[Primary aortoenteric fistula. Report of a case]. / Su un caso di fistola aorto-enterica primitiva.

Primary aortoenteric fistulas (PAEF) are rare entities associated with a high mortality. Although several causes have been reported, their occurrence is usually due to erosion of an abdominal aortic aneurysm into the intestinal tract. The most common sites for the fistula are the third and fourth portions of duodenum. The classical triad of gastrointestinal hemorrhage, abdominal mass and abdominal or back pain, though highly suggestive for PAEF, is uncommon. The typical bleeding pattern associated with PAEF is characteristically intermittent, starting with a brief "herald bleeding" followed eventually by major gastrointestinal hemorrhage, often with fatal outcome. The pre-operative examinations are often not helpful and can lead to delayed diagnosis and surgery. In a patient with risk factors for atherosclerosis and significant upper gastrointestinal bleeding in the absence of an evident source, PAEF should be suspected. A high index of suspicion of this condition allows correct diagnosis and definitive treatment to be carried out. If PAEF is suspected and the patient is unstable the surgeon should be prepared to skip the preoperative investigations in favour of early surgical exploration. Definitive treatment includes primary duodenal repair and aortic aneurismal resection with graft "in situ" replacement. The authors present a successfully treated case and stress the importance of clinical suspicion in order to achieve correct diagnosis and treatment.

(99)mTc-tetrofosmin SPET in the detection of both primary breast cancer and axillary lymph node metastasis.

The aim of this study was to evaluate the usefulness of (99m)Tc-tetrofosmin single-photon emission tomography (SPET) in the detection of both primary breast cancer and axillary lymph node metastasis. We studied 192 consecutive patients in whom primary breast cancer was suspected on the basis of mammography and/or physical examination. After intravenous injection of 740 MBq (99m)Tc-tetrofosmin, both planar and SPET scintimammography was performed in all patients using a rectangular dual-head gamma camera equipped with low-energy, high-resolution, parallel-hole collimators. In 175 patients with breast cancer at histology, the per-lesion overall sensitivity of SPET and planar imaging for the detection of breast cancer was 95.8% and 75.9% (P<0.0005), respectively. The sensitivity of SPET and planar imaging was, respectively, 96.5% and 79.5% in palpable (P<0.0005) and 90% and 45% in non-palpable lesions (P<0.01). With regard to lesion size, the sensitivity of SPET and planar imaging was, respectively, 90.5% and 45.2% in lesions < or =10 mm ( P<0.0005), 95.3% and 81.4% in lesions of 11-20 mm (P<0.005), 100% and 84.6% in lesions of 21-30 mm (P<0.05) and 100% and 95.8% in lesions >30 mm (P>0.05). In the remaining 17 patients with benign mammary lesions at histology, per-lesion overall specificity of SPET and planar imaging was 76.2% and 85.7% (P>0.05), respectively. Neither SPET nor planar imaging showed false-positive results in non-palpable lesions or in those < or =10 mm. In 173 breast cancer patients submitted to axillary lymph node dissection (ALND), per-axilla overall sensitivity of SPET and planar imaging in the detection of axillary lymph node metastasis was 93% and 52.3% ( P<0.0005), respectively. The sensitivity of SPET and planar imaging was, respectively, 100% and 82.6% in palpable nodes (P>0.05), 90.5% and 41.3% in non-palpable nodes (P<0.0005), 92.8% and 35.7% in the presence of < or =3 nodes ( P<0.0005) and 93.2% and 68.2% in the presence of >3 nodes (P<0.005). The specificity of SPET and planar imaging was 91% and 100% (P<0.05), respectively. (99m)Tc-tetrofosmin SPET appears to be a reliable method for the detection of both primary BC and axillary lymph node metastasis, and its diagnostic accuracy exceeds that of (99m)Tc-tetrofosmin planar scintimammography. The use of SPET is particularly important in the identification of small non-palpable primary carcinomas and metastatic axillae with < or =3 non-palpable lymph nodes. More extensive use of SPET appears warranted in the management of breast cancer patients.

Domains of apolipoprotein E involved in the binding to the protein core of biglycan of the vascular extracellular matrix: potential relationship between retention and anti-atherogenic properties of this apolipoprotein.

