Preventive medicine and screening in older adults.

PURPOSE: To review important current issues, studies, recommendations and controversies relating to preventive medicine and screening in older people. STUDY SELECTION/DATA ABSTRACTION: MEDLINE searches for literature on prevention and screening with regard to older adults as well as each individual condition reviewed; bibliographical reviews of textbooks, journal articles, government and advocacy organization task force reports, and recommendations. Important information synthesized and discussed qualitatively. DATA SYNTHESIS: Data and recommendations are presented for most common preventive services, including primary prevention and screening for cardiovascular diseases and risk factors, common malignancies, endocrine and infectious diseases, osteoporosis, sensory deficits, and dementia. CONCLUSIONS: The goal of preventive medicine in older people should be not only reduction of premature morbidity and mortality but preservation of function and quality of life. Attempts to prevent diseases of old age should start in youth; the older the patient, the less likely the possibility or value of primary and secondary prevention, and the greater the stress must be on tertiary prevention. Age 85 is proposed as a general cutoff range beyond which conventional screening tests are unlikely to be of continued benefit; however, care must always be individualized. Emphasis should be on offering the best proven and most effective interventions to the individuals at highest risk of important problems such as cardiovascular diseases, malignancies, infectious and endocrine diseases, and other important threats to function in older people. Breast cancer screening, smoking cessation, hypertension treatment, and vaccination for infectious diseases are thus far among the most firmly proven and well accepted specific preventive measures, with physical exercise also being particularly promising. Although more research is needed, a current working approach is necessary and possible. A summary table of recommendations and information tools such as reminders or flowsheets may be valuable in helping the physician carry out prevention and screening programs.

Inactivation of factor VIII by activated protein C and protein S.

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.

The size of human factor VIII heterodimers and the effects produced by thrombin.

The heterodimeric structure of factor VIII was demonstrated by two approaches. First, the native molecular weights of several partially purified fractions of factor VIII were determined by measurement of Stokes radii and sedimentation coefficients to be approx. 237 500, 201 000 and 141 000. These measured molecular weights correlated with those derived from polypeptide chain composition, in which each molecule would consist of a doublet polypeptide of Mr 83 000/81 000 plus one predominant high-Mr polypeptide of either 146 000, 120 000 or 93 000. In addition, immunoadsorption using a monoclonal antibody specific for the light-chain doublet removed all of the heavy chains. Separation of the heavy chains from the light chain by EDTA further illustrated the non-covalent nature of the heterodimers. All forms had coagulant activity which was potentiated 13-15-fold by an equimolar amount of human alpha-thrombin. Thrombin converted the Mr 83 000/81 000 doublet to one of Mr 73 000/71 000, and cleaved the largest polypeptides to a transient intermediate form of Mr 93 000 which was further cleaved to polypeptides of Mr 51 000 and 43 000. Potentiation of coagulant activity was correlated with proteolytic cleavage of either or both the doublet and the Mr 93 000 polypeptides. These data indicate that human factor VIII purified from plasma consists of a group of heterodimers, composed of a light chain of Mr 83 000 (81 000) and a heavy chain which varies in size between Mr 170 000 and 93 000, each form of which is similarly potentiated and cleaved by thrombin.

Propolypeptide of von Willebrand factor circulates in blood and is identical to von Willebrand antigen II.

The generally mild bleeding disorder of von Willebrand disease is associated with abnormalities of two distinct plasma proteins, the large multimeric von Willebrand factor (vWF), which mediates platelet adhesion, and von Willebrand antigen II (vW AgII), which is of unknown function. The two proteins were found to have a common biosynthetic origin in endothelial cells and megakaryocytes, which explains their simultaneous absence in the severe form of this hereditary disease. Shared amino acid sequences from a 100-kilodalton plasma glycoprotein and from vW AgII are identical to amino acid sequences predicted from a complementary DNA clone encoding the 5' end of vWF. In addition, these proteins have identical molecular weights and immunologic cross reactivities. Monoclonal antibodies prepared against both proteins recognize epitopes on the pro-vWF subunit and on a 100-kilodalton protein that are not present on the mature vWF subunit in endothelial cell lysates. In contrast, polyclonal antibodies against vWF recognize both pro-vWF and vWF subunits. Thus, the 100-kilodalton plasma glycoprotein and vW AgII are identical proteins and represent an extremely large propolypeptide that is first cleaved from pro-vWF during intracellular processing and then released into plasma.

Biosynthesis of von Willebrand protein by human megakaryocytes.

Immunofluorescence staining of buffy coat smears from a patient with chronic myelogenous leukemia in accelerated phase showed that approximately 13% of all nucleated cells contained von Willebrand protein and, therefore, appeared to be of megakaryocytic origin. This was confirmed by positive staining with antisera against platelet factor 4 and platelet glycoproteins. Short-term cultures of the buffy coat, which lacked endothelial cells, were metabolically labeled with [35S]methionine, and von Willebrand protein was immunopurified from cell lysates and culture medium. Cultures from this patient synthesized and secreted von Willebrand protein, in contrast with cultures from other patients with leukemia, who lacked circulating megakaryocytes, and from normal volunteers. The subunit composition of the megakaryocytic von Willebrand protein was very similar to that of human umbilical vein endothelial cells. The size of the processed subunit (220 kD) and of the cellular (260 kD) and secreted (275 kD) precursors from the two cell types were indistinguishable by gel electrophoresis. Furthermore, the ratio of precursor to processed subunit and the pattern of cellular and secreted nonreduced multimers were very similar. It appears, therefore, that the processing steps in biosynthesis of von Willebrand protein used by the megakaryocytes are very similar to those of umbilical vein endothelial cells.

