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Interaction of lipoprotein(a) with the platelet LDL receptor
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Poster session abstracts / Atheroselerosis 115 (Suppl.) 11995) $45-S129 Pl l Lipoprotein (a)
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329
INTERACTION OF LIPOPROTEIN(a) WITH THE PLATELET LDL RECEPTOR J. Pedrefio and R. Fern:~ndez Dept Biochemistry, Hospital Son Dureta, Palma de Majorca, Spain
APOL1POPROTEIN(A) KpnI G E N O T Y P E IS A RISK F A C T O R FOR C O R O N A R Y H E A R T DISEASE IN D A N I S H M E N L. Lemming. I.C. Klausen, P.S. Hansen, L.U. Gerdes, O. F~ergeman Department of Internal Medicine and Cardiology A, Aarhus Amtssygehus University Hospital, 8000 Aarhus C
Recently, it has been described that Lp(a) binds to human intact platelets and further studies have showed that Lp(a) bound to the individual platelet GP liB. On the other hand, it is well kwnon that platelets contain a LDL receptor which is different from the classical LDL receptor, and the thductible fibrinogen receptor (GP liB-IliA complex) has been involved in the binding of LDL to platelets. However, there are not data about the interaction between Lp(a) and the platelet LDL receptor. In the present study we have investigated both the interaction of Lp(a) with the platelet LDL receptor and the implication of individual GP liB on platelet LDL binding. Under our experimental conditions, platelet LDL binding seem to be different compared with platelet Lp(a) binding. Displacement experiments showed that the ~I-LDL binding (0.0I g/I) to intact resting platelets was inhibited with the same apparent affinity by both unlabelled LDL and apo(a)-free lipoprotein particle [Lp(a), an LDL-like particle prepared from Lp(a)[. The Hill coefftcients for displacement curves of labeted-LDL binding by unlabeled LDL and Lp(a) particles were -1.05 and 43.96. respectively; suggesting that a single set of binding sites are involved. In contrast, under the same experimental conditions, Lp(a) particles were unable to inhibit the binding of labeled LDL, even at a 50-fuld excess. Furthermore, out results revealed thai human platelets bound labeled Lp(a) to a class of saturable binding sites numbering approximateh 1,958±235 binding sites per platelet with a dissociation constant (Kr~) of 48.3±12 nM. These values are similar to those obtained for native LDL particles (I.367±209 binding sites per platelet with a KD of 51±8 nM). Finally, the implication of individual GP lIB on the platelet LDL binding was investigated by ligand-binding assays. Our results demonstrated a fully lack of effect of a wide panel of monoclonal and polyclonal antibodies against GP liB, indicating that the individual GP lIB is not involved in tile binding of LDL to intact resting platelets. Therefore, we speculated that first Lp(a)-, a LDL-like particle obtained from Lp(a) particles, and LDL are bound by human intact resting platelets with similar affinity. Second platelet LDL receptor not recognizes Lp(a) particles and finally, individual GP liB is not involved in the platelel LDL binding to intact resting platelets.
W e have compared apo(a) Kpnl genotype distributions and Lp(a) concentrations in 111 Danish men with coronary heart disease aged 35-55 years (mean age 49.4 years), selected among the Danish participants in the Scandinavian Simvastatin Survival Study (4S) with those in 455 Danish reference men (age 40 years). Apo(a) KpnI genotypes were determined by pulsed-field gel electrophoresis followed by hybridisation with a kringle-4 specific probe, and Lp(a) concentrations were measured with a two-site immunoradiometric assay. The distribution of Kpnl genotypes was significantly different in C H D patients and reference men; the genotypes, comprising any a l l e l e < n u m b e r 13, which correspond to protein phenotypes F, B, S I and $2, were more frequent in C H D patients than in reference men ( P = 0 . 0 0 7 1 ) . The median Lp(a) concentration was significantly higher in C H D patients (21.6 mg/dl) than in reference men (6.33 rng/dl) ( P < 0 . 0 0 0 0 1 ) . W e conclude that Lp(a) is a risk factor for C H D in a Danish population.
