Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors.

The regulation of human Factor Xa was studied in vitro in human and mouse plasma, and in vivo in mouse. In human plasma, 125I-Factor Xa bound to alpha 1-proteinase inhibitor, antithrombin III, and alpha 2-macroglobulin in a ratio of 4.9:1.9:1 as determined by gel electrophoresis and by adsorption to IgG-(antiproteinase inhibitor)-Sepharose beads. The distribution of Factor Xa in mouse plasma was similar. The clearance of Factor Xa in mice was rapid (50% clearance in 3 min) and biphasic. alpha 1-Proteinase inhibitor-trypsin, even at a 2,000-fold molar excess, failed to inhibit the clearance of Factor Xa, while alpha 2-macroglobulin-trypsin inhibited only the later phase of clearance. The plasma clearance of diisopropylphosphoryl-Factor Xa was more rapid than native Factor Xa (50% clearance in 2.5 min), and the clearance was blocked by diisopropylphosphoryl-thrombin. Electrophoresis experiments confirmed that by 2 min after injection into the murine circulation, 90% of the bound Factor Xa was on alpha 2-macroglobulin, in marked contrast to the in vitro results. Organ distribution studies at 3 and 15 min with 125I-Factor Xa demonstrated that the majority of radioactivity was in the liver, with significant radioactivity also present in lung and kidney. Autopsies performed 30 s after injection of 125I-Factor Xa also demonstrated significant binding to the aorta and vena cava. These studies indicate that Factor Xa binds to specific thrombin-binding sites on endothelial cells, and that this binding alters its proteinase inhibitor specificity. Factor Xa binds to alpha 2-macroglobulin in vivo, whereas the predominant in vitro inhibitor of Factor Xa is alpha 1-proteinase inhibitor.     

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin

Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.     

Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator.

Vascular endothelium possesses multiple procoagulant properties, including synthesis and expression of Factor V. We studied the effects of homocysteine on the regulation of endothelial cell Factor V activity. Elevated levels of homocysteine are associated with the congenital thrombotic disorder homocystinuria. Treatment of cultured endothelial cells with 0.5-10 mM homocysteine had no effect on cell morphology, but did increase Factor V activity and prothrombin activation by Factor Xa. A radioimmunoassay for endothelial cell Factor V demonstrated that homocysteine treatment did not increase Factor V antigen levels. 125I-prothrombin was activated by treated endothelial cells and Factor Xa in the presence of thrombin inhibitors. Exogenous 125I-Factor V was cleaved by homocysteine-treated but not control endothelial cells. 125I-Factor V cleavage products distinct from those generated by thrombin and Factor Xa were identified. These data provide evidence for regulation of endothelial cell Factor V activity, and indicate that increased Factor V activity associated with homocysteine-treated vascular endothelium results primarily from induction of an activator of Factor V.     

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.     

Formation of factor Va by atherosclerotic rabbit aorta mediates factor Xa-catalyzed prothrombin activation.

Vascular cell procoagulant activity may be important in the pathogenesis of atherosclerosis. In previous studies, we described the ability of the atherogenic metabolite homocysteine to activate endothelial cell Factor V, a key coagulation cofactor for thrombin generation. The present study was designed to investigate Factor V activity and Factor Xa-catalyzed prothrombin activation by control and atherosclerotic aorta from normal and hypercholesterolemic rabbits. Factor Xa generated ninefold more thrombin on atherosclerotic aortic segments than on control segments. Atherosclerotic segments activated 125I-prothrombin with Factor Xa in the presence of the thrombin inhibitor dansyl arginine-4-ethylpiperidine amide and cleaved 125I-Factor V. This suggests that increases in vessel-wall Factor V activity and Factor Xa-catalyzed prothrombin activation result from activation of vessel-wall Factor V. 125I-Factor Va peptides generated by atherosclerotic aorta were very similar in molecular weight to those generated by homocysteine-treated cells. When vascular endothelium was mechanically removed by brushing, atherosclerotic vessels still generated four- to fivefold more thrombin than control vessels. These data and results from immunocytochemical studies suggest that Factor V in atherosclerotic vessels is associated with both endothelium and other cells of the lesion. In contrast, Factor V in control vessels is associated primarily with endothelium. The increases in Factor V activity and thrombin formation in the blood vessel wall of hypercholesterolemic rabbits may contribute to the development of atherosclerosis and its complications.     

