Ira I. Sussman

LONG-TERM CULTURE OF HUMAN ENDOTHELIAL CELLS

SummaryHuman umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (103/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.

Role of A and B blood group antigens in the expression of adhesive activity of von Willebrand factor

ABO (H) blood group antigens are covalently linked to the oligosaccharide side‐chains of von Willebrand factor (VWF). In this study, we investigated the role of the A and B antigens in the expression of VWF adhesive activity. VWF of type A, B or O was purified from fresh frozen plasma. Presence of A or B antigen on the VWF was confirmed by enzyme‐linked immunosorbent assay (ELISA) and by immunoblotting with monoclonal anti‐A or anti‐B. The A or B antigen was also detected in the 48/52‐kDa fragment of the respective VWF after trypsin digestion. Removal of A antigen with α‐N‐acetylgalactosaminidase or B antigen with α‐galactosidase did not affect its multimer size or antigenic level, but decreased the ristocetin cofactor (RCoF) activity of the respective VWF by 33–39% (P < 0·01–0·002). Removal of A or B antigen from VWF did not affect the binding of the VWF to immobilized type III collagen. A and B antigens were not detected in platelet VWF. These results indicate that AB structures play a role in platelet aggregating activity of VWF.

Subunit composition of plasma von Willebrand factor multimers: Evidence for a non-proteolytic mechanism resulting in apparent increase in proteolytic fragments

Plasma von Willebrand factor (vWf) consists of a series of multimers of different molecular sizes. When analyzed for subunit composition, plasma vWf contained a major polypeptide of approximately 225 kD, and at least three smaller proteolytic fragments. In the present study, the subunit composition of each of these multimers was analyzed using a two dimensional electrophoresis technique. Although nearly all the multimers, as separated by SDS 1.5% agarose gel electrophoresis, contained both the major polypeptide and the smaller fragments, the relative amount of the fragments varied among the multimers. The smaller multimers contained relatively more proteolytic fragments. After the larger multimers were depleted by incubation of plasma with formaldehyde-fixed platelets and ristocetin, the intact subunit decreased, while the amount of the proteolytic fragments remained relatively unchanged. The findings of this study are consistent with the scheme that plasma multimers undergo proteolytic cleavage after multimer assembly. Furthermore, an apparent increase in the amount of these proteolytic fragments may result from mechanisms unrelated to proteolysis, such as selective adsorption of the larger multimers onto platelets.

Pathology service line: a model for accountable care organizations at an academic medical center

Accountable care is designed to manage the health of patients using a capitated cost model rather than fee for service. Pay for performance is an attempt to use quality and not service reduction as the way to decrease costs. Pathologists will have to demonstrate value to the system. This value will include (1) working with clinical colleagues to optimize testing protocols, (2) reducing unnecessary testing in both clinical and anatomic pathology, (3) guiding treatment by helping to personalize therapy, (4) designing laboratory information technology solutions that will promote and facilitate accurate, complete data mining, and (5) administering efficient cost-effective laboratories. The pathology service line was established to improve the efficiency of delivering pathology services and to provide more effective support of medical center programs. We have used this model effectively at the Montefiore Medical Center for the past 14 years.

Sub-aggregatory doses of catecholamines prevent prostacyclin-induced inhibition of platelet aggregation

To assess the effect of subaggregatory concentrations of catecholamines on the antiaggregatory effect of prostacyclin (PGI2), platelets from normal human volunteers were exposed sequentially in vitro to epinephrine (less than or equal to 50 nM)- or norepinephrine (less than or equal to 1 microM) followed by PGI2 and adenosine diphosphate (ADP). Platelets thus pretreated did not manifest the normal inhibitory response to PGI2, aggregating to a similar extent as platelets exposed to ADP alone. This effect was unaffected by aspirin but was abolished by exposure to phentolamine. Catecholamine pretreatment similarly blocked the PGI2-induced increase in intracellular cyclic AMP, an effect which was also reversed by phentolamine. These data suggest that platelets exposed in vivo to elevated catecholamine concentrations, such as are seen clinically during myocardial infarction, might be similarly unresponsive to endogenous PGI2.

The destabilization of factor VIII by a vitamin K dependent protein.

Summary Since a vitamin K dependent protein, protein C, can inactivate factor VIII, a study was undertaken to determine if the level and stability of factor VIII in plasma are influenced by such a protein. Factor VIII lability was determined by incubating citrated plasma, diluted 1:10 and 1:20 in pH 7.2 imidizole buffer, for 6 h at 37°C. Normal plasma had a mean factor VIII of 98 ± 61 U/100 ml. The amount of factor VIII remaining after 6 h of incubation was 68 ± 14% of the original factor VIII level. In warfarinized patients, factor VIII (218± 65 U/100 ml) and VWF:AGN (331 ±102 U/100 ml) were elevated (P<0.001). Following incubation, their residual activity was 103 ±20% of the original factor VIII level. In samples taken after warfarin was discontinued, normal factor VIII lability returned, while plasma levels of factor VIII and VWF: AGN remained elevated. Similarly, in the plasma of a vitamin K deficient patient, increased factor VIII stability was also evident; lability was restored following vitamin K replacement. We conclude that factor VIII stability is determined in part by a vitamin K dependent protein. In clinical states in which this protein is functionally absent, factor VIII is elevated and more stable.