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PAF-ACETYLHYDROLASE ACTIVITY ON Lp(a). INVOLVEMENT IN THE PHOSPHATIDYLCHOLINE HYDROLYSIS DURING OXIDAq IVE MODIFICATION IN VITRO S A.P. Karabina ~, M. Elisaf2, J. Goudevenos 3, K. Siamopoulos 2. D. Sideris3 and A.D. Tselepsis ~ ~Laboratory of Biochemistry Department of Chemistry, ~-Department of Internal Medicine and 3Department of Cardiology, Medical School, University of loannina, Greece
A PENTANUCLEOTIDE REPEAT POLYMORPHISM 1N THE 5' CONTROL REGION OF THE APOLIPOPROTEIN(A) GENE IS ASSOCIATED WITH LIPOPROTEIN(A) PLASMA LEVELS IN CAUCASIANS M. Trommsdorff~, S. K6chl I, A. Lingenhel I, F, Kronenberg t, R. Delpord, H. Vermaak 2, L. Lemming3, O. Faergeman ~, G. Uterrnann ~, H.G. Kraft t qnst of Medical Biology and Human Genetics, Innsbruek, Austria; 21nst of Chemical Pathology, University of Pretoria, South Africa; 3Dept of Internal Medicine and Cardiology, Univ Hospital, 8000 Aarhus, Denmark
In the present study we investigated the existence of Platelet Activating Factor acetylhydrolase (PAF-AH) activity on Lp(a) and its possible involve ment in the hydrolysis of the phosphatidylcholine (PC) content of Lp(a) during its oxidation. The distribution of PAF-AH activity in human plasma with Lp(a) levels =35 mg/dl was studied with gradient ultracentrifugation. Furthermore, Lp(a) was isolated by sequential ultracentrifugation, purified by Sepharose 6B column chromatography and oxidized with Cu 2÷. PAF AH activity was determined by the trichloroacetic acid precipitation method and PC hydrolysis by phosphorus analysis and expressed as Lyso-PC/Sphingo myelin (Sph) molar ratio. The percentage of total plasma PAF-AH activity distributed on Lp(a) was 34.1%, on LDL+VLDL 52.6% and on HDL 13.2%, whereas in plasma with not detectable amounts of Lp(a), the distribution is 70% on LDL and 30% on HDL. During Lp(a) oxidation. PAF-AH activity was decreased from 10.9+2.3 nmol/mg/min to 4.6_+ 1.2 nmol/mg/min p<0.0001 and 2.6_+0.6 nmol/mg/min p<0.0001, 90 min and 4h from the onset of the oxidation respectively. Lyso-PC/Sph ratio was increased from 0.17 +0.02 (before oxidation) to 0.52 +0.09, 4h afterwards, p<0.001. Complete inactivation of PAF-AH by diisopropylfluorophosphate (DFP) before oxidation, resulted in inhibition of PC hydrolysis, (lysoPC/Sph ratio 0.29+0.06, 4h from the onset of the oxidation). Our results show that in plasma with Lp(a) levels-35 mg/dl, a relatively high amount of total plasma PAF-AH activity is distributed on Lp(a). This enzyme seems to play the major role in PC hydrolysis during Lp(a) oxidation since it is inhibited when PAF-AH has been inactivated.
The enormous variation in Lp(a) plasma levels is almost entirely controlled by the apo(a) locus. A variable number of transcribed kringle4 repeats (K4VNTR) in the gene explains a large fraction of this variation, whereas the rest is presently unexplained. We here have analyzed by PCR the effect of the K4-VNTR and of a pentanucleotide repeat polymorphisrn (TTTTA)n (n= 6-11) in the 5' control region of the apo(a) gene on plasma Lp(a) levels in unrelated healthy Tyroleans (n=130), Danes (n=154) and Black South Africans (n= 112). The K4-VNTR had a significant effect on plasma Lp(a) levels in Caucasians. Both, the pentanucleotide repeat (PNR) allele frequencies and their effects on Lp(a) concentrations were heterogeneous among populations. A significant negative correlation between the number of pentanucleotide repeats and the plasma Lp(a) concentration was observed in Tyroleans and Danes. The effect of the 5' PNRP on plasma Lp(a) concentrations was independent from the K4-VNTR and explained from 10% to 14% of the variation in Lp(a) concentrations in Caucasians. No significant effect of the PNRP was present in Black Africans. This suggests allelic associations between PNR alleles and sequences affecting Lp(a) levels in Caucasians. Thus, in Caucasians but not in Blacks, concentrations of the atherogenic Lp(a) particle are strongly associated with two repeat polymorphisms in the apo(a) gene.
