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Biomedical subjects

G J Broze

Publications and source records attributed to G J Broze.

At least 19 recordsLinked to original sources
PubMed2001

Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis.

Lipoprotein (a) [Lp(a)] has been associated with both anti-fibrinolytic and atherogenic effects. However, no direct link currently exists between this atherogenic lipoprotein and intravascular coagulation. The current study examined the binding and functional effects of Lp(a), its lipoprotein constituents, apoliprotein (a) [apo(a)] and low-density lipoprotein (LDL), and lysine-plasminogen (L-PLG), which shares significant homology with apo(a), on tissue factor pathway inhibitor (TFPI), a major regulator of tissue factor-mediated coagulation. Results indicate that Lp(a), apo(a), and PLG but not LDL bound recombinant TFPI (rTFPI) in vitro and that apo(a) bound to a region spanning the last 37 amino acid residues of the c-terminus of TFPI. The apparent binding affinity for TFPI was much higher for Lp(a) (KD approximately 150 nM) compared to PLG (KD approximately 800 nM) and nanomolar concentrations of apo(a) (500 nM) inhibited PLG binding to TFPI. Lp(a) also inhibited in a concentration-dependent manner rTFPI activity and endothelial cell surface TFPI activity in vitro, whereas PLG had no such effect. Moreover physiologic concentrations of PLG (2 microM) had no effect on the concentration-dependent inhibition of TFPI activity induced by Lp(a). In human atherosclerotic plaque, apo(a) and TFPI immunostaining were shown to coexist in smooth muscle cell-rich areas of the intima. These data suggest a novel mechanism whereby Lp(a) through its apo(a) moiety may promote thrombosis by binding and inactivating TFPI.

Animals↗
PubMed2001

Deficiency of tissue factor pathway inhibitor promotes atherosclerosis and thrombosis in mice.

BACKGROUND: Tissue factor initiates blood coagulation after atherosclerotic plaque disruption. Tissue factor pathway inhibitor (TFPI) inhibits tissue factor activity and may reduce thrombus formation in this setting. We evaluated the effect of heterozygous TFPI deficiency on the development of atherosclerosis and thrombosis in atherosclerosis-prone mice. METHODS AND RESULTS: Mice with a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency (TFPI(+/-)/apoE(-/-)) were generated by crossbreeding, and they were analyzed for atherosclerosis throughout the vascular tree. Compared with mice with a normal TFPI genotype (TFPI(+/+)/apoE(-/-)), mice with a TFPI deficiency exhibited a greater atherosclerotic burden involving the carotid and common iliac arteries. Staining for active tissue factor within the plaque revealed more activity in TFPI(+/-)/apoE(-/-) mice compared with TFPI(+/+)/apoE(-/-) mice. Consistent with increased plaque tissue factor activity, the time to occlusive thrombosis after photochemical carotid plaque injury was significantly decreased in TFPI(+/-)/apoE(-/-) mice. CONCLUSIONS: These observations indicate that TFPI protects from atherosclerosis and is an important regulator of the thrombosis that occurs in the setting of atherosclerosis.

Animals↗
PubMed2001

Protein Z circulates in plasma in a complex with protein Z-dependent protease inhibitor.

Protein Z (PZ) is a vitamin K-dependent plasma protein that forms a Ca++-dependent complex with factor Xa at phospholipid surfaces. This interaction between PZ and factor Xa enhances by >1,000-fold the inhibition of factor Xa by the serpin called protein Z-dependent protease inhibitor (ZPI). These experiments show that PZ also binds ZPI in a process that does not require Ca++ or phospholipids. In pooled normal plasma, which contains excess ZPI relative to PZ, all the PZ appears to be bound in a complex with ZPI. The binding of PZ to ZPI reduces the rate and extent of factor XIa inhibition produced by ZPI. During the course of these studies, it was noted that a PZ purification procedure, that included NaSCN (2.0 M) elution of PZ from an immunoaffinity column, produced aggregated, inactive forms of PZ.

Blood Protein Electrophoresis↗
PubMed2001

Mouse protein Z-dependent protease inhibitor cDNA.

