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PubMed1987

Proteolysis of factor Va by factor Xa and activated protein C.

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.

Amino Acid Sequence↗
PubMed2006

Residues Tyr253 and Glu255 in strand 3 of beta-sheet C of antithrombin are key determinants of an exosite made accessible by heparin activation to promote rapid inhibition of factors Xa and IXa.

We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 of beta-sheet C, which promote the reaction of the inhibitor with the target proteases, factor Xa and factor IXa. To determine which residues comprise the exosites, we mutated strand 3C residues that are conserved in all vertebrate antithrombins. Combined mutations of the three conserved surface-accessible residues, Tyr253,Glu255, and Lys257, or of just Tyr253 and Glu255, but not any of these residues alone, was sufficient to reproduce the exosite defects of a strand 3C antithrombin-alpha1-proteinase inhibitor chimera in reactions of the heparin-activated variants with both factor Xa and factor IXa. Importantly, the exosite-defective antithrombins bound heparin with nearly wild-type affinities, and the heparin-activated mutants showed near normal reactivities with thrombin, a protease that does not utilize the exosite. Mutation of the conserved but partially buried strand 3C residue, Gln254, the reactive loop P6' residue, Arg399, which interacts with Glu255, or a residue proposed to constitute the exosite from modeling studies, Glu237, all produced minimal effects on antithrombin reactivity with thrombin, factor Xa, and factor IXa in the absence or presence of heparin. Together, these results indicate that Tyr253 and Glu255 are key exosite determinants responsible for promoting the reactions of conformationally activated antithrombin with both factor Xa and factor IXa.

Amino Acid Sequence↗
PubMed2003

Localization of an antithrombin exosite that promotes rapid inhibition of factors Xa and IXa dependent on heparin activation of the serpin.

We have previously shown that exosites in antithrombin outside the P6-P3' reactive loop region become available upon heparin activation to promote rapid inhibition of the target proteases, factor Xa and factor IXa. To identify these exosites, we prepared six antithrombin-alpha 1-proteinase inhibitor chimeras in which antithrombin residues 224-286 and 310-322 that circumscribe a region surrounding the reactive loop on the inhibitor surface were replaced in 10-16-residue segments with the homologous segments of alpha1-proteinase inhibitor. All chimeras bound heparin with a high affinity similar to wild-type, underwent heparin-induced fluorescence changes indicative of normal conformational activation, and were able to form SDS-stable complexes with thrombin, factor Xa, and factor IXa and inhibit these proteases with stoichiometries minimally altered from those of wild-type antithrombin. With only one exception, conformational activation of the chimeras with a heparin pentasaccharide resulted in normal approximately 100-300-fold enhancements in reactivity with factor Xa and factor IXa. The exception was the chimera in which residues 246-258 were replaced, corresponding to strand 3 of beta-sheet C, which showed little or no enhancement of its reactivity with these proteases following pentasaccharide activation. By contrast, all chimeras including the strand 3C chimera showed essentially wild-type reactivities with thrombin after pentasaccharide activation as well as normal full-length heparin enhancements in reactivity with all proteases due to heparin bridging. These findings suggest that antithrombin exosites responsible for enhancing the rates of factor Xa and factor IXa inhibition in the conformationally activated inhibitor lie in strand 3 of beta-sheet C of the serpin.

Amino Acid Sequence↗
PubMed2006

Mature hepatocyte growth factor/scatter factor on the surface of human granulocytes is released by a mechanism involving activated factor Xa.

Serum hepatocyte growth factor (HGF) is rapidly increased in patients suffering from various tissue injuries including arterial occlusive diseases. However, the cellular sources of the HGF increase remain largely unknown. In the present study, we showed that bioactive mature HGF is constitutively present on the surface of granulocytes in human peripheral blood. Exogenously added 125I-labeled iodo-HGF efficiently bound to granulocyte surface, whereas only a scarce amount of HGF mRNA was detected in granulocytes, indicating that the mature HGF on granulocytes is likely to be derived from other cell types. Interestingly, treatment of granulocytes with human serum rapidly induced the release of the cell surface-associated HGF. In vivo, thromboplastin injection into mice increased HGF release from transplanted human granulocytes, which was inhibited by the pretreatment with DX9065a, a specific inhibitor of factor Xa. Furthermore, DX9065a also inhibited the serum-induced HGF release from human granulocytes in vitro, suggesting that the HGF-releasing factor(s) in serum is associated with factor Xa activation. Thus, human granulocytes may function as a transporter of HGF in the peripheral blood, releasing HGF at the injured sites caused by blood coagulation, where HGF may promote tissue repair.

