Louis B. Jaques

The observation of heparin on endothelium after injection

Abstract Heparin was demonstrated by the microelectrophoresis procedure of Jaques & Wollin in endothelial powders obtained after acetone extraction of Hautchen preparations after injection of 1,000 units in rats. Concentrations of heparin calculated for the endothelium was 30 to 7,500 times greater than the concentration of heparin in blood at a given time with a comparable dose. An electron micrograph of preparations stained with Ruthenium Red taken at five minutes after intravenous injection of heparin, demonstrated a marked increase in electron density, as compared to normal endothelium, at the cell membrane and in vesicles near the luminal surface of the cell.

Vascular sequestration of heparin.

Abstract Heparin was administered intravenously and by intratracheal instillation to rats. With 3 H-heparin, radioactivity accumulated in endothelium and vessel wall of aorta and vena cava. These vessels were also removed from rats receiving higher doses of cold heparin, extracted for mucopolysaccharides and heparin identified and measured by microelectrophoresis. This procedure revealed the presence of more heparin on the endothelium of the aorta than of the vena cava. Despite the presence of high radioactivity in the vessel wall denuded of endothelium, no heparin was obtained by the chemical extraction, suggesting a heparin metabolizing activity in the endothelium and/or vessel wall. Heparin was also administered intravenously to rats and by intratracheal instillation to mice. The drug was identified by light and electron microscopy in the endothelial tissue of rat aorta and of mouse pulmonary vessels.

INTRAPULMONARY HEPARIN: A New Procedure for Anticoagulant Therapy

Heparin was administered by the intrapulmonary route to dogs, mice, and human volunteers. In all species, a single administration of a large quantity of heparin via the lung resulted in a prolonged state of moderate hypocoagulability (3 days in dogs, 14 days in man) due to the production of a sustained low concentration of heparin in plasma. The lengthening of clotting-time and duration of this response increased with dosage. Effective doses are above 8 mg (1300 units) per kg body-weight. Examination of the lungs, body-fluids, and tissues shows that the heparin is cleared rapidly from the lung and enters a body cellular compartment (probably the macrophages) from which it is slowly released to plasma. No evidence was found (symptomatic or on gross or histological postmortem examination) of haemorrhage or any heparin-related pathological change indicating either immediate or long-term toxic effects in the lung or other tissues. Suggestions are made for the clinical use of the unique features of anticoagulant treatment by intrapulmonary heparin.

Spontaneous Hemorrhage with Anticoagulants

When dicumarolized animals are subjected to various types of stress, a high mortality from spontaneous internal hemorrhage occurs. This phenomenon is observed with various combinations of the treatments—Dicumarol, phenylindanedione, heparin, frostbite, insulin convulsions, hypertonic saline intraperitoneally, adrenalectomy, adrenocorticotropic hormone, corticosteroids, histamine, adrenalin, salicylates, P32, reserpine. Hemostasis depends on blood coagulation, platelets, and vascular integrity. Experimental results demonstrate that spontaneous hemorrhage resultswhen any two of these mechanisms are deranged simultaneously. Hormonal and neural factors affect the blood vessels and in this way determine spontaneous hemorrhage with anticoagulants. Spontaneous hemorrhage has a multiple causation.

Orally administered dextran sulfate is absorbed in HIV-positive individuals

Preliminary in vivo studies suggested that oral dextran sulfate was poorly absorbed, but investigations were limited by inadequate methods for measuring the drug in the body. To determine absorption in HIV-positive subjects, hydrogenated dextran sulfate, average molecular weight 8000 (Usherdex 8), was orally administered in a short-term (single dose, 4 g/day for 5 days, 7 subjects) and in a long-term study (1 g, 4 times per day for 29 to 335 days, 8 subjects), which was a continuation of the short-term study with the inclusion of an additional subject. When an agarose gel electrophoresis technique with toluidine blue staining was used, the drug was recovered from plasma (67%, peak 2.2 microg/mL) and circulating peripheral blood lymphocyte (PBL) samples (50%, peak 333 microg/L blood) obtained at 5 and 15 minutes and 1, 3, 6, and 24 hours after the first days dose and from plasma (56%) and PBL samples (38%) obtained 5 minutes after administration on 4 subsequent days in the short-term study. In the long-term study, the drug was found in plasma (67%, peak 2.4 microg/mL) and PBL samples (25%, peak 126 microg/L blood) obtained at monthly visits within 4 hours of the last dose. The drug was found in all urine samples from all subjects in both studies (short-term study, 24-hour samples up to 4 days after the final dose; long-term study, monthly samples within 4 hours of the last dose). In the long-term study, bone marrow preparations from 3 subjects showed metachromatic inclusions present in reticular cells when the cells were stained with toluidine blue, indicating the presence of sulfated polyanions. A significant rise in activated partial thromboplastin time and a drop in platelet count (P < .025) were demonstrated, with thrombocytopenia developing in 3 patients. Mild-to-moderate gastrointestinal disturbances were experienced by 6 subjects in the short-term study and by all subjects in the long-term study. One subject experienced mild central nervous system symptoms in the short-term study. These results indicate that dextran sulfate is absorbed after oral administration; therefore, further studies on its efficacy, particularly in the early stages of the disease, along with additional observations on its toxicity, are warranted.

