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Biomedical subjects

B N Bouma

Publications and source records attributed to B N Bouma.

At least 37 records · Page 2Linked to original sources
PubMed2001

The role of protein S in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) and regulation of fibrinolysis.

Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxy-peptidase B-like proenzyme that after activation by thrombin downregulates fibrinolysis. Thrombomodulin stimulates the activation of both TAFI and protein C whereas activated protein C inhibits the activation of TAFI by downregulation of thrombin formation, a process in which protein S acts as a cofactor. Here we determined the role of protein S in the activation of TAFI and regulation of fibrinolysis. Depletion of protein S from plasma or inhibition of protein S by specific antibodies resulted in an increased rate of TAFI activation and in an increased maximum of TAFIa activity generated. The effect on the rate of TAFI activation could be attributed to the APC-independent anticoagulant function of protein S whereas the effect on the maximum activity could be attributed to the APC cofactor function of protein S. Therefore it is concluded that protein S inhibits TAFI activation in two ways. On one hand, protein S functions as a cofactor for APC which results in a reduction of the maximum induced TAFI activity and on the other hand protein S inhibits the initial thrombin formation independently of APC which results in a decreased rate of TAFI activation. The effect of the APC-independent anticoagulant activity of protein S on the activation of TAFI provides a new mechanism for the regulation of fibrinolysis in the early stages of clot formation.

Activated Protein C Resistance↗
PubMed2001

Protein C inhibitor regulates the thrombin-thrombomodulin complex in the up- and down regulation of TAFI activation.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a carboxy-peptidase B-like proenzyme that after activation by thrombin down regulates fibrinolysis. Thrombomodulin (TM) stimulates the activation of both TAFI and protein C whereas activated protein C (APC) inhibits the activation of TAFI by down regulating thrombin generation. Recently, protein C inhibitor (PCI) was identified as a potent inhibitor of thrombin bound to TM and it can thereby regulate the balance between TAFI activation, and inhibition of TAFI activation by APC. Both in a purified system and in plasma, activation of TAFI and protein C by [Ia-TM could be inhibited by PCI. Previously we found in plasma that at low concentrations (approximately 1 nM), TM predominantly stimulated the activation of TAFI whereas at higher concentrations of TM (approximately 10 nM) the activation of protein C resulted in inhibition of the activation of TAFI. In agreement with this. PCI inhibited the activation of TAFI at 1 nM TM whereas at 10 nM TM PCI inhibited the activation of protein C resulting in an increase in the activation of TAFI. This suggests that PCI can up regulate TAFI activation by inhibiting the protein C activation. PCI may therefore be an important regulator in the balance between coagulation and fibrinolysis by differentially inhibiting the activation of TAFI and of protein C. The local TM concentration plays an important role in the outcome of this process.

Blood Coagulation↗
PubMed2000

C4b-binding protein protects coagulation factor Va from inactivation by activated protein C.

We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.

Animals↗
PubMed2000

Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage.

Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperature-dependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg(302). Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg(302).

Carboxypeptidase B2↗
PubMed2000

High levels of coagulation factor XI as a risk factor for venous thrombosis.