Protein Z-dependent protease inhibitor (ZPI) is plasma proteinase inhibitor in the serpin superfamily that produces rapid inhibition of factor Xa in the presence of phospholipids, Ca++ and protein Z (PZ). Mouse ZPI cDNA was isolated and cloned from mouse liver RNA using RT-PCR. The cDNA contains 100 nucleotides 5' of a translation initiation codon and an open reading frame of 1344 nucleotides followed by a 163 nucleotide 3' untranslated sequence with a poly (A) tail. The cDNA predicts a signal peptide containing 21 amino acids and a mature protein of 427 residues with 8 potential sites for N-linked glycosylation. The oligonucleotide and predicted amino acid sequences of mouse ZPI are 72% and 81% homologous with those of human ZPI. Like human ZPI, mouse ZPI contains tyrosine-serine (P1-P1') at its reactive center in contrast to the rat molecule which contains tyrosine-cysteine. By Northern analysis, mouse ZPI mRNA is 1.6 kb in size and, similar to both human and rat, it is detectable in liver, but not in heart, brain, spleen, lung, kidney, skeletal muscle or testes.

Animals↗
PubMed2001

Protein Z-dependent regulation of coagulation.

Protein Z (PZ) is a 62 kDa vitamin K-dependent plasma protein that serves as a cofactor for the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). ZPI is a recently identified 72 kDa member of the serpin superfamily of proteinase inhibitors that contains a tyrosine at its reactive center. PZ circulates in plasma in a complex with ZPI. Inhibition of factor Xa by ZPI in the presence of phospholipids and Ca++ is enhanced 1000-fold by PZ, but ZPI also inhibits factor XIa in a process that does not require PZ, phospholipids or Ca++. ZPI activity is consumed during coagulation through proteolysis mediated by factor Xa with PZ and factor Xla. Concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor VLeiden mice. Studies to determine the potential roles of PZ and ZPI deficiency in human thrombosis are in progress.

Animals↗
PubMed2000

Characterization of the protein Z-dependent protease inhibitor.

Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca(++) (t(1/2) less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa-ZPI complex is not stable to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but is detectable by alkaline-polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca(++). In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca(++). Heparin (0.2 U/mL) enhances the rate (t(1/2) = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P(1) residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.

Blood Coagulation↗
PubMed2000

Prothrombotic phenotype of protein Z deficiency.

Protein Z (PZ) is a vitamin K-dependent plasma protein whose function has been uncertain. The structure of PZ is very similar to that of the coagulation-related factors VII, IX, and X and PC, but PZ differs from these other proteins in that it is not the zymogen of a serine protease. We have shown recently that PZ forms a calcium ion-dependent complex with activated factor X at phospholipid surfaces and that this interaction leads to the inhibition of activated factor X activity through, in part, the action of a previously unidentified plasma protein named PZ-dependent protease inhibitor. Herein, we report that the presence of PZ dampens the coagulation response in human plasma and that concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor V(Leiden) mice. The results indicate that PZ plays a physiologically important role in the regulation of coagulation.

Animals↗
PubMed2000

Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo.

Tissue factor pathway inhibitor (TFPI) plays a key role in the regulation of tissue factor-initiated blood coagulation secondary to loss of the integrity of the blood vessel wall. TFPI is a naturally occurring Kunitz-type protease inhibitor that inhibits coagulation factor Xa and, in a factor Xa-dependent manner, mediates feedback inhibition of the factor VIIa/tissue factor catalytic complex. In vivo full-length TFPI is thought to be primarily bound to the vascular endothelium and the high affinity binding requires an intact carboxy terminus. Here we describe a full-length TFPI molecule, expressed in mouse C127 cells (TFPI(C127)), which exhibits virtually no cellular binding yet contains the intact carboxy terminus. This TFPI (TFPI(C127)) is neither internalized nor degraded via the TFPI endocytic receptor, LDL-receptor-related protein. Pharmacokinetic studies of TFPI(C127 )in vivo demonstrate a 10-fold prolongation in the plasma half-life, compared with that of bacterial recombinant TFPI. (Blood. 2000;95:1973-1978)

Animals↗
PubMed1999

The protein Z-dependent protease inhibitor is a serpin.

