Marion I. Barnhart

DDAVP: Does the drug have a direct effect on the vessel wall?

Evidence is presented that 1-deamino-8-d-arginine vasopressin (DDAVP), a vasopressin analog, has a direct effect on isolated vessel segments. The most significant finding is increased platelet adhesion and spreading at injury sites. An isologous human umbilical vein perfusion model was used to compare effects of DDAVP with those of epinephrine or zero drug controls. Scanning electron microscopy, in conjunction with morphometry, permitted quantification of platelet adhesion to subendothelium exposed by minimal injury in the model. In addition, umbilical vein effluents were tested for levels of factor VIII moieties (F VIII:C, F VIII:Rag, F VIII:RCof) and the prostanoids, 6 keto PGF1 alpha (stable metabolite of prostacyclin) and TXB2 (stable metabolite of thromboxane A2. Only F VIII:C from DDAVP treated segments was significantly (p less than 0.01) changed from controls.

Platelet hyperaggregability in patients with chest pain and angiographically normal coronary arteries

Forty-one patients with chest pain and angiographically normal coronary arteries were studied for platelet abnormalities. Patients with conditions known or suspected to be associated with chest pain or platelet dysfunction were excluded. After coronary angiography and 2-week withdrawal from all medications, platelet aggregometry was performed using peripheral venous plasma samples and 3 concentrations of adenosine diphosphate, 2.34, 1.17 and 0.58 microM, and epinephrine, 11, 1.1 and 0.55 microM, as stimuli. Platelet morphology in response to surface contact (adhesion) was evaluated by transmission electron microscopy to determine the percentage of platelets in the round/abortive (inactive), dendritic (intermediate) and spread (activated) forms. Plasma specimens obtained from healthy volunteers of similar age and sex were analyzed in parallel and served as control subjects. Compared with control subjects, patients had increased aggregation at all concentrations of both adenosine diphosphate and epinephrine (p less than 0.001). Patients also had fewer platelets in the dendritic form and more in the round/abortive and spread forms. Thus, patients with chest pain and normal coronary arteries have platelet hyperaggregability in vitro, although the clinical relevance of this finding is unclear.

PROTEASES IN INFLAMMATION

Inflammation is a descriptive term for several events that are provoked by injury.*s2 The resulting biochemistry as well as cellular and tissue responses vary with the stressor but underlying mechanisms and adaptive benefits remain poorly understood. A common feature among various types of inflammation is the accumulation of protein at or near the site of injury. Local conditions frequently favor denaturation, heat aggregation or overt fibrin formation. These altered proteins must ultimately be digested or removed for the completion of healing. Thus, proteolysis whether by cellular or extracellular means occupies a central position in the resolution of inflammation. The present communication considers the protease potential of inflammatory fluids, the sources of such proteases and further defines certain biochemical properties of cathepsin, the acid protease system of cells. It has become apparent in this work that extreme hydrogen ion concentrations are not prerequisite for measurable proteolysis to occur. Furthermore, the proteolysis products eventuating from partial hydrolysis may be of consequence in the continuation or termination of the inflammatory responses. Most of the work by other investigators has made good use of synthetic substrates or foreign proteins, such as casein or edestin, for evaluating the enzymatic properties of various biological materials. Our study of proteases attempts to reproduce the circumstances that might be encountered in the body when proteases exercise their capabilities extracellularly. Accordingly, we confined our materials to one species (man) and selected natural proteins (albumin, fibrinogen, fibrin and gamma globulin) as substrates for proteolysis. Such proteins frequently assume an extravasculai location and their accumulation at an inflammatory site must be controlled for wound healing to progress. Inflammatory fluids from joint disease had measurable amounts of acid protease and alkaline protease activity which could be ascribed to the enzymes cathepsin and fibrinolysin respectively. The proteolytic magnitude seemed more related to the degree of inflammation than to specific types of joint disease.

Scanning Electron Microscopy of Human Reproductive Physiology

Recent findings on the scanning electron microscopical (SEM) structures of epithelium of female and male reproductive organs spermatozoa placenta amniotic cells umbilical cord neonatal blood cells and pathologies of their structures - carcinoma of the endometrium cervix vagina and vulva - are reviewed. The methodology in preparation of soft tissue cervical mucus and amniotic tissues is described including pinning fixation dehydration and drying mounting and metal coating and viewing with the SEM. There are striking morphological differences between the tissue organization of the mucosa of different segments of the reproductive tract. These morphological di fferences are discussed in terms of observed physiological functions. SEM is a useful tool in studying infertility the status of the newborn monitoring fetal maturity and carcinoma diagnosis.

