Polycyclic aromatic hydrocarbons induce endothelial injury through miR-155 to promote atherosclerosis.

Polycyclic aromatic hydrocarbons (PAHs) are considered as an external factor that induces atherosclerotic cardiovascular disease. Although miR-155 is known to be involved in cardiovascular disease, whether it is involved in PAH-induced arteriosclerosis remains unclear. We evaluated the effects of PAHs on vascularization, permeability, and miR-155 expression in HUVECs. We found that PAHs-induced sclerosis of HUVECs was characterized by increasing permeability, decreasing proliferation, and vascular lumen number. The expression of miR-155 was upregulated by PAHs treatment, and transfection with miR-155 inhibitor could reverse above effect of PAHs-induced sclerosis. Meanwhile, transcriptome sequencing revealed that 63 genes were downregulated in the group of PAHs treatment alone, and were then upregulated in the miR-155 inhibitor group. These genes were mainly involved in complement and coagulation cascades, cytokine-cytokine receptor interaction, TNF signaling pathway, and NF-kappa B signaling pathway. Among these 63 genes, SERPIND1 was directly targeted and regulated by miR-155. Further in vivo experiments in ApoE-/- mice confirmed that PAH accelerates the development of arteriosclerosis by promoting the expression of miR-155 to downregulate the SERPIND1. Therefore, PAH exaggerates atherosclerosis by activating miR-155-dependent endothelial injury. This study provides a fundamental insight on the miR-155 mechanism for PAHs enhancing atherosclerosis and miR-155 potentially serving as a novel drug target.

Identification and characterization of a novel isoform of heparin cofactor II in human liver.

Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C-terminal region to give a novel protein, HCII-N. MD simulations of HCII-N and three-dimensional structure showed a unique 51 amino acid sequence at the C-terminal having unique RCL-like structure. The HCII-N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence-based analysis revealed a more compact structure of HCII-N that was in a more hydrophilic environment. The HCII-N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII-N may have alternate protease specificity or a non-inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene.

Glial cells maintain synapses by inhibiting an activity-dependent retrograde protease signal.

Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system.

Label-free quantitative proteomics reveals fibrinopeptide B and heparin cofactor II as potential serum biomarkers in respiratory syncytial virus-infected mice treated with Qingfei oral liquid formula.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.

Analysis of the gene expression profile in response to human epididymis protein 4 in epithelial ovarian cancer cells.

Currently, there are emerging multiple studies on human epididymis protein 4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717 genes were upregulated and 898 genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166 genes were upregulated and 285 were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16 genes consistently upregulated and 8 genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4 cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107 paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer.

Prothrombotic SERPINC1 gene polymorphism may affect heparin sensitivity among different ethnicities of Chinese patients receiving heart surgery.

The purpose of this study was to investigate a possible correlation between single-nucleotide polymorphisms (SNPs) of the antithrombin (gene, SERPINC1, and perioperative sensitivity to heparin in patients receiving heart surgery. The SERPINC1 genotype and allele frequency, coagulation parameters 24 hours before and after surgery, and clinical findings were compared among 3 ethnic groups, Han, Uighur, and Kazakh, patientswho received heart surgery. In Han patients, longer coagulation time as well as higher heparin and protamine dosage was observed. SERPINC1 gene sequencing identified 2 mutations in exon 5, g.981A>G (rs5877) and g.1011A>G (rs5878). The minor allele frequency of allele (A>G) for rs5877 and rs5878 was higher in the Han patients and was significantly different among the ethnic groups (P = .004 and P = .006, respectively). The increased SERPINC1 SNP frequency among Han patients receiving heart surgery might contribute to the differences in their perioperative sensitivity to heparin.

Heparin co-factor II enhances cell motility and promotes metastasis in non-small cell lung cancer.

Using the Serial Analysis of Gene Expression (SAGE) database from the Cancer Genome Anatomy Project, we identified heparin co-factor II (HCII), which is over-expressed in non-small cell lung cancer (NSCLC). Here, we investigated the clinical significance of HCII and provided molecular evidence to support the suggestion that HCII could enhance cancer metastasis in NSCLC. We found that high HCII expression in tumour tissue was associated with increased cancer recurrence and shorter overall survival times in 75 clinically operable NSCLC patients. High pretreatment plasma concentration of HCII was associated with reduced overall survival in 57 consecutive NSCLC patients. We over-expressed and knocked down HCII expression in lung cancer cell lines and confirmed that HCII could promote cell motility, invasion ability and filopodium dynamics in NSCLC cells in vitro and increased metastatic colonization in an in vivo mouse model. Exogenous treatment of HCII promoted cancer cell migration, and this promigratory effect of HCII was independent of thrombin. We further showed that HCII could up-regulate cancer cell migration through the activation of PI3K, which acts upstream of Rac1 and Cdc42, and this effect could be blocked by heparin. We suggest that HCII is a novel metastasis enhancer and may be used as a prognostic predictor for heparin treatment in NSCLC.