In the presence of phospholipid vesicles and calcium ions, protein Z (PZ) serves as a cofactor for the inhibition of coagulation factor Xa by a plasma protein called PZ-dependent protease inhibitor (ZPI). To further characterize ZPI, its cDNA has been isolated and cloned from a human liver cDNA library. The ZPI cDNA is 2.44 kb in length and has a relatively long 5' region (466 nt) that contains six potential ATG translation start codons. ATG's 1-4 are followed by short open reading frames, whereas ATG(5) and ATG(6) are in an uninterrupted open reading frame that includes the encoded ZPI protein. In vitro experiments show that ATG(6) is sufficient for the expression of rZPI in cultured Chinese hamster ovary cells. Northern analysis suggests the liver is a major site of ZPI synthesis. The predicted 423 residue amino acid sequence of the mature ZPI protein is 25-35% homologous with members of the serpin superfamily of protease inhibitors and is 78% identical to the amino acid sequence predicted by a previously described cDNA isolated from rat liver, regeneration-associated serpin protein-1 (rasp-1). Thus, ZPI is likely the human homologue of rat rasp-1. Alignment of the amino acid sequence of ZPI with those of other serpins predicts that Y387 is the P(1) residue at the reactive center of the ZPI molecule. Consistent with this notion, rZPI(Y387A), an altered form of ZPI in which tyrosine 387 has been changed to alanine, lacks PZ-dependent factor Xa inhibitory activity.

Adult↗
PubMed1999

Structural requirements for TFPI-mediated inhibition of neointimal thickening after balloon injury in the rat.

The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK(1), a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI(WT), the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI(K36I), an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI(13-161), a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK(1), TFPI(WT), and high levels of TFPI(K36I) begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI(13-161) was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK(1) approximately TFPI(WT)>TFPI(K36I)>>TFPI(13-161). The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action.

Angioplasty, Balloon↗
PubMed1999

Factor VII deficiency rescues the intrauterine lethality in mice associated with a tissue factor pathway inhibitor deficit.

Mice doubly heterozygous for a modified tissue factor pathway inhibitor (TFPI) allele (tfpi delta) lacking its Kunitz-type domain-1 (TFPI+/delta) and for a deficiency of the factor VII gene (FVII+/-) were mated to generate 309 postnatal and 205 embryonic day 17.5 (E17. 5) offspring having all the predicted genotypic combinations. Progeny singly homozygous for the tfpidelta modification but with the wild-type fVII allele (FVII+/+/TFPIdelta/delta), and mice singly homozygous for the fVII deficiency and possessing the wild-type tfpi allele (FVII-/-/TFPI+/+), displayed previously detailed phenotypes (i.e., a high percentage of early embryonic lethality at E9.5 or normal development with severe perinatal bleeding, respectively). Surprisingly, mice of the combined FVII-/-/TFPIdelta/delta genotype were born at the expected mendelian frequency but suffered the fatal perinatal bleeding associated with the FVII-/- genotype. Mice carrying the FVII+/-/TFPIdelta/delta genotype were also rescued from the lethality associated with the FVII+/+/TFPIdelta/delta genotype but succumbed to perinatal consumptive coagulopathy. Thus, the rescue of TFPIdelta/delta embryos, either by an accompanying homozygous or heterozygous FVII deficiency, suggests that diminishment of FVII activity precludes the need for TFPI-mediated inhibition of the FVIIa/tissue factor coagulation pathway during embryogenesis. Furthermore, the phenotypes of these combined deficiency states suggest that embryonic FVII is produced in mice as early as E9.5 and that any level of maternal FVII in early-stage embryos is insufficient to cause a coagulopathy in TFPIdelta/delta mice.

Animals↗
PubMed1998

Isolation of a protein Z-dependent plasma protease inhibitor.

