PubMed Health⌕ Search

Biomedical subjects

R M Bertina

Publications and source records attributed to R M Bertina.

At least 19 recordsLinked to original sources
PubMed1999

A reduced sensitivity for activated protein C in the absence of factor V Leiden increases the risk of venous thrombosis.

Activated protein C (APC) resistance caused by the factor V Leiden mutation is associated with an increased risk of venous thrombosis. We investigated whether a reduced response to APC, not due to the factor V point mutation, is also a risk factor for venous thrombosis. For this analysis, we used the Leiden Thrombophilia Study (LETS), a case-control study for venous thrombosis including 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. All carriers of the factor V Leiden mutation were excluded. A dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis: the lower the normalized APC sensitivity ratio, the higher the associated risk. The risk for the lowest quartile of normalized APC-SR (<0.92), which included 16.5% of the healthy controls, compared with the highest quartile (normalized APC-SR > 1.05) was greater than fourfold increased (OR = 4.4; 95% confidence interval, 2.9 to 6.6). We adjusted for VIII:C levels, which appeared to affect our APC resistance test. The adjusted (age, sex, FVIII:C) odds ratio for the lowest quartile was 2.5 (95% confidence interval, 1.5 to 4.2). So, after adjustment for factor VIII levels, a reduced response to APC remained a risk factor. Our results show that a reduced sensitivity for APC, not caused by the factor V Leiden mutation, is a risk factor for venous thrombosis.

Activated Protein C Resistance↗
PubMed1999

Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation.

Tissue Factor (TF) gene expression is transiently induced in human monocytic THP-1 cells by lipopolysaccharide (LPS). We characterized the transcription factor complexes binding to the TF gene promoter LPS response element (LRE) (-220 to -172), which contains binding sites for nuclear factor kappaB (NFkappaB) and activator protein 1 (AP1) transcription factors, and examined the nature of the activation of these factors during a 24-h time course of LPS stimulation. We found proteolysis of the cytoplasmic inhibitory protein IkappaBalpha and nuclear translocation of the NFkappaB/Rel family proteins p65 and c-Rel, corresponding to the transient binding of a p65/c-Rel heterodimer to the kappaB-like site of the LRE. AP1 binding to the LRE was found to be constitutive, with the majority of the AP1 complexes being JunD/Fra-2 heterodimers. A change in the activation state of the AP1 complexes was, however, found to be transient, as determined by JunD phosphorylation of AP1 bound to the proximal binding site. This directly correlates to the transient activation of Jun N-terminal kinase (SAPK/JNK). These data indicate that LPS induction of TF gene expression in monocytic THP-1 cells is regulated by both the transient phosphorylation of Jun-family proteins and the nuclear translocation and transient binding of NFkappaB/Rel proteins.

Base Sequence↗
PubMed1999

Cholesterol or triglyceride loading of human monocyte-derived macrophages by incubation with modified lipoproteins does not induce tissue factor expression.

Macrophages/foam cells localized in cholesterol- and triglyceride-rich regions of atherosclerotic plaques express high levels of tissue factor (TF), the essential cofactor and receptor of factor VIIa. It is not clear whether modified lipoproteins, for which several agonistic effects on macrophages have been described, are independent stimuli of TF expression in these cells. Therefore, we studied the effect of short-term (1 day) and long-term (4 to 7 days) incubation of human monocyte-derived macrophages cultured in suspension with modified and native LDLs or VLDLs on the expression of TF mRNA, antigen, and activity. We used native LDL or VLDL, moderately oxidized LDL or VLDL, severely oxidized LDL or VLDL, acetylated LDL, and beta-VLDL at a protein concentration of 100 microg/mL. Cholesterol loading occurred within 9 hours after the addition of acetylated LDL and continued during long-term incubation. Incubation of severely oxidized LDL for 7 days resulted in a slight increase in cholesterol content. Triglyceride loading was observed during short-term and long-term incubation with native and modified VLDLs. Neither cholesterol nor triglyceride loading resulted in expression of TF. Bacterial LPS still could induce TF expression in lipid-laden macrophages. Our results show that incubation with modified lipoproteins or lipid loading does not lead to TF expression in monocyte-derived macrophages cultured in suspension. This suggests that induction of TF expression in foam cells in the atherosclerotic lesion is triggered by additional or other components.

