PCSK9 association with lipoprotein(a)professional.heart.org/idc/groups/ahamah-public/@wcm/...PCSK9 association with lipoprotein(a) Hagai Tavori Center for Preventive Cardiology, KCVI,
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PCSK9 association with lipoprotein(a) Hagai Tavori Center for Preventive Cardiology, KCVI, OHSU, Portland, OR.
Center for Preventive Cardiology, KCVI, OHSU, Portland, OR.
Presenter
Presentation Notes
First I want thank the scientific committee for giving me the opportunity preset to you our work on PCSK9 and its interaction with Lp(a).
Background
Kringle repeats
Lipoprotein (a) Plasminogen
Protease
Unlike plasminogen, Lp(a) is only present in Humans, Apes, Old World Monkeys and the Hedgehog.
Lipoprotein(a) Structure
Presenter
Presentation Notes
I’m sure this audience is familiar with this lipoprotein that is called Lp(a), it is an LDL size particle that contains the apo(a) proteins. Apo(a) protein is made out of kringle repeats and is simlar in structure to plasminogen. Apo(a) is very heterogenious in size due to different number of KIV type2 repeats
• Lp(a) confers CVD risk both through the atherogenic LDL and the thrombogenic apolipoprotein(a).
• Lp(a) levels in patients with FH are generally 2-fold higher compared to non-affected relatives, despite the fact that the LDL receptor is likely not involved in its clearance.
• Current pharmacological lipid lowering therapies are ineffective in treating elevated Lp(a) levels.
Lipoprotein(a) and Familial Hypercholesterolemia
Presenter
Presentation Notes
Lp(a) levels are determent genetically, mainly be levels of expression and size (we will get back to the size issue of apo(a) later). Lp(a) is known for along time and in recent years it came to a consensus that its an independent and casual risk factor for CVD. Despite all the we know about Lp(a) it is still an enigmatic lipoprotein as it is not clear where it is forming and how is it cleared. Many receptors have been suggested to be involved in it clearance including, CD36, SRBI, LRP1, VLDLR, plasminogen-R and LDLR – evidence for all of these receptors is weak. In recent years it have been shown the FH pateitns have higher Lp(a) levels, which again may be puzzling as evidence do not clearly support a role for LDLR. Due to the lack of understanding of its mechanism of clearance therapeutic approaches to reduce Lp(a) are limited
BackgroundTherapeutic Approaches for Elevated Lipoprotein (a) levels
• Apheresis - Most effective therapy. Average of ~70% acute reduction (not FDA approved for elevated Lp(a) levels).
• Nicotinic acid - Most effective drug therapy. 20-30% significant reduction.
• Statins, Ezetimibe, Bile acid-binding resins, Fibrates – no effect.
• Mipomersen - ~25% significant reduction.• CETP inhibitors – no effects; in phase III testing• PCSK9 inhibitors – 20% -30% significant reduction; in
phase III in testing.
BackgroundProprotein Convertase Subtilisin/Kexin type 9 (PCSK9)
Fan D. et al. Biochemistry 2008.Tavori H. et al. Circulation 2013.Tavori H. et al. Circulation Research 2013.
Furin-Cleaved (55+13kDa)
Mature (62+13kDa)
Presenter
Presentation Notes
I’m sure you all know PCSK9, so I”ll just make a brief introduction to this proteins. It is made primarily, but not exclusively by the liver as a proprotein that under go auto-catalytic cleavage to form a heterodimer that is secreted to the circulation where it can be further proceed (inactivated?). Once in is outside of the cell PCSK9 can interact with the LDLR and destined both protein for lysosomal degradation. An additional piece of information that is gaining more more acknowledgment in the literature is that part of PCSK9 associate with LDL in the circulation
Aim and Methods
We aim to study whether PCSK9 associates with Lp(a) particles and whether this association depends on Lp(a) levels and/or apo(a) size.
• Nine subjects with FH and elevated Lp(a).
• Natural gradient ultracentrifugation separation of LDL and Lp(a).
• Measurements of apoB, apo(a) and PCSK9, and their interactions.
• Direct measurement of PCSK9 association with Lp(a) in plasma.
