**2. Structural arrangement and catabolism of Lp(a)**

The major protein component of this LDL-like particle is apolipoprotein B (apo-B-100) which carries an additional protein called apolipoprotein-a [apo-a] linked to apo-B-100 via disulphide bridges (Fig.1) the lipid moiety however being almost indistinguishable from that of LDL [12]. Human apo-a itself consists of multiple so-called kringle repeats, sequences consisting of 80-90 amino acids arranged in a tripleloop tertiary structure and tandemly arrayed resembling kringles IV and V of plasminogen and a protease domain [13]. Copy number variants in the LPA gene on chromosome 6 coding for apo-a are responsible for a variation of plasma Lp(a) levels of up to 1000-fold among individuals. The most influential is the kringle IV-2 size polymorphism [14] while kringle IV types 1 and 3-10 as well as kringle V occur only once in Lp(a) [15]. The number of kringle IV type 2 structure repeats results in a large number of different sized isoforms of apo-a and correlates inversely with the plasma concentration of Lp(a) [16]. Although the exact mechanism responsible for this inverse correlation has not been elucidated so far an isoform dependent retention and degradation in the endoplasmatic reticulum has been implicated [17].

742 Lipoproteins – Role in Health and Diseases

far although the kidney is favourized to be implicated [11].

Chem. Composition [%]

**Table 1.** Chemical and physicochemical properties of LDL and Lp(a)

**2. Structural arrangement and catabolism of Lp(a)** 

metabolic behaviour completely different from other apo-B containing lipoproteins. Turnover studies in vivo performed with labelled VLDL confirmed these assumptions. Nearly all the activity of labelled VLDL could be detected in LDL whereas only trace amounts could be found in Lp(a) [9] confirming the hypothesis that unlike LDL, Lp(a) probably has no triglyceride-rich lipoproteins as precursors but seems to be secreted directly by the liver [10]. On the other hand the site of catabolism of Lp(a) in humans is unknown so

Despite extensive work on Lp(a) its possible physiological function remains unclear till now.

Hydr. Density [g/ml] 1.034 1.085 Mol. Wt. [x 106] 2.4 5.5 Diameter [Å] 210 250 E. Mobility β pre-β1

Free cholesterol 11 10 Cholesterolester 40 30 Triglycerides 4 4 Phospholipids 21 20 Protein 22 28 Carbohydrates 2 8

The major protein component of this LDL-like particle is apolipoprotein B (apo-B-100) which carries an additional protein called apolipoprotein-a [apo-a] linked to apo-B-100 via disulphide bridges (Fig.1) the lipid moiety however being almost indistinguishable from that of LDL [12]. Human apo-a itself consists of multiple so-called kringle repeats, sequences consisting of 80-90 amino acids arranged in a tripleloop tertiary structure and tandemly arrayed resembling kringles IV and V of plasminogen and a protease domain [13]. Copy number variants in the LPA gene on chromosome 6 coding for apo-a are responsible for a variation of plasma Lp(a) levels of up to 1000-fold among individuals. The most influential is the kringle IV-2 size polymorphism [14] while kringle IV types 1 and 3-10 as well as kringle V occur only once in Lp(a) [15]. The number of kringle IV type 2 structure repeats results in a large number of different sized isoforms of apo-a and correlates inversely with the plasma concentration of Lp(a) [16]. Although the exact mechanism responsible for this

LDL Lp(a)

Contradictory results have been reported about the clearance of Lp(a) and till now it remains unclear whether Lp(a) binds to the B/E receptor via apo-B like LDL or whether it is catabolised independently of the LDL-receptor mediated pathway. Whereas in one study using fibroblasts from normal subjects and from subjects with autosomal dominant hypercholesterolemia the conclusion was reached that Lp(a) enters fibroblasts independently of the LDL-receptor [18] others concluded that Lp(a) is also bound to the LDL-receptor, internalized and degraded but with a degradation capacity of only 25% of that of LDL [19]. Binding studies of native and reduced Lp(a) with different monoclonal antibodies against apolipoprotein B revealed that there was no antibody that failed to react with native Lp(a) but some of the antibodies recognized apoB of Lp(a) to a lesser degree than that of LDL. This favoured the idea that certain regions on apo-B of Lp(a) could be different from those on LDL and led to the assumption that certain domains close to the binding domain of Lp(a) to the B/E-receptor could be covered by apo-a or that apo-a causes conformational changes in the binding region of apo-B thereby constricting the binding of Lp(a) to the LDL-receptor [20] being in agreement with the fact that normal unreduced Lp(a) seemed to be taken up by fibroblasts through B/E-receptor-mediated endocytosis but showed poorer specificity for the receptor than LDL [21].

