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Biomedical subjects

A Hillarp

Publications and source records attributed to A Hillarp.

At least 37 records · Page 2Linked to original sources
PubMed1999

Factor V Q506 (resistance to activated protein C) and prognosis after acute coronary syndrome.

Factor V:Q506 causing resistance to activated protein C (APC-resistance), is a risk factor for venous thrombosis. Some studies have indicated an association with arterial disease, especially in women. We investigated the prevalence of the FV:Q506 allele prospectively in 295 patients with acute coronary syndrome. Mortality and myocardial infarction rate were evaluated after 30 days and after 2 years. The FV:Q506 allele was found in 38 patients. In a Cox proportional hazards model, smokers carrying FV:Q506 had a higher risk of infarction or death within 30 days, compared to non-smokers with a normal genotype (relative risk 2.9 [95% CI 1.2-7.0]). The difference remained significant after 2 years (relative risk 2.8 [95% CI 1.2-6.5]). The effect of the FV:Q506 allele on clinical outcome in acute coronary syndrome has not previously been described. Our results demonstrate a gene-environment interaction between smoking and the FV:Q506 allele, with an increased risk of early complications after an acute ischemic event.

Activated Protein C Resistance↗
PubMed1998

Geographic distribution of the 20210 G to A prothrombin variant.

A variant in prothrombin (clotting factor II), a G to A transition at nucleotide position 20210, has recently been shown to be associated with the prothrombin plasma levels and the risk of both venous and arterial thrombosis. The purpose of this study was to investigate the prevalence of carriership of this mutation in various populations. We combined data from 11 centres in nine countries, where tests for this mutation had been performed in groups representing the general population. We calculated an overall prevalence estimate, by a precision-weighted method, and, since the distribution of the prevalences did not appear homogeneous, by an unweighted average of the prevalences. We examined differences in the prevalences by geographical location and ethnic background as a possible explanation for the heterogeneity. Among a total of 5527 individuals who had been tested, 111 heterozygous carriers of the 20210A mutation were found. The prevalence estimates varied from 0.7 to 4.0 between the centres. The overall prevalence estimate was 2.0 percent (CI95 1.4-2.6%). The variation around the summary estimate appeared more than was expected by chance alone, and this heterogeneity could be explained by geographic differences. In southern Europe, the prevalence was 3.0 percent (CI95 2.3 to 3.7%), nearly twice as high as the prevalence in northern Europe (1.7%, CI95 1.3 to 2.2%). The prothrombin variant appeared very rare in individuals from Asian and African descent. The 20210A prothrombin variant is a common abnormality, with a prevalence of carriership between one and four percent. It is more common in southern than in northern Europe. Since this distribution within Europe is very different to that of another prothrombotic mutation (factor V Leiden or factor V R506Q), founder effects are the most likely explanation for the geographical distribution of both mutations.

Brazil↗
PubMed1998

A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen.

A new method to determine the concentration of free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1:20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specificity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies.

Antibodies, Monoclonal↗
PubMed1997

The amino-terminal module of the C4b-binding protein alpha-chain is crucial for C4b binding and factor I-cofactor function.

C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (alpha-chains) and one unique one (beta-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the alpha-chain contains eight CCPs and the beta-chain three. Each alpha-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the alpha-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP alpha-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP beta-chain. Furthermore, seven different monoclonal antibodies were raised against C4BP and characterized using the recombinant chimeric proteins. Whereas all three recombinant chimeras bind protein S with the same affinity as plasma-purified C4BP, none of them bound to C4b. Three of the antibodies, which were found to bind to alpha-chain CCP 1 and CCP 2, completely inhibited the binding of plasma-purified C4BP to immobilized C4b. In addition, two of these antibodies totally blocked the factor I-cofactor activity of C4BP in a C4b-degradation assay. The binding site for one of the monoclonal antibodies was also studied using electron microscopy where it was confirmed that this antibody bound to the amino-terminal tip of the alpha-chain. These results show that the amino-terminal CCP of the C4BP alpha-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.

Animals↗
PubMed1997

Activated protein C resistance caused by a common factor V mutation has a single origin.

