High Concentrations Of Heparin Research Articles

THE HEPARIN CO-FACTOR ACTIVITY IN PLASMA AND ITS RELATION TO THE ANTICOAGULANT EFFECT OF INTRAVENOUSLY INJECTED HEPARIN.

AbstractBlombäck, B., M. Blombäck, H. Lagergren and P. Olsson. The heparin co‐factor activity in plasma and its relation to the anticoagulant effect of intravenously injected heparin. Acta physiol. stand. 1963. 58. 306—318. — The heparin co‐factor activity in plasma, defined as the antithrombin activity at high heparin concentrations, was measured with two thrombin‐titration methods in dogs and human subjects following single or repeated intravenous injections of heparin. The co‐factor activity was found to decrease continuously during the period of elimination of heparin from the plasma, after which it rose slowly to the initial level. In dogs, the anticoagulant effect of intravenous heparin was studied at different levels of co‐factor activity in the plasma. The co‐factor activity was increased by infusion of bovine plasma fraction IV—1, prepared according to Cohn. The rise in co‐factor activity was always accompanied by an increase in heparin effect (prolongation of the coagulation time). The co‐factor alone exerted no effect on the coagulation time.

Studies on Thrombin Inactivation in Normal Plasma, Serum and Plasma Fractions, and its Relation to Heparin

SummaryThe antithrombin activity of plasma, serum and plasma fractions has been studied under different experimental conditions. Two different methods have been used for the assay of antithrombin activity.Under the experimental conditions described the following antithrombin activities have been found to be of importance in the inactivation of thrombin:1. Progressive inactivation in the absence of heparin can take place in plasma and to a less extent in defibrinated plasma and serum.2. Inactivation of thrombin in presence of fibrinogen and heparin in low concentration (about 0.03 I.U./ml). This effect was abolished at high heparin levels.3. Inactivation of thrombin by plasma, defibrinated plasma and serum at high concentrations of heparin (about 2—15 I.U./ml). This activity is tentatively named the heparin co-factor.

Mast cells and fibrinolysin.

DURING the investigation of human tissues for fibrinolytic activity the presence of a fibrinolytic substance was detected in a specimen of a spleen. This was a case of splenic mastocytosis previously reported1 in which there were many mast cells and a high concentration of histamine and heparin in the splenic tissue.

Isolation and Purification of a Coagulant from Snake Venom of the Species Bothrops jararaca and the Study of its Properties

Summary1. A material active in coagulating fibrinogen has been purified thirty-fold from crude venom of the species Bothrops jararaca. This material exhibits clotting and esterase activities, but no measurable proteolytic activity if measured by its ability to digest denatured hemoglobin.2. The clotting activity of the purified material (but not its esterase activity) is greatly enhanced by the addition of 2—3 μM of calcium ion; no other divalent metallic ion can duplicate the accelerating effect of Ca++ ion.3. The purified material is stable to dialysis, standing at room temperature for 18—20 hours, or heating at 50° C for 10 minutes; it loses both its clotting and esterase activities if heated at 70° C for 25 minutes or at 80° C for 10 minutes4. Although the powerful proteolytic enzyme inhibitors ovomucoid and soyabean trypsin inhibitor can block the clotting and esterase activities of thrombin, they are ineffective with respect to these properties of venom-coagulin. DFP, an inhibitor of far less specificity, can prevent the actions both of thrombin and of venom-coagulin5. An amount of heparin sufficient to block the clotting activity of thrombin is unable to prevent the action of venom-coagulin on fibrinogen; a much higher concentration of heparin is needed. In both cases the addition of 2—3 μM of Ca++ ion necessitates the introduction of considerably more heparin in order to block the actions of thrombin and venom-coagulin.6. While the antithrombin contained in serum can neutralize the clotting and esterase activities of thrombin, it has no effect on the actions of venom-coagulin.