Poster session abstracts / Atheroselerosis 115 (Suppl.) 11995) $45-S129 Pl l Lipoprotein (a)
327
329
INTERACTION OF LIPOPROTEIN(a) WITH THE PLATELET LDL RECEPTOR J. Pedrefio and R. Fern:~ndez Dept Biochemistry, Hospital Son Dureta, Palma de Majorca, Spain
APOL1POPROTEIN(A) KpnI G E N O T Y P E IS A RISK F A C T O R FOR C O R O N A R Y H E A R T DISEASE IN D A N I S H M E N L. Lemming. I.C. Klausen, P.S. Hansen, L.U. Gerdes, O. F~ergeman Department of Internal Medicine and Cardiology A, Aarhus Amtssygehus University Hospital, 8000 Aarhus C
Recently, it has been described that Lp(a) binds to human intact platelets and further studies have showed that Lp(a) bound to the individual platelet GP liB. On the other hand, it is well kwnon that platelets contain a LDL receptor which is different from the classical LDL receptor, and the thductible fibrinogen receptor (GP liB-IliA complex) has been involved in the binding of LDL to platelets. However, there are not data about the interaction between Lp(a) and the platelet LDL receptor. In the present study we have investigated both the interaction of Lp(a) with the platelet LDL receptor and the implication of individual GP liB on platelet LDL binding. Under our experimental conditions, platelet LDL binding seem to be different compared with platelet Lp(a) binding. Displacement experiments showed that the ~I-LDL binding (0.0I g/I) to intact resting platelets was inhibited with the same apparent affinity by both unlabelled LDL and apo(a)-free lipoprotein particle [Lp(a), an LDL-like particle prepared from Lp(a)[. The Hill coefftcients for displacement curves of labeted-LDL binding by unlabeled LDL and Lp(a) particles were -1.05 and 43.96. respectively; suggesting that a single set of binding sites are involved. In contrast, under the same experimental conditions, Lp(a) particles were unable to inhibit the binding of labeled LDL, even at a 50-fuld excess. Furthermore, out results revealed thai human platelets bound labeled Lp(a) to a class of saturable binding sites numbering approximateh 1,958±235 binding sites per platelet with a dissociation constant (Kr~) of 48.3±12 nM. These values are similar to those obtained for native LDL particles (I.367±209 binding sites per platelet with a KD of 51±8 nM). Finally, the implication of individual GP lIB on the platelet LDL binding was investigated by ligand-binding assays. Our results demonstrated a fully lack of effect of a wide panel of monoclonal and polyclonal antibodies against GP liB, indicating that the individual GP lIB is not involved in tile binding of LDL to intact resting platelets. Therefore, we speculated that first Lp(a)-, a LDL-like particle obtained from Lp(a) particles, and LDL are bound by human intact resting platelets with similar affinity. Second platelet LDL receptor not recognizes Lp(a) particles and finally, individual GP liB is not involved in the platelel LDL binding to intact resting platelets.
W e have compared apo(a) Kpnl genotype distributions and Lp(a) concentrations in 111 Danish men with coronary heart disease aged 35-55 years (mean age 49.4 years), selected among the Danish participants in the Scandinavian Simvastatin Survival Study (4S) with those in 455 Danish reference men (age 40 years). Apo(a) KpnI genotypes were determined by pulsed-field gel electrophoresis followed by hybridisation with a kringle-4 specific probe, and Lp(a) concentrations were measured with a two-site immunoradiometric assay. The distribution of Kpnl genotypes was significantly different in C H D patients and reference men; the genotypes, comprising any a l l e l e < n u m b e r 13, which correspond to protein phenotypes F, B, S I and $2, were more frequent in C H D patients than in reference men ( P = 0 . 0 0 7 1 ) . The median Lp(a) concentration was significantly higher in C H D patients (21.6 mg/dl) than in reference men (6.33 rng/dl) ( P < 0 . 0 0 0 0 1 ) . W e conclude that Lp(a) is a risk factor for C H D in a Danish population.