Blood coagulation factor XIa binds specifically to a site on activated human platelets distinct from that for factor XI

Binding of 125I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 37 degrees C. Factor XIa binding was specific: 50- to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversible and was saturable at concentrations of added Factor XIa of 0.2-0.4 microgram/ml (1.25-2.5 microM). The number of Factor XIa binding sites on activated platelets was estimated to be 225 per platelet (range, 110-450). We conclude that specific, high affinity, saturable binding sites for Factor XIa are present on activated platelets, are distinct from those previously demonstrated for Factor XI, and require the presence of high molecular weight kininogen.     

Inactivation of factor XII active fragment in normal plasma. Predominant role of C-1-inhibitor

To define the factors responsible for the inactivation of the active fragment derived from Factor XII (Factor XIIf ) in plasma, we studied the inactivation kinetics of Factor XIIf in various purified and plasma mixtures. We also analyzed the formation of 125I-Factor XIIf -inhibitor complexes by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In purified systems, the bimolecular rate constants for the reactions of Factor XIIf with C-1-inhibitor, alpha 2-antiplasmin, and antithrombin III were 18.5, 0.91, and 0.32 X 10(4) M-1 min-1, respectively. Furthermore, SDS-PAGE analysis revealed that 1:1 stoichiometric complexes were formed between 125I-Factor XIIf and each of these three inhibitors. In contrast, kinetic and SDS-PAGE studies indicated that Factor XIIf did not react with alpha 1-antitrypsin or alpha 2-macroglobulin. The inactivation rate constant of Factor XIIf by prekallikrein-deficient plasma was 14.4 X 10(-2) min-1, a value that was essentially identical to the value predicted from the studies in purified systems (15.5 X 10(-2) min-1). This constant was reduced to 1.8 X 10(-2) min-1 when Factor XIIf was inactivated by prekallikrein-deficient plasma that had been immunodepleted (less than 5%) of C-1-inhibitor. In addition, after inactivation in normal plasma, 74% of the active 125I-Factor XIIf was found to form a complex with C-1-inhibitor, whereas 26% of the enzyme formed complexes with alpha 2-antiplasmin and antithrombin III. Furthermore, 42% of the labeled enzyme was still complexed with C-1-inhibitor when 125I-Factor XII was inactivated in hereditary angioedema plasma that contained 32% of functional C-1-inhibitor. This study quantitatively demonstrates the dominant role of C-1-inhibitor in the inactivation of Factor XIIf in the plasma milieu.     

Cellular processing of bovine factors X and Xa by cultured bovine aortic endothelial cells

Previous studies have shown that Factor X and Factor Xa bind specifically to distinct sites on the endothelial cell surface. Since the coagulant activity of a cell-bound clotting protein is dependent on its remaining on the cell surface, endocytosis and degradation studies have been carried out. Cell-bound Factor X was internalized at 0.07 fmol/min/10(6) cells, a rate slower than its dissociation from the cell surface. Endocytosed Factor X was not degraded, but was returned to the cell surface. In contrast, Factor Xa was internalized at an initial rate of 0.38 fmol/min/10(6) cells and subsequently degraded at about the same rate. The degradation of Factor Xa was prevented by chloroquine. These results suggest that Factor Xa is internalized and degraded by a lysosomal-dependent pathway. Studies with Factor X- and Xa-colloidal gold conjugates showed endocytosis proceeding at coated pit regions, and accumulation of Factor Xa-gold particles in lysosome-like structures. Endocytosis was studied as a clearance pathway for cell-bound Factor Xa by activating Factor X with Factors IXa and VIII on the endothelial cell surface. Endocytosis of the Factor Xa formed was significant, as only 44% of the Factor Xa formed was released into the supernatant, whereas the remainder was internalized and degraded. Thus, endocytosis of Factor Xa bound to its specific endothelial cell sites may be an important factor in the balance of vessel wall hemostatic mechanisms.     

Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model.