Cell Line↗
PubMed1998

Type I protein C deficiency caused by disruption of a hepatocyte nuclear factor (HNF)-6/HNF-1 binding site in the human protein C gene promoter.

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals affected by protein C deficiency are at risk for venous thrombosis. One such affected individual was shown earlier to carry a -14 T --> C mutation in the promoter region of the protein C gene. It is shown here that the region around this mutation corresponds to a binding site for the transcription factor hepatocyte nuclear factor (HNF)-6 and that this site completely overlaps an HNF-1 binding site. HNF-6 and HNF-1 bound in a mutually exclusive manner. The -14 T --> C mutation reduced HNF-6 binding. In transient transfection experiments, HNF-6 transactivated the wild-type protein C promoter and introduction of the mutation abolished transactivation by HNF-6. Similar experiments showed that wild-type protein C promoter activity was reduced by cotransfection of an HNF-1 expression vector. This inhibiting effect of HNF-1 was reversed to a stimulatory effect when promoter sequences either upstream or downstream of the HNF-6/HNF-1 site were deleted. It is concluded that HNF-6 is a major determinant of protein C gene activity. Moreover, this is the first report describing the putative involvement of HNF-6 and of an HNF-6 binding site in human pathology.

Animals↗
PubMed1998

Interaction of coagulation defects and cardiovascular risk factors: increased risk of myocardial infarction associated with factor V Leiden or prothrombin 20210A.

BACKGROUND: A genetic variation located in the 3'-untranslated region of the prothrombin gene (prothrombin 20210 G-->A) was recently described as a risk factor for venous thrombosis. We examined how the presence of this mutation affected the risk of myocardial infarction in a population-based case-control study. Furthermore, we studied the risk of myocardial infarction associated with the simultaneous presence of a coagulation defect (ie, the 20210 AG genotype of prothrombin or the factor V Leiden mutation) and Major cardiovascular risk factors. METHODS AND RESULTS: Among 560 men with a first myocardial infarction before the age of 70 years, 1.8% were heterozygous carriers of the 20210 variant of the prothrombin gene. The control group consisted of 646 men who were frequency matched by age. In the latter group, the frequency of the 20210 AG genotype was 1.2%. The risk of myocardial infarction in the presence of the AG genotype was increased by 50% (odds ratio, 1.5; 95% confidence interval [95% CI], 0.6 to 3.8). The risk of myocardial infarction for carriership of factor V Leiden mutation was increased by 40% (odds ratio, 1.4; 95% CI, 0.8 to 2.2). When a coagulation defect was present (ie, the 20210 AG prothrombin genotype or the factor V Leiden mutation), the risk of myocardial infarction for carriers versus noncarriers was 1.4 (95% CI, 0.9 to 2.2). This risk was substantially increased when one of the major cardiovascular risk factors of smoking, hypertension, diabetes mellitus, or obesity also was present, with odds ratios varying between 3 and 6. These risks exceeded those of the single effects of the cardiovascular risk factors (ie, in the absence of the coagulation defect). CONCLUSIONS: We conclude that in men the 20210 G-->A variant of prothrombin is associated with an increased risk of myocardial infarction. The combined presence of major cardiovascular risk factors and carriership of a coagulation defect increases the risk considerably.

Adult↗
PubMed1998

Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain.

Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.

Animals↗
PubMed1998

Identification of residues in the Gla-domain of human factor IX involved in the binding to conformation specific antibodies.

The binding of Ca2+ induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(II) antibodies recognize a conformational epitope (FIX') that can be induced by several metal ions such as Ca2+, Mg2+, Mn2+ and Ba2+, while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX*) that can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) pp. 7605-7612). The latter conformation is essential for the function of factor IX. In this study we tried to identify residues in the Gla-domain of factor IX which are involved in binding to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For this we substituted residues in recombinant human factor IX for those of factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodies. Re-introduction of factor IX specific residues increased the binding to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicating that the structural integrity of the Gla-domain was not seriously affected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg(II) antibodies, while residues 1-11 are important for binding to anti-factor IX:Ca(II) antibodies.

Amino Acid Sequence↗
PubMed1998

Involvement of amino acid residues 423-429 of human protein S in binding to C4b-binding protein.

