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W Kisiel

Publications and source records attributed to W Kisiel.

At least 55 records · Page 3Linked to original sources
PubMed1998

The inhibitory specificity of human proteinase inhibitor 8 is expanded through the use of multiple reactive site residues.

Serine proteinase inhibitors function as regulators of serine proteinase activity in a variety of physiological processes. Proteinase inhibitor 8 (PI8) is a 45 kDa member of the ovalbumin family of serpins that is an inhibitor of trypsin-like proteinases through the use of Arg339 as the inhibitory P1 amino acid residue in its reactive site loop. In this study, we have described the inhibitory mechanism of recombinant human PI8 towards chymotrypsin. PI8 formed an SDS-stable complex with and inhibited the amidolytic activity of chymotrypsin via a two-step mechanism with an overall equilibrium inhibition constant of 1.7 nM and an overall second-order association rate constant of 1.0 x 10(4) M-1s-1, utilizing Ser341 as the P1 residue. The use of separate reactive site loop residues by PI8 to inhibit distinctly different classes of proteinases not only supports the hypothesis of the existence of the serpin reactive site as a highly mobile and flexible loop, but also suggests an evolved function in which separate amino acid residues can be used to broaden the inhibitory specificity of PI8.

Animals↗
PubMed1998

Inhibition of soluble recombinant furin by human proteinase inhibitor 8.

Furin is a ubiquitous prototypical mammalian kexin/subtilisin-like endoproteinase that is involved in the proteolytic processing of a variety of proteins in the exocytic and endocytic pathways, with cleavage occurring at the C terminus of the minimal consensus furin recognition sequence Arg-Xaa-Xaa-Arg. In this study, human proteinase inhibitor 8 (PI8), a widely expressed 45-kDa ovalbumin-type serpin that contains two sequences homologous to the minimal sequence for recognition by furin in its reactive site loop, was tested for its ability to inhibit a recombinant soluble form of human furin. PI8 formed an SDS-stable complex with furin and inhibited its amidolytic activity via a two-step mechanism with a kappa assoc of 6.5 x 10(5) M-1 S-1 and an overall Ki of 53.8 pM. Thus, PI8 inhibits furin in a rapid, tight binding manner that is characteristic of physiological serpin-proteinase interactions. PI8 is not only the first human ovalbumin-type serpin to demonstrate inhibitory activity toward furin, but it is also the first significant inhibitor of furin identified that is not a serpin reactive site loop mutant, either naturally occurring or engineered.

Amino Acid Sequence↗
PubMed1998

Selective anticoagulation with active site-blocked factor IXA suggests separate roles for intrinsic and extrinsic coagulation pathways in cardiopulmonary bypass.

BACKGROUND: Multiple stimuli converge in cardiopulmonary bypass to create a tremendous prothrombotic stimulus. The ideal anticoagulant for cardiopulmonary bypass should selectively target only the intravascular stimuli, thereby eliminating pathologic clotting in the bypass circuit while preserving hemostasis in the thoracic cavity. We propose the inhibition of factor IX as such a targeted anticoagulant strategy. METHODS: We prepared an inhibitor of activated factor IX and applied it to a primate model of cardiopulmonary bypass to confirm the anticoagulant efficacy of activated factor IX in this setting and to assess more subtle markers of thrombin generation, macrophage procoagulant activity, and cellular tissue factor expression. Seven baboons that received activated factor IX (460 microg/kg) and 7 that received heparin (300 IU/kg) and protamine underwent cardiopulmonary bypass for 90 minutes and were followed after the operation for 3 hours. RESULTS: Analysis of plasma factor IX activity demonstrated adequate inhibition (<20%) of factor IX throughout cardiopulmonary bypass. Activated factor IX-treated baboons demonstrated similar circuit patency to heparin-treated baboons but had significantly diminished intraoperative blood loss. Preservation of extravascular hemostasis was further demonstrated in activated factor IX-treated animals by (1) significantly increased levels of thrombin-antithrombin III complex and prothrombin activation peptide (F1+2) without intravascular thrombosis, (2) significantly greater macrophage procoagulant activity in pericardial-derived monocytes, and (3) immunohistochemical evidence of tissue factor expression in pericardial mesothelial cells and macrophages. CONCLUSIONS: Anticoagulation with activated factor IX allows for intravascular anticoagulation with maintenance of extravascular hemostasis. These findings suggest activated factor IX as an agent that not only exemplifies a targeted approach to selective anticoagulation in cardiac surgery but also further characterizes the procoagulant milieu during cardiopulmonary bypass.

