Robert D. Litwiller

Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women

While the risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened for enrollment into the Kronos Early Estrogen Prevention Study. Microparticles were evaluated by flow cytometry, and coronary artery calcification (CAC) was scored using 64-slice computed tomography scanners. The procoagulant activity of isolated microparticles was determined with a sensitive fluorescent thrombin generation assay. Chronological age, body mass index, serum lipids, systolic blood pressure (Framingham risk score < 10%, range 1-3%), and high-sensitivity C-reactive protein did not differ significantly among women with low (0 < 35; range, 0.3-32 Agatston units) or high (>50; range, 93-315 Agatston units) CAC compared with women without calcification. The total concentration and percentage of microparticles derived from platelets and endothelial cells were greatest in women with high CAC scores. The thrombin-generating capacity of the isolated microparticles correlated with phosphatidylserine expression, which also was greatest in women with high CAC scores. The percentages of microparticles expressing granulocyte and monocyte markers were not significantly different among groups. Therefore, the characterization of platelet and endothelial microparticles may identify early menopausal women with premature CAC who would not otherwise be identified by the usual risk factor analysis.

Alterations in Platelet Function and Cell-Derived Microvesicles in Recently Menopausal Women: Relationship to Metabolic Syndrome and Atherogenic Risk

A woman’s risk for metabolic syndrome (MS) increases at menopause, with an associated increase in risk for cardiovascular disease. We hypothesized that early menopause-related changes in platelet activity and concentrations of microvesicles derived from activated blood and vascular cells provide a mechanistic link to the early atherothrombotic process. Thus, platelet functions and cellular origin of blood-borne microvesicles in recently menopausal women (n = 118) enrolled in the Kronos Early Estrogen Prevention Study were correlated with components of MS and noninvasive measures of cardiovascular disease [carotid artery intima medial thickness (CIMT), coronary artery calcium (CAC) score, and endothelial reactive hyperemic index (RHI)]. Specific to individual components of the MS pentad, platelet number increased with increasing waist circumference, and platelet secretion of ATP and expression of P-selectin decreased with increasing blood glucose (p = 0.005) and blood pressure (p < 0.05), respectively. Waist circumference and systolic blood pressure were independently associated with monocyte- and endothelium-derived microvesicles (p < 0.05). Platelet-derived and total procoagulant phosphatidylserine-positive microvesicles, and systolic blood pressure correlated with CIMT (p < 0.05), but not with CAC or RHI. In summary, among recently menopausal women, specific platelet functions and concentrations of circulating activated cell membrane-derived procoagulant microvesicles change with individual components of MS. These cellular changes may explain in part how menopause contributes to MS and, eventually, to cardiovascular disease.

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3‐related autoimmune processes investigated in wild‐type‐, mNE‐ and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico‐chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc‐AAPV‐pNA and Suc‐AAPV‐pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by α1‐antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom‐designed hNE inhibitor, Val15‐aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species‐specific assessment of neutrophil protease function and inhibition.

Purification of Six Human Vitamin K-Dependent Proteins in a Single Chromatographic Step Using Immunoaffinity Columns1

The major human vitamin K-dependent proteins were purified from plasma using immunoadsorbents made with antibodies specific for each protein. Monoclonal antibodies to Factor VII, Factor IX, Factor X, Protein C, and Protein S were prepared from mice immunized with isolated vitamin K-dependent antigens. Purified monoclonal antibodies and a purified burro polyclonal anti-prothrombin immunoglobulin were individually coupled to Sepharose and used in a tandem series of columns to purify each of the vitamin K-dependent proteins from eluates of barium citrate precipitates of plasma. The proteins were eluted from the columns by sodium thiocyanate and retained functional activity following dialysis. Prothrombin, Factor VII, Factor IX, Factor X and Protein C were essentially homogeneous as judged by NaDodSO4-PAGE; Protein S was isolated as a Protein S-C4b binding protein complex. These results indicate the utility of monoclonal antibody immunoadsorbents for purifying the human vitamin K-dependent proteins and represent a considerable simplification over other purification schemes.

Circulating microparticles and endogenous estrogen in newly menopausal women

Background Estrogen modulates antithrombotic characteristics of the vascular endothelium and the interaction of blood elements with the vascular surface. A marker of these modulatory activities is formation of cell-specific microparticles. This study examined the relationship between blood-borne microparticles and endogenous estrogen at menopause. Methods Platelet activation and plasma microparticles were characterized from women being screened (n = 146) for the Kronos Early Estrogen Prevention Study. Women were grouped according to serum estrogen (< 20 pg/ml; low estrogen, n = 21 or > 40 pg/ml; high estrogen, n = 11). Results Age, body mass index, blood pressure and blood chemistries were the same in both groups. No woman was hypertensive, diabetic or a current smoker. Platelet counts, basal and activated expression of P-selectin on platelet membranes were the same, but activated expression of glycoprotein IIb/IIIa was greater in the high-estrogen group. Numbers of endothelium-, platelet-, monocyte- and granulocyte-derived microparticles were greater in the low-estrogen group. Of the total numbers of microparticles, those positive for phosphatidylserine and tissue factor were also greater in the low-estrogen group. Conclusion These results suggest that, with declines in endogenous estrogen at menopause, numbers of procoagulant microparticles increase and thus may provide a means to explore mechanisms for cardiovascular risk development in newly menopausal women.

Carboxypeptidase inhibitors from Ascaris suum: the primary structure

The carboxypeptidase A inhibitor from Ascaris suum was isolated from aqueous extracts by affinity chromatography toward immobilized carboxypeptidase A. The amino acid sequence is DQVRKCLSDT10DCTNGEKCVQ20KNKICSTIVE30IQRCEKEHFT40IPCKSNNDCQ50VWAHEKICN K60LPWGL65 . The carboxypeptidase A inhibitor is not homologous with the chymotrypsin/elastase or trypsin inhibitors from Ascaris, but shows homology in a 9-residue internal sequence with the 37/39-residue carboxypeptidase inhibitors from tomato and potato. The carboxy-terminal 5 (4) residues in the three inhibitors are similar, suggesting a common mechanism of inhibition.