Genetic variants and evolutionary analyses of heparin cofactor II.

Heparin cofactor II (HCII) belongs to serpin superfamily and it acts as a thrombin inhibitor in the coagulation cascade, in a glycosaminoglycan-dependent pathway using the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. This serpin belongs to multiple member group V2 of vertebrate serpin classification. However, there is no comprehensive study illustrating the exact phylogenetic history of HCII, to date. Herein, we explored phylogenetic traits of HCII genes. Structures of HCII gene from selected ray-finned fishes and lamprey varied in exon I and II with insertions of novel introns of which one in core domain for ray-finned fishes in exon II at the position 241c. We found HCII remain nested in the largest intron of phosphatidylinositol (PI) 4-kinase (PIK4CA) gene (genetic variants of this gene cause schizophrenia) at the origin of vertebrates, dated about 500MY old. We found that sequence features such as two acidic repeats (AR1-II), GAG-binding helix-D, three serpin motifs and inhibitory reactive center loop (RCL) of HCII protein are highly conserved in 55 vertebrates analyzed. We identified 985 HCII variants by analysis of 1092 human genomes with top three variation classes belongs to SNPs (84.3%), insertion (7.1%) and deletion (5.0%). We identified 37 deleterious mutations in the human HCII protein and we have described these mutations in relation to HCII sequence-structure-function relationships. These understandings may have clinical and medical importance as well.

Origin of serpin-mediated regulation of coagulation and blood pressure.

Vertebrates evolved an endothelium-lined hemostatic system and a pump-driven pressurized circulation with a finely-balanced coagulation cascade and elaborate blood pressure control over the past 500 million years. Genome analyses have identified principal components of the ancestral coagulation system, however, how this complex trait was originally regulated is largely unknown. Likewise, little is known about the roots of blood pressure control in vertebrates. Here we studied three members of the serpin superfamily that interfere with procoagulant activity and blood pressure of lampreys, a group of basal vertebrates. Angiotensinogen from these jawless fish was found to fulfill a dual role by operating as a highly selective thrombin inhibitor that is activated by heparin-related glycosaminoglycans, and concurrently by serving as source of effector peptides that activate type 1 angiotensin receptors. Lampreys, uniquely among vertebrates, thus use angiotensinogen for interference with both coagulation and osmo- and pressure regulation. Heparin cofactor II from lampreys, in contrast to its paralogue angiotensinogen, is preferentially activated by dermatan sulfate, suggesting that these two serpins affect different facets of thrombin's multiple roles. Lampreys also express a lineage-specific serpin with anti-factor Xa activity, which demonstrates that another important procoagulant enzyme is under inhibitory control. Comparative genomics suggests that orthologues of these three serpins were key components of the ancestral hemostatic system. It appears that, early in vertebrate evolution, coagulation and osmo- and pressure regulation crosstalked through antiproteolytically active angiotensinogen, a feature that was lost during vertebrate radiation, though in gnathostomes interplay between these traits is effective.

The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand.

BACKGROUND: Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli. RESULTS: Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 µM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1. CONCLUSIONS: Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): molecular characterization, cloning and expression analysis.

Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX™ genomic data of rock bream (Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly downregulated (p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant downregulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL™ system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII's thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibition activity of RbHCII with its temporal downregulation against immune stimulants, it could be suggested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation.

Heparin cofactor II protects against angiotensin II-induced cardiac remodeling via attenuation of oxidative stress in mice.

Heparin cofactor II (HCII), a serine protease inhibitor, inhibits tissue thrombin action after binding with dermatan sulfate proteoglycans in the extracellular matrix of the vascular system. We previously reported that heterozygous HCII-deficient (HCII(+/-)) humans and mice demonstrate acceleration of vascular remodeling, including atherosclerosis. However, the action of HCII on cardiac remodeling never has been determined. HCII(+/+) and HCII(+/-) mice at age 25 weeks were infused with angiotensin II (Ang II; 2.0 mg/kg/d) for 2 weeks by an osmotic mini-pump. Echocardiography revealed acceleration of cardiac concentric remodeling in HCII(+/-) mice and larger left atrial volume in HCII(+/-) mice than in HCII(+/+) mice. Histopathologic studies showed more prominent interstitial fibrosis in both the left atrium and left ventricle in HCII(+/-) mice than in HCII(+/+) mice. Daily urinary excretion of 8-hydroxy-2'-deoxyguanosine, a parameter of oxidative stress, and dihydroethidium-positive spots, indicating superoxide production in the myocardium, were markedly increased in Ang II-treated HCII(+/-) mice compared to those in HCII(+/+) mice. Cardiac gene expression levels of atrial natriuretic peptides and brain natriuretic peptides, members of the natriuretic peptide family, Nox 4, Rac-1, and p67(phox) as components of NAD(P)H oxidase, and transforming growth factor-beta1 and procollagen III were more augmented in HCII(+/-) mice than in HCII(+/+) mice. However, administration of human HCII protein attenuated all of those abnormalities in Ang II-treated HCII(+/-) mice. Moreover, human HCII protein supplementation almost abolished cardiac fibrosis in Ang II-treated HCII(+/+) mice. The results indicate that HCII has a protective role against Ang II-induced cardiac remodeling through suppression of the NAD(P)H oxidase-transforming growth factor-beta1 pathway.