We recently identified elements in the C-terminal domain of apolipoprotein E that are critical for the binding of this protein to the protein core of biglycan, a proteoglycan of the vascular extracellular matrix. This binding, ionic in nature, requiring no participation by glycosaminoglycans, suggests that protein-protein interactions may play an important role in the binding of apolipoprotein E to the extracellular matrix. These interactions may represent a potential mechanism for the anti-atherogenic potential of this apolipoprotein by preventing further anchoring to the vascular matrix of pro-atherogenic factors.

Stimulation of interleukin-8 production in human THP-1 macrophages by apolipoprotein(a). Evidence for a critical involvement of elements in its C-terminal domain.

In the vessel wall, macrophages are among the cells that upon activation contribute to the atherosclerotic process. Low density lipoproteins (LDL) can mediate this activation but only after enzymatic or oxidative modification. Lipoprotein(a) (Lp(a)) is an LDL variant that has been shown to have an atherogenic potential by no clearly established mechanisms. In the present study we examined whether native Lp(a) can activate macrophages and, if so, identify the structural elements involved in this action. For this purpose, we utilized human THP-1 macrophages, prepared by treating THP-1 monocytes with phorbol ester, and we exposed them to Lp(a) and its two derivatives, apo(a)-free LDL (Lp(a-)) and free apo(a). We also studied apo(a) fragments, F1 (N terminus) and F2 (C terminus) and subfragments thereof, obtained by leukocyte elastase digestion. By Northern blot analyses, Lp(a), but not Lp(a-), caused up to a 12-fold increase in interleukin 8 (IL-8) mRNA as compared with untreated cells. Free apo(a) also induced the production of IL-8 mRNA; however, the effect was 3-4-fold higher than that of Lp(a). The increase in mRNA was associated with the accumulation of IL-8 protein in the culture medium. F1 had only a minimal effect, whereas F2 was 1.5-2-fold more potent than apo(a), an activity mostly contained in the Kringle V-protease region. A monoclonal antibody specific for Kringle V inhibited the apo(a)-mediated effect on IL-8. We conclude that Lp(a) via elements contained in the C-terminal domain of apo(a) causes in THP-1 macrophages an increased production of IL-8, a chemokine with pro-inflammatory properties, an event that may be relevant to the process of atherosclerosis.

Oxidative events cause degradation of apoB-100 but not of apo[a] and facilitate enzymatic cleavage of both proteins.

Lipoprotein [a] (Lp[a]) contains equimolar amounts of apoB-100 and apolipoprotein [a] (apo[a]). Both proteins are amenable to degradation in vivo by mechanisms yet to be clearly defined. In this study, we examined the in vitro susceptibility of LDL and Lp[a], obtained from the same donor, to oxidation by either Cu(2)+ or the combined Crotalus adamanteus phospholipase A2 and soybean lipoxygenase system, monitoring the course of the reaction by the generation of conjugated dienes and fatty acids. In some experiments, treatment with leukocyte elastase (LE) or matrix metalloproteinase 12 (MMP-12) was administered before and after the oxidative step. In the case of Lp[a] we found that with both oxidizing systems, conditions that caused the breakdown of apoB-100 did not degrade apo[a] although oxidation-mediated changes were detected in the latter by intrinsic tryptophan fluorescence spectroscopy. Similar results were obtained with a reassembled Lp[a] obtained by incubating free apo[a] with LDL. Both apo[a] and apoB-100 were cleaved by LE and MMP-12 but the enzymatic cleavage was more marked when the preoxidized proteins were used as a substrate. Taken together, our in vitro studies indicate that apo[a] but not apoB-100 resists oxidative fragmentation, whereas both proteins are cleaved by enzymes of the serine and metalloproteinase families. We speculate that the fragments of apo[a] observed in vivo may be preferentially generated by proteolytic rather than oxidative events, whereas apoB-100 can be degraded by both mechanisms.

The role of lipoprotein(a) in the pathogenesis of atherosclerotic cardiovascular disease and its utility as predictor of coronary heart disease events.

