Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization.

BACKGROUND: Lipoprotein(a) [Lp(a)] level variability, related to atherothrombotic risk increase, is mainly attributed to LPA gene, encoding apolipoprotein(a), with kringle IV type 2 (KIV2) copy number variation (CNV) acting as the primary genetic determinant. Genetic characterization of Lp(a) is in continuous growth; nevertheless, the peculiar structural characteristics of this variant constitute a significant challenge to the development of effective detection methods. The aim of the study was to compare quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR) in the evaluation of KIV2 repeat polymorphism. METHODS: We analysed 100 subjects tested for cardiovascular risk in which Lp(a) plasma levels were assessed. RESULTS: Correlation analysis between CNV values obtained with the two methods was slightly significant (R = 0.413, p = 0.00002), because of the wider data dispersion in qPCR compared with ddPCR. Internal controls C1, C2 and C3 measurements throughout different experimental sessions revealed the superior stability of ddPCR, which was supported by a reduced intra/inter-assay coefficient of variation determined in this method compared to qPCR. A significant inverse correlation between Lp(a) levels and CNV values was confirmed for both techniques, but it was higher when evaluated by ddPCR than qPCR (R = -0.393, p = 0.000053 vs R = -0.220, p = 0.028, respectively). When dividing subjects into two groups according to 500 mg/L Lp(a) cut-off value, a significantly lower number of KIV2 repeats emerged among subjects with greater Lp(a) levels, with stronger evidence in ddPCR than in qPCR (p = 0.000013 and p = 0.001, respectively). CONCLUSIONS: Data obtained support a better performance of ddPCR in the evaluation of KIV2 repeat polymorphism.

The kringle IV type 2 domain variant 4925G>A causes the elusive association signal of the LPA pentanucleotide repeat.

Lipoprotein(a) [Lp(a)] concentrations are regulated by the LPA gene mainly via the large kringle IV-type 2 (KIV-2) copy number variation and multiple causal variants. Early studies suggested an effect of long pentanucleotide repeat (PNR) alleles (10 and 11 repeats, PNR10 and PNR11) in the LPA promoter on gene transcription and found an association with lower Lp(a). Subsequent in vitro studies showed no effects on mRNA transcription, but the association with strongly decreased Lp(a) remained consistent. We investigated the isolated and combined effect of PNR10, PNR11, and the frequent splice site variant KIV-2 4925G>A on Lp(a) concentrations in the Cooperative Health Research in the Region of Augsburg F4 study by multiple quantile regression in single-SNP and joint models. Data on Lp(a), apolipoprotein(a) Western blot isoforms, and variant genotypes were available for 2,858 individuals. We found a considerable linkage disequilibrium between KIV-2 4925G>A and the alleles PNR10 and PNR11. In single-variant analysis adjusted for age, sex, and the shorter apo(a) isoform, we determined that both PNR alleles were associated with a highly significant Lp(a) decrease (PNR10: ß = -14.43 mg/dl, 95% CI: -15.84, -13.02, P = 3.33e-84; PNR11: ß = -17.21 mg/dl, 95% CI: -20.19, -14.23, P = 4.01e-29). However, a joint model, adjusting the PNR alleles additionally for 4925G>A, abolished the effect on Lp(a) (PNR10: ß = +0.44 mg/dl, 95% CI: -1.73, 2.60, P = 0.69; PNR11: ß = -1.52 mg/dl, 95% CI: -6.05, 3.00, P = 0.51). Collectively, we conclude that the previously reported Lp(a) decrease observed in pentanucleotide alleles PNR10 or PNR11 carriers results from a linkage disequilibrium with the frequent splicing mutation KIV-2 4925G>A.

Lipoprotein(a) beyond the kringle IV repeat polymorphism: The complexity of genetic variation in the LPA gene.