Heparin cofactor II as a novel vascular protective factor against atherosclerosis.

Heparin cofactor II (HCII) specifically inhibits thrombin action at the site of vascular wall injury. We encountered a congenital HCII deficiency patient with advanced multiple atherosclerotic lesions. This patient led us to conduct clinical studies to examine the role of HCII against atherosclerosis. We found that the incidence of in-stent restenosis after percutaneous coronary intervention, severity of carotid atherosclerosis and prevalence of peripheral arterial disease are inversely associated with plasma HCII activity. In order to clarify the vascular protective action of HCII, we generated HCII- deficient mice by gene targeting. In contrast to a previous study, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, accelerated intimal hyperplasia and frequent thrombosis were observed after cuff or wire injury of femoral arteries. The number of protease-activated receptor-1 (PAR-1) -positive cells and the gene expression levels of inflammatory cytokines and chemokines were increased in the thickened vascular walls of HCII(+/-) mice. The accelerated intimal hyperplasia in HCII+/- mice with vascular injury was attenuated by human HCII administration. Furthermore, HCII deficiency exaggerated aortic plaque formation with increased oxidative stress in apolipoprotein E(-/-) mice. These results demonstrate that HCII protects against thrombin-induced vascular remodeling in both humans and mice and suggest that HCII is a predictive biomarker and therapeutic target for atherosclerosis.

The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of alpha1-proteinase inhibitor M358R for thrombin.

Natural inhibitors of coagulation or inflammation such as the serpins antithrombin (AT), heparin cofactor II (HCII), and alpha(1)-proteinase inhibitor (alpha(1)-PI) can be overwhelmed in thrombosis and/or sepsis. The reactive centre (P1-P1') variant alpha(1)-PI M358R inhibits not only procoagulant thrombin but also anticoagulant activated protein C (APC). We previously described HAPI M358R, comprising a fusion of HCII residues 1-75 to the N-terminus of a(1)-PI M358R that yielded increased anti-thrombin, but not anti-APC activity. We hypothesized that further alterations to the HAPI M358R reactive centre loop would yield additional refinements in specificity. The reactions with thrombin or APC of recombinant alpha(1)-PI M358R variants with or without the HCII extension were characterized electrophoretically and kinetically. Their extension of clotting times and inhibition of fibrin-bound thrombin were measured, and the survival of HAPI M358R in mice was determined. Replacing the P7-P3 and P2' residues of HAPI M358R with AT residues reduced APC inhibition rates by 140-fold, but those of thrombin less than two-fold;substituting the P16-P2 and P2'-P3' residues of HAPI M358R with HCII residues reduced APC inhibition rates by 180-fold, but those of thrombin 10.5-fold. Fused variants extended thrombin clotting times more effectively than unfused inhibitors, were at least as effective at inhibiting clot-bound thrombin, and remained intact in the murine circulation. The combination of modifications inside and outside the RCL resulted in a 1,360-fold increase in selectivity of HAPI M358R (AT P7-P3/P2') for thrombin versus APC relative to alpha(1)-PI M358R. Our results predict that this protein may be effective in limiting thrombosis in vivo.

Accelerated atherogenesis and neointima formation in heparin cofactor II deficient mice.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin when bound to dermatan sulfate or heparin. HCII-deficient mice are viable and fertile but rapidly develop thrombosis of the carotid artery after endothelial injury. We now report the effects of HCII deficiency on atherogenesis and neointima formation. HCII-null or wild-type mice, both on an apolipoprotein E-null background, were fed an atherogenic diet for 12 weeks. HCII-null mice developed plaque areas in the aortic arch approximately 64% larger than wild-type mice despite having similar plasma lipid and glucose levels. Neointima formation was induced by mechanical dilation of the common carotid artery. Thrombin activity, determined by hirudin binding or chromogenic substrate hydrolysis within 1 hour after injury, was higher in the arterial walls of HCII-null mice than in wild-type mice. After 3 weeks, the median neointimal area was 2- to 3-fold greater in HCII-null than in wild-type mice. Dermatan sulfate administered intravenously within 48 hours after injury inhibited neointima formation in wild-type mice but had no effect in HCII-null mice. Heparin did not inhibit neointima formation. We conclude that HCII deficiency promotes atherogenesis and neointima formation and that treatment with dermatan sulfate reduces neointima formation in an HCII-dependent manner.