BACKGROUND: Factor XI, a component of the intrinsic pathway of coagulation, contributes to the generation of thrombin, which is involved in both the formation of fibrin and protection against fibrinolysis. A deficiency of factor XI is associated with bleeding, but a role of high factor XI levels in thrombosis has not been investigated. METHODS: We determined factor XI antigen levels in the patients enrolled in the Leiden Thrombophilia Study, a large population-based, case-control study (with a total of 474 patients and 474 controls) designed to estimate the contributions of genetic and acquired factors to the risk of deep venous thrombosis. Odds ratios were calculated as a measure of relative risk. RESULTS: The age- and sex-adjusted odds ratio for deep venous thrombosis in subjects who had factor XI levels above the 90th percentile, as compared with those who had factor XI levels at or below that value, was 2.2 (95 percent confidence interval, 1.5 to 3.2). There was a dose-response relation between the factor XI level and the risk of venous thrombosis. Adjustment of the odds ratios for other risk factors such as oral-contraceptive use, homocysteine, fibrinogen, factor VIII, female sex, and older age did not alter the result. Also, when we excluded subjects who had known genetic risk factors for thrombosis (e.g., protein C or S deficiency, antithrombin deficiency, the factor V Leiden mutation, or the prothrombin G20210A mutation), the odds ratio remained the same, indicating that the risk of venous thrombosis associated with elevated levels of factor XI was not the result of one of the known risk factors for thrombosis. CONCLUSIONS: High levels of factor XI are a risk factor for deep venous thrombosis, with a doubling of the risk at levels that are present in 10 percent of the population.

Adolescent↗
PubMed2000

The interaction between anticoagulant protein S and complement regulatory C4b-binding protein (C4BP).

An important mechanism of regulation of blood coagulation is the anticoagulant protein C pathway. In this pathway, the anticoagulant activity of activated protein C is increased by its cofactor protein S. The cofactor activity of protein S can be regulated by binding to complement regulatory C4b-binding protein (C4BP). The sites of interaction of protein S and C4BP are discussed.

Animals↗
PubMed2000

Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is a disease that is distinguished from other leukaemias by the high potential for early haemorrhagic death. Several processes are involved, such as disseminated intravascular coagulation and hyperfibrinolysis. Recently, TAFI (thrombin-activatable fibrinolysis inhibitor) was identified as a link between coagulation and fibrinolysis. TAFI can be activated by thrombin, and in its activated form potently attenuates fibrinolysis by removing C-terminal lysine and arginine residues that are important for the binding and activation of plasminogen. Activation of TAFI by the coagulation system results in a down-regulation of fibrinolytic activity and, thereby, prevents a rapid dissolution of the fibrin clot. To establish whether TAFI was involved in the severity of the bleeding complications in APL, the TAFI antigen and activity levels were determined in a group of 15 patients. The TAFI antigen concentration was normal, but the activity of TAFI was severely reduced in APL by approximately 60%. The reduction of TAFI activity was most probably caused by the action of plasmin on TAFI because in vitro experiments revealed that plasmin slightly reduced antigen levels but severely reduced TAFI activity. The acquired functional TAFI deficiency in APL may contribute to the severity of the haemorrhagic diathesis because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis.

Adult↗
PubMed2000

Role of blood coagulation factor XI in downregulation of fibrinolysis.

Factor XI is a component of the intrinsic pathway of coagulation. A deficiency of factor XI is associated with a mild to moderate bleeding disorder especially from tissues with a high local fibrinolytic activity. In contrast, high levels of factor XI are a risk factor for venous thrombosis. The recent finding that factor XI can be activated by thrombin led to a revised model of coagulation. In this model the primary thrombin generation that results in fibrin formation takes place via the extrinsic pathway. Additional thrombin generation takes place inside the fibrin clot via the intrinsic pathway after the activation of factor XI by thrombin. High concentrations of thrombin are formed that are necessary for the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Activated TAFI protects the fibrin clot against lysis. The role of factor XI in hemostasis can therefore be seen as a combination of procoagulant and antifibrinolytic actions. The new insights in the role of factor XI in coagulation and fibrinolysis may lead to new strategies for the treatment of thrombotic disorders.

Animals↗
PubMed2000

Aging, physical conditioning, and exercise-induced changes in hemostatic factors and reaction products.