High lipoprotein(a) [Lp(a)] concentrations are one of the most important genetically determined risk factors for cardiovascular disease. Lp(a) concentrations are an enigmatic trait largely controlled by one single gene (LPA) that contains a complex interplay of several genetic elements with many surprising effects discussed in this review. A hypervariable coding copy number variation (the kringle IV type-2 repeat, KIV-2) generates >40 apolipoprotein(a) protein isoforms and determines the median Lp(a) concentrations. Carriers of small isoforms with up to 22 kringle IV domains have median Lp(a) concentrations up to 5 times higher than those with large isoforms (>22 kringle IV domains). The effect of the apo(a) isoforms are, however, modified by many functional single nucleotide polymorphisms (SNPs) distributed over the complete range of allele frequencies (<0.1% to >20%) with very pronounced effects on Lp(a) concentrations. A complex interaction is present between the apo(a) isoforms and LPA SNPs, with isoforms partially masking the effect of functional SNPs and, vice versa, SNPs lowering the Lp(a) concentrations of affected isoforms. This picture is further complicated by SNP-SNP interactions, a poorly understood role of other polymorphisms such as short tandem repeats and linkage structures that are poorly captured by common R2 values. A further layer of complexity derives from recent findings that several functional SNPs are located in the KIV-2 repeat and are thus not accessible to conventional sequencing and genotyping technologies. A critical impact of the ancestry on correlation structures and baseline Lp(a) values becomes increasingly evident. This review provides a comprehensive overview on the complex genetic architecture of the Lp(a) concentrations in plasma, a field that has made tremendous progress with the introduction of new technologies. Understanding the genetics of Lp(a) might be a key to many mysteries of Lp(a) and booster new ideas on the metabolism of Lp(a) and possible interventional targets.

Apo(a) and ApoB Interact Noncovalently Within Hepatocytes: Implications for Regulation of Lp(a) Levels by Modulation of ApoB Secretion.

BACKGROUND: Elevated plasma Lp(a) (lipoprotein(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease and aortic valve stenosis. However, the cell biology of Lp(a) biosynthesis remains poorly understood, with the locations of the noncovalent and covalent steps of Lp(a) assembly unclear and the nature of the apoB-containing particle destined for Lp(a) unknown. We, therefore, asked if apo(a) and apoB interact noncovalently within hepatocytes and if this impacts Lp(a) biosynthesis. METHODS: Using human hepatocellular carcinoma cells expressing 17K (17 kringle) apo(a), or a 17KΔLBS7,8 variant with a reduced ability to bind noncovalently to apoB, we performed coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays to document intracellular apo(a):apoB interactions. We used a pulse-chase metabolic labeling approach to measure apo(a) and apoB secretion rates. RESULTS: Noncovalent complexes containing apo(a)/apoB are present in lysates from cells expressing 17K but not 17KΔLBS7,8, whereas covalent apo(a)/apoB complexes are absent from lysates. 17K and apoB colocalized intracellularly, overlapping with staining for markers of endoplasmic reticulum trans-Golgi, and early endosomes, and less so with lysosomes. The 17KΔLBS7,8 had lower colocalization with apoB. Proximity ligation assays directly documented intracellular 17K/apoB interactions, which were dramatically reduced for 17KΔLBS7,8. Treatment of cells with PCSK9 (proprotein convertase subtilisin/kexin type 9) enhanced, and lomitapide reduced, apo(a) secretion in a manner dependent on the noncovalent interaction between apo(a) and apoB. Apo(a) secretion was also reduced by siRNA-mediated knockdown of APOB. CONCLUSIONS: Our findings explain the coupling of apo(a) and Lp(a)-apoB production observed in human metabolic studies using stable isotopes as well as the ability of agents that inhibit apoB biosynthesis to lower Lp(a) levels.

Frequent LPA KIV-2 Variants Lower Lipoprotein(a) Concentrations and Protect Against Coronary Artery Disease.