Role of blood coagulation in acute inflammation.

Abstract The inflammatory process is a response to many different kinds of tissue injury. One event thay may be common to many apparently unrelated irritants is the deposition of fibrin. Three experimental designs have considered the significance of fibrin in acute inflammation, but only the third will be discussed extensively (details of the other approaches have been published). In the first approach, the biochemical profile of proteins concerned in fibrin formation was defined quantitatively for inflammatory fluids obtained from acutely inflamed joints of humans with arthritic diseases. Quantitative differences appeared to reflect degress of inflammation rather than to define a profile unique for the types of arthritis. The second experimental approach attempted to correlate the type of exudative leucocytes with the presence of fibrin in human joint disease and experimental skin windows on dogs. The granulocyte phase of inflammation was maintained and prolonged by the presence of fibrin. The third experimental approach was a quantitative study of leucocyte response to the chemotactic power of fibrin, fibrinogen, and their proteolysis products. By a skin window collection chamber technique with dogs, the leucocyte responses to fibrin-related material. other protein aggregates (albumin, γ-globulin, antigen-antibody complexes), and several inert particulate or structured preparations were compared. Fibrin-related materials were the most potent chemotactic agents for granulocyte (predominantly neutrophil) emigration in acute inflammation.

THE CELLULAR LOCALIZATION OF FIBRINOGEN AS REVEALED BY THE FLUORESCENT ANTIBODY TECHNIQUE.

Although much attention has been given to the liver as concerned in fibrinogen production, the precise cells responsible for synthesis have not been clearly defined. Studies on perfusates from isolated and functioning liver exposed to radioactive amino acids have emphasized the importance of the liver in fibrinogen synthesis (1, 21. Immunoprecipitin methods were applied by STHAUB [3] in a successful demonstration of fibrinogen production by liver slices incubated in vitro. However, the fluorescent antibody technique, a method for direct visualization of cellular localizations of proteins, has not been especially rewarding until now in either supporting the importance of the liver or revealing the specific cell type responsible for the synthesis of fibrinogen. For example, GITLIN, LANDING and WIIIPPLE [4] demonstrated much extracellular fibrin but were unsuccessful in finding cellular fibrinogen. More recently, HAMASHIMA, HARTER and COONS [5] using fluorescent anti-fibrin reported fibrinogen predominantly in Kupffer cells. An intracellular localization for fibrinogen in liver parenchymal cells was found in this laboratory by BARNHART and ANDERSON [6] using immunochemical and cellular fractionation procedures. In none of these cellular studies was the experimental design adequate for a clear-cut distinction between synthesis and storage of fibrinogen. In the present report we describe an attempt to study the dynamic aspects of fibrinogen physiology. Defibrination was employed to alter the rate of fibrinogen production in clogs and information was gained relating t o its biosynthesis, storage and release. The liver parenchymal cell was revealed by the fluorescent antibody technique as the site for fibrinogen synthesis and storage under appropriate conditions. In the

Platelet responses in health and disease

SummaryThis article summarizes recent ultrastructure findings from our laboratory and documents some of the information accumulated primarily since 1975 from many laboratories. Special attention is given to documentation by scanning electron microscopy which affords insight into platelet activation (adhesion, aggregation, release/secretion) and especially platelet-vessel wall interactions. Structural physiology of platelets is considered in some detail as a basis for understanding platelet disorders contributing to clinical problems of thrombosis and hemorrhage. The impaired ability of vonWillebrand platelets to adhere to injured vessel wall is reported using the human umbilical vein perfusion model. Relationships between platelets and blood coagulation factors focus on the exquisite sensitivity of platelets to minute amounts of thrombin. Unmasking of platelet factor 3 sites is identified on activated platelets, after glutaraldehyde fixation, by their reaction to latex bearing anti-platelet factor 3 markers. The basis for platelet-collagen interactions is reviewed. Conditions for and possible mechanisms behind platelet interaction with vessel wall are discussed. Ex vivo flowing blood-vessel wall models offer opportunities for improved understanding of the platelets role(s) in vascular diseases.