A point mutation (FV:R506Q) in the human coagulation factor V gene is associated with resistance to activated protein C and life-long increased risk of venous thrombosis. The mutation is common in populations of Caucasian origin but virtually absent among other populations. In this study of 140 healthy Swedish volunteers and 110 homozygotes for the FV:R506Q mutation, we determined the allele frequencies of the FV:R506Q mutation and four other dimorphisms, C/T at nucleotide positions 2298 and 2325, and A/G at nucleotide positions 2379 and 2391. Manifest linkage disequilibrium was found between the FV:R506Q mutation and the four different dimorphisms. The finding of a single FV:R506Q haplotype in all homozygotes constitutes strong evidence of a common ancestor of Swedish individuals with the FV:R506Q mutation.

Adult↗
PubMed1997

Molecular cloning of rat C4b binding protein alpha- and beta-chains: structural and functional relationships among human, bovine, rabbit, mouse, and rat proteins.

The C4b binding protein (C4BP) functions as a regulator of the complement system by interacting with the activated form of the fourth complement component, C4b. Human C4BP also interacts with the anticoagulant protein S and the serum amyloid P component (SAP). It is composed of seven identical 70-kDa alpha-chains and one 45-kDa beta-chain. The alpha-chain contains a binding site for C4b, whereas the beta-chain contains the protein S binding site. Recent studies have shown rabbit and bovine plasma to lack a C4BP-protein S complex, and the mouse beta-chain gene to have evolved into a pseudogene. Using a gel filtration chromatography system in combination with Western blotting, we detected a complex between C4BP and protein S in rat plasma, similar to the complex known in human plasma. Using purified rat C4BP and SAP we were unable to detect any complex between the two proteins, but rat C4BP was able to form a complex with human SAP. Rat cDNA clones encoding the C4BP alpha- and beta-chains were isolated from a rat liver cDNA library. The rat alpha-chain cDNA predicted a mature polypeptide chain of 545 amino acid residues, whereas the beta-chain cDNA predicted a mature polypeptide of 243 amino acid residues. The overall amino acid sequence identities between the rat alpha-chain and the mouse, human, rabbit, and bovine alpha-chains were 64, 60, 59, and 52%, respectively. The identities between the rat beta-chain and the human and bovine beta-chains were 68 and 57%, respectively. The rat represents the first non-primate species in which the C4BP-protein S interaction has been found to be conserved.

Amino Acid Sequence↗
PubMed1997

Activated protein C resistance due to a common factor V gene mutation is a major risk factor for venous thrombosis.

Inherited resistance to activated protein C (APC) was recently discovered to be a cause of familial thrombophilia and is now known to be the most common genetic risk factor for venous thrombosis. It is caused by a single point mutation in the gene for factor V, which predicts substitution or arginine (R) at position 506 with a glutamine (Q). Accordingly, the activated form of mutated factor V (FVa:Q506) is more slowly degraded by activated protein C than normal FVa (FVa:R506) is, resulting in hypercoagulability and a lifelong 5- to 10-fold increased risk of venous thrombosis. Previously known inherited hypercoagulable states, i.e. deficiencies of the anticoagulant proteins antithrombin III, protein S, and protein C, are found fewer than 10-15% of thrombosis patients in western countries, whereas inherited APC resistance is present in 20-60% of such patients. The FV mutation is common in populations of Caucasian origin, with prevalences ranging from 1-15%, whereas it is not found in certain other ethnic groups such as Japanese and Chinese. The high prevalence of APC resistance, in combination with the availability of simple laboratory tests, will have a profound influence on the development of therapeutic and prophylactic regimens for thrombosis and will, it is hoped, result in a decreased incidence of thromboembolic events.

DNA Mutational Analysis↗
PubMed1997

The 20210 A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis.

A dimorphism in the 3'-untranslated region of the prothrombin gene (G to A transition at position 20210) has recently been reported to be associated with increases in plasma prothrombin levels and in the risk of venous thrombosis. We have examined the prothrombin dimorphism among 99 unselected outpatients with phlebography verified deep venous thrombosis, and in 282 healthy controls. The prevalence of the 20210 A allele was 7.1% (7/99) in the patient group, and 1.8% (5/282) in the healthy control group (p = 0.0095). The relative risk of venous thrombosis was calculated to be 4.2 (95% CI, 1.3 to 13.6), and was still significant when adjustment was made for age, sex and the factor V:R506Q mutation causing APC resistance [odds ratio 3.8 (95% CI, 1.1 to 13.2)]. As previously reported, 28% of the patients were carriers of the factor V:R506Q mutation. Thus, 34% (one patient carried both traits) of unselected patients with deep venous thrombosis were carriers of an inherited prothrombotic disorder. To sum up, our results confirm the 20210 A allele of the prothrombin gene to be an important risk factor for venous thrombosis.

