Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

Rhesus lipoprotein(a) (Lp[a]) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K4(37) plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp(a).

Enzymatic and chemical modifications of lipoprotein(a) selectively alter its lysine-binding functions.

The pathogenicity of lipoprotein(a) [Lp(a)] as a risk factor for cardiovascular disease may depend upon its lysine binding sites (LBS) which impart unique functions to Lp(a) not shared with low density lipoprotein. Biologically relevant modifications of Lp(a) were tested for alterations of LBS activity using two previously described functional assays, a LBS-Lp(a) immunoassay and a lysine-Sepharose bead assay. In the LBS-Lp(a) immunoassay, minimal changes in the LBS activity of Lp(a) were observed after modification with lipoprotein lipase, sphingomyelinase, or phospholipase C. In contrast, a significant (p<0.003) increase in the LBS activity of Lp(a) occurred after phospholipase A2 (PLA2) treatment, and this increase was confirmed using the lysine-Sepharose bead assay. The increase depended upon the release of fatty acids from Lp(a) by PLA2. A decrease in the LBS activity of Lp(a) occurred after oxidation of Lp(a) with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) (44% decrease), but CuSO4 oxidation increased LBS activity (210%). N-acetylcysteine (NAC) treatment of Lp(a) decreased (48%) LBS activity while homocysteine treatment had no (89%) effect. Thus, modification of phospholipids and protein moieties can alter the LBS-activity of Lp(a). Such enzymatic and chemical modifications may contribute to the variability in LBS function of Lp(a) seen within the population.

Apo(a) promotes thrombosis in a vascular injury model by a mechanism independent of plasminogen.

OBJECTIVE: Structural similarity between apolipoprotein(a) [apo(a)], the unique apoprotein of lipoprotein(a), and plasminogen (Plg), the zymogen for plasmin, results in inhibition of functions of Plg by apo(a) in vitro. The objective of this study was to evaluate the interaction of Plg and apo(a) in vivo. METHODS AND RESULTS: Vascular injury was induced in the carotid artery with a perivascular cuff in: (i) wild-type (WT); (ii) Plg deficient (Plg-/-); (iii) apo(a) (6 KIV construct) transgenic [apo(a)tg]; and (iv) apo(a) transgenic and Plg deficient [apo(a):Plg-/-] mice. At 10 days after cuff placement, the media and adventitia area were increased in the injured carotids compared with the uninjured carotids, and collagen deposition was greater in apo(a)tg, Plg-/- and apo(a):Plg-/- mice compared with WT mice. The incidence of a thrombus was greater (P < 0.05) in apo(a):Plg-/- mice (83%) than WT (20%), Plg-/- (12%), and apo(a)tg mice (9%). In the thrombi from apo(a)tg and apo(a):Plg-/- mice, P-selectin and von Willebrand factor immunostaining, indicating a platelet-rich thrombi, was greater than in WT and Plg-/- mice. The presence of fibrin(ogen) in the thrombi was greater in Plg-/- and apo(a):Plg-/- mice than apo(a)tg and WT mice. Of the four genotypes, only the apo(a):Plg-/- mice had both increased platelet and increased fibrin(ogen) deposition. CONCLUSIONS: The major finding of this study is the high incidence of thrombosis after vascular injury in apo(a)transgenic mice in a Plg deficient background, providing strong evidence for a prothrombotic role of apo(a) independent of Plg in vivo.

Polymorphisms in human apolipoprotein(a) kringle IV-10 and coronary artery disease: relationship to allele size, plasma lipoprotein(a) concentration, and lysine binding site activity.

Elevated plasma levels of lipoprotein(a) [Lp(a)] represent a major independent risk factor for the development of atherosclerosis. The kringle IV type 10 of apolipoprotein(a) [apo(a)] is the primary lysine binding site (LBS) of Lp(a) and is associated with lesion formation in transgenic mice. The purpose of this study was to search for mutations in the apo(a) kringle IV type 10 which could alter the LBS activity of Lp(a) from patients with coronary artery disease. We found the DNA region of kringle IV type 10 of apo(a) to be mutable but relatively well preserved in the Spanish population. We identified a novel mutation which probably leads to a truncated form of apo(a) in a patient heterozygous for the mutation and with low lysine binding activity and low plasma Lp(a) concentration. Two other mutations have been previously identified in humans, the substitutions W81R and M75T. The W81R was not found in our sample, but the M75T mutation was present in 43% of patients with coronary artery disease and 23% of age-matched controls. The genotype TT conferred a significant risk for myocardial infarction (odds ratio 2.53). This association was not due to linkage disequilibrium with kringle IV repeats. The M75T polymorphism was not associated with the LBS function of apo(a), but it influenced plasma Lp(a) concentration.