BACKGROUND: Lipoprotein(a) (Lp(a)) concentrations are a major independent risk factor for coronary artery disease (CAD) and are mainly determined by variation in LPA. Up to 70% of the LPA coding sequence is located in the hypervariable kringle IV type 2 (KIV-2) region. It is hardly accessible by conventional technologies, but may contain functional variants. OBJECTIVES: This study sought to investigate the new, very frequent splicing variant KIV-2 4733G>A on Lp(a) and CAD. METHODS: We genotyped 4733G>A in the GCKD (German Chronic Kidney Disease) study (n = 4,673) by allele-specific polymerase chain reaction, performed minigene assays, identified proxy single nucleotide polymorphisms and used them to characterize its effect on CAD by survival analysis in UK Biobank (n = 440,234). Frequencies in ethnic groups were assessed in the 1000 Genomes Project. RESULTS: The 4733G>A variant (38.2% carrier frequency) was found in most isoform sizes. It reduces allelic expression without abolishing protein production, lowers Lp(a) by 13.6 mg/dL (95% CI: 12.5-14.7; P < 0.0001) and is the strongest variance-explaining factor after the smaller isoform. Splicing of minigenes was modified. Compound heterozygosity (4.6% of the population) for 4733G>A and 4925G>A, another KIV-2 splicing mutation, reduces Lp(a) by 31.8 mg/dL and most importantly narrows the interquartile range by 9-fold (from 42.1 to 4.6 mg/dL) when compared to the wild type. In UK Biobank 4733G>A alone and compound heterozygosity with 4925G>A reduced HR for CAD by 9% (95% CI: 7%-11%) and 12% (95% CI: 7%-16%) (both P < 0.001). Frequencies in ethnicities differ notably. CONCLUSIONS: Functional variants in the previously inaccessible LPA KIV-2 region cooperate in determining Lp(a) variance and CAD risk. Even a moderate but lifelong genetic Lp(a) reduction translates to a noticeable CAD risk reduction.

Genetic and nutritional factors determining circulating levels of lipoprotein(a): results of the "Montignoso Study".

Increasing evidence shows an association between high lipoprotein(a) [Lp(a)] levels and atherothrombotic diseases. Lp(a) trait is largely controlled by kringle-IV type 2 (KIV-2) size polymorphism in LPA gene, encoding for apo(a). Environmental factors are considered to determinate minor phenotypic variability in Lp(a) levels. In the present study, we investigated the possible gene-environment interaction between KIV-2 polymorphism and Mediterranean diet adherence or fish weekly intake in determining Lp(a) levels. We evaluated Lp(a), KIV-2 polymorphism, fish intake and Mediterranean diet adherence in 452 subjects [median age (range) 66 (46-80)years] from Montignoso Heart and Lung Project (MEHLP) population. In subjects with high KIV-2 repeats number, influence of Mediterranean diet adherence in reducing Lp(a) levels was observed (p = 0.049). No significant difference in subjects with low KIV-2 repeats according to diet was found. Moreover, in high-KIV-2-repeat subjects, we observed a trend towards influence of fish intake on reducing Lp(a) levels (p = 0.186). At multivariate linear regression analysis, high adherence to Mediterranean diet remains a significant and independent determinant of lower Lp(a) levels (ß = - 64.97, standard error = 26.55, p = 0.015). In conclusion, this study showed that only subjects with high KIV-2 repeats can take advantage to lower Lp(a) levels from correct nutritional habits and, in particular, from Mediterranean diet.

The constitutive high-affinity Met-binding site in the kringle domain is dispensable for the signalling activity of hepatocyte growth factor.

Activation of a tyrosine kinase receptor Met by hepatocyte growth factor (HGF) requires binding of proteolytically activated, two-chain (tc) HGF, but the biochemical detail of this ligand-receptor interaction specificity remains elusive because biologically inactive single chain (sc) HGF can also bind to Met with high affinity. We found that this proteolysis-independent Met binding can be eliminated by mutagenesis introduced in the kringle domain without losing the ability to bind and activate cellular Met receptor after proteolytic activation, arguing against this site's involvement in the physiological signalling. This non-signal producing Met-HGF interaction can also be eliminated by addition of a heparin mimetic sucrose octasulphate (SOS). By including SOS in the running buffer, we succeeded in detecting cleavage-dependent tcHGF-Met complex formation by size exclusion chromatography.

Role of lipoprotein (a) and LPA KIV2 repeat polymorphism in bicuspid aortic valve stenosis and calcification: a proof of concept study.