Adult↗
PubMed1996

Resistance to activated protein C, the FV:Q506 allele, and venous thrombosis.

Vitamin K-dependent protein C is an important regulator of blood coagulation. After its activation on the endothelial cell surface by thrombin bound to thrombomodulin, it cleaves and inactivates procoagulant cofactors Va and VIIIa, protein S and intact factor V working as cofactors. Until recently, genetic defects of protein C or protein S were, together with antithrombin III deficiency, the established major causes of familial venous thromboembolism, but they were found in fewer than 5-10% of patients with thrombosis. In 1993, inherited resistance to activated protein C (APC) was described as a major risk factor for venous thrombosis. It is found in up to 60% of patients with venous thrombosis. In more than 90% of cases, the molecular background for the APC resistance is a single point mutation in the factor V gene, which predicts substitution of an arginine (R) at position 506 by a glutamine (Q). Mutated factor V (FV:Q506) is activated by thrombin or factor Xa in normal way, but impaired inactivation of mutated factor Va by APC results in life-long hypercoagulability. The prevalence of the FV:Q506 allele in the general population of Western countries varies between 2 and 15%, whereas it is not found in several other populations with different ethnic backgrounds. Owing to the high prevalence of FV:Q506 in Western populations, it occasionally occurs in patients with deficiency of protein S, protein C, or antithrombin III. Individuals with combined defects suffer more severely from thrombosis, and often at a younger age, than those with single defects, suggesting severe thrombophilia to be a multigenetic disease.

Alleles↗
PubMed1996

Inherited resistance to activated protein C caused by presence of the FV:Q506 allele as a basis of venous thrombosis.

Inherited resistance to activated protein C (APC) was recently discovered as a cause of familial thrombophilia and is now known to be the most common genetic risk factor for venous thrombosis. In a majority of cases, APC resistance is associated with a single point mutation in the factor V gene, which results in substitution of arginine (R) at position 506 by glutamine (Q) (FV:Q506). The mutation renders factor Va partially resistant to degradation by activated protein C (APC), which leads to a hypercoagulable state and a life-long 5-10-fold increased risk of venous thrombosis. The previously known inherited deficiencies of antithrombin, protein S or protein C, are in western societies together found in less than 10-15% of thrombosis patients, whereas APC resistance is present in 20 to 60% of the patients. A functional APC resistance test, which includes predilution of the patient plasma with factor V deficient plasma, is 100% sensitive and specific for the presence of FV:Q506. The FV:Q506 allele is common in populations of Caucasian origin (prevalence ranging between 1 and 15%), whereas it is not found in certain other ethnic groups such as in Japanese and Chinese. The thrombotic risk in individuals with APC resistant may be further increased by other genetic defects such as protein C or protein S deficiency and by exposure to circumstantial risk factors such as oral contraceptives, pregnancy, immobilisation and surgery.

Adolescent↗
PubMed1995

Activated protein C resistance: from phenotype to genotype and clinical practice.

The anticoagulant protein C system is an important regulator of the blood coagulation process. Its targets are the procoagulant cofactors factor Va and factor VIIIa, which are cleaved and inactivated by activated protein C, protein S and intact factor V working as cofactors. Genetic defects of protein C or protein S were, together with antithrombin III deficiency, the previously established major causes of familial venous thromboembolism. However, these abnormalities are found in less than 5-10% of patients with thrombosis. Inherited resistance to activated protein C was recently identified as a major risk factor for venous thromboembolism. The activated protein C-resistance phenotype is found in 20-60% of the patients with venous thrombosis, depending on selection criteria and on the prevalence of activated protein C-resistance in the population. The frequency of activated protein C-resistance is 2-10% in the normal populations studied so far. In more than 90% of cases, the molecular background for the activated protein C-resistance is a single point mutation in the factor V gene, which predicts substitution of an arginine at position 506 by a glutamine. Mutated factor V is activated by thrombin or factor Xa in the normal way, but impaired inactivation of mutated factor Va by activated protein C results in a life-long hypercoagulability. Owing to the high prevalence of activated protein C-resistance in the population, it occasionally occurs in patients with deficiency of protein S, protein C or antithrombin III. Individuals with combined defects suffer more severely from thrombosis, and often at a younger age, than those with single defects, suggesting thrombophilia to be a multigenetic disease.