The glycemic response to meals with six different fruits in insulin-dependent diabetics using a home blood-glucose monitoring system.

Glycemic effect of adding 10-g carbohydrate portions of apple, banana, grapes, honeydew, orange, or strawberries to a standard meal on separate occasions was measured in 10 insulin-dependent diabetics monitored at home. The meal comprised 29% of total daily caloric intake and contained green beans, rice, turkey, and margarine (50% carbohydrate, 20% protein, and 30% fat). Blood-glucose response to meals containing grapes, honeydew, orange, or strawberries was slightly higher than meals containing apple, banana, or no fruit and the small amount of starch in apple and banana may have contributed to their lower blood-glucose response compared to the other fruits tested. Age, duration of diabetes, or insulin regimen did not correlate with subjects' responses to fruit.

Plasminogen regulates pro-opiomelanocortin processing.

BACKGROUND: Plasminogen-deficient mice exhibit behavioral differences in response to stress, including a markedly reduced acoustic startle reflex response compared with wild-type (WT) littermates. The acoustic startle reflex activates the hypothalamic-pituitary axis and is modulated by these hormones. OBJECTIVES: The purpose of this study was to investigate whether plasminogen plays a role in the processing of hormones in the hypothalamic-pituitary axis. METHODS: In this study the concentration of plasma, pituitary, and brain hypothalamic-pituitary axis hormones and precursor processing was examined in WT and plasminogen deficient (Plg-/-) mice before and after acoustic startle reflex testing. RESULTS: Plasma adrenocorticotropic hormone (ACTH), beta-endorphin and alpha-melanocyte stimulating hormone were elevated after acoustic startle reflex testing in both WT and (Plg-/-) mice. However, in the Plg-/- mice, beta-endorphin values were 43, 35, and 45% lower in the plasma, pituitary, and whole brain, respectively, compared with the WT mice. Plasmin readily degraded precursor peptides, the 23-kDa precursor, beta-lipotropin, and ACTH, when presented as purified proteins or as the secretory products of mouse pituitary cells (AtT-20). The precursor peptide, 23 kDa, for beta-endorphin and alpha-melanocyte stimulating hormone was reduced in the pituitaries from the Plg-/- mice, and the mRNA for Plg was found in pituitaries from WT mice. Infusion of beta-endorphin and alpha-melanocyte stimulating hormone into the brain of Plg-/- mice increased acoustic startle reflex. CONCLUSIONS: The results of this study show that plasmin is involved in the processing of hormones derived from the pro-opiomelanocortin precursor in the intermediate pituitary. A deficiency of plasminogen reduces processing of beta-endorphin and alpha-melanocyte stimulating hormone, and interferes with normal brain function.

The role of plasminogen in angiogenesis in vivo.

UNLABELLED: Plasminogen, by virtue of its role in the degradation of extracellular matrix proteins and by facilitation of cell migration, may contribute to angiogenesis. OBJECTIVE: the purpose of this study was to evaluate the contribution of plasminogen to angiogenesis in vivo. METHODS: Angiogenesis was assessed in gene-targeted mice with deficiencies of plasminogen, urokinase plasminogen activator (uPA), and urokinase receptor (uPAR) in a mouse corneal model. In wild-type mice, female and young mice showed a trend toward increased angiogenesis compared to males and old mice. Because of this influence of age and gender on angiogenesis, young, female mice (6-13 weeks of age) were used for this study. RESULTS: In response to angiogenic stimulation by basic fibroblast growth factor (bFGF), uPA deficient mice exhibited a decrease in new vessel formation as reflected by vessel length (0.47 in control vs. 0.33 mm in uPA-/- mice, P = 0.043), but new vessel formation was not altered (P = 0.107) in the uPAR deficient mice compared to control mice. A significantly decreased angiogenic response of new vessel formation to both vascular endothelial growth factor (VEGF) (P < 0.02) and bFGF (P < 0.007) was observed in Plg deficient (Plg-/-) mice (VEGF - 0.36 mm, bFGF - 0.67 mm) compared to Plg+/+ mice (VEGF - 0.56 mm, bFGF - 0.85 mm). CONCLUSIONS: These results demonstrate the importance of plasminogen, as well as of uPA, in angiogenesis in vivo.