Unchecked thrombin is bad news for troubled arteries.

Thrombin is clearly a key trigger of thrombosis, the proximal cause of most morbidity and mortality in atherosclerotic cardiovascular disease. Might thrombin also contribute to longer-term, structural changes in the arterial wall that promote narrowing and clotting? A study in this issue of the JCI argues that it can. Aihara et al. report that haploinsufficiency of heparin cofactor II, a glycosaminoglycan-dependent thrombin inhibitor, exacerbates injury- or hyperlipidemia-induced arterial lesion formation in mice, possibly by excessive thrombin signaling through protease-activated receptors (see the related article beginning on page 1514).

Strain-dependent embryonic lethality and exaggerated vascular remodeling in heparin cofactor II-deficient mice.

Heparin cofactor II (HCII) specifically inhibits thrombin action at sites of injured arterial wall, and patients with HCII deficiency exhibit advanced atherosclerosis. However, the in vivo effects and the molecular mechanism underlying the action of HCII during vascular remodeling remain elusive. To clarify the role of HCII in vascular remodeling, we generated HCII-deficient mice by gene targeting. In contrast to a previous report, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, prominent intimal hyperplasia with increased cellular proliferation was observed after tube cuff and wire vascular injury. The number of protease-activated receptor-1-positive (PAR-1-positive) cells was increased in the thickened vascular wall of HCII(+/-) mice, suggesting enhanced thrombin action in this region. Cuff injury also increased the expression levels of inflammatory cytokines and chemokines in the vascular wall of HCII(+/-) mice. The intimal hyperplasia in HCII(+/-) mice with vascular injury was abrogated by human HCII supplementation. Furthermore, HCII deficiency caused acceleration of aortic plaque formation with increased PAR-1 expression and oxidative stress in apoE-KO mice. These results demonstrate that HCII protects against thrombin-induced remodeling of an injured vascular wall by inhibiting thrombin action and suggest that HCII is potentially therapeutic against atherosclerosis without causing coagulatory disturbance.

Adenovirus-mediated expression of heparin cofactor II inhibits thrombin-induced cellular responses in fibroblasts and vascular smooth muscle cells.

Heparin cofactor II functions as a physiological inhibitor of thrombin activity. The rate of inactivation of thrombin by heparin cofactor II is increased in the presence of dermatan sulfate, which is produced by fibroblasts or smooth muscle cells. To elucidate the role of heparin cofactor II in the extravascular cells, we induced expression of heparin cofactor II in cultured human fibroblasts or vascular smooth muscle cells using adenovirus-mediated gene transfer. After infection of adenovirus vector, these cells secreted heparin cofactor II protein into culture medium. The expressed heparin cofactor II formed the complex with exogenous thrombin and inhibited the proteolytic activity of thrombin. Expression of heparin cofactor II by infection of adenovirus vector inhibited thrombin-induced tissue-type plasminogen activator and interleukin-6 releases from fibroblasts and thrombin-induced interleukin-6 release from vascular smooth muscle cells. These findings show that fibroblasts and vascular smooth muscle cells expressing heparin cofactor II are resistant to thrombin-induced cellular responses.

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition.

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.

Homozygous deficiency of heparin cofactor II: relevance of P17 glutamate residue in serpins, relationship with conformational diseases, and role in thrombosis.

BACKGROUND: Heparin cofactor II (HCII) is a hepatic serpin with significant antithrombin activity that has been implicated in coagulation, inflammation, atherosclerosis, and wound repair. Recent data obtained in mice lacking HCII suggest that this serpin might inhibit thrombosis in the arterial circulation. However, the clinical relevance and molecular mechanisms associated with deficiency of HCII in humans are unclear. METHODS AND RESULTS: We studied the first family with homozygous HCII deficiency, identifying a Glu428Lys mutation affecting a conserved glutamate at the hinge (P17) of the reactive loop. No carrier reported arterial thrombosis, and only 1 homozygous HCII-deficient patient developed severe deep venous thrombosis, but she also had a de novo Glu100Stop nonsense truncation in the antithrombin gene. CONCLUSIONS: Our results confirm the key structural role of the P17 glutamate in serpins. The same mutation causes conformational instability and polymerization in 3 serpins: Drosophila necrotic, human alpha1-antitrypsin, and human HCII, which explains their plasma deficiency. In the family under study here, however, plasma HCII deficiency was not associated with a significant clinical phenotype.