Animals↗
PubMed2002

Differential mitogenic effects of single chain hepatocyte growth factor (HGF)/scatter factor and HGF/NK1 following cleavage by factor Xa.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.

Alternative Splicing↗
PubMed1989

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction.

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.

Antithrombin III↗
PubMed1997

Comparative effects of two direct and indirect factor Xa inhibitors on free and clot-bound prothrombinase.

Factor Xa, as with thrombin, binds to the clot and contributes to the propensity of thrombi to activate the coagulation system. The aim of this work was to compare the extent of prothrombinase inhibition produced by two factor Xa inhibitors: the antithrombin III-dependent synthetic pentasaccharide (SR 90107/Org 31540) and DX-9065A, a direct factor Xa inhibitor. When incubated together with prothrombin, factor Xa, phospholipids, antithrombin III and calcium, clots formed from human plasma exhibited a prothrombinase activity as measured through fragment 1-2 (F1+2) generation. Ten washes of the clot were required to achieve complete removal of unbound factor Xa. The absence of F1+2 generation brought about by washed clots in buffer when factor V was omitted, or in the presence of annexin V, indicated that they contained bound factor Xa and phospholipids but no factor V/Va. In all tested experimental conditions, clot-bound-factor Xa-induced F1+2 generation was inhibited by SR 90107/AT and DX-9065A with IC50 in the same range of concentrations (0.5 microM). In contrast, the inhibition of prothrombinase formed with factor Xa, factor Va phospholipids and calcium in buffer was observed at significantly lower concentrations of DX-9065A than of SR 90107/AT (respective IC50 concentrations: 0.1 and 70 microM). In vivo, fibrin accretion onto a preformed thrombus as well as venous thrombosis induced in the jugular vein of rabbits was inhibited by SR 90107 and DX-9065A in the same range of concentrations therefore showing that inhibition of clot-bound factor Xa is a predominant factor for the antithrombotic activity of both direct and indirect inhibitors for factor Xa.

Animals↗
PubMed1993

Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface.

Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti-factor V IgG and to a large extent (approximately 70%) was calcium-independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2-bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L-arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti-tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti-factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti-TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.

Amyloid beta-Protein Precursor↗
PubMed1978

Binding of bovine coagulation factor Xa to platelets.

The binding of highly purified bovine coagulation factor Xa to washed bovine platelets was studied. 125I-labeled factor Xa underwent binding to a platelet receptor that became accessible only after induction of the platelet release reaction by thrombin or by the calcium ionophore A 23187. The zymogen factor X did not bind to platelets. The factor Xa binding was saturable, reversible, and correlated with the rate of thrombin formation. The number of factor Xa binding sites per platelet was 290--420 and the apparent association constant was estimated to be 2.8 x 109 to 1.0 x 1010 M-1. Diisoprophyl fluorophosphate-factor Xa bound to the same platelet receptor as factor Xa indicating that limited proteolysis of a receptor protein was not required for binding. The rate of factor Xa binding was rapid (2.1 x 10(6) to 2.9 x 10(6) M-1 s-1) and similar to that preveiously found for the rate of binding of polypeptide hormones to their receptors. Displacement of factor Xa from the platelet receptor by diisopropyl fluorophosphate-factor Xa effectively blocked thrombin formation. Low concentrations of factor Xa catalyze prothrombin activation more effectively in the presence of platelets than in the presence of phospholipid and factor V.

Animals↗
PubMed1998

Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding.

The single-chain procofactor factor V is cleaved by thrombin (FVaIIa) at Arg709, Arg1018, and Arg1545 and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/HNE. The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa. Significant differences in cofactor function were observed between the two assay systems. Whereas FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus, cleavage at Arg1545 optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.

Arginine↗
PubMed1999

Crystallization and preliminary X-ray diffraction data of the complex of recombinant tick anticoagulant peptide (rTAP) and bovine factor Xa.

The complex of bovine factor Xa and recombinant tick anticoagulant peptide (rTAP) was crystallized in two different crystal forms using polyethylene glycol as a precipitant. Form I belongs to space group P42212 with unit-cell dimensions a = b = 133.1, c = 68.8 A. It contains one complex per asymmetric unit and diffracts to 3.0 A resolution. Form II belongs to P41212 (or P43212) with dimensions a = b = 126.5, c = 146.7 A; it contains two complexes per asymmetric unit and diffracts to 2.5 A. The crystals of both forms consist of factor Xa (MW = 45.3 kDa) and rTAP (MW = 6.7 kDa).

Animals↗
PubMed2002

Comparative study of the active site caging of serine proteases: thrombin and factor Xa.