**Figure 1.** Schematic model of Lp(a) in comparison to LDL

#### **3. Free apolipoprotein-a in human serum**

In the beginning of "Lp(a)-research" this lipoprotein was believed to represent a genetically polymorphic form of LDL [22]. According to this assumption apo-a should distribute uniformly between all apo-B-containing lipoproteins. Investigation of this problem in more detail revealed that Lp(a) forms a particular lipoprotein class found primarily in the HDL-2 density region [23] but can also be detected in LDL class (d = 1.019-1.063 g/ml) [24] and even in chylomicrons induced by fat feeding [25]. The fact however that a portion of the Lp(a) specific antigen can be found in the d > 1.21 g/ml lipoprotein free bottom fraction after ultracentrifugation of plasma [26] led to further investigation of this phenomenon. Apo-a is virtually absent in the VLDL fraction (d < 1.006 g/ml) of freshly drawn fasting human sera while 95% of total Lp(a) can be obtained in the d > 1.006 g/ml bottom fraction. Approximately 5% of the total serum Lp(a) are found in the d > 1.125 g/ml bottom fraction after ultracentrifugation as well as with polyanionic precipitation agents irrespective of the Lp(a) concentration in serum [8]. Due to the lack of Sudan Black staining this bottom Lp(a) is considered as a lipid free "apo-a protein" raising the question whether or not free apo-a can reassociate with LDL to form "native Lp(a)".

### **4. Lp(a) and platelet aggregation**

One of the physiological roles of platelets involves binding to subendothelial tissue after vascular injury [27]. The adherence of platelets to the exposed connective tissue, preferably collagen, leads to aggregation followed by the release of ADP, 5-hydroxytryptamine and Ca2+ from their dense granules, causing passing platelets to adhere to the primary clot [28].

There is little doubt that lipoproteins interfere with platelets in vivo being reflected by the fact that platelets from hyperlipoproteinemic patients are hyperreactive [29]. This is confirmed by the fact that incubation of platelets with physiological concentrations of atherogenic apoB-containing lipoproteins such as LDL or VLDL results in enhanced platelet aggregability [30] while antiatherogenic lipoproteins such as HDL exert the opposite effect [31]. Concerning Lp(a) it is generally accepted that elevated plasma concentrations of this lipoprotein are connected with premature atherosclerosis [32] but much uncertainty remains about the influence of Lp(a) on platelet activation, a phenomenon that is believed to be involved not only in long-term processes of plaque formation but also in acute events such as stroke and myocardial infarction [33]. Moreover a two-fold increase in the risk of coronary heart disease (CHD) and ischaemic stroke could be demonstrated especially in subjects with small apolipoprotein(a) phenotypes [34] and prospective findings in the Bruneck study have revealed a significant association specifically between small apolipoprotein(a) phenotypes and advanced atherosclerotic disease involving a component of plaque thrombosis [35]. Indeed, Lp(a) is a "sticky" lipoprotein that self-aggregates, attaches to all sorts of surfaces [36], and precipitates not only in vitro but possibly in vivo. Moreover, Lp(a) binds to proteoglycans and glycosaminoglycans [37] and it has high affinity for fibronectin [38], tetranectin [39], collagen [40], and other connective-tissue structures [41]. Therefore the influence of Lp(a) on platelet aggregation induced with various triggers was investigated measuring serotonin release and thromboxane A2 formation during collagen-triggered aggregation as well as adhesion of platelets to collagen in flowing blood under the influence of Lp(a). As Lp(a) represents an LDL-like particle an elevated platelet reactivity was expected under the influence of this lipoprotein similar to that described for LDL [42].

744 Lipoproteins – Role in Health and Diseases

reassociate with LDL to form "native Lp(a)".