To assess the contribution of Factor IX/IXa, to intravascular thrombosis, a canine coronary thrombosis model was studied. Thrombus formation was initiated by applying current to a needle in the circumflex coronary artery. When 50% occlusion of the vessel developed, the current was stopped and animals received an intravenous bolus of either saline, bovine glutamyl-glycyl-arginyl-Factor IXa (IXai), a competitive inhibitor of Factor IXa assembly into the intrinsic Factor X activation complex, bovine Factor IX, or heparin. Animals receiving saline or Factor IX developed coronary occlusion due to a fibrin/platelet thrombus in 70 +/- 11 min. In contrast, infusion of IXai prevented thrombus formation completely (greater than 180 min) at doses of 460 and 300 micrograms/kg, and partially blocked thrombus formation at 150 micrograms/kg. IXai attenuated the accumulation of 125I-fibrinogen/fibrin at the site of the thrombus by approximately 67% (P less than 0.001) and resulted in approximately 26% decrease in serotonin release from platelets in coronary sinus (P less than 0.05). Hemostatic variables in animals receiving IXai, remained within normal limits. Animals given heparin in a concentration sufficient to prevent occlusive thrombosis had markedly increased bleeding, whereas heparin levels that maintained extravascular hemostasis did not prevent intracoronary thrombosis. This suggests that Factor IX/IXa can contribute to thrombus formation, and that inhibition of IXa participation in the clotting mechanism blocks intravascular thrombosis without impairing extravascular hemostasis.     

An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice.

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.     

Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium: POSSIBLE ROLE IN THE INACTIVATION OF THROMBIN BY ANTITHROMBIN III

The clearance of ¹²⁵I-thrombin and diisopropylphosphoryl-¹²⁵I-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was ~90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-¹²⁵I-thrombin resulted in the rapid reappearance of the DIP-¹²⁵I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-¹²⁵I-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to high-affinity binding sites, since dissociation constants (K_D) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively.     

Importance of factor Xa in determining the procoagulant activity of whole-blood clots.

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.     

Monoclonal immunoglobulin M lambda coagulation inhibitor with phospholipid specificity. Mechanism of a lupus anticoagulant.

Prolongation of all phospholipid-dependent coagulation tests was found in a patient with macroglobulinemia, despite absence of bleeding manifestations. The purified monoclonal IgM lambda protein and its Fabmu tryptic fragment induced similar changes in normal plasma. Patient IgM and Fabmu completely inhibited Ca++-dependent binding of radiolabeled prothrombin and Factor X to mixed phospholipid micelles. The patient's IgM lambda paraprotein reacted with phosphatidylserine and, to a lesser extent, with phosphatidylinositol and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine. Prior incubation of phospholipid with patient Fabmu blocked the positive reactions. Substitution of washed platelets for phospholipid led to normalization of patient coagulation tests and corrected all abnormalities produced in normal plasma by patient IgM. Furthermore, binding of 125I-Factor Xa to thrombin-treated platelets was entirely normal in the presence of patient IgM. These studies support the concept that platelets, rather than phospholipid micelles, are the primary locus of prothrombin and Factor X activation in normal hemostasis.     

Interaction of antithrombin III with bovine aortic segments. Role of heparin in binding and enhanced anticoagulant activity

Bovine antithrombin III (AT III) interaction with the luminal surface of bovine aortic segments with a continuous layer of endothelium was examined. Incubation of 125I-AT III with vessel segments, previously washed free of endogenous AT III, demonstrated specific, time-dependent binding to the protease inhibitor to the endothelium. Half-maximal binding was observed at an added AT III concentration of 14 nM. Binding of 125I-AT III to the vessel wall was reversible (50% dissociated in 4 min), and addition of either heparin or Factor Xa accelerated displacement of 125I-AT III from the vessel segment. Dissociation of 125I-AT III from the vessel segment in the presence of factor Xa coincided with the formation of a Factor Xa-125I-AT III complex. Inactivation of Factor IXa and Factor Xa by AT III was facilitated in the presence of vessel segments. Pretreatment of vessel segments with highly purified Flavobacterium heparinase precluded the vessel-dependent augmentation of AT III anticoagulant activity as well as specific binding of 125I-AT III to the vessel endothelium. In contrast, pretreatment of the vessel segments with chrondroitinases (ABC or AC) had no detectable effect on 125I-AT III binding or on AT III anticoagulant activity. AT III binding to vessel segments was competitively inhibited by increasing concentration of platelet factor 4. Binding of the protease inhibitor to vessel segments was inhibited by chemical modification of AT III lysyl or tryptophan residues. These AT III derivatives retained progressive inhibitory activity. These data suggest that heparin-like molecules are present on the aortic vessel wall and mediate binding of AT III to the vessel surface, as well as enhancing the anticoagulant activity of AT III at these sites.     