Presenter
Presentation Notes
In fact back in 2008 we were the first to demonstrate the PCSK9 associates with LDL and here we aim to study whether the same is also rue also for Lp(a), an LDL size particle. We used plasma samples from nine FH patients from our lipoprotein apheresis unit that have elevated Lp(a) based (<30mg/dL). We used UC to seprate the lipoproteins and I’m using the term natural gradient not because we are veggies/organic/tree huggers from Portland but simply to emphasize that this in not the standard KBr isolation. We measured the proteins levels ,molecular form and interaction.
Results – PCSK9 is associated with Lp(a)
Lp(a)
LDL apoB100
apoB48
apo(a)VLDL
LDL
Lp(a)apoB
Coomassie
apo(a)
PCSK9
LDL
Lp(a)
LDL and Lp(a) isolation Reducing gel WB Native gel WB
Isolation and characterization of LDL and Lp(a) using natural gradient ultracentrifugation.
PCSK9
Presenter
Presentation Notes
First, as I said we isolated the particle sing this natural gradient UC and show that we obtain relatively clean faction of LDL and Lp(a). Runnig these fractions on reducing gel we found the expected presence of PCSK9 (mainly the 62kDa) in the LDL faction but also in the Lp(a) fraction. This however does not tell us that PCSK9 in fact associate with the particles. Using native gel (upper band Lp(a) and lower band is LDL) we were able to show that PCSK9 is indeed associated with LDL and Lp(a).
Results – PCSK9 prefers Lp(a) over LDL
Levels of Lp(a)-bound PCSK9 do not correlate with total Lp(a) levels
Higher PCSK9/apoB ratio in Lp(a) suggests preferential association
Measurement of Lp(a)-bound PCSK9 and PCSK9/apoB
Presenter
Presentation Notes
We next wanted to investigate what drive the association of PCSK9 to Lp(a). The y-axis use a method we have developed togther with Sam Tsimikas f UCSD to directly measure the amount of PCKS9 in Lp(a) and as you can see it is not determined by Lp(a) levels the. It is also not affected by PCSK9 or LDL levels (not showing for the interest of time). However during this investigation we stumbled across an interesting phenomenon that in FH patients with high Lp(a), PCSK9 “prefers” to be on Lp(a) rather than on LDL, as measured by the ration of PCSK9 to apoB.
Results – PCSK9 prefers larger apo(a) isoforms
460
268
238
Ladder (kDa)
Total serum
PCSK9 antibodyIsotype
control
apo(a)
Lp(a)-PCSK9/LDL-PCSK9 ratio is unexpectedly inversely correlated with Lp(a) levels.
PCSK9 associates with Lp(a) particles carrying the higher molecular weight apo(a).
PCSK9 association as a function of Lp(a) levels and apo(a) size.
Presenter
Presentation Notes
We than asked what drive this preferential association and found the the relative levels of PCSK9 on Lp(a) compare to LDL are inversely associated with Lp(a). This results seems unexpected, but we have to remember that Lp(a) levels are also inversely associated with apo(a) size. So in other words this graph suggest that PCSK9 association with Lp(a) is directly correlated to apo(a) size. To test this we used anti-PCSK9 antibody to Co-IP apo(a) and found that PCSK9 is almost exclusively associated with the larger apo(a)
• PCSK9 is physically associated with Lp(a) particles.
• An 18-fold higher PCSK9/apoB ratio for Lp(a) compared with LDL suggests a preferential distribution of PCSK9 with Lp(a).
• PCSK9 preferentially associates with the higher molecular weight of apo(a).
Summary
Conclusions
Our results suggest that Lp(a)-bound PCSK9 exists in plasma of patients with FH and high Lp(a) and that this association depends on the size of apo(a). This provides a possible mechanism for the reduction in Lp(a) caused by PCSK9mAb.
Acknowledgments
• Sergio Fazio• Nathalie Pamir• Ilaria Giunzioni• Irene Predazzi• Cathleen Mosibrocki• Anna Shivinsky• Deanna Plubell• Michael D. Shapiro• Barton P. Duell
• Sotirios Tsimikas• Calvin Yeang
UCSDOHSU
This study is supported by the National Institutes of Health (NHLBI) through grant R01-HL106845