Hemodynamic valvular impairment is a frequent determinant of the natural history of bicuspid aortic valve (BAV). The role of elevated Lp(a) levels and LPA Kringle IV type 2 (KIV-2) size polymorphism in influencing aortic valve calcification and stenosis development in patients with tricuspid aortic valve was recognized. In this study, we investigate the association between Lp(a) and LPA KIV-2 repeat number, and the presence of calcification and stenosis in BAV patients. Sixty-nine patients [79.7% males; median age 45(30-53) yrs], consecutively referred to Center for Cardiovascular Diagnosis or Referral Center for Marfan syndrome or related disorders, AOU Careggi, from June to November 2014, were investigated. For each patient, clinical (ECG and echocardiography) and laboratory [Lp(a) (Immunoturbidimetric assay) and LPA KIV-2 repeat number (real-time PCR)] evaluation were performed. Patients were compared with 69 control subjects. No significant association between Lp(a) circulating levels and LPA KIV-2 repeat number and BAV was evidenced. Among BAV patients, significantly higher Lp(a) levels according to calcification degree were found [no calcifications:78(42-159) mg/L, mild/moderate: 134(69-189) mg/L; severe: 560(286-1511) mg/L, p = 0.008]. Conversely, lower LPA KIV-2 repeat numbers in subjects with more severe calcification degree were observed. Furthermore, higher Lp(a) levels in patients with aortic stenosis [214(67-501) mg/L vs 104(56-169) mg/L, p = 0.043] were also found. In conclusion, present data suggest the potential role for Lp(a) as a possible risk marker useful to stratify, among BAV patients, those with a higher chance to develop valvular calcifications and aortic stenosis.

A comprehensive map of single-base polymorphisms in the hypervariable LPA kringle IV type 2 copy number variation region.

Lipoprotein (a) [Lp(a)] concentrations are among the strongest genetic risk factors for cardiovascular disease and present pronounced interethnic and interindividual differences. Approximately 90% of Lp(a) variance is controlled by the LPA gene, which contains a 5.6-kb-large copy number variation [kringle IV type 2 (KIV-2) repeat] that generates >40 protein isoforms. Variants within the KIV-2 region are not called in common sequencing projects, leaving up to 70% of the LPA coding region currently unaddressed. To completely assess the variability in LPA, we developed a sequencing strategy for this region and report here the first map of genetic variation in the KIV-2 region, a comprehensively evaluated ultradeep sequencing protocol, and an easy-to-use variant analysis pipeline. We sequenced 123 Central-European individuals and reanalyzed public data of 2,504 individuals from 26 populations. We found 14 different loss-of-function and splice-site mutations, as well as >100, partially even common, missense variants. Some coding variants were frequent in one population but absent in others. This provides novel candidates to explain the large ethnic and individual differences in Lp(a) concentrations. Importantly, our approach and pipeline are also applicable to other similar copy number variable regions, allowing access to regions that are not captured by common genome sequencing.

Overexpressing kringle 1 domain of hepatocyte growth factor with adeno-associated virus inhibits the pathological retinal neovascularization in an oxygen-induced retinopathy mouse model.

Current clinical treatments for ocular neovascularization are characterized by high possibility of damaging healthy tissues and high recurrence rates. It is necessary to develop new treatment methods to control neovascularization with a stable and effective effect. Kringle1 domain of hepatocyte growth factor (HGFK1) has anti-angiogenesis activity. Here, we established oxygen-induced retinopathy (OIR) model to study if using adeno-associated virus (AAV) as a delivery system to overexpression HGFK1 in retinal cells could benefit retinal neovascularization. We show that, overexpressed exogenous gene was mainly expressed in the inner and outer nuclear layer of the retina. Compared with control mice, the mice pretreated with rAAV-HGFK1 at P3 showed relatively normal vascular branches examined by fluorescence fundus angiography. Subsequent H&E staining and immunohistochemical staining of CD31 of the eye tissue sections showed that the mice received rAAV-HGFK1 had a relatively normal distribution of vascular endothelial cells. Additionally, immunohistochemical staining indicated a lower expression of VEGF in the eye tissues of rAAV-HGFK1 treated OIR mice. Further in vitro studies showed that HGFK1 could inhibit the proliferation but promote the apoptosis of bovine retinal microvascular endothelial cells (BRECs) under the presence of VEGF. Moreover, HGFK1 could inhibit VEGF induced ERK activation but promote p38 activation in BRECs. Therefore, we propose that intravitreal injection of rAAV-HGFK1 might be used to improve the retinal neovascularization and HGFK1 may function through regulating VEGF signaling pathway to inhibit neovascularization.