Antithrombotic activity of oral unfractionated heparin.

Although heparin is believed to be poorly absorbed orally, we recently demonstrated that oral heparin rapidly enters the circulation, with most of the drug being taken up by endothelium. To determine the effective antithrombotic dose of oral heparin, we induced thrombosis by applying 10% formalin in 65% methanol to exposed rat jugular vein. Saline or heparin, at doses ranging from 3.25 to 60 mg/kg, was immediately placed in the stomach; 4 h later, the vein was inspected for a thrombus. A dose-dependent decrease in thrombosis was observed with oral heparin. Although there was little change in anticoagulant activity as measured by the activated partial thromboplastin time (APTT) of plasma samples taken 4 h after administration, a significant dose effect was demonstrated by regression analysis. Heparin could be demonstrated chemically in 52% of plasma samples and in 38% of aortic or vena caval endothelial samples. A significant dose effect was observed in aortic endothelial heparin concentrations, with amounts 1,000-fold that determined in plasma. These results indicate that oral heparin exhibits antithrombotic activity in a dose-dependent manner, with low levels in plasma.

Appearance of heparin antithrombin-active chains in vivo after injection of commercial heparin and in anaphylaxis.

Abstract Recently it has been demonstrated that only one-third of commercial heparin binds with antithrombin III to enhance antithrombin activity, and that heparin is rapidly taken up by the RES. Commercial heparin was given to rabbits, dogs and men by subcutaneous and intravenous injection. Plasma samples were deproteinized with phenol, urine samples precipitated with benzidine, and the separated heparin measured for both antithrombin and metachromatic (dye-binding) activity compared to a reference heparin. Urinary excretion as heparin and uroheparin accounted for approximately 5% of the injected heparin. The extraction of heparin added to blood in vitro gave identical values in metachromatic and antithrombin assays, as did heparin from blood samples immediately after intravenous injection. However, later blood samples after injection gave much higher values in the test for antithrombic activity than in the metachromatic test. Subcutaneous injection of heparin subjected to acid inactivation in vitro, reducing its antithrombin activity, likewise resulted in heparin appearing in vivo with higher antithrombin activity. Endogenous heparin released to the circulation in canine anaphylaxis also gave much higher values in anti-thrombin assays. It is concluded that the difference in values is due to the conversion of antithrombin-inactive heparin chains to antithrombin-active heparin chains.

Metachromasia: An explanation of the colour change produced in dyes by heparin and other substances

Abstract Previous explanations given for metachromasia have been the dye-stacking model of Bradley and Wolf and the pairing concept of Young, Phillips and Balazs. The special properties of heparin and heparinoids in producing metachromasia are reviewed. To explain these observations, a model combining the dye-stacking and pairing concepts is proposed. The dye-dimer interacts with suitable ionic sites provided by the chromotrope. The loose ionic interaction alters the electron mobility of the dye-dimer, and this results in the colour change. This is illustrated with the absorption spectra recorded as differences between dye and dye-chromotrope in a double beam recording spectrophotometer. A metachromatic band shift was found with the dye in 3.8 M NaCl so that the chromotrope can be either a polyanion such as heparin or an ion cage formed by a strong electrolyte.

Variation in commercial heparin and its relation to the problem of heparin standardization for clinical use

Abstract This paper reports values for chemical and biological analyses for eight recently obtained commercial heparins, an acetic acid treated heparin and sheep heparin and compares these with values found in a previous study. While there is less variation in chemical analytical values, a lower sulfur content and higher acetyl value is characteristic. When potency estimations were determined by in vitro assay systems for heparin, inconsistency was found in potency values obtained. The results obtained were compared with values obtained with an in vivo assay in dogs, similar to clinical control methods for heparin and showing on statistical testing adequate design and reliability. A beef lung heparin and a pig mucosa heparin preparation showed the same potency by these in vivo tests, while a sheep lung heparin and pig mucosa heparin partially inactivated by exposure to acetic acid show much lower potencies. USP, BP, Howell assays did not give these results for this series of heparin preparations and thus do not predict their potency in vivo . Two types of pmr spectra of heparin are observed, those with an N-acetyl peak such as pig mucosa heparin, and those with no N-acetyl peak, such as beef lung heparin. Beef lung heparin appears to have one component on microelectrophoresis while pig mucosa heparin in most cases has two. The importance of these results for the standardization of heparin are discussed.

STRESS AND MULTIPLE-FACTOR ETIOLOGY OF BLEEDING*

Surgical bleeding means spontaneous hemorrhage. It is a common observation that patients or animals can have a deficient coagulation system n r a lack of circulating platelets without showing severe hemorrhage. Previous authors have described bleeding due to defects in platelets, in coagulation factors, in blood vessels. Our studies indicate that severe hemorrhage depends on the simultaneous occurrence of several such defects. The basis of our work has been the finding that defects in hemostasis can be produced in animals which result in such severe spontaneous hemorrhage as to result in a high mortaliy.14 In a series of investigations, we have observed spontaneous hemorrhage in rabbits and rats so severe as to result in 30-100 per cent mortality. With any given treatment we observe the same variety of hemorrhage as with any other, so I will first summarize the types of hemorrhage observed.