Human protein S binds to C4b-binding protein (C4BP) both in plasma and in a system using purified proteins. Amino acid residues 420-434 of the first disulfide loop of the sex hormone binding globulinlike domain of protein S are involved in the interaction of protein S with C4BP. To define the involvement of specific polar amino acids within residues 420-434, we studied in parallel synthetic protein S peptides and recombinant protein S variants containing the same amino acid replacements, K423E, E424K, Q427E and K429E. Synthetic peptide analogs of peptide PSP-420 (residues 420-434) were assayed for binding C4BP and as inhibitors of complex formation. The PSP-420 peptide and the analogous peptide with the substitution E424K, but not the peptides containing the substitutions K423E and K429E, were able to bind C4BP. Recombinant proteins with mutations of K423E, Q427E and K429E showed reduced affinity for C4BP compared to plasma protein S, recombinant wild type protein S, or E424K-protein S. These results suggest that Lys-423, Gln-427 and Lys-429 of protein S are important for normal binding to C4BP. The anti-protein S monoclonal antibody LJ-56, raised against peptide PSP-420, recognizes only free protein S and inhibits complex formation with C4BP. Antibody LJ-56 recognized the E424K and Q427E peptides but not the K423E or K429E peptides. Similarly, the E424K and Q427E protein S mutants were recognized by LJ-56, whereas the K423E and K429E protein S mutants were not recognized. This suggests that both in the peptide PSP-420 and in protein S, Lys-423 and Lys-429 significantly contribute to binding to antibody LJ-56. These results demonstrate that protein S residues 423, 427 and 429, but not residue 424, are involved in binding to both the antibody LJ-56 and to C4BP. When peptides PSP 420 and SL-6 (residues 447-460) with carboxyterminal amide or carboxylate moieties were compared to their ability to inhibit C4BP-protein S complexation, PSP-420-amide was the most potent. This finding together with the other results described here supports the hypothesis that the residues 420 and 434 in protein S provides a major binding site for C4BP.

Amino Acid Sequence↗
PubMed1998

Identification of evolutionarily invariant sequences in the protein C gene promoter.

Recent studies on human protein C gene expression have revealed the presence of three transcription factor binding sites in close proximity to the transcription start site. Binding sites for the liver-enriched hepatocyte nuclear factors 1 and 3 (HNF-1 and HNF-3, respectively) are located immediately upstream of the transcription start site, whereas just downstream of the start site a presently unidentified transcription factor may bind. To identify other candidate transcription factor binding sites in the protein C promoter, we studied the promoter sequence identity in a number of evolutionarily close and more distant species: Gorilla gorilla, Pongo pygmaeus, Pan troglodytes, Homo sapiens, Cebus apella, Macaca mulatta, Callithrix jacchus, Papio hamadryas, Macaca fascicularis, and Rattus norvegicus. This analysis showed that a high degree of identity (78%) exists among the different primates. Comparison of the primate consensus sequence with the Rattus norvegicus protein C promoter sequence revealed the presence of seven identical regions (I to VII). Two of these regions overlap with established regulatory sequences for HNF-3 and HNF-1 (region VI) and for PCE-1 (region VII), respectively. The functional importance and the transcription factors that may bind to the other five identical regions are now to be determined.

Animals↗
PubMed1998

Activation of the protein C system during cardiopulmonary bypass with and without aprotinin.

BACKGROUND: The protein C system is important in the regulation of hemostasis. We studied its behavior during coronary artery bypass grafting procedures with and without aprotinin treatment using assays sensitive for activation of the protein C system. METHODS: In a prospective, double-blind, randomized study of 48 patients we investigated the levels of antigen to proteins C and S and of the complexes between activated protein C with its two major plasma inhibitors, protein C inhibitor and alpha1-antitrypsin in patients treated with placebo (n = 17), low-dose (n = 15), and high-dose (n = 16) aprotinin during elective coronary artery bypass grafting. RESULTS: The levels of proteins C and S showed a rapid decrease after heparinization, decreased greatly after start of cardiopulmonary bypass, and remained stable during cardiopulmonary bypass. Activated protein C inhibitor complexes were markedly elevated at the start of the procedure. Activated protein C-alpha1-antitrypsin decreased greatly after the start of cardiopulmonary bypass and remained stable during cardiopulmonary bypass. A significant peak was observed at the intensive care unit. Activated protein C-protein C inhibitor levels showed a peak after heparinization in accordance with the accelerating effect of heparin on the complex formation but decreased thereafter. Treatment with aprotinin did not notably alter any of the measured patterns. CONCLUSIONS: In this study no evidence was found for increased activation of the protein C system during coronary artery bypass grafting. Administration of aprotinin did not result in different patterns of activation of the protein C system.