Lipoprotein(a), is a highly heterogeneous lipoprotein, due to variations in the size of apolipoprotein(a), and the density of the apoB100-containing particles to which apo(a) is linked. Although high plasma levels of Lp(a) have been associated with an increased risk for atherosclerotic cardiovascular disease, the mechanism underlying this association is still largely undetermined, as is the potential role played by the particle's heterogeneity. Lp(a) pathogenicity may also be influenced by the action of environmental factors and post-translational events relating to oxidative processes, and the action of lipolytic and proteolytic enzymes. Complicating the study of Lp(a) are the competing methods for its quantification due to its complex structure, and the lack of standardized methodologies. The recognition that Lp(a) particles may not all be alike in atherogenic potential should encourage studies to identify genetic and nongenetic factors underlying its heterogeneity, in order to reach a better understanding of its actual impact on atherosclerotic cardiovascular disease.

Changes in plasma triglyceride levels shift lipoprotein(a) density in parallel with that of LDL independently of apolipoprotein(a) size.

Lipoprotein(a) [Lp(a)] represents a class of low density lipoprotein (LDL) particles that have as a protein moiety apolipoprotein B-100-linked covalently to a single molecule of apolipoprotein(a) [apo(a)], a specific multikringle protein of the plasminogen family. Lp(a) is polymorphic in density because of either the density heterogeneity of constitutive LDL, apo(a) size, or both. Authentic LDL also represents a set of heterogeneous particles whose density is affected by metabolic events. Whether in vivo these events may also affect Lp(a) density is not clearly established. To this effect, we studied 75 subjects with plasma Lp(a) protein levels between 7 and 50 mg/dL and containing a single apo(a) size isoform. We used density gradient ultracentrifugation to simultaneously monitor the changes in the peak density of LDL and Lp(a) at entry and during the course of treatments directed at reducing plasma triglyceride levels. In each case, we found that at entry, Lp(a) peak density was correlated with LDL peak density (r=0.71, P<0.0001) and that during treatment, changes in plasma triglycerides were associated with shifts of Lp(a) peak density that paralleled those of LDL peak density. A high correlation (r=0.94, P<0.0001) was particularly evident in subjects with initial plasma triglycerides in the 300-mg/dL range. In vitro assembly studies showed that an apo(a) isoform containing 14 kringle IV type 2 repeats, exhibited, on incubation with LDL, a comparable degree of incorporation into LDL species varying in density between 1.035 and 1.057 g/mL Taken together, our results indicate that metabolically dependent changes in the peak density of Lp(a) can occur independently of apo(a) size. These changes may have to be taken into account in assessing the cardiovascular pathogenicity of this lipoprotein particle in hypertriglyceridemic subjects.

Apolipoprotein(a): structure and biology.

Apolipoprotein(a), apo(a), the distinctive glycoprotein constituent of lipoprotein(a), Lp(a), is synthesized in the liver, links covalently to apoB100-lipoprotein, and travels so linked in the plasma to tissue sites where removal mechanisms are yet undetermined. Depending on the redox status of the surrounding milieu, apo(a) may re-acquire its unbound state shown to have structural and functional properties different from those of the bound form. Apo(a) is potentially athero-thrombogenic, a property which may be influenced by its size, sequence polymorphism, type of lipoprotein it is linked to and the inflammatory state of the vessel wall. This set of variables must be taken into account when assessing the cardiovascular pathogenicity of free and bound apo(a).

Lipoprotein(a), Friedewald formula, and NCEP guidelines. National Cholesterol Education Program.

The Friedewald low-density lipoprotein cholesterol formula, which is commonly used in clinical chemistry laboratories, comprises both low-density lipoprotein and lipoprotein(a) cholesterol. This confounder must be recognized and appropriately corrected when dealing with subjects with high plasma lipoprotein(a) levels.

Properties of human free apolipoprotein(a) and lipoprotein(a) after either freezing or lyophilization in the presence and absence of cryopreservatives.

