Irving B. Mink

STUDIES ON THE MECHANISM OF ACTION OF INHIBITORS OF THE FIBRINOLYSIN SYSTEM

With increasing awareness of fibrinolytic phenomena, improved diagnostic methods and the availability of antifibrinolytic fibrinolytic hemorrhage is progressing from the obscure register of rare syndromes to an important emergency condition. From a long list of potential inhibitors of the fibrinolysin system, three have reached the stage of clinical trials. Epsilon-aminocaproic acid (EACA) was introduced by Okamoto and associate^^,^ as was 4-aminomethyl cyclohexane carboxylic acid (AMCHA) .5,6 The latter compound was originally studied as the naturally occurring mixture of stereoisomers. Later, it was found that only the trans isomer is a~tive.~-lO Laskowski and Laskowskill reviewed the literature on trypsin inhibitors isolated from organs and blood. The pancreatic inhibitor of Kunitz and Northrop12 was later isolated also from bovine salivary glands and lungs and was shown to inhibit the kallikrein-kinin system and the fibrinolysin system as well.13-15 This agent is presently available for investigation under the trade name TrasyloP. The purpose of this study was to investigate the mechanism of action of these inhibitors of the fibrinolysin system. Several authors proposed that EACA and AMCHA are primarily inhibitors of the activation of plasminogen, plasmin itself being inhibited only at much higher concentrations.1°J6-18 Others reported that these compounds act chiefly by inhibiting plasmin or that the inhibitory concentrations are equal for plasmin and for the activation of p l a s m i n ~ g e n . ~ J ~ ~ ~ The present study suggests that additional mechanisms are also involved.

Effect of cigarette smoking on blood coagulation

Eighteen medical students who were not smokers were asked to smoke a cigarette, inhaling as much as possible. Blood samples were obtained before and 5 minutes after smoking. A significant increase was observed in platelet adhesiveness. Whole blood coagulation time, recalcified plasma coagulation time, platelet count, partial thromboplastin time, thromboplastin generation time, and tensile strength of the clot showed changes compatible with increased coagulability; however, these were statistically not significant.

Distribution and Fate of I 131 -Labeled Components of the Fibrinolysin System

The distribution, metabolism, and excretion of intravenously administered I131-labeled components of the fibrinolysin system were studied in dogs. lodination of plasmin tended to decrease slightly its in vitro fibrinolytie activity. Inaetivation appeared to depend on degree of iodination. Todination of strepto-kinase or urokinase did not decrease their plasminogen activator potential. All plasmins and plasminogen, regardless of the position of the iodine tag, adsorbed to preformed clots in the dog. Spontaneously activated human and chloroform-activated bovine plasmin adsorbed to a lesser extent than streptokinase-or urokinase-activated human plasmin. Clots removed from piasmin-treated dogs lysed in vitro within 24 hours. However, streptokinase or urokinase alone, on the basis of radioactivity measurements, did not exhibit any affinity for clots, nor did they cause any clot lysis. Plasma clearance curves revealed the presence of at least two processes in all preparations: a fast one (average half-life four minutes) responsible for the removal of over 60 per cent of the injected radioactivity and a slower process with a biological half-life greater than four hours. Pour hours after the injection of labeled compounds, the thyroid and stomach were the only organs which contained significant percentages of the injected radioactivity. Major excretory routes for the radioactivity were the urinary and biliary systems. Persistence of circulating radioactivity at a time when no free fibrinolytie activity could be determined in the blood was interpreted as complex formation between plasmin and antiplasmin.

Progestational agents and blood coagulation: IV. Changes induced by progestogen alone☆

To evaluate the effects of chlormadinone acetate upon the coagulation of blood and fibrinolysin systems, 35 healthy, young women voluntarily using some form of birth control were studied. 10 women who served as controls used intrauterine devices; 25 women took either a progestin-estrogen (1 mg norethindrone acetate and 1 mg mestranol) combination or a synthetic progestational agent (0.5 mg chlormadinone acetate) on a coded, double-blind basis. Platelet counts, thrombelastograms, and plasma assays were performed prior to and after 3 and 6 months of treatment. After 3 months, those taking progestin-estrogen showed a highly significant increase toward hypercoagulability in Quick time, Factors II, VII, and X, and increased levels in the thromboplastin generation time (TGT), Factors V and IX, and plasminogen. At 6 months all levels remained elevated except for TGT. Those on chlormadinone acetate had only a slightly significant change toward hypercoagulability in Quick time and Factor VIII, an increase in Factor IX, and a decrease in Factor X. In the control group only TGT was elevated. The progestin alone induced only minimal changes in comparison to the marked rises accompanied with progestin-estrogen therapy.

