Litwiller, Robert D.

Heterogeneous, phospholipid‐rich sub‐micron vesicles (“microvesicles”) shed by activated vascular and blood cells may reflect or influence the coagulant propensity of blood. To determine how the anticoagulant used to collect the blood affects microvesicle (Mv) recovery and characteristics, blood was collected from laboratory volunteers and participants of the Kronos Early Estrogen Prevention Study (KEEPS) into tubes containing either sodium citrate, EDTA or hirudin plus tick anticoagulant peptide (H&T). Microvesicles prepared by double centrifugation were analyzed by flow cytometry to define size and surface markers. There were 2–3 times the number of platelet‐derived (CD61 positive) Mv in preparations from citrate and H&T plasma, relative to numbers in EDTA plasmas. Procoagulant activity of Mv was determined by surface phosphatidylserine (PS) expression using annexin‐V‐fluorescein. Annexin‐V (PS) positive Mv did not differ, but total (PS positive and negative) Mv was 2 fold lower from EDTA plasma compared to citrate and H&T. These results suggest that anticoagulation of blood itself alters microvesicle characteristics. Therefore, standardization and specification of the anticoagulant is warranted to identify potential contribution of microvesicles to cardiovascular pathophysiology. Funded by, NIH HL78638, Kronos Research Institute and Mayo Foundation.

Risk of CAD increases at menopause and platelets contribute to the etiology of CAD. We hypothesized that individual variation in platelet procoagulant activity at menopause correlates with the extent of CAD as reflected by coronary artery calcification ( CAC ). We developed several novel and global assays of platelet procoagulant activity: dense (ATP release) and alpha (P‐selectin expression) granule secretion and GPα IIb β 3 fibrinogen‐binding (PAC1 binding) to ADP stimulation, and prostaglandin E 1 (PEG 1 ) sensitivity as a surrogate for prostacyclin sensitivity. Platelets were prepared from venous blood collected from women screened for the Kronos Early Estrogen Prevention Study (n=125; mean±SD age=52.5±2.4 years) within 19.4±9.2 months of last menses. Baseline CAC score was determined. Of conventional CAD risk factors, only systolic blood pressure correlated with positive CAC score (n=23). ATP secretion tended to be less in women with positive CAC scores and was negatively correlated with blood glucose (P<0.001). P‐selectin expression and PAC1 binding increased with total cholesterol (P<0.05). PEG 1 sensitivity increased with increasing age and waist circumference (P<0.05). These results indicate that platelet functions change with age and in association with conventional risk factors for the disease in women at menopause. Supported by the Kronos Longevity Research Institute and NIH HL90639.

Abstract Factor VIII (FVIII) inhibitors have been classified as Type I, linear inactivation kinetics and complete inhibition of FVIII at saturation, or Type II, non-linear kinetics and residual FVIII activity. To investigate the hypothesis that "inhibitor-blocking antibodies" contribute to the characteristics of Type II inhibitors, mixing studies were performed between fractionated Type II antiserum and various Type I inhibitor preparations. Type II IgG fractions were tested for inhibitory capacity and ability to prevent inhibition mediated by inhibitors from other hemophiliacs. The IgG from Type II plasma (II) (29 BU, 8% FVIII) and several Type I plasmas ((I) plasmas) were fractionated with DEAE resin. Late-eluting Type II fractions (II):6 and (II):7, showed slight protection of FVIII from the inhibiting activity of several Type I plasmas and additive inhibition with others. When tested against Type I IgG fractions instead of type I plasmas, (II):6 and (II):7 provided significant protection of FVIII from the inhibitory action of some immunoglobulins and additive inhibition with others. When (II):6, which contained slightly less than 1 BU of inhibitory activity, was preincubated with normal plasma, it blocked about 3 BU of inhibitory activity in an equal volume of fraction (I):1; residual FVIII increased from 11.4% to 27.2%. IgG fraction (II):6 provided protection from DEAE fraction 2, (I):2, from another type I patient (residual FVIII went from 15.1% to 31.7%) but was additive with (I):4 from the same patient (residual FVIII went from 8.0% to 3.2%). Preincubation with (II):7 increased residual FVIII activity in normal plasma incubated with (I):1 from 10.1% to 20.7%. Fraction (II):7 was processed further by hydrophobic interaction chromatography on phenyl sepharose and two fractions, Phen(II):5 and Phen(II):6 provided significant protection of FVIII from inhibition by (I):1; residual FVIII activity increased from 11.5% to 34.1% and from 7.9% to 28.2%, respectively. DEAE fractions (II):6 and (II):7 were shown to contain anti-FVIII IgG. Depletion of mixtures of plasma or rFVIII and (II):6 with immobilized Protein A, reduced FVIII activity by 42.8% and 68.8%, respectively. In addition, adsorption of (II):6 with immobilized Protein A, eliminated its ability to block FVIII inhibition by (I):1. The co-existence of these antibodies in "acquired hemophiliac" plasma would contribute to the complex kinetics seen in these patients. If a sufficiently diverse panel of such blocking IgGs occurred naturally, or could be produced in the laboratory, and their interaction with the target antigen did not adversely affect the half-life, then these blocking antibodies might prove useful to alleviate the effects of inhibitory antibodies in hemophilia and other diseases.

Solutions of anionic liquid ion-exchangers in organic solvents are potentially useful for extracting steroidal glucosiduronic acids from biological fluids and for purifying mixtures of these acids by chromatography. If a glucosiduronic acid is to be isolated in pure form after either of these procedures, it is necessary to separate it from the ion-exchanger. Separation from an organic solution of tetraheptylammonium chloride may be accomplished by extraction with water under the following conditions, which promote transfer of a glucosiduronate to the aqueous phase: (1) an appropriate solvent (diluent) as the organic phase, (2) the presence in the two-phase mixture of an anion such as myristate or dodecyl sulphate to combine with the tetraheptylammonium ion, and (3) an increase of the pH of the aqueous phase in association with the presence of myristate or dodecyl sulphate. The foregoing factors apply also to removal of glucosiduronates from organic solutions of ion exchangers that are hydrochlorides of tertiary, secondary, or primary amines. Since these amines exert progressively less solubilizing effect for glucosiduronates as the pH of the aqueous phase is increased, the conjugates can be released from the organic phase by adjusting the pH to 10 and omitting the myristate or dodecyl sulphate.

A pilot study on the extraction of three steroidal glucosiduronic acids from water into organic solutions of liquid ion-exchangers is reported. A single extraction of a 0.5mm aqueous solution of either 11-deoxycorticosterone 21-glucosiduronic acid or cortisone 21-glucosiduronic acid with 0.1m-tetraheptylammonium chloride in chloroform took more than 99% of the conjugate into the organic phase; under the same conditions, the very polar conjugate, β-cortol 3-glucosiduronic acid, was extracted to the extent of 43%. The presence of a small amount of chloride, acetate, or sulphate ion in the aqueous phase inhibited extraction, but making the aqueous phase 4.0m with ammonium sulphate promoted extraction strongly. An increase in the concentration of ion-exchanger in the organic phase also promoted extraction. The amount of cortisone 21-glucosiduronic acid extracted by tetraheptylammonium chloride over the pH range of 3.9 to 10.7 was essentially constant. Chloroform solutions of a tertiary, a secondary, or a primary amine hydrochloride also will extract cortisone 21-glucosiduronic acid from water. The various liquid ion exchangers will extract steroidal glucosiduronic acid methyl esters from water into chloroform, although less completely than the corresponding free acids. The extraction of the glucosiduronic acids from water by tetraheptylammonium chloride occurs by an ion-exchange process; extraction of the esters does not involve ion exchange.