Animals↗
PubMed1998

Heparinless cardiopulmonary bypass with active-site blocked factor IXa: a preliminary study on the dog.

OBJECTIVE: Cardiopulmonary bypass is a potent stimulus for activation of procoagulant pathways. Heparin, the traditional antithrombotic agent, however, is often associated with increased perioperative blood loss because of its multiple sites of action in the coagulation cascade and its antiplatelet and profibrinolytic effects. Furthermore, heparin-mediated immunologic reactions (that is, heparin-induced thrombocytopenia) may contraindicate its use. Cardiopulmonary bypass with a selective factor IXa inhibitor was tested to see whether it could effectively limit bypass circuit/intravascular space thrombosis while decreasing extravascular bleeding, thereby providing an alternative anticoagulant strategy when heparin may not be safely administered. METHODS: Active site-blocked factor IXa, a competitive inhibitor of the assembly of factor IXa into the factor X activation complex, was prepared by modification of the enzyme's active site by the use of dansyl glutamic acid-glycine-arginine-chlormethylketone. Twenty mongrel dogs (five were given standard heparin/protamine; 15 were given activated site-blocked factor IXa doses ranging from 300 to 600 microg/kg) underwent 1 hour of hypothermic cardiopulmonary bypass, and blood loss was monitored for 3 hours after the procedure. RESULTS: Use of activated site-blocked factor IXa as an anticoagulant in cardiopulmonary bypass limited fibrin deposition within the extracorporeal circuit as assessed by scanning electron microscopy, comparable with the antithrombotic effect seen with heparin. In contrast to heparin, effective antithrombotic doses of activated site-blocked factor IXa significantly diminished blood loss in the thoracic cavity and in an abdominal incisional bleeding model. CONCLUSION: These initial studies on the dog suggest that administration of activated site-blocked factor IXa may be an effective alternative anticoagulant strategy in cardiopulmonary bypass when heparin is contraindicated, affording inhibition of intravascular/extracorporeal circuit thrombosis with enhanced hemostasis in the surgical wound.

Animals↗
PubMed1998

The effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa--tissue factor activity by antithrombin III.

We investigated the effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa-tissue factor activity by antithrombin III (ATIII) on a human bladder carcinoma (J82) cell line and an ovarian carcinoma (OC-2008) cell line, two tumor cell lines which constitutively synthesize and express high levels of cell surface tissue factor. We observed that ATIII inactivated factor VIIa-tissue factor more readily on OC-2008 cells than on J82 cells in the absence of added heparin. Likewise, factor Xa was more effectively inactivated on OC-2008 cells than on J82 cells. The ability of ATIII to inactivate factor VIIa-tissue factor activity on the OC-2008 cell was reduced following treatment of the cells with heparinase. This indicated that heparin-like GAGs were expressed on the OC-2008 cell surface, and that these GAGs were important for the inhibition of factor VIIa-tissue factor activity by ATIII. In addition, we demonstrated that the ability of ATIII to inactivate factor VIIa-tissue factor activity was markedly reduced following treatment of cells with calcium ionophore (A23187). However, the effect of cell surface GAGs on the inhibition of factor Xa by ATIII remained even after treatment of OC-2008 cells with A23187. In contrast to the manner of inhibition by ATIII/heparin, TFPI effectively inactivated factor VIIa-tissue factor activity on the cell surfaces even after induced physical damage or disruption of the cell by treatment with A23187. Our collective findings suggest that GAGs on cell surfaces play an important role in regulating factor VIIa-tissue factor activity by ATIII under normal conditions, or in the early phases of physical damage or destruction of the cell. However, TFPI may play a more important role than ATIII in regulating the activity of factor VIIa-tissue factor in a vascular trauma site following extensive cell injury.