328
330
PAF-ACETYLHYDROLASE ACTIVITY ON Lp(a). INVOLVEMENT IN THE PHOSPHATIDYLCHOLINE HYDROLYSIS DURING OXIDAq IVE MODIFICATION IN VITRO S A.P. Karabina ~, M. Elisaf2, J. Goudevenos 3, K. Siamopoulos 2. D. Sideris3 and A.D. Tselepsis ~ ~Laboratory of Biochemistry Department of Chemistry, ~-Department of Internal Medicine and 3Department of Cardiology, Medical School, University of loannina, Greece
A PENTANUCLEOTIDE REPEAT POLYMORPHISM 1N THE 5' CONTROL REGION OF THE APOLIPOPROTEIN(A) GENE IS ASSOCIATED WITH LIPOPROTEIN(A) PLASMA LEVELS IN CAUCASIANS M. Trommsdorff~, S. K6chl I, A. Lingenhel I, F, Kronenberg t, R. Delpord, H. Vermaak 2, L. Lemming3, O. Faergeman ~, G. Uterrnann ~, H.G. Kraft t qnst of Medical Biology and Human Genetics, Innsbruek, Austria; 21nst of Chemical Pathology, University of Pretoria, South Africa; 3Dept of Internal Medicine and Cardiology, Univ Hospital, 8000 Aarhus, Denmark
In the present study we investigated the existence of Platelet Activating Factor acetylhydrolase (PAF-AH) activity on Lp(a) and its possible involve ment in the hydrolysis of the phosphatidylcholine (PC) content of Lp(a) during its oxidation. The distribution of PAF-AH activity in human plasma with Lp(a) levels =35 mg/dl was studied with gradient ultracentrifugation. Furthermore, Lp(a) was isolated by sequential ultracentrifugation, purified by Sepharose 6B column chromatography and oxidized with Cu 2÷. PAF AH activity was determined by the trichloroacetic acid precipitation method and PC hydrolysis by phosphorus analysis and expressed as Lyso-PC/Sphingo myelin (Sph) molar ratio. The percentage of total plasma PAF-AH activity distributed on Lp(a) was 34.1%, on LDL+VLDL 52.6% and on HDL 13.2%, whereas in plasma with not detectable amounts of Lp(a), the distribution is 70% on LDL and 30% on HDL. During Lp(a) oxidation. PAF-AH activity was decreased from 10.9+2.3 nmol/mg/min to 4.6_+ 1.2 nmol/mg/min p<0.0001 and 2.6_+0.6 nmol/mg/min p<0.0001, 90 min and 4h from the onset of the oxidation respectively. Lyso-PC/Sph ratio was increased from 0.17 +0.02 (before oxidation) to 0.52 +0.09, 4h afterwards, p<0.001. Complete inactivation of PAF-AH by diisopropylfluorophosphate (DFP) before oxidation, resulted in inhibition of PC hydrolysis, (lysoPC/Sph ratio 0.29+0.06, 4h from the onset of the oxidation). Our results show that in plasma with Lp(a) levels-35 mg/dl, a relatively high amount of total plasma PAF-AH activity is distributed on Lp(a). This enzyme seems to play the major role in PC hydrolysis during Lp(a) oxidation since it is inhibited when PAF-AH has been inactivated.
The enormous variation in Lp(a) plasma levels is almost entirely controlled by the apo(a) locus. A variable number of transcribed kringle4 repeats (K4VNTR) in the gene explains a large fraction of this variation, whereas the rest is presently unexplained. We here have analyzed by PCR the effect of the K4-VNTR and of a pentanucleotide repeat polymorphisrn (TTTTA)n (n= 6-11) in the 5' control region of the apo(a) gene on plasma Lp(a) levels in unrelated healthy Tyroleans (n=130), Danes (n=154) and Black South Africans (n= 112). The K4-VNTR had a significant effect on plasma Lp(a) levels in Caucasians. Both, the pentanucleotide repeat (PNR) allele frequencies and their effects on Lp(a) concentrations were heterogeneous among populations. A significant negative correlation between the number of pentanucleotide repeats and the plasma Lp(a) concentration was observed in Tyroleans and Danes. The effect of the 5' PNRP on plasma Lp(a) concentrations was independent from the K4-VNTR and explained from 10% to 14% of the variation in Lp(a) concentrations in Caucasians. No significant effect of the PNRP was present in Black Africans. This suggests allelic associations between PNR alleles and sequences affecting Lp(a) levels in Caucasians. Thus, in Caucasians but not in Blacks, concentrations of the atherogenic Lp(a) particle are strongly associated with two repeat polymorphisms in the apo(a) gene.