Low LPA gene kringle IV-2 repeat copy number association with elevated lipoprotein (a) concentration as an independent risk factor of coronary atherosclerotic heart disease in the Chinese Han population.

BACKGROUND: Lipoprotein (a) [Lp(a)], which is genetically determined by the LPA gene kringle IV type 2 (KIV-2) repeat copy number, has previously been reported in different populations. However, it is uncertain if the same occurs in the Chinese Han population. This study explored the correlation of Lp(a) mass or particle concentration with KIV-2 repeat copy number and application for coronary atherosclerotic heart disease (CAHD) risk assessment. METHODS: A cross-sectional study including 884 subjects was conducted. The Lp(a) level and routine risk factors of CAHD were compared. The KIV-2 copy number distribution, relationship with Lp(a), and assessment for CAHD risk were explored. RESULTS: The mean of Lp(a) mass or particle concentration in the CAHD group was higher than that in the non-CAHD group, while the KIV-2 copy number in the CAHD group was lower. Lp(a) had auxiliary values in gauging the type of plaque and was significantly higher in the soft-plaque group than that in the other two groups (200 mg/L [21.5 nmol/L], 166 mg/L [18.6 nmol/L], 149 mg/L [17.1 nmol/L], respectively, P < 0.05). Kappa test indicated divergence for the same individual using two Lp(a) concentrations (kappa value was 0.536 [< 0.75]). Elevated Lp(a) was an independent CAHD risk factor, whatever mass or particle concentration, and large KIV-2 copy number was a protective factor. CONCLUSION: Lp(a) level and small KIV-2 copy number are risk factors for CAHD in the Chinese Han population; furthermore, elevated Lp(a) may gauge the type of coronary plaque.

A novel but frequent variant in LPA KIV-2 is associated with a pronounced Lp(a) and cardiovascular risk reduction.

AIMS: Lp(a) concentrations represent a major cardiovascular risk factor and are almost entirely controlled by one single locus (LPA). However, many genetic factors in LPA governing the enormous variance of Lp(a) levels are still unknown. Since up to 70% of the LPA coding sequence are located in a difficult to access hypervariable copy number variation named KIV-2, we hypothesized that it may contain novel functional variants with pronounced effects on Lp(a) concentrations. We performed a large scale mutation analysis in the KIV-2 using an extreme phenotype approach. METHODS AND RESULTS: We compiled an discovery set of 123 samples showing discordance between LPA isoform phenotype and Lp(a) concentrations and controls. Using ultra-deep sequencing, we identified a splice site variant (G4925A) in preferential association with the smaller LPA isoforms. Follow-up in a European general population (n = 2892) revealed an exceptionally high carrier frequency of 22.1% in the general population. The variant explains 20.6% of the Lp(a) variance in carriers of low molecular weight (LMW) apo(a) isoforms (P = 5.75e-38) and reduces Lp(a) concentrations by 31.3 mg/dL. Accordingly the odds ratio for cardiovascular disease was reduced from 1.39 [95% confidence interval (CI): 1.17-1.66, P = 1.89e-04] for wildtype LMW individuals to 1.19 [95%CI: 0.92; 1.56, P = 0.19] in LMW individuals who were additionally positive for G4925A. Functional studies point towards a reduction of splicing efficiency by this novel variant. CONCLUSION: A highly frequent but until now undetected variant in the LPA KIV-2 region is strongly associated with reduced Lp(a) concentrations and reduced cardiovascular risk in LMW individuals.

Mechanism of plasmin generation by S100A10.

Plasminogen (Pg) is cleaved to form plasmin by the action of specific plasminogen activators such as the tissue plasminogen activator (tPA). Although the interaction of tPA and Pg with the surface of the fibrin clot has been well characterised, their interaction with cell surface Pg receptors is poorly understood. S100A10 is a cell surface Pg receptor that plays a key role in cellular plasmin generation. In the present report, we have utilised domain-switched/deleted variants of tPA, truncated plasminogen variants and S100A10 site-directed mutant proteins to define the regions responsible for S100A10-dependent plasmin generation. In contrast to the established role of the finger domain of tPA in fibrin-stimulated plasmin generation, we show that the kringle-2 domain of tPA plays a key role in S100A10-dependent plasmin generation. The kringle-1 domain of plasminogen, indispensable for fibrin-binding, is also critical for S100A10-dependent plasmin generation. S100A10 retains activity after substitution or deletion of the carboxyl-terminal lysine suggesting that internal lysine residues contribute to its plasmin generating activity. These studies define a new paradigm for plasminogen activation by the plasminogen receptor, S100A10.

