarction and in 100 healthy controls. FV:C (P<0.0001) and FVII:C (P<0.0001) were found to be independent risk factors for myocardial infarction. High FV:C or high FVII:C combined with smoking or arterial hypertension increased the relative risk for myocardial infarction up to 50-fold. One of 177 patients (0.6%) and 4 of 89 controls (4.5%) had the prothrombin 20210 AG genotype. Eleven of 177 patients (6.2%) and 6 of 89 controls (6.7%) were heterozygous for the factor V Leiden mutation. No homozygous carrier for these mutations was found. Neither the prothrombin gene 20210G-->A transition (odds ratio [OR], 0.1; 95% confidence interval [CI], 0.01 to 1.1) nor the factor V Leiden mutation (OR, 1.0; 95% CI, 0.4 to 2.8) were associated with an increased relative risk for myocardial infarction. In conclusion, our data indicate that neither the prothrombin gene 20210G-->A transition nor the factor V Leiden mutation are risk factors for myocardial infarction. High FVII:C was confirmed to be an independent risk factor for myocardial infarction. Moreover, we describe for the first time that high FV:C is an independent risk factor for myocardial infarction. (+info)
Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry.
Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Osterlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420; Contreras, J. A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic alpha/beta-hydrolase fold core. (+info)
The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity.
Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Recently, we showed that isolated A2 subunit enhanced the kcat for factor IXa-catalyzed activation of factor X by approximately 100-fold ( approximately 1 min-1), whereas isolated A1 or A3-C1-C2 subunits showed no effect on this rate (Fay, P. J., and Koshibu, K. J. (1998) J. Biol. Chem. 273, 19049-19054). However, A1 subunit increased the A2-dependent stimulation by approximately 10-fold. The Km for factor X in the presence of A2 subunit was unaffected by A1 subunit, whereas the kcat observed in the presence of saturating A1 and A2 subunits ( approximately 15 min-1) represented 5-10% of the value observed for native factor VIIIa (approximately 200 min-1). An anti-A1 subunit antibody that blocks the association of A2 eliminated the A1-dependent contribution to factor IXa activity. Inclusion of both A1 and A2 subunits resulted in greater increases in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa than that observed for A2 subunit alone and approached values obtained with factor VIIIa. These results indicate that A1 subunit alters the A2 subunit-dependent modulation of the active site of factor IXa to synergistically increase cofactor activity, yielding an overall increase in kcat of over 1000-fold compared with factor IXa alone. (+info)
Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.
Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary. (+info)
BACKGROUND: The mechanisms by which lipid lowering reduces the incidence of acute thrombotic complications of coronary atheroma in clinical trials remains unknown. Tissue factor (TF) overexpressed in atheroma may accelerate thrombus formation at the sites of plaque disruption. A cell surface cytokine CD40 ligand (CD40L) enhances TF expression in vitro. METHODS AND RESULTS: To test the hypothesis that lipid lowering reduces TF expression and activity, we produced atheroma in rabbit aortas by balloon injury and cholesterol feeding for 4 months (Baseline group, n=15), followed by either a chow diet (Low group, n=10) or a continued high-cholesterol diet for 16 months (High group, n=5). Immunolocalization of TF, CD40L, and its receptor CD40 was quantified by computer-assisted color image analysis. Macrophages in atheroma of the Baseline and High groups strongly expressed TF. Intimal smooth muscle cells and endothelial cells also contained immunoreactive TF. Regions of expression of CD40L and CD40 colocalized with TF. Protein expression of TF diminished substantially in the Low group in association with reduced expression of CD40L and CD40. In situ binding of TF to factors VIIa and X, detected by digoxigenin-labeled factors VIIa and X, colocalized with TF protein in atheroma and decreased after lipid lowering. We also determined reduced TF biological activity in the Low group by use of a chromogenic assay. The level of TF mRNA detected by reverse transcription-polymerase chain reaction also decreased after lipid lowering. CONCLUSIONS: These results suggest decreased expression and activity of TF as a novel mechanism of reduced incidence of thrombotic complications of atherosclerosis by lipid lowering. (+info)
"Normal" thrombin generation.
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)