Inhibition by human thrombomodulin of factor Xa-mediated cleavage of prothrombin.

Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by Factor Xa in the presence of phospholipid or platelets, calcium, and Factor Va. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-prothrombin activation revealed that thrombomodulin reduced the rate of prothrombin activation but did not alter the cleavage pattern. The inhibition was reversed by the inclusion of a highly specific rabbit antithrombomodulin antibody. If thrombomodulin was replaced by hirudin, the rate of thrombin generation was not decreased excluding the possibility that the inhibition by thrombomodulin was secondary to the binding of small amounts of thrombin formed early in the reaction and the prevention of feedback breakdown of prothrombin by thrombin. The inhibitory activity of thrombomodulin was overcome by increasing the concentration of Factor Xa and specific, saturable binding of thrombomodulin to Factor Xa was demonstrated. These results indicate that thrombomodulin binds to Factor Xa and thereby inhibits the activity of the prothrombinase complex.     

Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX by a modification of a method described for bovine Factor IX activation by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707). Initial rates of trichloroacetic acid-soluble 3H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl2 (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3H preceded the appearance of Factor IXa activity, and the percentage of 3H released remained constant when the mole fraction of 3H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of 3H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include Km (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (kcat) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707) for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.     

Functions of AAV-CMV-F.IX And AAV-EF1alpha-F.IX in gene therapy for hemophilia B

There has been substantial progress in using gene therapy to treat animals with hemophilia. Adeno-associated viral (AAV) gene transfer of coagulation factor IX to skeletal muscle and liver of murine and canine models of hemophilia has resulted in sustained systemic expression and, in several studies, in complete cure of the bleeding disorder. Two AAV vectors widely used at present are AAV-CMV-F.IX and AAV-EF1alpha-F.IX. This work compares the predicted molecular functions of AAV-CMV-F.IX and AAV-EF1alpha -F.IX by sequence docking and gene ontology. It is shown that both AAV-CMV-F.IX and AAV-EF1alpha -F.IX induce coagulation factor IXa activity; however, AAV-CMV-F.IX administration also yields coagulation factor XIa activity and AAV-EF1alpha -F.IX treatment results in coagulation factor Xa activity. Therefore, AAV-CMV-F.IX might be useful for factor XI deficiency. AAV-CMV-F.IX has several additional molecular functions and processes compared with AAV-CMV-F.IX.     

Role of human factor VIII in factor X activation.

The cofactor function of human Factor VIII in Factor X activation was investigated by an initial-rate assay of 3H-Factor X activation in the presence of human factor IXa, Ca2+, and either phospholipid or fresh washed human platelets. Purified Factor VIII that has not been activated by thrombin or Factor Xa supports Factor X activation after a lag of several minutes. A specific inhibitor of Factor Xa, which had no inhibitory activity against Factor IXa, markedly prolonged this lag, whereas specific thrombin inhibitors did not prolong the lag. These data support the conclusion that unactivated Factor VIII has no ability to support Factor X activation in a purified system until it is activated by Factor Xa feedback during the lag period. When Factor VIII was optimally preactivated by thrombin, the lag was completely abolished, regardless of the order of addition of the other reactants or the phospholipid source. These data indicate that there is no slow, time-dependent ordering of the reactants at the phospholipid or activated platelet surface if Factor VIII has been preactivated. Unactivated platelets did not support Factor X activation by Factors IXa and VIII. The effect of activated Factor VIII on the kinetics of bovine Factor X activation was primarily to increase the Vmax (54-fold), whereas with human Factor X, Factor VIII both increased the Vmax 56-fold and decreased the Km sixfold to 0.14 microM, similar to the plasma concentration of Factor X. Therefore, a change in the plasma factor X concentration would be expected to have a major effect on the rate of Factor X activation in vivo.     

The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor.

The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints.     

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.