Lipoprotein (a): impact by ethnicity and environmental and medical conditions.

Levels of lipoprotein (a) [Lp(a)], a complex between an LDL-like lipid moiety containing one copy of apoB, and apo(a), a plasminogen-derived carbohydrate-rich hydrophilic protein, are primarily genetically regulated. Although stable intra-individually, Lp(a) levels have a skewed distribution inter-individually and are strongly impacted by a size polymorphism of the LPA gene, resulting in a variable number of kringle IV (KIV) units, a key motif of apo(a). The variation in KIV units is a strong predictor of plasma Lp(a) levels resulting in stable plasma levels across the lifespan. Studies have demonstrated pronounced differences across ethnicities with regard to Lp(a) levels and some of this difference, but not all of it, can be explained by genetic variations across ethnic groups. Increasing evidence suggests that age, sex, and hormonal impact may have a modest modulatory influence on Lp(a) levels. Among clinical conditions, Lp(a) levels are reported to be affected by kidney and liver diseases.

Elevated Lipoprotein(a) Levels, LPA Risk Genotypes, and Increased Risk of Heart Failure in the General Population.

OBJECTIVES: This study sough to test whether elevated lipoprotein(a) levels and corresponding LPA risk genotypes (low number of kringle IV type 2 repeats, rs3798220 and rs10455872, minor allele carriers) are associated with an increased risk of heart failure (HF). BACKGROUND: Elevated lipoprotein(a) levels represent a genetically determined risk factor for myocardial infarction (MI) and aortic valve stenosis (AVS). It is presently unknown whether elevated lipoprotein(a) levels also cause heart failure (HF). METHODS: We combined 2 general population studies, the Copenhagen City Heart Study (n = 10,855) and the Copenhagen General Population Study (n = 87,242), which totaled 98,097 Danish participants, of whom 4,122 were diagnosed with HF (1976 to 2013). We conducted observational and genetic instrumental variable analyses in a Mendelian randomization study design, assessing evidence of causality, and we performed mediation analyses. RESULTS: Elevated lipoprotein(a) levels were associated with multivariable adjusted hazard ratios for HF of 1.10 (95% CI: 0.97 to 1.25) for the 34th to 66th percentiles (8 to 19 mg/dl), 1.24 (95% CI: 1.08 to 1.42) for the 67th to 90th percentiles (20 to 67 mg/dl), 1.57 (95% CI: 1.32 to 1.87) for the 91st to 99th percentiles (68 to 153 mg/dl), and 1.79 (95% CI: 1.18 to 2.73) for levels >99th percentile (>153 mg/dl) versus levels <34th percentile (<8 mg/dl) (trend, p < 0.001), corresponding to a population-attributable risk of 9%. By combining all LPA risk genotypes, instrumental variable analysis yielded a genetic relative risk for HF of 1.18 (95% CI: 1.04 to 1.34) per 10-fold higher lipoprotein(a) levels, which was comparable to the corresponding observational hazard ratio of 1.22 (95% CI: 1.11 to 1.35). Upon exclusion of participants diagnosed with MI or AVS, risk estimates were attenuated. Accordingly, 63% (95% CI: 45% to 99%) of HF risk was mediated via MI and AVS combined. CONCLUSIONS: Elevated lipoprotein(a) levels and corresponding LPA risk genotypes were associated with an increased risk of HF consistent with a causal association. The association appeared to be partly mediated by MI and AVS.

Sequence variation within the KIV-2 copy number polymorphism of the human LPA gene in African, Asian, and European populations.