Anticoagulants↗
PubMed1994

Bovine C4b binding protein. Molecular cloning of the alpha- and beta-chains provides structural background for lack of complex formation with protein S.

C4b binding protein (C4BP) regulates the complement system. It also interacts with anticoagulant protein S and with serum amyloid P component. Human C4BP is composed of seven identical 70-kDa alpha-chains and one 45-kDa beta-chain. The binding site for C4b is located on the alpha-chain, whereas the beta-chain binds protein S. Nothing is known about the structure and function of bovine C4BP. No complexed form of protein S was detected by using a gel filtration chromatography system combined with Western blotting. Bovine cDNA clones encoding the C4BP alpha- and beta-chains were isolated from a bovine liver cDNA library. Three overlapping alpha-chain clones predicted a 562-amino acid residues-long mature polypeptide. The overall amino acid sequence similarity with the human alpha-chain was 61%. Like its human counterpart, the bovine alpha-chain is composed of eight contiguous short consensus repeat units, each of approximately 60 amino acid residues, and a carboxyl-terminal nonrepeat region. One bovine beta-chain clone was found and characterized. It predicted a mature bovine beta-chain of 181 amino acid residues. The identity with the human beta-chain was 65% at the amino acid level. A noteworthy difference between bovine and human beta-chains was that the bovine beta-chain only contained two short consensus repeats compared with three in human beta-chain. Sequence alignment indicates that the region corresponding to residues 1-60 (repeat 1) in the human beta-chain is absent in the homologous bovine polypeptide. Because the short consensus repeats of the human beta-chain contain the binding site for protein S, the lack of one repeat unit in the bovine beta-chain may provide a clue to the lack of complex formation between C4BP and protein S in bovine plasma.

Amino Acid Sequence↗
PubMed1993

The human C4b-binding protein beta-chain gene.

Human complement component C4b-binding protein (C4BP) is composed of seven alpha-chains and one beta-chain. The alpha- and beta-chains are homologous and both contain multiple copies of short consensus repeats (SCR) and in addition carboxyl-terminal non-repeat regions. Each of the alpha-chains contains a binding site for C4b, whereas the beta-chain binds protein S, a vitamin K-dependent protein involved in the regulation of blood coagulation. The alpha- and beta-chain genes are closely linked in the regulators of complement activation gene cluster on the long arm of human chromosome 1, band 1q32. The human beta-chain gene which has now been characterized was found to span more than 10 kilobases of DNA. The presence of at least two different beta-chain gene transcripts was suggested by the isolation of two new cDNA clones which contained different sequences in their extended 5'-untranslated regions. Northern blot analysis demonstrated that the two clones represented distinct beta-chain mRNAs with different 5' end sequences. One class of beta-chain mRNA (denoted A19) was found to be encoded by six exons and primer extension, and S1 nuclease protection assays revealed multiple closely spaced transcription start sites for this mRNA class. Its 5'-untranslated region and signal peptide was encoded by the first exon. The second class of mRNA (denoted A12) had a different transcription start site and its 5'-untranslated region was derived from at least three exons out of which the last one was formed by utilization of an acceptor splice site within the first A19 exon. Exons encoding the mature beta-chain and the 3'-untranslated region were common to both classes of mRNA. The beta-chain contains three SCRs, out of which the first and second are encoded by individual exons, whereas two exons encode the third SCR. The exon encoding the carboxyl-terminal part of the third SCR also encodes 14 amino acids of the non-repeat region. The last exon encodes the remaining 46 carboxyl-terminal amino acids and the entire 3'-untranslated region. The elucidation of the organization of the beta-chain gene provides insight into the sophisticated molecular structure of C4BP and a basis for future structural and functional studies.

Amino Acid Sequence↗
PubMed1991

Molecular analysis of the beta-chain of human C4b-binding protein.