The Effect of Heparin on Components of the Human Fibrinolytic System

Abstract It was demonstrated with human urokinase, human fibrinolytic euglobulins and human pyrogen plasma as enzyme source, and with bovine fibrin plates and human plasma plates as substrate, that high heparin concentrations inhibit fibrinolysis of preformed fibrin, in contrast to small ones which may enhance this phenomenon, provided albumin is present. No interference could be observed in the absence of plasma, serum or albumin fraction. Albumin or substances associated with the albumin fraction are required for interference of heparin with fibrinolysis. This interference probably takes place with the action of plasmin on fibrin.

Studies on isolated cell components.

1. The addition of heparin to rat liver, kidney, or brain nuclei has been found to bring about the release of a gel. Chemical analysis and histochemical studies on whole homogenates and isolated nuclei demonstrated that the material released by heparin contained desoxyribonucleic acid (DNA) and protein. The action of heparin on nuclei is interpreted as the result of a combination with the basic proteins of the nucleus with a consequent displacement of DNA. 2. The addition of heparin to a finely divided dilute liver homogenate prepared in a phosphate-sucrose solution at pH 7.1 brings about a marked increase in viscosity which reaches a maximum in 6 to 8 minutes at 23 degrees and then declines. 3. The concentration threshold for the viscosity effect was 0.1 mg. per 100 mg. fresh rat liver, with further increases in viscosity at higher heparin concentrations. Over a period of several hours a marked decrease in response to heparin was observed in homogenates stored at 0 degrees . 4. Fractionation of the homogenate demonstrated that the viscosity increase was due to the presence of the nuclei alone, other components showing no effect. Microscopic observation showed that the increase in viscosity was associated with the appearance of a clear gel around nuclei treated with heparin. 5. Heparin brought about the release of DNA from the nuclei of incubated rat liver, kidney, and brain homogenates. In some instances over half the DNA is found in the supernatant after high speed centrifugation (20 minutes, 21,000 x g). 6. No correlation was found between anticoagulant activity of heparin preparations and their effectiveness in causing an increase in the viscosity of liver homogenates. Desulfated heparin produced none of the results described here for heparin.

Assay of Heparin. I. Method and Qualitative and Quantitative Aspects Using Beef and Sheep Plasmas

A method of assay for heparin based on the use of frozen beef or sheep plasma is described. The clotting curves of both plasmas are essentially the same except for high concentrations of heparin for which slight differences were noted. The visual estimation of degree of coagulation is easier with beef plasma than with sheep plasma because of the greater density of the clots of the former.

STUDIES ON PLATELETS: VI. The Isolation of Platelets from Human and Dog Blood

Abstract 1. Two methods for the isolation of platelets are described in detail. Employing both methods, 55 isolations were made from 31 human beings and 20 isolations from 20 dogs. Both methods are based on the principle of using the platelet layer obtained by repeated fractional centrifugation. 2. The Blood Anticoagulant Platelet Isolation (A) differs from the Blood Saline Platelet Isolation (B). In Method A the following anticoagulants were used: sodium heparin, in various concentrations; sodium citrate; sodium oxalate; and magnesium sulfate. In Method B 10 to 15 parts of saline were added to 1 part blood to keep the blood incoagulable. 3. Special Platelet Isolation (P-I) tubes were designed to isolate the platelets in the final steps. 4. In both methods the platelets were washed with chilled 0.9 per cent sodium chloride solution. The final platelet suspension in saline was obtained free from red and white cells and plasma after six to seven centrifugations and washings. 5. Method A has the advantage of being less laborious. Method B may be preferred for studies of platelets obtained without anticoagulants. 6. The approximate platelet yield in each method varied greatly. It was found that high concentrations of heparin did not produce the highest yields of platelets. 7. Decreases of more than 25 per cent of the initial count were considered significant. The platelets isolated from blood containing sodium heparin, sodium citrate, and sodium oxalate did not significantly decrease in number following storage for a fairly long time. However, platelets isolated using magnesium sulfate and saline did decrease significantly after one to three days’ storage at 1 to 4°C. 8. The isolated platelets did not appear to be morphologically altered. The cell outline was regular and the size did not vary extremely. 9. Both methods are recommended for securing pure platelet suspensions. Such suspensions should prove to be very satisfactory for studies of the physiology, the chemical constitution, and the physical properties of the mammalian blood platelet.