The influence of age on training-induced changes in resting and stimulated hemostatic potential was studied in three age categories (Cat I-III; 20-30 yr, 35-45 yr, and 50-60 yr, respectively) of sedentary men before and after 12 wk of training. Coagulation, fibrinolytic activity, and activation markers (reflecting fibrin formation and degradation) were determined. Physical conditioning resulted in a more pronounced increase in von Willebrand factor (vWF) and factor VIII clotting activity (FVIII:c) in Cat I and II and a more pronounced shortening of the activated partial thromboplastin time in all categories at maximal exertion and during recovery. Enhanced increases in tissue-type plasminogen activator (t-PA) antigen and activity and single-chain (sc) urokinase-type plasminogen activator (u-PA) at maximal exercise and 5 min of recovery were observed in all age groups after training. The effects on FVIII:c, vWF, and scu-PA were most pronounced in the youngest age group (Cat I). Increases in the marker of thrombin generation were highest in Cat III; no effect was seen on thrombin-antithrombin complex, plasmin-antiplasmin complex, and D-dimer in any of the age groups. We concluded that training enhances both coagulation and fibrinolytic potential during strenuous exercise. The effect on FVIII/vWF and t-PA/u-PA is most pronounced in younger individuals, whereas thrombin formation is most pronounced in older individuals.

Adult↗
PubMed2000

Effects on coagulation of levonorgestrel- and desogestrel-containing low dose oral contraceptives: a cross-over study.

Combined oral contraceptives (OC) are known to increase the risk of venous thromboembolism. The aim of this randomized, cycle-controlled, cross-over study in 28 healthy volunteers was to assess potential differences between the effects of an OC containing 150 microg levonorgestrel (as representative of the so-called second generation OC) and an OC containing 150 microg desogestrel (as representative of the third generation OC) in combination with 30 microg ethinylestradiol on several coagulation factors and markers of thrombin formation. All participants used each OC for two cycles, and were switched to the other OC after a washout period of two menstrual cycles. The plasma concentrations of factors II, VII, X, and fibrinogen significantly increased during use of both the levonorgestrel- and desogestrel-containing OC's. The plasma concentrations of factor VIII increased, and of factor V decreased, changes which only reached statistical significance during the use of the desogestrel-containing OC. During exposure to the desogestrel-containing OC, as compared with the levonorgestrel-containing OC, both factor VII and factor II showed a greater increase (FVII: 32% and 12% respectively; p <0.0001; FII: 16% and 12% respectively; p = 0.048), whereas factor V showed a greater decrease (-11% and -3% respectively; p = 0.010). Only one of the markers for ongoing coagulation (prothrombin fragment 1+2) showed a significant increase during OC use, whereas concentrations of thrombin-antithrombin complexes and soluble fibrin remained unchanged. For these markers, there was no difference between the tested OC's. We conclude that there are differences between the effects of levonorgestrel and desogestrel-containing OC's on some coagulation factors. Whether these changes provide a biological explanation for the reported differences in venous thromboembolic risk is as yet unclear. The real challenge now becomes to define a pattern of changes in the various systems which, if affected simultaneously, may tip the hemostatic balance towards a prethrombotic state and may lead to overt clinical venous thromboembolism.

Adolescent↗
PubMed2000

Increased fibrinolytic activity during use of oral contraceptives is counteracted by an enhanced factor XI-independent down regulation of fibrinolysis: a randomized cross-over study of two low-dose oral contraceptives.

The effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 microg ethinylestradiol, 150 microg levonorgestrel) or third generation OC (30 microg ethinylestradiol, 150 microg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-alpha2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor- (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.

Adolescent↗
PubMed2000

A randomized cross-over study on the effects of levonorgestrel- and desogestrel-containing oral contraceptives on the anticoagulant pathways.

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cycle-controlled randomized cross-over study, in which we determined the effects of so-called second and third generation OC's on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of alpha2-M, alpha1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETP-based (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.

Activated Protein C Resistance↗
PubMed1999

Low-dose oral contraceptives and acquired resistance to activated protein C: a randomised cross-over study.