**4. Lp(a) and platelet aggregation** 

uniformly between all apo-B-containing lipoproteins. Investigation of this problem in more detail revealed that Lp(a) forms a particular lipoprotein class found primarily in the HDL-2 density region [23] but can also be detected in LDL class (d = 1.019-1.063 g/ml) [24] and even in chylomicrons induced by fat feeding [25]. The fact however that a portion of the Lp(a) specific antigen can be found in the d > 1.21 g/ml lipoprotein free bottom fraction after ultracentrifugation of plasma [26] led to further investigation of this phenomenon. Apo-a is virtually absent in the VLDL fraction (d < 1.006 g/ml) of freshly drawn fasting human sera while 95% of total Lp(a) can be obtained in the d > 1.006 g/ml bottom fraction. Approximately 5% of the total serum Lp(a) are found in the d > 1.125 g/ml bottom fraction after ultracentrifugation as well as with polyanionic precipitation agents irrespective of the Lp(a) concentration in serum [8]. Due to the lack of Sudan Black staining this bottom Lp(a) is considered as a lipid free "apo-a protein" raising the question whether or not free apo-a can

One of the physiological roles of platelets involves binding to subendothelial tissue after vascular injury [27]. The adherence of platelets to the exposed connective tissue, preferably collagen, leads to aggregation followed by the release of ADP, 5-hydroxytryptamine and Ca2+ from their dense granules, causing passing platelets to adhere to the primary clot [28].

There is little doubt that lipoproteins interfere with platelets in vivo being reflected by the fact that platelets from hyperlipoproteinemic patients are hyperreactive [29]. This is confirmed by the fact that incubation of platelets with physiological concentrations of atherogenic apoB-containing lipoproteins such as LDL or VLDL results in enhanced platelet aggregability [30] while antiatherogenic lipoproteins such as HDL exert the opposite effect [31]. Concerning Lp(a) it is generally accepted that elevated plasma concentrations of this lipoprotein are connected with premature atherosclerosis [32] but much uncertainty remains about the influence of Lp(a) on platelet activation, a phenomenon that is believed to be involved not only in long-term processes of plaque formation but also in acute events such as stroke and myocardial infarction [33]. Moreover a two-fold increase in the risk of coronary heart disease (CHD) and ischaemic stroke could be demonstrated especially in subjects with small apolipoprotein(a) phenotypes [34] and prospective findings in the Bruneck study have revealed a significant association specifically between small apolipoprotein(a) phenotypes and advanced atherosclerotic disease involving a component of plaque thrombosis [35]. Indeed, Lp(a) is a "sticky" lipoprotein that self-aggregates, attaches to all sorts of surfaces [36], and precipitates not only in vitro but possibly in vivo. Moreover, Lp(a) binds to proteoglycans and glycosaminoglycans [37] and it has high affinity for fibronectin [38], tetranectin [39], collagen [40], and other connective-tissue structures [41]. Therefore the influence of Lp(a) on platelet aggregation induced with various triggers was investigated measuring serotonin release and thromboxane A2 formation during collagen-triggered aggregation as well as adhesion of platelets to collagen in flowing blood Unlike LDL, Lp(a) revealed no proaggregatory effects on platelets, contrary collageninduced platelet aggregation was inhibited by up to 54% and the aggregation rate was attenuated by 47% compared with platelets incubated with Tyrode's solution (Fig. 2), being accompanied by a significant reduction of serotonin release and TXA2 formation. Furthermore Lp(a) significantly reduced platelet adhesion to collagen by about 20% and the size of platelet aggregates up to 63% especially at high shear rates (Fig. 3) suggesting that Lp(a) exerts antiaggregatory effects at least under well-defined in vitro conditions [43]. If these observations are relevant for the in vivo situation, a variety of potential plateletcollagen binding sites such as GPIa/IIa or GPIV could be covered by Lp(a) the more that binding of Lp(a) to platelets could be demonstrated [44]. As there is conflicting evidence on the role of Lp(a) in thrombosis in vivo and in vitro work has been done to elucidate the mechanisms whereby Lp(a) is influencing platelet aggregation and a variety of mechanisms is suggested how Lp(a) interferes with platelet aggregation and hence fibrin bound clot formation. Lp(a) binds to resting, non-stimulated platelets on the IIb subunit of the fibrinogen (IIb/IIIa) receptor via binding sites distinct from the arginyl-glycyl-aspartyl (RGD) epitope of apo-a [45]. By this way the RGD binding site of Lp(a) could be exposed via conformational change induced by platelet agonist stimulation leading to binding of the RGD epitope of apo-a to the RGD binding site on the IIb protein of the fibrinogen (IIb/IIIa) receptor of the platelet [46] thereby reducing fibrinogen binding to the platelet [47]. Low concentrations of Lp(a) (1-25 mg/100 ml washed platelets) increase intracellular levels of c-AMP of in vitro resting platelets leading to an antiaggregatory condition [48] while at higher in vitro levels of Lp(a) (50-100 mg/100 ml washed platelets) resting platelet intracellular c-AMP levels return to normal [49] which cannot explain the reported progressive Lp(a) mediated decrease in collagen-induced aggregation [43, 50]. Further investigations strongly support an apo-a mediated, Lp(a) induced reduction of collagen and ADP-stimulated platelet aggregation via diminished production of thromboxane A2 [43, 51]. Concerning the in vivo situation only one study has been published to date looking at adult human type 2 diabetics all of whom where obese (BMI >30). In this in vivo study of human type 2 diabetics there is a positive correlation between fasting serum concentrations of Lp(a) and bleeding time, a strong correlate of in vivo platelet aggregation [52] favouring the inhibitory effect of Lp(a) on platelet aggregation. On the other hand there are studies reporting an apparent proaggregatory action of Lp(a) possibly mediated by the apo-a subunit. While no effect of recombinant apo-a [r-apoa] derivatives on primary ADP-induced platelet aggregation was observed weak platelet responses stimulated by the thrombin receptor-activating peptide SFLLRN were significantly enhanced by the r-apo-a derivatives accompanied by a significant enhancement of [14C]serotonin release of the dense granules [53]. Further investigations showed that r-apo-a isoforms and Lp(a) do not cause platelet aggregation by themselves but preincubation of platelets with r-apo-a derivatives promotes an aggregation response to otherwise subaggregant doses of thrombin receptor activation peptide (TRAP) and arachidonic acid while inversely platelet stimulation with arachidonic acid enhanced platelet binding of apo-a [54]. In both studies it turned out that the size of r-apo-a determined by the number of KIV type 2 modules seems not to play a crucial role in its proaggregant effect.