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII.

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.

Factor VIII: structure and function in blood clotting.

Factor VIII (antihemophilic factor) is the protein that is deficient or defective in patients with classical hemophilia and Von Willebrand syndrome. Factor VIII in plasma is thought to be associated in a complex with the highest molecular weight multimers of another glycoprotein, Von Willebrand protein. Highly purified human factor VIII appears to have an Mr of between 200,000 and 300,000 and to consist of several polypeptide chains. The concentration of factor VIII in plasma is around 100-200 ng/ml, equivalent to around 1 nM. The purified proteins retain one or more of the known properties of factor VIII, including the acceleration of factor IXa-mediated activation of factor X, ability to be activated by thrombin and factor Xa, inactivation by activated protein C, and by human antibodies to factor VIII. Among the known clotting factors, factors VIII and V are exceptional in not possessing enzymatic activity. Factors IXa and VIII and X appear to form a functional complex, all of which need to be present and active simultaneously for optimal activation of factor X. The mechanism by which factor VIII promotes activation of factor X by factor IXa is not known, but the major effect is to increase the rate of the reaction. Following treatment of factor VIII with thrombin, a new and smaller polypeptide Mr around 70,000 +/- 5,000 is produced. Factors IXa and Xa also have been reported to activate factor VIII. It is not known whether limited proteolytic cleavage is required absolutely for the expression of factor VIII activity or if it only increases an activity already expressed by the uncleaved protein. Factor VIII is inactivated by thrombin and by activated protein C. Thus, factor VIII can be modulated by at least four of the serine proteases in the clotting system. A major goal for future research is to increase our understanding of the role in blood clotting played by factor VIII, and to apply this information to clinical problems which result from inherited abnormalities of factor VIII.

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues.

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.

Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain.

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VIII.

Fibrinogen New Orleans: hereditary dysfibrinogenemia with an A alpha chain abnormality.

An abnormal fibrinogen (Fibrinogen New Orleans, or FNO) has been found in a 30 year-old woman, her mother and daughter, but not her father. The propositus suffered mild bleeding, but not thrombo-embolism or abnormal wound healing. Plasma and purified fibrinogen from the propositus caused a prolongation in the clotting time of normal plasma. FNO had increased anodal migration when studied by immunoelectrophoresis. We have been able to follow the release of fibrinopeptides A and B using sensitive SDS-PAGE. In FNO, release of the A peptide was markedly delayed, whereas the cleavage of B peptide was much less delayed, compared to that of A peptide.

A human milk formula.

In view of the possible deficits in the energy value and protein content of human milk when used for feeding low birth weight preterm neonates, a method has been devised suitable for use in a human milk bank for making milk formulae from human milk products. Human milk formula (HMF) is produced by adding, to whole human milk, human cream, obtained by separation by centrifugation, together with salt-free and lactose-free human milk protein, extracted by simple dialysis and freeze-drying. This human milk formula is, therefore, enriched in energy, human milk fat, protein and salts (which may be added), to approach the current concept of an ideal milk formula(e) for preterm infants. In addition, the increased concentration of antimicrobial proteins achieved in HMF may offset any losses in these proteins caused by pasteurisation.

Plama-thromboglobulin concentrations in diabetes mellitus.

Abnormally high plasma-concentrations of the platelet protein thromboglobulin were demonstrated in a group of 72 diabetic patients. The highest concentrations were found in patients with clinical evidence of tissue damage. Many diabetic patients, without such complications have elevated thromboglobulin levels, and this blood abnormality may predate recognisable clinical lesions.

Changes in electrophoretic mobility of human factor VIII-related antigen: evidence for subunit structure.

Therapeutic factor-VIII concentrates were found to have factor VIII-related antigen (FVIIIRAg) with an electrophoretic mobility faster than that of plasma using two-dimensional crossed immunoelectrophoresis. Immediately after infusion of factor-VIII concentrates into patients, the electrophoretic mobility of FVIIIRAg in the patients' plasmas was increased to that of the antigen in the infused concentrates. Two hours after infusion, a proportion of the antigen had an electrophoretic mobility intermediate between that of the pre-infusion antigen and that of the concentrate antigen, and by 24 h after infusion the reversion of electrophoretic mobility to pre-infusion values was complete. The return of electrophoretic mobility to normal did not occur in vitro after 24 h. In vitro mixing experiments between pre-infusion plasma and concentrate resulted in antigen with a range of intermediate mobilities which were related to the relative proportions of slow and fast antigen in the mixture. In vitro mixing experiments with slow and fast antigen separated from intermediate purity factor VIII concentrate by agarose gel filtration resulted in the formation of a relatively large proportion of antigen with intermediate electrophoretic mobility. The most reasonable interpretation of the results is the formation of hybrids between the two electrophoretically different populations of antigen. This implies that VIIIRAg normally exists in a polymeric form which can spontaneously dissociate into the exist in equilibrium with a pool of partially and/or completely dissociated subunits.

Reconstitution of pig lymphocyte plasma membranes from solubilized components, with particular reference to membrane-associated immunoglobulins.

Plasma membranes of lymphocytes obtained from pig mesenteric lymph nodes were reconstituted after solubilization with bile salts. The proportion by weight of immunoglobulin in the reconstituted membrane was no greater than about 5-10% of that in the original membrane. Possible reasons for the low reincorporation of immunoglobulin are discussed.