Phospholipase A2 modification enhances lipoprotein(a) binding to the subendothelial matrix.

Lipoprotein(a), Lp(a), is found in the extracellular matrix in atherosclerotic plaques, but with a different localization than LDL. A two-compartment system, with a monolayer of endothelial cells forming a barrier, was used to compare the transport, cell binding, and retention of Lp(a) and LDL into the subendothelial matrix. Baseline values for transport and retention of Lp(a) and LDL were not significantly different. Incubation with lipoprotein lipase or sphingomyelinase caused modest and similar increases in transport and retention of the two lipoproteins. In contrast, incubation with phospholipase A2 (PLA2) resulted in a marked (4-fold) increase in retention of Lp(a) on the subendothelial matrix, but a lesser (2-fold) increase in LDL retention. Moreover, PLA2 treatment of Lp(a) enhanced its binding to individual matrix proteins (fibronectin, laminin, or collagen) by 4-10 times above that of LDL. The enzymatic activity of PLA2 was responsible for its effect on Lp(a) binding. The lysine binding sites of Lp(a) contributed to the increased binding of PLA2-modified Lp(a) to the matrix, and the enhanced lysine binding functions of PLA2-modified Lp(a) was demonstrated by two independent approaches. Thus, PLA2 modification leads to enhanced interactions of lipoproteins with the extracellular matrix, and this effect is more pronounced with Lp(a).

Impact of apolipoprotein(a) isoform size heterogeneity on the lysine binding function of lipoprotein(a) in early onset coronary artery disease.

Elevated plasma Lp(a) is an independent risk factor for cardiovascular disease. Unique to Lp(a) is the apoprotein, apo(a) which can vary from 250 to 800 kDa in molecular weight. Small isoforms are also associated with the risk of cardiovascular disease. The purpose of this study was to examine the association of Lp(a) concentration, apo(a) size, and Lp(a) lysine-binding site(s) (LBS) function in patients with early onset heart disease, and age-matched controls. Mean values of Lp(a) were significantly higher in the patients than for the age-matched group. The smallest molecular weight isoform for each subject had significantly fewer kringles for the patients than the age-matched controls. There was a significant correlation between LBS activity and kringle number in the single-banded phenotypes of the patients, but not the controls. LBS activity was significantly higher in patients with small isoforms (< or =18 kringles) compared to controls. The odds ratio for coronary artery disease for high LBS activity and high Lp(a) concentration was 4.4 (p = 0.002) and for high LBS activity and small isoforms was 10.1 (p = 0.002). In the patients, Lp(a) concentration was higher, apo(a) size was smaller, and LBS activity higher in the small isoforms compared to the controls. This study suggests an association of high LBS activity in small isoforms of Lp(a) with disease in humans.

Interaction of Lp(a) with plasminogen binding sites on cells.

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Selective behaviors altered in plasminogen-deficient mice are reconstituted with intracerebroventricular injection of plasminogen.

In vitro studies demonstrate a role for the plasminogen (Plg) system in neurological function and recently in vivo studies show a role of the Plg system in neurodegeneration after the injection of an excitotoxic agent. Differences in the development of neurological function, however, have not been demonstrated in the Plg-deficient (Plg-/-) mice compared to wild-type (WT) mice. The role of Plg system in neurological function may relate to remodeling which occurs in response to various environmental challenges. In this study, behaviors (open field, grooming, hind-leg gait, water maze, and acoustic startle reflex) were tested in the Plg-deficient and WT mice at 6-8 weeks of age. Grooming, a response to the stress of an open field or fur moistening, was increased in the Plg-/--deficient mice compared to WT mice, and the acoustic startle reflex (ASR) was markedly decreased in the Plg-/- mice. The reduced ASR in Plg-/- mice occurred in mice with a mixed C57BL:129 background or in mice with a C57BL background. Plg was required for the ASR, since a deficiency of the Plg activators, urokinase (uPA) or tissue Plg activator (tPA), did not cause a reduction in the ASR compared to their WT control. Infusion of Plg directly into the brain was effective in restoring the ASR in the Plg-/- mice, but had no effect on the ASR of WT mice. Peripheral bolus injections of Plg or infusion into the jugular vein were ineffective in restoring the ASR in the Plg-/- mice. These results indicate that Plg is required for the appropriate response to the environmental challenge of a sudden loud sound, and that the response can be restored in Plg-/- mice by directly infusing Plg into the brain.

Growth and behavioral development in plasminogen gene-targeted mice.