Apolipoprotein(a), apo(a), the specific multikringle glycoprotein constituent of lipoprotein(a), Lp(a), occurs in the plasma mostly bound to apoB100-containing lipoproteins but also in a free form. Often the properties of these products are determined after storage in the cold; yet limited information is available on their stability at low temperatures. To shed light on this subject, we examined the effect of two parameters, freezing and lyophilization, in either the absence or the presence of cryopreservatives. Lp(a)s each having a single apo(a) size isoform containing either 14 or 17 kringle (K) IVs were isolated from the plasma of healthy donors by combining density gradient ultracentrifugation and lysine-Sepharose column chromatography using solutions containing both antioxidants and proteolytic inhibitors. Apo(a) was obtained from parent Lp(a) by a mild limited reductive procedure. Either freezing at -20 degrees C or lyophilization in the presence of 5% sucrose did not change the electrophoretic, immunochemical, and lysine-binding properties of Lp(a) including its ability to generate free apo(a). Irrespective of source, apo(a) remained stable when either frozen at -20 and -80 degrees C or lyophilized in the presence of 125 mM trehalose. In all cases, the absence of cryopreservatives caused the samples to aggregate irreversibly. Thawed or reconstituted samples of both free and bound apo(a) kept at 4 degrees C under sterile conditions in the presence of antioxidants, proteolytic inhibitors, and cryopreservative exhibited no significant changes in properties within the time of observation. Both apo(a) isoforms gave comparable results. We conclude that apo(a), either free or bound, can be kept stable at low temperatures in the presence of appropriate cryopreservatives.

Three cases of cardiac hydatidosis: diagnosis, surgical treatment, and complications.

Three cases of cardiac hydatid disease from among the many cases of hydatidosis (>300) in various organs observed by the authors are reported. The sites of the cysts and the complications that arose are described. The first case developed hydatid pulmonary embolism caused by rupture into the right ventricular cavity, the second suffered peripheral hydatid embolism caused by rupture into the left ventricular cavity, and the third, whose diagnosis was fortuitous, had no complications. The first patient died shortly after admission. The other two underwent radical pericystectomy and partial pericystectomy with cardiopulmonary bypass. The best result was obtained in the third case where rupture had not occurred. The second patient recovered but developed hemiparesis. The various diagnostic tools available are discussed, as well as some technical aspects of pericystectomy, which has a high mortality rate. The importance of early diagnosis and treatment of this rare localization of Echinococcus granulosus is emphasized, and echocardiography is recommended even for nonspecific cardiac symptoms in areas where the parasite is endemic.

99mTc-Tetrofosmin pinhole-SPECT (P-SPECT) and radioguided sentinel node (SN) biopsy and in breast cancer axillary lymph node staging.

We compared 99mTc-Tetrofosmin P-SPECT with radioguided SN biopsy in 101 T1/T2 BC pts to predict axillary lymph node status. The day before surgery all pts underwent lymphoscintigraphy (LS) to mark the SN, following subdermal injection of 99mTc-colloidal sulphur surrounding the breast lesion. LS was combined with pre and intraoperative gamma probe. Previously, all pts had also undergone P-SPECT. ALND was performed in all cases. The SN(s) was detected in 97/101 cases (96%) by LS and gamma probe; in the 4 missed cases P-SPECT predicted lymph node status. In the 97 comparable cases, radioguided SN biopsy showed a slightly higher accuracy than P-SPECT (94.8% vs 93.8%), but a higher false-negative rate (14.3% vs 8.6%); P-SPECT had a higher NPV (95.2% vs 92.5%). The two procedures when combined achieved 100% accuracy. Radioguided SN biopsy alone had 100% accuracy only in pts with BC < 15 mm. P-SPECT had 3 false negative cases, 2 of which were micrometastatic SNs, and 3 false positives. P-SPECT identified 81.2% of cases with a single node, determined the exact number of nodes in 82.6% of cases with 1 to 3 node and correctly classified 93.7% of pts as having < or = 3 or > 3 metastatic nodes. Radioguided SN biopsy seems indicated in selected, early stage, small BC pts, while P-SPECT shows a high sensitivity independent of primary tumor size, giving additional important preoperative prognostic information. The two procedures combined provided a better axillary lymph node status prediction in T1/T2 carcinomas, and could thus improve ALND pt selection.

Use of a reference material proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a).