Antithrombin III↗
PubMed1998

Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a serine protease inhibitor consisting of three tandemly-arranged Kunitz-type protease inhibitor domains. While TFPI-2 is a potent inhibitor of trypsin, plasmin, kallikrein, and factor XIa in the test tube, the function of this inhibitor in vivo remains unclear. In the present study, we investigated the synthesis and secretion of TFPI-2 by cultured endothelial cells derived from human umbilical vein, aorta, saphenous vein, and dermal microvessels to gain insight into its biological function. While all endothelial cells examined synthesized and secreted TFPI-2, dermal microvascular endothelial cells synthesized threefold to sevenfold higher levels of TFPI-2. Approximately 60% to 90% of the TFPI-2 secreted by endothelial cells was directed to the subendothelial extracellular matrix (ECM). When cultured human umbilical vein endothelial cells were stimulated with inflammatory mediators such as phorbol 12-myristate,13-acetate; endotoxin; and tumor necrosis factor-alpha, TFPI-2 synthesis by these cells increased twofold to 14-fold. Recombinant TFPI-2 bound to dermal microvascular endothelial cell monolayers and its ECM in a specific, dose-dependent, and saturable manner with Kd values of 21 and 24 nmol/L, respectively. TFPI-2 interacted with 4.5 X 10(10) sites/cm2 (3 X 10[5] sites/cell) and 2.3 X 10(11) sites/cm2 on endothelial cells and ECM, respectively. In the presence of rabbit anti-TFPI-2 IgG, but not preimmune IgG, endothelial cells dissociated from the culture flask in a time- and IgG concentration-dependent manner. Our findings provide evidence that endothelial cell-derived TFPI-2 is primarily secreted into the abluminal space and presumably plays an important role in maintaining the integrity of the ECM essential for cell attachment.

Cell Adhesion↗
PubMed1997

Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.

Journal Article↗
PubMed1997

Human proteinase inhibitor 9 (PI9) is a potent inhibitor of subtilisin A.

Serine proteinase inhibitors function as regulators of serine proteinase activity in a variety of physiological processes. Proteinase inhibitor 9 (PI9) is a 42 kDa member of the ovalbumin family of serpins that is expressed in placenta, lung, and cytotoxic lymphocytes. In this study, we have described the inhibitory mechanism of recombinant human PI9 towards the bacterial endoproteinase subtilisin A. PI9 inhibited the amidolytic activity of subtilisin A via a rapid, single step mechanism with an equilibrium inhibition constant of 3.6 pM and an overall second-order association rate constant of 2.4 x 10(6) M-1s-1, which is the strongest inhibitory mechanism of PI9 that has been described. The inhibitory action of PI9 towards subtilisin as a model proteinase may yield some indication of potential proteinases that may be regulated by PI9 in vivo.

Enzyme Inhibitors↗
PubMed1997

Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site.

Membrane-binding properties of human and bovine forms of vitamin K-dependent proteins Z, S, and C were characterized. Each of these proteins showed unique properties and interspecies differences that correlated with specific amino acid sequence variations in the amino-terminal 45 residues. Protein Z showed 100-fold slower membrane binding and dissociation kinetics relative to other vitamin K-dependent proteins that have been tested. This property seemed to correlate with an extra gamma-carboxyglutamic acid (Gla) residue at position 11 of protein Z. The interspecies difference for protein Z consisted of a higher packing density for the bovine protein on the membrane and a 9-fold slower dissociation rate. Higher affinity correlated with Asp at position 34 of bovine protein Z, where the human protein contains Asn. While both protein S species showed high affinity for the membrane, it was significantly greater for the human protein versus bovine protein S. Again, higher affinity correlated with an Asp (vs Asn) at position 34. Protein C was characterized by binding affinities that were 100-1000-fold lower than the other proteins. Low affinity appeared to be related to loss of Gla-32 (homologous to Gla-33 of protein Z). Interspecies differences of protein C appeared to be related to proline at position 10 (homologous to position 11 of protein Z) of bovine protein C, which produced at least 10-fold lower affinity than the human protein. Comparable substitutions at positions homologous to 11, 33, and 34 of protein Z may also underlie membrane binding behaviors of other vitamin K-dependent proteins. The three-dimensional structure of strontium-prothrombin fragment 1 [Seshadri et al. (1994) Biochemistry 33, 1087] shows that these positions are clustered on the protein surface near strontium-8, another possible candidate for membrane contact. A membrane contact mechanism consisting of an isolated protein-lipid ion pair is proposed. Comparison of naturally occurring vitamin K-dependent proteins has provided possible bases for divergent membrane binding and suggested future approaches to determine biological function.

Amino Acid Sequence↗
PubMed1997

Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation.

Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.