Ultrastructural Changes in Human Endometrium with Copper and Nonmedicated Iuds in Utero

Scanning and transmission electron microscopy were used for a study of the surface and glandular ultrastructure of human endometrium in the presence of different types of IUDs at comparable phases of the menstrual cycle. The aim of the study was to compare the effect of the nonmedicated with the copper and multiload copper devices to further explain the differences in their contraceptive potencies and their mechanism of action. The endometrium was evaluated at and away from the IUD; emphasis was put on the ultrastructure of endometrial gland openings, secretory activity, cellular glycogen content, ciliated cells, microvillous pattern, and kinocilia. The changes of the surface ultrastructure of the endometrium in the presence of copper IUDs were more extensive in this study than those previously reported. There seems to be a direct relationship between the amount of copper incorporated in the device, the degree of ultrastructure changes, and the area of endometrium involved. Copper devices affect the endometrial cells away from the IUD. The altered secretory function with disturbed macroapocrine secretion, the abnormality of ciliated cells, and the defective microvillous growth seem to interfere with the physiologic and functional integrity of the endometrium, reducing the chances of contraception in the presence of copper IUDs.

Canine plasminogen: purification and a demonstration of multimolecular forms.

Abstract A method for the purification of canine plasminogen has been developed with yields to 86% and a consistent 714-fold concentration over plasma. The procedure capitalized on the affinity of plasminogen for fibrin. This induced complex association was easily washed free of inert proteins and the proenzyme, plasminogen, was freed by eluting the complex with e-aminocaproic acid. Methanol then precipitated the partially purified proenzyme which was chromatographed on hydroxyapatite. The final product was judged homogeneous by criteria of ultracentrifugation, immunology, electron microscopy, and cellulose-acetate electrophoresis. The plasminogen was soluble and highly stable at physiological pH and did not contain assayable spontaneous proteolytic activity. This method was applicable to equine plasma and gave similar yields of high quality plasminogen. An isoproenzyme system of plasminogen was suggested by starch-gel electrophoresis. This procedure, using hydroxyapatite plasminogen, revealed four distinct electrophoretic components, which were activable with urokinase to plasmin (EC 3.4.4.14). A new assay technique on strips of fibrin-impregnated cellulose acetate demonstrated in situ the lysis of fibrin by each of the electrophoretic components. These isoproenzymes when separated and re-electrophoresed retained their individuality and capacity for activity.

INTRACELLULAR LOCALIZATION OF PROTHROMBIN.

Prothrombin formation and storage are clearly functions of liver parenchymal cells according to information obtained with the fluorescent antibody technique (1,2). The present report extends the information on prothrombin localization to the intracellular level in dogs and cattle. Materials and methods. Isolated parenchymal cell concentrates were obtained with the technique of St. George (3) on Dowex 50 W resin columns. Evaluation of the homogeneity of the fractions was made by differential cell counts on Leishman stained smears. A typical cell concentrate contained 96.5% parenchymal cells, 3% free nuclei and 0.5% red blood cells. In cell counts up to 600 cells neither Kupffer cells nor platelets were found. Subcellular fractions were prepared in 0.25 m sucrose by the procedures of Schneider and Hogeboom(4) and de Duve and associates (5). The following fractions were studied: total cell homogenate (TCH), cytoplasmic extract (CE), nuclei (N), mitochondria (M), lysosomes (L), microsomes (Mic) and soluble fraction (S). Further purification of the nuclear fraction was achieved by centrifugation at 345 g for 5 minutes to sediment the associated whole cells and other subcellular particulates. The resulting supernatant containing nuclei was recentrifuged at 750 g for 5 minutes. The nuclei in this precipitate were then resuspended in 0.25 m sucrose and washed twice. All fractions were finally suspended in sucrose so that the stock solutions were 10% by volume. (TCH) and soluble (S) fraction from a normal dog. B. Identical antigenic substances exist in mierosomes (Mic) and soluble (S) fraction of normal dogs. C. Note the preeipitin band indicative of prothrombin localization in soluble fraction of dicumarolized dogs (SD) as well as in soluble fraction from Vit. K1 stimulated (SK) animals, D. Microsomes obtained from a dicumarolized dog (MicD) did not react with antiprothrombin.