C4b-binding protein (C4BP) is a multimeric glycoprotein in plasma with important regulatory functions in the complement system. It occurs in two forms, as free protein and in a non-covalent bimolecular complex with the vitamin K-dependent protein S. Protein S is an important anticoagulant and enhances the rate of inactivation by activated protein C of blood coagulation factors, Va and VIIIa. Protein S bound to C4BP is inactive as an anticoagulant, indicating C4BP to have a regulatory function in the blood coagulation process. Approximately 50% of C4BP in plasma circulates in complex with protein S, but little has been known about as to how these proteins interact. This report describes the structure of C4BP and its relation to protein S binding. A novel C4BP subunit, designated the beta-chain, which in all likelihood contains the protein S binding site, has been identified, isolated and characterized. The major form of C4BP is composed of seven alpha-chains and one beta-chain, and the subunits are covalently linked by their carboxy-terminal regions giving the molecule a spider-like quaternary structure. A subpopulation of C4BP, which does not bind protein S, was found to lack the beta-chain. This provides support for the concept that the single protein S binding site is located on the beta-chain. The beta-chain is structurally related to the alpha-chain of C4BP, and both subunits belong to the superfamily of C3b/C4b-binding proteins. The genes coding for the alpha- and beta-chains of C4BP were found to be closely linked within a cluster of genes, coding for structurally related proteins, on the long arm of human chromosome 1.

Amino Acid Sequence↗
PubMed1990

Assembly of protein S and C4b-binding protein on membranes.

The interaction of protein S with membranes and subsequent combination with complement C4b-binding protein (C4BP) was studied. Protein S interacted with phospholipid vesicles in a calcium-dependent manner typical of other vitamin K-dependent proteins. Association of C4BP with protein S showed no apparent selectivity for membrane-bound or solution phase protein S. When bound to the membrane, the protein complexes projected out from the vesicle surface and induced vesicle radius changes of 11.4 nm for tightly packed protein S alone and 17.5 nm for the protein S-C4BP complex. Due to a low density of the protein S-C4BP on the membrane at saturation, the actual projection of this complex out from the membrane surface would be much greater than 17.5 nm. A low saturation density suggested that the protein complex had a large two-dimensional hydrodynamic radius in the plane of the membrane that prevented tight packing of protein. In the presence of calcium, the protein-protein interaction was rapid (ka greater than or equal to 1.10(6) M-1 s-1) and had very high affinity (KD less than or equal to 10(-10) M). The dissociation rate was slow with an estimated rate constant of less than or equal to 2.10(-4) s-1 at 25 degrees C. Protein-protein interaction was much slower in the absence of calcium with an estimated association rate constant of only 2.10(4) M-1 s-1. Consequently, the protein-protein interaction was greatly enhanced by calcium. The very high affinity interaction between protein S and C4BP suggested specificity and an important function for the protein S-C4BP complex in blood. In this regard it was important that C4BP which was bound to protein S on the phospholipid surface could interact with complement protein C4b. These results suggested that protein S may serve an important role in localizing C4BP to negatively charged phospholipid. This would provide regulation of complement activation at sites where the coagulation system is activated such as on the surface of activated platelets.

Animals↗
PubMed1990

Genes for C4b-binding protein alpha- and beta-chains (C4BPA and C4BPB) are located on chromosome 1, band 1q32, in humans and on chromosome 13 in rats.

C4b-binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical alpha-chains and a single copy of a unique beta-chain. The latter was identified only recently and its structure determined by cDNA cloning. Both subunits in C4b-binding protein belong to the same superfamily of proteins composed predominantly of tandemly arranged short consensus repeats (SCR) approximately 60 amino acid residues in length. The gene for the human alpha-chain is known to be located in a gene cluster on chromosome 1, band 1q32, which is called the regulators of complement activation (RCA) gene cluster. We have used cDNA probes for both alpha- and beta-chains of human C4b-binding protein to localize their genes with an in situ hybridization technique. We find the genes for both chains to be located on chromosome 1, band 1q32, in the human. This suggests that the beta-chain gene is also a member of the RCA gene cluster and that the alpha- and beta-chain genes are located close to each other. The cDNA probes for the alpha- and beta-chains also were used to screen mouse-rat somatic cell hybrids using Southern blotting to localize their genes in the rat. Both the alpha- and beta-chain genes were shown to be located on chromosome 13 in the rat. These are the second and third genes to be located on rat chromosome 13, and the results suggest that the genes for the alpha- and beta-chains together with the gene for coagulation factor V represent a conserved chromosomal region in rat and man.

Animals↗