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↗
PubMed1996

Extracellular matrix-associated serine protease inhibitors (Mr 33,000, 31,000, and 27,000) are single-gene products with differential glycosylation: cDNA cloning of the 33-kDa inhibitor reveals its identity to tissue factor pathway inhibitor-2.

Recently, we reported the identification and partial characterization of three serine protease inhibitors (M(r) 33,000, 31,000, and 27,000) from the extracellular matrix (ECM) of human umbilical vein endothelial cells and skin cells. Here, we report that a full-length cDNA clone for the 33-kDa inhibitor from SV-40 transformed human skin fibroblasts (t12FB) is identical to a recombinant trypsin/tissue factor pathway inhibitor called TFPI-2 from placenta. By immunoblotting, the three inhibitors from ECM and cell lysates demonstrated cross-reactivity with an antiTFPI-2 IgG. To further elucidate how these inhibitors are related, pulse-chase labeling of t12FB with [35S]methionine followed by immunoprecipitation with antiTFPI-2 IgG was performed on ECM and cytosolic proteins. A precursor-product relationship did not exist between the three inhibitors from ECM. In contrast, the various species of inhibitors from cytosolic fractions demonstrated a precursor-product relationship. Within the cytosolic fraction, 26-, 29-, and 30-kDa inhibitors were detected in the early chases (0 and 15 min) but they form precursors to the synthesis of the 33-kDa inhibitor which accumulated in the later chases (30 min to 1 h). When pulse-chase experiments were performed in the presence of tunicamycin, synthesis as well as sequestration of the three inhibitors into ECM was completely inhibited. In the presence of tunicamycin, the cells synthesized and sequestered a single 25.5-kDa inhibitor into ECM. Peak quantities of the 25.5-kDa inhibitor appeared in the ECM after 6 h chase while they were 1 h for the 27- and 31-kDa inhibitors and 3 h for the 33-kDa inhibitor. To further support that the three inhibitors are related but only differ in the extent of glycosylation, the 33-kDa inhibitor from the t12FB ECM was deglycosylated with N-glycosidase F and the products were identified by immunoblotting with antiTFPI-2 IgG. The enzyme released the 31-, 27-, and 25.5-kDa inhibitors from the 33-kDa inhibitor. Collectively, these results demonstrate that the ECM-associated 33-, 31-, and 27-kDa inhibitors are biosynthetic products of a single gene with differential glycosylation. The 25.5-kDa inhibitor is unglycosylated, whereas 27- and 30- to 31-kDa inhibitors are partially glycosylated and the 33-kDa inhibitor is fully glycosylated. Inhibition of glycosylation significantly retarded the rate of secretion of the inhibitor but did not prevent its association with ECM. Quantitation of the inhibitors with cell-conditioned medium and ECM fractions reveals that 70-75% were ECM-associated and 25-30% cell-associated. None or very little of the inhibitors (0-2%) remained in a conditioned medium. Because they are primarily associated with ECM, the inhibitors may play a major role in ECM remodeling and turnover.

Base Sequence↗
PubMed1996

Immunohistochemical localization of tissue factor pathway inhibitor-1 (TFPI-1), a Kunitz proteinase inhibitor, in Alzheimer's disease.

Senile plaques in Alzheimer's disease (AD) are composed principally of A beta, a 4 kDa fragment of the amyloid precursor protein (APP). Longer forms of APP which contain a Kunitz proteinase inhibitor (KPI) domain are elevated in aged and in AD brains. Tissue factor pathway inhibitor-1 (TFPI) contains three tandem KPI domains and has been well characterized for its role as a natural anticoagulant in the extrinsic coagulation pathway. Functionally, the first two KPI domains of TFPI bind and inhibit the activity of factor Xa and VIIa respectively. In addition, TFPI and APP-KPI share a common clearance mechanism through the low density lipoprotein receptor-related protein (LRP). As part of an ongoing study of the role of KPI-containing proteins in AD, the current study examines TFPI localization in the brain. We report here that TFPI is immunohistochemically localized to microglia in both AD and non-AD individuals and is localized to some senile plaques in AD. Western blot analyses indicate that the amount of TFPI is elevated in frontal cortex samples from AD brains. We propose that TFPI may play a cell specific role in proteinase regulation in the brain.

Alzheimer Disease↗
PubMed1996

Sphingosine-containing phospholipid vesicles support human factor VII autoactivation in the absence of tissue factor.