Progestational agents and blood coagulation. V. Changes induced by sequential oral contraceptive therapy.

Blood coagulation changes induced by sequential oral contraceptive (OC) therapy were studied. 112 healthy women volunteers were monitored over a 2-year period while on a regimen of sequential OC therapy (50 mcg ethinyl estradiol daily from Day 5 through 14 and 50 mcg ethinyl estradiol plus 1 mg morethindrone acetate on Day 15 through 25). The treated group showed marked increases toward hypercoagulability in the Hicks and Pitney thromboplastin generation time screening test at 3 and 9 months, Factor 5 at 9 months, Factor 8 at 3 and 9 months, and fibrinogen at 3 months. Decreases from base lines were seen in antiplasmins at 24 months and in alkaline phosphatase at all intervals (3, 9, and 24 months). It was suggested that the decrease in serum antiplasmin indicates a compensatory tesponse in the fibrinolytic system to the hyperactivity of the coagulation system.

Progestational agents and blood coagulation: VIII. Effect of low-dose, alternate-day, estrogen-progestin combinations on blood coagulation factors in man, with a special note on the effect of freezing of blood samples

Changes in the blood coagulation system were studied in three groups of 20 patients each. The first group received 0.5 mg. of norethindrone daily, plus 0.06 mg. of ethinyl estradiol on alternate days from cycle Day 5 through 25. The second group, all of whom had been fitted with an intrauterine contraceptive device (IUD), received no hormonal treatment and served as a control group. The third group received 0.5 mg. of norethindrone daily, combined with 0.045 mg. of ethinyl estradiol given on alternate days from cycle Day 5 through 25. Blood samples were drawn prior to the initiation of the study and after three months of treatment. Tests of the following parameters of the blood coagulation system were performed: direct platelet count; platelet adhesiveness; prothrombin time; thrombin time; fibrinogen; factor II assay; activity of factors V, VII, VIII, IX, and X; antithrombin III; and fibrin/fibrinogen degradation products. For a number of these factors, both fresh and frozen blood samples were examined. It was concluded that the two treatment regimens, with the use of alternate-day estrogen administration over a three-month period, had no clinically significant effect on the blood coagulation system.

Progestational agents and blood coagulation: VI. Relationship of ABO blood types to changes induced by oral contraceptives☆

Data from the authors previous studies regarding changes which occur in coagulation and fibrinolytic levels in the blood of women using oral contraceptives have been compared with ABO blook groups in 307 women. Although a tendency toward hypercogulability in group A was noted, it was not statistically significant. Patients had been treated with 4 different dosage combinations of estrogen and progestins over periods varying from 3 months to 24 months. The steroid therapy had increased the levels of activity of some of the coagulation and fibrinolytic factors regardless of progestin component dosage. No differences among blood groups was shown.

CLINICAL AND EXPERIMENTAL STUDIES ON ADENINE, VARIOUS NUCLEOSIDES AND THEIR ANALOGS IN HEMATOLOGY*

In red blood cells as well as in platelets there appears to be a decrease in adenine nucleotides during storage under blood bank conditions. This can be decreased by use of anticoagulant preservatives with higher phosphate content than the standard ACD solution, through the addition of adenine and inosine. Maintenance of higher ATP levels appears to be related to longer circulating life span after transfusion into patients in the case of red blood cells but not platelets. Inosine and more alkaline preservative medium also contribute to the maintenance of 2,3-DPG levels in red blood cells, and with it to the maintenance of normal hemoglobin dissociation curves and thus oxygen-carrying capacity. Certain nucleoside analogs may contribute to the preservation of platelets and of whole blood by their platelet-aggregation inhibitory activity. Platelet-aggregation inhibitors may also be useful in preventing thromboembolic episodes with potentially greater safety than anticoagulants.