Amazingly little sequence variation is reported for the kringle IV 2 copy number variation (KIV 2 CNV) in the human LPA gene. Apart from whole genome sequencing projects, this region has only been analyzed in some detail in samples of European populations. We have performed a systematic resequencing study of the exonic and flanking intron regions within the KIV 2 CNV in 90 alleles from Asian, European, and four different African populations. Alleles have been separated according to their CNV length by pulsed field gel electrophoresis prior to unbiased specific PCR amplification of the target regions. These amplicons covered all KIV 2 copies of an individual allele simultaneously. In addition, cloned amplicons from genomic DNA of an African individual were sequenced. Our data suggest that sequence variation in this genomic region may be higher than previously appreciated. Detection probability of variants appeared to depend on the KIV 2 copy number of the analyzed DNA and on the proportion of copies carrying the variant. Asians had a high frequency of so-called KIV 2 type B and type C (together 70% of alleles), which differ by three or two synonymous substitutions respectively from the reference type A. This is most likely explained by the strong bottleneck suggested to have occurred when modern humans migrated to East Asia. A higher frequency of variable sites was detected in the Africans. In particular, two previously unreported splice site variants were found. One was associated with non-detectable Lp(a). The other was observed at high population frequencies (10% to 40%). Like the KIV 2 type B and C variants, this latter variant was also found in a high proportion of KIV 2 repeats in the affected alleles and in alleles differing in copy numbers. Our findings may have implications for the interpretation of SNP analyses in other repetitive loci of the human genome.

The linker connecting the two kringles plays a key role in prothrombin activation.

The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146-167.

High-level production of Fc-fused kringle domain in Pichia pastoris.

Recently, as a new non-immunoglobulin-based protein scaffold, a human kringle domain was successfully engineered toward biologically functional agonists and antagonists. In this study, the fed-batch cultivation conditions were optimized for enhanced production of an Fc-fused kringle domain (KD548-Fc) in Pichia pastoris. Fed-batch cultivations were performed in 5-l laboratory-scale bioreactors, and in order to find the optimal conditions for high-level production of KD548-Fc, several parameters including the initial carbon source (glycerol) concentration, temperature, and pH were investigated. When cells were cultivated at pH 4.0 and 25 °C with 9.5 % glycerol in the initial medium, the highest production yield (635 mg/l) was achieved with high productivity (7.2 mg/l/h). Furthermore, functional KD548-Fc was successfully purified from the culture broth using a simple purification procedure with high purity and recovery yield.

Cloning and expression of hybrid streptokinase towards clot-specific activity.

Streptokinase (SK) is a thrombolytic agent that is widely used to treat myocardial infarction and pulmonary embolism. The lack of fibrin specificity of SK for the clot lysis is one of the limitations of SK. In this study, we have incorporated the finger and Kringle 2 domains from the human tissue type plasminogen activator gene (t-PA) at the 5' end of the SK gene. These domains are responsible for specific binding to fibrin. We have used the pRSETB vector in an attempt to express the hybrid streptokinase possessing specificity for fibrin. On this regard, three hybrid streptokinase were constructed and expressed in Escherichia coli BL21 (DE3): the finger domain with SK (FSK), the Kringle 2 domain with SK (KSK) and the finger domain+Kringle 2 with SK (FKSK). The activities of the hybrid SKs were assessed by caseinolytic assay and clot lysis assay. All hybrid SKs were found to activate plasminogen in the caseinolytic plate assay. In the clot lysis assay, KSK and FSK were able to dissolute human blood and artificial clots in a fibrin-dependent manner unlike the SK and FKSK proteins.

Testing for apolipoprotein(a) phenotype using isoelectric focusing and immunoblotting technique.

Increased levels of lipoprotein(a) are known as an independent risk factor for atherosclerosis, heart disease, and stroke in man. Even in children it could show that elevated levels of Lp(a) are an independent thromboembolic risk factor. Levels of Lp(a) are influenced by several factors like nutrition, kidney or liver function, or acute-phase reaction. But the most important factors are genetically determined. About 45% of genetic variation depends on polymorphisms and mutations in the promotor region. About 50% are dependent on the size polymorphism of Lp(a). The number of Kringle 4 domains varies between 12 and over 40. The number of Kringle 4 repeats correlates negatively with the level of Lp(a) in plasma. The determination of apo(a) phenotype is able to estimate thromboembolic risk due to this risk factor.