Liquid ion exchangers in paper chromatography of steroidal glucosiduronic acids, glucosiduronic esters and free steroids : Influence of concentration of exchanger and counterion

The chromatographic mobility of steroidal glucosiduronic acids on paper in chloroform-formamide increases as the concentration of ion exchanger in the chloroform phase increases; mobility decreases as the concentration of counterion in formamide increases. Mobility of glucosiduronic esters and of hydroxylated free steroids increases with an increase in concentration of exchanger; small changes in concentration of counterion in the stationary phase do not alter the migration of these nonionizable compounds. Data are presented which suggest that partition of the glucosiduronic acids between the two phases occurs predominantly by an ion-exchange process and that hydrogen bonding plays a secondary role. Partition of the glucosiduronic esters and hydroxylated free steroids appears to occur primarily by a hydrogen-bonding process.

The amino acid sequence of the NH2-terminal portion of rat and human vitamin D binding protein: Evidence for a high degree of homology between rat and human vitamin D binding protein

We determined the amino terminal sequence of rat and human vitamin D binding protein (VDBP). The sequences of the two proteins are: Rat VDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysGlnGluLeuSerThrLeuGlyLys Human VDBP: LeuGluArgGlyArgAspTyrGluLysAsnLysValCysLysGluPheSerHisLeuGlyLys AspAspPhe GluAspPhe There are 19 matches out of a total of 24 residues sequenced giving a percent match/length of 79.2%. Differences in the composition of the two proteins at residue 10, 14, 16, and 22 can be accounted for by single base changes in the the gene for the proteins. The difference (Thr----His) at residue 18 requires a change in two bases in the respective genes. We conclude that the sequence of the amino terminus of rat and human VDBP is similar with a high degree of homology between the two proteins. Vitamin D sterols, such as 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 25,26-dihydroxyvitamin D3, are bound with high affinity by a plasma alpha-globulin - VDBP, also known as group-specific component (Gc). Other vitamin D sterols, such as 1,25-dihydroxyvitamin D3 and vitamin D3 itself, are bound to this protein with a lesser affinity. VDBP also binds actin with high affinity. Its role in vitamin D physiology is unclear, although it may play a role in the bioavailability of different D sterols. Svasti et al. have shown that human group specific component (Gc) exists as different isoforms that have rapid or slow mobility on gel electrophoresis. The different human Gc isoforms have similar NH2-terminal and COOH-terminal amino acid sequences. The difference in the mobility of the various isoforms is due to post-translational modification of the protein by various carbohydrate residues; treatment of the protein with neuraminidase results in the conversion of the different isoforms to a single isoform. The amino acid sequence of the amino terminus of rat VDBP is not known. Recently, Cooke reported preliminary data from the analysis of cDNA clones showing that rat and human VDBP are partially homologous and that rat and human VDBP exhibit homology with rat and human albumin and alpha-fetoprotein. The NH2-terminal sequence of the rat VDBP, however, has not been reported. In order to learn more about the nature of the NH2-terminal sequence of human and rat VDBP, we isolated these proteins in relatively pure form and determined the NH2-terminal amino acid sequence of both of them.(ABSTRACT TRUNCATED AT 400 WORDS)

Liquid ion exchangers in reversed-phase systems for chromatography of steroidal glucosiduronic acids.

Steroidal glucosiduronic acids were chromatographed on paper by the reversed-phase technique using five different liquid ion exchangers as stationary phase and aqueous KCl as mobile phase. The relationship of mobility of the acids (Rm) to both the amount of exchanger on the paper and the concentration of KCl in the mobile phase is discussed: the relationships may be expressed as Rm=n.log [exchanger] + const. and RM=-N.LOG [KCl] + const., respectively. Migration of the acids in the presence of different exchangers is correlated by use of the equation Rm (exchanger Y)=a.Rm (exchanger X) + b. The lack of appreciable correlation between migration of the acids in a reversed-phase system and a corresponding straight-phase system is discussed and expressed by means of regression equations. The correlation coefficients and standard errors of estimate from these equations provide useful indices for selecting two solvent systems that are to be used sequentially to obtain maximal resolution of a group of compounds. deltaRm values obtained for various functional groups with reversed-phase and straight-phase techniques are compared.

Liquid ion exchangers in paper chromatography of steroidal glucosiduronic acids : Influence of different exchangers on the mobility in chloroform-formamide and correlation of chromatographci data

A group of 25 steroidal glucosiduronic acids was chromatographed on paper chloroform-formamide in the presence of several different liquid ion exchangers. Chromatograms were run also in three Bush-type systems. RF values were converted into RM values and the data were correlated by use of a series of regression equations of the type RM(Y) = a-RM(X) + b, in which X designates a standard system to which each other system (Y) is compared. The ratio of the slope a to the correlation coefficient r (i.e., a/r) is a measure of the resolving power of system Y relative to the standard system; intercept b, in association with slope a, is an indication of the polarity of system Y relative to X. The correlation coefficient r and the standard error of estimate sy-x are indications of whether solvent systems Y and X have very similar or relatively different resolving properties for a group of solutes. The regression equations are useful for correlating chromatographic data obtained from a group of compounds in several solvent systems. Properties of the chromatography systems are discussed and the relative importance of ion exchange and hydrogen bonding with the various solvent systems is pointed out. Delta RMg and delta RMr values are given for functional groups at several locations in the conjugates for ten of the chromatography systems.