The plasminogen system, in addition to its major role in fibrinolyis, is believed to play a key role in development of the nervous system. The purpose of this study was to directly examine and quantify the importance of plasminogen in physical and behavioral development in plasminogen deficient mice (Plg-/-), plasminogen heterozygous mice (Plg+/-), and wild-type mice (Plg+/+, WT) at 2-21 days of age. Remarkably, little difference in growth and behavioral development was observed between Plg-/- and WT mice. Body weight gain and the milestones of physical development-ear detachment, eye opening and teeth eruption were similar from 2-21 days of age. Differences were found in physical development only after 4 wks of age, body weight gain was less and vaginal patency was delayed in the Plg-/- mice compared to WT mice. Behaviors, assessed during the 2-21 days of age period, developed in the Plg-/- mice in a pattern similar to WT mice. Specifically, no differences were found between Plg-/- and WT mice in the development of reflexes, neuromotor ability, motor coordination, locomotor activity, reaction to gravitational positioning, integration of motor and vestibular systems, olfactory development, and incidence of audiogenic seizure susceptibility. However, Plg-/- mice demonstrated a faster surface righting response and a faster latency for audiogenic seizure susceptibility, as well as an increase in the number of grooming bouts at age 17-21 days. These differences indicate that a plasminogen deficiency alters reactivity and the response to stress. The weight of the pituitary was smaller and pituitary and plasma corticotrophin releasing hormone were elevated in the Plg-/- mice compared to the WT mice. The results of this study suggest a role for the plasminogen system in hormone processing and neuroendocrine regulation.

Strain and model dependent differences in inflammatory cell recruitment in mice.

OBJECTIVE AND DESIGN: The objective of this study was to determine genetic differences in inflammation in these distinct inbred mouse strains. METHODS: Peritoneal leukocyte recruitment, matrix metalloproteinases and cytokines were quantified in A/J, 129/svJ, C57BL/6J, using thioglycollate or biomaterial implants as inflammatory stimuli. RESULTS: In response to thioglycollate, A/J had significant decreases compared to C57BL/6J in both neutrophil (86 %) and macrophage (62 %) recruitment, and 129/svJ had a significant (43 %) decrease compared to C57BL/6J in macrophage recruitment. The reduced leukocyte recruitment corresponded to reduced matrix metalloproteinase-9. In the bioimplant model, 129/svJ had a 2-fold increase in neutrophil and macrophage recruitment compared to C57BL/6J, and the increased leukocyte recruitment corresponded to elevated cytokines, monocyte inhibitory protein-2 and monocyte chemoattractant protein-1, in the lavage compared to the values for C57BL/6J. CONCLUSION: Not only was leukocyte recruitment strain dependent, but the three strains had marked differences in metalloproteinases and cytokine response. In addition, there were model specific differences in the metalloproteinase and cytokine response to the two inflammatory stimuli. Thus, inflammatory cell recruitment is genetically determined and stimulus specific and may determine the susceptibility to complex diseases.

Plasminogen binding is increased with adipocyte differentiation.

The purpose of this study was to examine the role of the plasminogen system in the development of adipose tissue. Plasminogen binding capacity was determined in differentiated and undifferentiated cells from adipose tissue of plasminogen deficient mice and 3T3 cells, a well-characterized tissue culture model. In 3T3 cells, plasminogen binding was fivefold higher in differentiated cells compared to the undifferentiated cells. Inhibition of binding by carboxyl-terminal lysine analogs was similar for the differentiated and undifferentiated cells with tranexamic acid > EACA > lysine. The binding of plasminogen was concentration-dependent and approaches saturation in the both cell types. The number of plasminogen binding sites was tenfold higher in the differentiated compared to the undifferentiated cells. In isolated mature fat cells and stromal cell cultures from mouse adipose tissue, plasminogen binding was also higher in the differentiated mature fat cells and differentiated stromal cells compared to undifferentiated stromal cells. Plasminogen binding was elevated in the differentiated cells from the Plg-/- mice compared to cells from the WT mice. These results suggest that the plasminogen system plays an important role in adipose tissue development.

Oxygen consumption in mice (I strain) after feeding.