BACKGROUND: As part of the NIH/National Heart, Lung and Blood Institute Contract for the Standardization of Lipoprotein(a) [Lp(a)] Measurements, a study was performed in collaboration with the IFCC Working Group for the Standardization of Lp(a) Assays. The aims of the study, performed with the participation of 16 manufacturers and 6 research laboratories, were to evaluate the IFCC proposed reference material (PRM) for its ability to transfer an accuracy-based value to the immunoassay calibrators and to assess concordance in results among different methods. METHODS: Two different purified Lp(a) preparations with protein mass concentrations determined by amino acid analysis were used to calibrate the reference method. A Lp(a) value of 107 nmol/L was assigned to PRM. After uniformity of calibration was demonstrated in the 22 evaluated systems, Lp(a) was measured on 30 fresh-frozen sera covering a wide range of Lp(a) values and apolipoprotein(a) [apo(a)] sizes. RESULTS: The among-laboratory CVs for these samples (6-31%) were, in general, higher than those obtained for PRM (2.8%) and the quality-control samples (14%, 12%, and 9%, respectively), reflecting the broad range of apo(a) sizes in the 30 samples and the sensitivity of most methods to apo(a) size heterogeneity. Thus, although all of the assays were uniformly calibrated through the use of PRM, no uniformity in results was achieved for the isoform-sensitive methods. CONCLUSIONS: Linear regression analyses indicated that to various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a). We have also shown that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease.

[Unusual case of benign neoplasm of the breast mimicking a carcinoma: granular cell tumor. Case report]. / Su un raro caso di neoplasia benigna della mammella simulante un carcinoma: il tumore a cellule granulose. Case report.

Granular cell tumors are rare, usually benign neoplasms of soft tissues which most commonly occur in the tongue, skin and subcutaneous tissue. Although the histogenesis is still object of debate, recent immunohistochemical studies and ultrastructural findings support the origin of this neoplasm from the peripheral nervous tissue, most likely from Schwann's cells. Occurrence of this neoplasm in the breast, although uncommon, warrants special attention, since its clinical, mammographic and pathological appearances on frozen sections "may often closely resemble" hose of breast malignancy. The authors analyze and commenton, with special reference to clinical aspects and surgical treatment, a case of benign granular cell tumor of the breast occurring in a 42 year-old woman. The mammographic and clinical findings suggested a breast carcinoma. The correct diagnosis was established by definitive microscopic examination of the paraffin-embedded specimens and the treatment was a simple local excision of the tumour and a small rim of surrounding breast parenchyma. Although the granular cell tumor of the breast is a rare entity, surgeons and pathologists should be aware of its existence in order to avoid inappropriate radical surgery not justified by the benign behavior of the neoplasm.

Apolipoprotein (a) fragments in relation to human carotid plaque instability.

PURPOSE: An elevated plasma level of lipoprotein (a) is an independent risk factor for atherothrombotic cardiovascular disease by yet undefined mechanisms. We have previously reported that matrix metalloproteinases cleave apolipoprotein (a) into 2 main fragments, F1 and F2, the latter (the C-terminal domain) exhibiting in vitro a high-affinity binding to extracellular matrix components, including fibrin(ogen). We therefore tested the hypothesis that the lipoprotein (a) matrix metalloproteinase-derived F2 is localized in potentially or morphologically unstable human carotid plaque at regions of increased matrix metalloproteinase activity. METHODS: Carotid plaques removed after endarterectomy (n = 18) were evaluated for structural features indicative of instability (thin fibrous cap, inflammation, and proximity of the necrotic core to the lumen); each plaque was classified as unstable (n = 10) or stable (n = 8). Western blot analysis was performed to quantitate apolipoprotein (a) and its fragments F1 and F2 in plaque extracts. Immunohistochemical staining was used to localize apolipoprotein (a) and its fragments within the atherosclerotic plaque. In situ zymography was used to determine regions of gelatinase (matrix metalloproteinase 2 and matrix metalloproteinase 9) activity. RESULTS: Western blot analyses demonstrated a 2.5-fold higher density of F2 in unstable plaques than in stable plaques (3.07 +/- 1.9 vs 1.18 +/- 0.8; P <.05). In morphologically unstable plaques, there was preferential distribution of F2 within regions of fibrous cap inflammation and/or foam cell accumulation and within abluminal necrotic cores. In morphologically stable plaques, however, localization was predominantly found in the medial smooth muscle cells. Regions of enhanced matrix metalloproteinase 2 and matrix metalloproteinase 9 activity co-localized with the transmural distribution of F2 within the plaque. CONCLUSIONS: These findings suggest that F2 in regions of increased matrix metalloproteinase activity is a potential mechanism for superimposed thrombotic events in morphologically unstable human carotid plaques. The relationship between plasma lipoprotein (a) levels and accumulation of F2 and the potential correlation of F2 to human plaque disruption and thrombosis warrant further study.

Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan.

Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitro with the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined the in vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE.DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs.

Macrophage metalloelastase, MMP-12, cleaves human apolipoprotein(a) in the linker region between kringles IV-4 and IV-5. Potential relevance to lipoprotein(a) biology.

In this study we found that macrophage metalloelastase, MMP-12 cleaves, in vitro, apolipoprotein(a) (apo(a)) in the Asn3518-Val3519 bond located in the linker region between kringles IV-4 and IV-5, a bond immediately upstream of the Ile3520-Leu3521 bond, shown previously to be the site of action by neutrophil elastase (NE). We have also shown that human apo(a) injected into the tail vein of control mice undergoes degradation as reflected by the appearance of immunoreactive fragments in the plasma and in the urine of these animals. To define whether either or both of these enzymes may be responsible for the in vivo apo(a) cleavage, we injected intravenously MMP-12(-/-), NE -/- mice and litter mates, all of the same strain, with either lipoprotein(a) (Lp(a)), full-length free apo(a), or its N-terminal fragment, F1, obtained by the in vitro cleavage of apo(a) by NE. In the plasma of Lp(a)/apo(a)-injected mice, F1 was detected in control and NE -/- mice but was virtually absent in the MMP-12(-/-) mice. Moreover, fragments of the F1 type were present in the urine of the animals except for the MMP-12(-/-) mice. These fragments were significantly smaller in size than those observed in the plasma. All of the animals injected with F1 exhibited small sized fragments in their urine. These observations provide evidence that, in the mouse strain used, MMP-12 plays an important role in the generation of F1 from injected human Lp(a)/apo(a) and that this fragment undergoes further cleavage during renal transit via a mechanism that is neither NE- nor MMP-12-dependent. Thus, factors influencing the expression of MMP-12 may have a modulating action on the biology of Lp(a).

Effect of phospholipase A2 digestion on the conformation and lysine/fibrinogen binding properties of human lipoprotein[a].

In vitro hydrolysis of human lipoprotein[a] (Lp[a]) by phospholipase A2 (PLA2) decreased the phosphatidylcholine (PC) content by 85%, but increased nonesterified fatty acids 3.2-fold and lysoPC 12.9-fold. PLA2-treated Lp[a] had a decreased molecular weight, increased density, and greater electronegativity on agarose gels. In solution, PLA2-Lp[a] was a monomer, and when assessed by sedimentation velocity it behaved like untreated Lp[a], in that it remained compact in NaCl solutions but assumed the extended form in the presence of 6-amino hexanoic acid, which was shown previously to have an affinity for the apo[a] lysine binding site II (LBS II) comprising kringles IV5-8. We interpreted our findings to indicate that PLA2 digestion had no effect on the reactivity of this site. This conclusion was supported by the results obtained from lysine Sepharose and fibrinogen binding experiments, in the presence and absence of Tween 20, showing that phospholipolysis had no effect on the reactivity of the LBS-II domain. A comparable binding behavior was also exhibited by the free apo[a] derived from each of the two forms of Lp[a]. We did observe a small increase in affinity of PLA2-Lp[a] to lysine Sepharose and attributed it to changes in reactivity of the LBS I domain (kringle IV10) induced by phospholipolysis. In conclusion, the extensive modification of Lp[a] caused by PLA2 digestion had no significant influence on the reactivity of LBS II, which is the domain involved in the binding of apo[a] to fibrinogen and apoB-100. These results also suggest that phospholipids do not play an important role in these interactions.

Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance.

The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead.

Dominant role of the C-terminal domain in the binding of apolipoprotein(a) to the protein core of proteoglycans and other members of the vascular matrix.

The C-terminal domain of apolipoprotein(a) binds in vitro to the protein core of proteoglycans as well as fibrinogen and fibronectin, suggesting that this domain plays a role in anchoring lipoprotein(a) or free apolipoprotein(a) to the vascular subendothelial matrix.