Bovine thrombin and human factor Xa were acylated at their active site selectively with inhibitors derived from the parent compound 4-guanidinophenyl (E)-4-diethylamino-2-hydroxy-alpha-methylcinnamate hydrochloride, 1b. Peptidyl side chains were attached to the phenol ring via amide connection, which served as a recognition motif in inhibiting different serine proteases. Upon irradiation with 366 nm light, the trans-cinnamate attached to the active-site serine isomerizes to the cis isomer which then rapidly lactonizes to release the free enzyme. The peptidyl side chain sequences specific for each serine protease were revealed via constructing and screening a library of homologous compounds. This methodology may be applied to other proteases. One application based on enzyme-specific, photoactivatable inhibitors is to isolate a designated active protease from a mixture of several proteases. Thus, a cinnamate inhibitor with a biotin moiety, 1d, was synthesized. A solution of enzyme-specific, biotinylated inhibitor was added into a mixture of proteases containing a target enzyme. The target enzyme was acylated at the active site and subsequently bore a biotin tail. An avidin column was used to separate the biotinylated enzyme from the unmodified ones, by a strong binding between biotin and avidin. After a brief irradiation on the avidin column, the retained enzymes were released from the biotin tag and eluted off the column. To demonstrate the idea, thrombin and factor Xa have been separated from each other by this strategy.

Acylation↗
PubMed1997

Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1.

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.

Binding Sites↗
PubMedDate not supplied

Coagulant factor Xa inhibits prostacyclin formation in human endothelial cells. Role of factor V.

Thrombin stimulates prostacyclin formation in cultured human endothelial cells. However, a countervailing process that prevents prostacyclin overproduction has not been described previously. In this study, we demonstrate that Factor Xa can inhibit prostacyclin synthesis induced by thrombin or sodium arachidonate. The required concentration of Factor Xa represents activation of only 2% of the Factor X in plasma. The inhibition is reversed by a human monoclonal antibody directed against the light chain of Factor Va (Mr = 78,000), which suggests that Factor Va is required for this down-regulation of prostacyclin production. Confluent human endothelial cells (10(7)) contained 1.4 to 2.2 micrograms of Factor V antigen as measured by a competitive enzyme-linked immunosorbent assay. The results indicate that in endothelial cells Factor Xa may play a regulatory role in prostacyclin formation through interaction with Factor Va.

Arachidonic Acid↗
PubMed1993

Specificity of factor Xa in the cleavage of fusion proteins.

The precursor protein honey bee prepromelittin has been expressed as a fusion protein in Escherichia coli joined to the C-terminus of a truncated form of the bacteriophage gene 10 protein via an engineered recognition sequence for Factor Xa. Factor Xa was found to cleave poorly at the engineered site, giving a low yield of the required prepromelittin. In contrast, cleavage on the C-terminal side of the sequence VLGR at residue 67 in the gene 10 sequence proceeded in high yield. Factor Xa may be inhibited by adjacent hydrophobic sequences on the C-terminal side of a potential cleavage site.

Amino Acid Sequence↗
PubMed2006

[Tyrosine kinase dependent lectin-like oxidized LDL receptor-1 upregulation by thrombin and factor Xa in vascular smooth muscle cells].

OBJECTIVE: Thrombin and factor Xa are key players in the process of arterial thrombi formation and lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a cell surface endocytosis receptor for atherogenic oxidized LDL (ox-LDL). Here we investigate whether thrombin and factor Xa can induce LOX-1 protein expressions in cell-associated forms and soluble forms in cultured bovine aortic smooth muscle cells (BSMCs). METHODS: BSMCs were treated with thrombin or factor Xa in the presence or absence of AG1478, an epidermal growth factor (EGF) receptor-associated tyrosine kinase inhibitor. Total cell lysates and concentrated culture medium were then analyzed by Western blot using a mouse anti-LOX-1 monoclonal antibody. RESULTS: LOX-1 protein levels in cell lysates and culture medium were significantly increased by thrombin and factor Xa in a concentration- and time-dependent manner. Upregulation of LOX-1 protein expressions in cell lysates and concentrated culture medium was observed at concentrations above 2.0 and 3.0 U/ml of thrombin and 50 and 100 nmol/L of factor Xa, respectively. Increased LOX-1 protein expressions in cell lysates and cell culture medium were detectable as early as 4 h and peaked at 12 h after treatment with thrombin or factor Xa. LOX-1 expression induced by thrombin and factor Xa could be blocked by pretreatment with AG1478. CONCLUSIONS: Thrombin and factor Xa can act as LOX-1 inducers via tyrosine kinase activation.

Animals↗
PubMed1996

Factor VII central. A novel mutation in the catalytic domain that reduces tissue factor binding, impairs activation by factor Xa, and abolishes amidolytic and coagulant activity.

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.

Adult↗