BACKGROUND: We have reported previously that, compared with use of second-generation oral contraceptives, the use of third-generation oral contraceptives is associated with increased resistance to the anticoagulant action of activated protein C (APC). Owing to the cross-sectional design of that study, these observations may have been subject to unknown bias or uncontrolled effects of the menstrual cycle. We aimed to overcome these sources of bias by doing a cycle-controlled randomised cross-over trial. METHODS: The response to APC in plasma was assessed in 33 women who received two consecutive cycles of a second-generation oral contraceptive (150 microg levonorgestrel and 30 microg ethinyloestradiol) or a third-generation oral contraceptive (150 microg desogestrel and 30 microg ethinyloestradiol), and who switched preparations after two pill-free cycles. Normalised APC sensitivity ratios were calculated by measurement of the effect of APC on thrombin generation in the plasma of these women and in pooled plasma from 90 controls. FINDINGS: Of the 33 women, five were excluded because not all required plasma samples were available. In the remaining 28 women, the normalised APC sensitivity ratio increased during treatment with both preparations. Compared with levonorgestrel, desogestrel-containing oral-contraceptive treatment caused a highly significant (p<0.0001) additional increase in normalised APC sensitivity ratio (0.51 [95% CI 0.37-0.66]). Normalised APC sensitivity ratios during oral-contraceptive treatment correlated with the values before oral-contraceptive use. INTERPRETATION: Oral-contraceptive treatment diminishes the efficacy with which APC down-regulates in-vitro thrombin formation. This phenomenon, designated as acquired APC resistance, is more pronounced in women using desogestrel-containing oral contraceptives than in women using levonorgestrel-containing preparations. Whether acquired APC resistance induced by oral contraceptives explains the increased risk of venous thromboembolism in oral-contraceptive users remains to be established.

Activated Protein C Resistance↗
PubMed1999

Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats.

Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.

Complement C4b↗
PubMed1999

The role of alcohol in the anti low density lipoprotein oxidation activity of red wine.

Oxidation of low density lipoprotein cholesterol (LDL-c) is supposed to play a role in the generation of atherosclerotic lesions. Grape derived beverages supply a large number of nutritional antioxidants because of their high content of polyphenols. This might be one of the mechanisms behind the supposed beneficial effect of red wine. Wine also contains alcohol and its role in oxidation processes especially in vivo is unclear. In this study the effect of daily red wine consumption for 2 weeks on oxidizability status of LDL was investigated. The role of alcohol in LDL oxidation was further explored in in vitro experiments. After abstinence from alcoholic beverages, grape juices and tea for a week, seven healthy male volunteers consumed 375 ml of red wine (30 g alcohol) per day during 2 weeks. At the start and end of the drinking period blood samples were taken and the susceptibility of LDL-c to copper-induced oxidation was analyzed with the addition of distilled water (control) and dilutions of a 12% alcohol solution, white wine and red wine. Although red wine at concentrations achievable in vivo caused a significant prolongation of the lag-time of metal ion dependent LDL oxidation in vitro (85.9+/-23.0-114.1+/-30.8 min, P<0. 001), a significant shortening of lag-time was found in vivo after the 2 weeks of wine consumption (56.3+/-13.0 min, P<0.001). A shorter lag-time compared to the control was found for both alcohol and white wine in vitro. The changed oxidizability status of LDL after 2 weeks of wine consumption made it more susceptible for the in vitro antioxidant effect of red wine. At low dilutions red grape juice extended lag-time as well, which was not influenced by the addition of alcohol. Red wine has a strong inhibitory effect on copper-induced oxidation of LDL in vitro, while red grape juice has a minor effect, an effect which should be attributed to the non alcohol components in the beverages. In vivo, however, this effect can be overshadowed by the prooxidant influence of alcohol. The balance between alcohol and polyphenols of a wine may be critical for its in vivo effect on LDL oxidation.