Human protein Z (PZ) is a 62,000-Mr, vitamin K-dependent plasma protein whose structure is similar to coagulation factors VII, IX, X, protein C, and protein S, but whose function is not known. The procoagulant activity of factor Xa in a one-stage plasma coagulation assay is reduced when factor Xa is first incubated with PZ. This apparent inhibitory effect is time dependent, requires the presence of calcium ions and procoagulant phospholipids (rabbit brain cephalin), and appears predominantly related to the incubation period of PZ with cephalin. In serum the initial rate of inhibition of factor Xa with calcium ions and cephalin also is enhanced in the presence PZ. A PZ-dependent protease inhibitor (ZPI) has been isolated from plasma. ZPI is a 72,000-Mr single-chain protein with an N-terminal amino acid sequence of LAPSPQSPEXXA (X = indeterminate) and an estimated concentration in citrate-treated plasma of 1.0-1.6 microg/ml. In systems using purified components, the factor Xa inhibition produced by ZPI is rapid (>95% within 1 min by coagulation assay) and requires the presence of PZ, calcium ions, and cephalin. The inhibitory process appears to involve the formation of a factor Xa-PZ-ZPI complex at the phospholipid surface.

Amino Acid Sequence↗
PubMed1998

Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor.

Tissue factor (TF) pathway inhibitor (TFPI) regulates factor X activation through the sequential inhibition of factor Xa and the VIIa.TF complex. Factor Xa formation was studied in a purified, reconstituted system, at plasma concentrations of factor X and TFPI, saturating concentrations of factor VIIa, and increasing concentrations of TF reconstituted into phosphatidylcholine:phosphatidylserine membranes (TF/PCPS) or PC membranes (TF/PC). The initial rate of factor Xa formation was equivalent in the presence or absence of 2.4 nM TFPI. However, reaction extent was small (<20%) relative to that observed in the absence of TFPI, implying the rapid inhibition of VIIa.TF during factor X activation. Initiation of factor Xa formation using increasing concentrations of TF/PCPS or TF/PC in the presence of TFPI yielded families of progress curves where both initial rate and reaction extent were linearly proportional to the concentration of VIIa.TF. These observations were consistent with a kinetic model in which the rate-limiting step represents the initial inhibition of newly formed factor Xa. Numerical analyses of progress curves yielded a rate constant for inhibition of VIIa.TF by Xa.TFPI (>10(8) M-1.s-1) that was substantially greater than the value (7.34 +/- 0.8 x 10(6) M-1.s-1) directly measured. Thus, VIIa.TF is inhibited at near diffusion-limited rates by Xa.TFPI formed during catalysis which cannot be explained by studies of the isolated reaction. We propose that the predominant inhibitory pathway during factor X activation may involve the initial inhibition of factor Xa either bound to or in the near vicinity of VIIa.TF on the membrane surface. As a result, VIIa.TF inhibition is unexpectedly rapid, and the concentration of active factor Xa that escapes regulation is linearly dependent on the availability of TF.

Animals↗
PubMed1998

Tissue factor pathway inhibitor gene disruption.

Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type plasma proteinase inhibitor that regulates the initiation of coagulation by producing activated factor X (FXa)-dependent feedback inhibition of the catalytic complex of FVIIa and tissue factor (TF). The second Kunitz domain of TFPI binds and inhibits FXa, whereas the first Kunitz domain is responsible for the inhibition of FVIIa in the TF-FVIIa complex. To define further the physiological role of TFPI, gene targeting was used to disrupt exon 4 encoding the first Kunitz domain of TFPI in the mouse TFPI gene. Mice heterozygous for TFPI gene disruption, TFPI(K1)(+/-), have plasma TFPI activity approximately 50% that of wild-type mice based on a functional assay that measures TF-FVIIa inhibition, and have a normal phenotype. Sixty per cent of TFPI(K1)(-/-) mice die between embryonic days 9.5 and 11.5 with signs of yolk sac haemorrhage. Organogenesis is normal in TFPI(K1)(-/-) animals that progress beyond embryonic day 11.5, but haemorrhage, particularly in the central nervous system and tail, is evident during later gestation, and none of the TFPI(K1)(-/-) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated TF-FVIIa action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate as an individual with TFPI deficiency has not been identified.

Animals↗
PubMed1997

Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

Amino Acid Sequence↗
PubMed1997

Tissue factor pathway inhibitor gene disruption produces intrauterine lethality in mice.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPI(K1)(+/-), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration approximately 40% that of wild-type TFPI. TFPI(K1)(+/-) animals have plasma TFPI activity approximately 50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPI(K1)(-/-) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPI(K1) null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPI(K1)(-/-) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.

Abnormalities, Multiple↗