Aprotinin↗
PubMed1998

Severe coagulation factor V deficiency caused by a 4 bp deletion in the factor V gene.

Factor V (FV) deficiency (parahaemophilia) is an autosomal recessive bleeding disorder with an incidence of 1:10(6). We have studied a young girl with very mild bleeding symptoms and undetectable levels of plasma factor V antigen and activity (<0.3% and <1.6% of normal, respectively). Both parents showed plasma levels of factor V activity of about 50% of normal. Sequence analysis of the 5'- and 3'-untranslated, coding and adjacent regions of the factor V gene revealed the presence of a 4 bp deletion in exon 13. Subsequent screening of members of the family for the mutation showed that both parents were heterozygous for the mutation, that one healthy sister carried only normal alleles, and that the patient was homozygous for the mutated allele. The mutation introduced a frameshift and a novel premature stop codon in codon 1303, and would predict the synthesis of a truncated factor V molecule that lacks part of the B domain and the complete light chain. However, no factor V heavy chain could be detected in the plasma of the patient. Furthermore, factor V activity could not be detected in the patients' platelets. This is the first reported mutation in the factor V gene that predicts a type I quantitative factor V deficiency. Surprisingly, the patient, who is homozygous for the mutation, so far has only a very mild bleeding tendency.

Amino Acid Sequence↗
PubMed1998

Two novel mutations in the prothrombin gene cause severe bleeding in a compound heterozygous patient.

Hypoprothrombinemia is a rare hereditary coagulation defect characterized by low levels of biologically active prothrombin. In this paper we report the laboratory and genetic analysis of a patient with a severe hypoprothrombinemia and some of her relatives. Laboratory analysis showed very low levels of prothrombin antigen. Molecular analysis of the prothrombin genes of the patient resulted in the identification of two novel sequence variations in heterozygous state, a 20079 G to A transition, which predicts a Trp 569-->Stop mutation, and a 1261C-->G change within intron B near the acceptor splice site. A cosegregation of prothrombin deficiency in family members with the two genetic defects was observed.

Adult↗
PubMed1998

The prothrombin 20210 G to A variation and thrombosis.

The 20210 G to A variation in the prothrombin gene was first described in 1996 and found to be associated with both elevated plasma prothrombin levels and a moderately increased risk for first venous thrombotic events. These findings generally have been confirmed in several independent studies. The 20210A allele seems to be rather common in white subpopulations, among whom the prevalence of heterozygous carriers may vary from 1% to 5%. The variant prothrombin allele contributes to the risk of thrombosis in carriers of other genetic risk factors (eg, factor V Leiden); also, this allele seems not to be an important risk factor for arterial thrombosis, unless risk factors for cardiovascular disease (eg, smoking) also are present.

Alleles↗
PubMed1998

Role of monocytes and bacteria in Staphylococcus epidermidis endocarditis.

The endocardial vegetation which is formed in the course of bacterial endocarditis (BE) contains tissue factor (TF)-dependent procoagulant activity. Earlier studies showed that monocytes are the main source of TF in the vegetations. The TF activity (TFA) of vegetations isolated from Streptococcus sanguis-infected rabbits depended on the numbers of bacteria as well as monocytes in the vegetation. In this study, we investigated whether for Staphylococcus epidermidis, a frequent pathogen in BE, an effect similar to that found for S. sanguis could be shown. In vitro, S. epidermidis was found to stimulate TFA of fibrin adherent monocytes significantly. This stimulation was maximal at a bacterium-to-monocyte ratio of 7. In vivo, TFA was found to be significantly higher in S. epidermidis-infected than in sterile catheter-induced vegetations. Reduction of vegetational bacterial numbers by teicoplanin treatment lead to a small but significant decrease of TFA. Reduction of monocyte numbers by etoposide did not affect vegetational TFA. Comparison of data for S. epidermidis and S. sanguis revealed that at equivalent bacterial numbers, vegetational TFAs were approximately the same for both microorganisms. Combining the results of the present study with those of a previous study using S. sanguis, we conclude that the main factor determining monocyte-dependent vegetational TFA is the number of vegetation-associated bacteria. The lower TFA found for S. epidermidis-infected than for S. sanguis-infected vegetations can be explained by the significantly lower bacterial numbers in the infected vegetations and consequently a lower stimulation of vegetation-associated monocytes.

Animals↗