To determine if excess heat production can account for the lower fat accumulation in I strain mice, oxygen consumption, a measure of energy expenditure, was measured in I mice and C57BL mice (a control strain) to determine basal metabolic rate (BMR) and resting metabolic rate (RMR) and response to food consumption and acute cold exposure. Oxygen consumption was higher in the I strain than in the C57BL mice only after the dark cycle (feeding period). No difference between I and C57BL mice in spontaneous activity was found during the dark or light cycle. Body temperature was also not different in I and C57BL mice. Oxygen consumption in response to norepinephrine was similar in the two strains. These results indicate oxygen consumption is greater in I than C57BL mice only in response to feeding. Differences in glucose utilization by I and C57BL mice, including a lower glucose tolerance curve, greater deposition of glucose to muscle glycogen and lactate production in I mice also indicate differences in nutrient processing. Higher oxygen consumption after feeding in I mice than in C57BL mice indicates inefficient food utilization and accounts for their lower ability to store energy as fat.

Increased insulin mediated glucose metabolism in fat cells from I versus C57BL mice.

The purpose of this study was to determine whether adipocytes from I strain mice, which are characterized by a greater in vivo glucose tolerance than most other strains, had a higher capacity to utilize glucose in response to physiological concentrations of insulin. Using C57BL mice as a control strain, we examined the effect of insulin on glucose metabolism in epididymal and inguinal adipocytes from 2-month-old male mice. Body weight was only slightly less (7%) for the I mice than for the C57BL mice, but fat pad sizes were 60 and 20% less for epididymal and inguinal depots, respectively, in the I mice. Fat cell size was also smaller in epididymal adipocytes from the I mice than from the C57BL mice. Fat cell size of inguinal adipocytes was similar in the two strains. Without insulin the rates of [U-14C]glucose incorporation into CO2 or lipids were twofold higher in cells from the I mice than in those from the C57BL mice. Maximal insulin concentration (2.5 nM) increased glucose metabolism by 140 and 500% in epididymal and inguinal adipose cells, respectively, in the I mice versus 30 and 50% in the C57BL mice. The maximal effect of insulin was reached at a much higher insulin concentration in the I mice than in the C57BL mice. The activity of fatty acid synthetase was four- to sixfold higher in fat cells from I than in those from C57BL mice. These results demonstrate an increased insulin responsiveness of glucose metabolism in fat cells from the I mice related to an increased lipogenic capacity. Furthermore, they show that adipose tissue in mice exhibits significant regional differences in terms of insulin responsiveness of glucose metabolism.

Reproductive performance in C57BL and I strain mice.

Mice of the I strain are regarded as difficult to breed. To characterize and elucidate the cause of poor reproductive performance in I strain mice, records of reproductive performance were analyzed from Cancer Research Laboratory, Kirschbaum Memorial Laboratory and Strong Research Laboratories which had bred the I strain mice and a control strain, C57BL, and compared with data from Jackson Laboratory and from three dietary studies conducted in this laboratory. Reproductive performance in the I strain mice is characterized by fewer productive matings, fewer litters and smaller litters than C57BL mice. Also, fewer mice of the I strain survive to weaning. These factors result in the production of only half as many mice per dam in the I strain as the C57BL mice. The fewer number of litters is not due to a greater proportion of resorptions in the I mice, nor are there differences in the survival of either sex. Diets which contain slightly higher (8-12 mg per kg of diet) amounts of pyridoxine (PN) than recommended (1-6 mg per kg of diet) improve performance in the I strain. However, 410 or 1230 mg PN does not increase productivity further. That survival rate of the C57BL mice improves with higher amounts of PN (410 and 1230 mg diets), suggests another dietary factor may be limiting for I strain mice. The reproductive performance in I strain mice is better with diets which result in a higher body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of surgical trauma on muscle protein turnover in rats.

The rate of synthesis and catabolism of sarcoplasmic- and myofibrillar-muscle protein was measured in operated, sham-operated and food-restricted rats by using Na2 14CO3. The food-restricted group underwent sham operations and were limited to the food intake of the operated animals. Protein synthesis and catabolism were increased in the sarcoplasmic-muscle fraction in operated rats compared with that in sham-operated or food-restricted rats. The rate of synthesis of the myofibrillar protein decreased in operated animals, but the rate of catabolism was not altered in the myofibrillar-muscle fraction of the operated animals compared with that in food-restricted and sham-operated animals. In the operated animals, there was a net loss of protein from the muscle. Thus the rats that underwent surgery lost muscle protein, primarily as a result of a decrease in synthesis of myofibrillar protein. The changes in protein turnover in operated animals were not due to decreases in food intake, since protein turnover in sham-operated animals that were restricted to the food intake of the operated rats was not different from that in sham-operated rats fed ad libitum.