We studied the effects of lipid membrane composition on factor VII autoactivation and observed that factor VII was activated in the presence of phospholipid vesicles containing sphingosine. The time course for the factor VII activation was sigmoidal and the duration of the initial lag phase was decreased by the addition of exogenous factor VIIA. Kinetic studies revealed that factor VII activation in the presence of sphingosine-containing phospholipids can be defined by a second-order reaction mechanism with an apparent second-order rate constant of 1.1x10(4) M(-1)s(-1). The sphingosine-mediated factor VII autoactivation rate was dependent on the concentration of calcium ions and sphingosine content of the vesicles. Neither bovine serum albumin-conjugated sphingosine nor sphingosine analogues (ceramide, sphingomyelin) affected the factor VII autoactivation rate.

Calcium↗
PubMed1996

Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor.

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.

Amino Acid Sequence↗
PubMed1996

The inhibition of human factor VIIa-tissue factor by antithrombin III-heparin is enhanced by factor X on a human bladder carcinoma cell line.

Previous studies have shown that antithrombin III-heparin effectively inhibited the factor VIIa-tissue factor complex. Herein, we show that the neutralization of factor VIIa in complex with the cell surface tissue factor by antithrombin III-heparin was markedly enhanced by plasma levels of factor X. Active site-mutated factor X (S376A factor X) and factor Xa previously inactivated with dansyl-Glu-Gly-Arg-chloromethyl ketone were as effective as plasma-derived factor X in this reaction, indicating that the active site serine residue of factor Xa was not involved in this mechanism. Furthermore, Gla-domainless factor X had no effect in this system, emphasizing the importance of the factor X Gladomain in this reaction. Antibody experiments revealed that this effect was not due to trace levels of a tissue factor pathway inhibitor contaminating either the factor X or antithrombin III preparations. The presence of heparin in this system was essential, as deletion of heparin resulted in a factor VIIa-tissue factor neutralization rate essentially identical to that observed for antithrombin III alone. Plasma levels of factor IX also accelerated the inhibition of factor VIIa-tissue factor by antithrombin III-heparin, although its effect was not as pronounced as that of factor X. Other vitamin K-dependent plasma proteins including protein S, protein C and prothrombin failed to augment the inhibition of factor VIIa-tissue factor by antithrombin III-heparin. Factor X did not enhance the neutralization rate of factor VIIa-tissue factor by antithrombin III-heparin when a carboxyl-terminal truncated tissue factor construct (TF1-219) was used, even in the presence of mixed phospholipids. Our collective finding suggest that antithrombin III and factor X bind to heparin at distinct sites on the heparin molecule resulting in a transient ternary complex of antithrombin III-heparin-factor X that represents the anticoagulant species. Factor X conceivably guides complex to a phosphatidylserine-rich site on the cell surface in close proximity to the factor VIIa-tissue factor complex and facilitates rapid neutralization of factor VIIa. Our findings also suggest that the effect of heparin on the regulation of the extrinsic pathway of blood coagulation may be more profound than previously recognized.

Antithrombin III↗
PubMed1996

The effect of heparin on the regulation of factor VIIa-tissue factor activity by tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits factor VIIa-tissue factor and factor Xa through its first and second Kunitz-type domains, respectively. Previous studies have shown that heparin binds to TFPI at two sites and enhances the inhibitory activity of TFPI towards factor Xa. We have studied the effect of heparin of the TFPI-mediated inhibition of factor VIIa-relipidated tissue factor amiodolytic activity and factor VIIa-tissue factor proteolytic activity towards factor IX and factor X on a human bladder carcinoma cell line, J82. The inhibitory activity of full-length TFPI on factor VIIa-tissue factor amidolytic and proteolytic activities was greatly enhanced by heparin, whereas the inhibitory activity of a truncated form of TFPI lacking the third Kunitz-type domain and carboxy-terminal tail (TFPI1-161) was not affected by heparin. Optimal inhibition of factor VIIa-tissue factor by TFPI was observed at 0.1 U/ml heparin. Heparin enhanced the inhibitory activity of TFPI towards factor Xa even in the presence of factor VIIa-tissue factor complexes in solution phase. In addition, heparin augmented the ability of TFPI to inhibit factor VIIa-tissue factor amidolytic activity in the presence of active-site mutated factor X (S376A factor X). Our collective results suggest that heparin may play a significant role in augmenting the physiological regulation of factor VIIa-tissue factor activity by TFPI.

Animals↗