Adult↗
PubMed1999

Factor XI dependent and independent activation of thrombin activatable fibrinolysis inhibitor (TAFI) in plasma associated with clot formation.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as plasma procarboxypeptidase B is activated by relatively high concentrations of thrombin in a reaction stimulated by thrombomodulin. In plasma an intact factor XI-dependent feed back loop via the intrinsic pathway is necessary to generate sufficient thrombin for TAFI activation. This thrombin generation takes place after clot formation with consequent down-regulation of fibrinolysis. We developed a specific and sensitive assay for activated TAFI (TAFIa) and studied its factor XI-dependent generation during clot formation. In the absence of thrombomodulin, addition of 20 nM thrombin to normal plasma generated 5-10% of the amount of TAFIa generated by 20 nM thrombin in the presence of 8 nM thrombomodulin. Minimal activation of TAFI was detected in factor II deficient plasma when clotting was initiated by 20 nM thrombin. Addition of 320-640 nM of thrombin to factor II deficient plasma resulted in the same amount of TAFIa as in normal plasma, suggesting that approximately 50% of factor II has to be converted to thrombin for extensive activation of TAFI. A Mab that neutralizes activated factor XII had no effect on TAFI activation indicating that an intact contact system is not necessary for the activation of TAFI. The dependency of TAFI activation of factor XI was tested using a Mab that neutralizes activated factor XI. When plasmas from 13 healthy individuals were tested, this Mab reduced TAFI activation by 65% (range 35-89%). Our results indicate that activation of TAFI in serum after clot formation can be quantitated and that it takes place in both factor XI-dependent and factor XI-independent mechanisms.

Blood Coagulation↗
PubMed1999

The identification of polymorphisms in the coding region of the apolipoprotein (a) gene--association with earlier identified polymorphic sites and influence on the lipoprotein (a) concentration.

Lipoprotein (a) [Lp(a)] is a quantitative genetic trait in human plasma and elevated levels represent a major inherited risk factor for the development of atherosclerotic disease. In our search for sequence polymorphisms in the coding region of the apolipoprotein(a) [apo(a)] gene that may affect the Lp(a) concentration, four new polymorphic sites were identified. These include two coinciding polymorphisms with an allele frequency of 38% located at amino acid positions 87 and 101 (Leu87,101-->Val) in the interkringle region of kringle IV (K.IV) type 7 and two polymorphisms located in K.IV type 7 (Arg60-->Ser) and in K.IV type 10 (Tyr2-->Phe) both with estimated allele frequencies of about 1%. The linkage between the newly identified K.IV type 7 Leu87,101 -->Val polymorphism and earlier described polymorphic sites in the non-coding and coding regions of the apo(a) gene, its distribution over the apo(a) isoform sizes and its possible influence on the Lp(a) concentration was analysed in 201 healthy unrelated Caucasians. The earlier described polymorphic sites included in this study were the variable number of a TTTTA pentanucleotide repeat (7-11 PNR) starting at -1231 bp, the -772 bp G/A polymorphism, the +93 bp C/T polymorphism and the +121 bp G/A polymorphism in the non-coding region, and the K.IV type 8 Thr12/Pro polymorphism and the K.IV type 10 Thr66/Met polymorphism in the coding region of the apo(a) gene. Linkage disequilibria were observed between the polymorphic sites in the 5' non-coding region and the sites in K.IV type 7 and 8 in the coding region of the apo(a) gene, confirming that the expansion of the variable number of K.IV type 2 repeats results from intrachromosomal recombinational events. The distribution over the apo(a) isoform sizes of the K.IV type 7 Val87,101 subtype was not significantly different from that of the K.IV type 7 Leu87,101 wild-type, suggesting a relative ancient mutational event. No influence of the K.IV type 7 Leu87,101-->Val polymorphism on the Lp(a) level was observed. In fact, of all the polymorphic sites studied, only the +121 A subtype could be associated with an increased, and the K.IV type 8 Pro12 and the 10 PNR subtypes with a reduced, Lp(a) concentration corrected for apo(a) isoform size (p <0.05).

Alleles↗