Clara M. Ambrus

CLINICAL AND EXPERIMENTAL STUDIES ON FIBRINOLYTIC ENZYMES

In vitro fibrinolytic activity has been reported for many proteolytic enzymes of animal and plant origin. A fibrinolytic factor was described in blood as early as 1893 (Dastre, 1893). For several years our group has been engaged in developing quantitative methods for the evaluation of potential fibrinolytic agents in oivo. Pharmacological properties, toxicology, and histopathological effects of these agents were studied. Promising preparations were selected for clinical trial in cancer patients with thromboembolic complications. On the basis of clinical experience, attempts are being made to produce improved preparations and to work out optimal dose schedules. Although this work is still in progress, it may be appropriate to review our completed experiments and to present a preliminary report on current studies.

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.

On the Mechanism of Thrombolysis by Plasmin

I131-labeled purified human fibrin clots were introduced into the circulation of dogs at various time intervals after an infusion of human plasmin. One hour later, the clots were removed and incubated in saline at 37 C. In most cases, at the time when these clots were in the circulation, there was no significant “free” plasmin activity demonstrable in the blood. Nevertheless, the clots removed from these animals exhibited in vitro lysis upon incubation. Plasmin-antiplasmin complexes were formed in vitro with no demonstrable caseinolytic activity. These complexes were as effective in dissolving fibrin clots in vivo as the same amounts of plasmin were alone. The activator (streptokinase) alone used to activate plasminogen to plasmin was significantly less effective than streptokinase-activated-plasmin, alone or in complex form with antiplasmin. These experiments were interpreted as pointing toward the ability of fibrin to compete effectively with antiplasmin for plasmin. Thus, antiplasmin may serve as a transport form or reservoir of plasmin, releasing it when fibrin clots are available, but protecting other plasma proteins from its proteolytic effect. Other phenomena apparently confirming this hypothesis have been mentioned.

Quantitative Method for the In Vivo Testing of Fibrinolytic Agents: Effect of Intravenous Trypsin on Radioactive Thrombi and Emboli

Quantitative methods for testing fibrinolytic agents in vivo are described. Methods based on the incorporation into the clot of P32 labeled blood cells or Cr51 labeled erythrocytes were found to be unsatisfactory. Clots were produced with I131 labeled fibrinogen. Dissolution of a clot resulted in decrease of radioactivity. This was recorded by placing segments of blood vessels in a special lead shield containing a scintillation detector head, connected to a radiation rate meter and an Esterline recorder. Pulmonary, peripheral and coronary emboli were produced by injecting radioactive clots produced and ground, in vitro. Using these methods it was found that maximal tolerated doses of trypsin showed no significant fibrinolytic effect.

Controlled study of the treatment of coronary occlusion with urokinase-activated human plasmin

Abstract One hundred and twenty patients were treated with urokinase-activated human plasmin or placebo in a double-blind study. Anti-coagulation with heparin and Dicumarol-type drugs was employed in both groups. Eighty-four patients were adequate for evaluation from the point of view of all criteria established (43 plasmin-treated, 41 controls). The two groups were found comparable from the point of view of age and sex distribution, body build, history and physical and laboratory findings on admission. No significant difference was seen between the two groups in mortality, evolution of the electrocardiogram, SGOT and LDH and other therapeutic criteria. Of the patients classified into the worst prognostic group on admission, 47 per cent died in the control group and 24 per cent in the plasmin-treated group. However, since only 32 patients were classified in this category, the numbers are too small to draw definite conclusions. No difference was seen in the occurrence of side effects between the control and plasmin-treated groups, indicating that this enzyme is safe at the dosage employed in the treatment of patients with myocardial infarction.

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.

REGULATION OF REGENERATION AND TRANSPLANTATION OF HEMIC TISSUES

Many hypotheses have been advanced on the regulation of the development, differentiation, growth, and function of hemic tissue. Great many technical problems hinder experimental studies in this field. We have attempted to approach some of these problems by studying the regeneration of hemic tissue and examining the fate of auto-, isoand honiografts. We thought that information on related pathologic processes (e.g., splenosis) and potential therapeutic applications might represent additional dividends from these experiments. This is a preliminary report on work in progress; some of these studies will be presented in more detail elsewhere.

Panel discussion: assay technics. The problems of correlation with results of treatment.

Abstract Dr. Baumgarten : In summarizing this discussion, it is apparent that standardization of fibrinolytic agents should be carried out by measuring the lysis of a fibrin clot. Most investigators agree on this. However, in addition, other types of assays may well be considered as adjuncts. Breakdown of an assay into proteolytic and activator activities can be carried out by employing casein as the substrate of choice. Some investigators prefer heated and unheated fibrin plates for such differential assays. It is a matter of preference of the investigator as to which assay procedure is employed. However, for purposes of standardization, one method should be stipulated as the primary assay. I recommend casein as the substrate of choice, as the results obtained with it are more reproducible than those obtained with the fibrin plate. Apparently casein has been used extensively in many laboratories throughout the world. Thus, while the clot lysis test assays the sum of proteolytic and activator activity, individual assay of the proteolytic and activator activity can be achieved with casein substrate.

Treatment of fibrinolytic hemorrhage with proteinase inhibitors: a preliminary report.

TABLE 1 shows the antifibrinolytic agents studied and the distribution of patients treated. In early experiments,2 we have investigated antiplasmin preparations isolated from human and bovine blood and the lima bean inhibitor. When epsilon-aminocaproic acid (EACA) and TrasylolB became available for clinical investigation, we concentrated our efforts on these agents. Of 151 patients treated, 80 received EACA and 57 Trasylol. In the series where conventionally treated controls were included in the study, 75 patients were entered. EACA was obtained from the Lederle C0.t and Trasylol from the Metachem C0.S Methods employed to estimate members of the fibrinolysin system have been reported previou~ly.~.~

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.