Summarizing, in vitro studies indicate that Lp(a) induced decreases, increases or no change at all in platelet aggregation [43, 45, 50, 51, 53, 54]. In all cases the mechanisms involved are quite unclear and only speculative. A recent work strongly supports the evidence to suggest that Lp(a) binds to platelets via its arginyl-glycyl-aspartyl (RGD) epitope of the apo(a) but not via apo(a)'s lysine binding region in both strong and weak agonist-stimulated platelets and inhibits the binding of fibrinogen thus reducing aggregation [55]. On the other side there are in vivo studies published quite recently suggesting that Lp(a) concentrations greater than 30 mg/dl are a frequent and independent risk factor for venous thrombosis [56] and that high levels of Lp(a) could be a more frequently thrombophilic risk factor in young women [57]. To date disagreement exists about increased arterial thrombosis due to elevated blood levels of Lp(a). The fact that this procedure is a result of collagen-exposed platelets in case of plaque rupture followed by clot formation argues against the proaggregatory nature of Lp(a) and maybe procedures others than platelet activation account for the atherogenic nature of Lp(a).

**Figure 2.** Aggregation curves showing the influence of lipoproteins on collagen-induced platelet aggregation. Gel filtered platelets (200 μl; 2x108/ml) were incubated for 30 min at 37°C with a) LDL 5 mg/ml, b) Tyrodes's buffer or c) Lp(a) 0.5 mg/ml. Aggregation was triggered with 10 μl collagen (final concentration 4 μg/ml).

**Figure 3.** Aggregate formation of fibrillar collagen at a shear rate of 1600/s for a control (top) and under addition of 1 mg/ml Lp(a) (bottom). Aggregates are shown in black.
