A. S. Douglas

Studies on Blood Coagulation, Fibrinolysis and Platelet Function Following Exercise in Normal and Splenectomized People

The effect of a short period of strenuous exercise on the haemostatic mechanism has been studied. Following exercise there was an increase of factor VIII which was not affected by prior splenectomy or by aminocaproic acid, an inhibitor of plasminogen activator. Although exercise produced increased plasminogen activator concentrations, as measured by the euglobulin lysis time, there was no alteration in the FR‐antigen (fibrinolytic degradation product) concentration. Platelet adhesiveness, and ADP‐induced aggregation were significantly increased after exercise and in the latter test the phase of platelet disaggregation was impaired.

Effects on blood coagulation, fibrinolysis, and platelet aggregation of normal and atheromatous aortic tissue

A comparison was made between the effects of atheromatous and normal areas of the same aorta on coagulation, fibrinolytic, and platelet aggregating systems. In the thromboplastin generation test it was found that atheromatous aorta possessed significantly greater ability to generate intrinsic prothrombin activator than did normal aortic tissue. But, by the one-stage prothrombin time technique, the content of extrinsic thromboplastin in both types of aorta was similar. Aortic preparations, consisting of intimal and medial layers only, were not found to possess fibrinolytic ability and did not contain inhibitors of the fibrinolytic system. The adhesion of platelets to ulcerated atheromatous areas of aorta was significantly greater than to normal or non-ulcerated atheromatous areas. However, homogenates of atheromatous and normal aorta did not differ in their ability to accelerate platelet aggregation and fibrin clot formation when tested by a modified Chandlers tube technique. The significance of the findings is discussed and the suggestion made that the mechanisms by which atheromatous aortic tissue might predispose towards intravascular thrombosis in vivo are the ability of such tissue to enhance intrinsic prothrombin activator formation and platelet aggregation.

In-Vitro Studies with Ancrod (‘Arvin’)

Summary. The effect of ancrod on the fibrinolytic enzyme system in vitro and on artificial thrombi in the Chandler tube system has been studied. Ancrod has no direct lytic activity and does not influence the rate of lysis of artificial thrombi in the Chandler tube. Clots formed by the action of ancrod are more susceptible to the lytic action of urokinase compared to those formed by thrombin but are equally as susceptible to spontaneous lysis as comparable thrombin clots. The rapidity with which the fibrin formed in vivo by ancrod is removed must be attributed to some process other than the generalized activity of the plasminogen–plasmin system.

Amyloidosis Associated with the Nephrotic Syndrome and Transfusion Reactions in a Haemophiliac

Summary. It is suggested that the immunological response in a severe haemophiliac to longstanding incompatible plasma infusions may have been responsible for the development of amyloidosis which culminated in the nephrotic syndrome. Serological studies showed that the patient had a circulating anti‐Gm(1) antibody, and also that possibly he was on occasions reacting violently towards the infusion of immune complexes formed between the IgG1 (Fc region) antigens, Gm(1) and ISf(1) and their antibodies. Despite the fact that there was a marked reduction in the number and severity of transfusion reactions after treatment solely with selected Gm(‐1) plasmas, there was a progressive deterioration in the patients renal function.

Urokinase therapy: dosage schedules and coagulant side effects.

The effect of a new preparation of urokinase (Roche) has been studied, in the laboratory and in 10 patients and three volunteers. It is a non‐toxic and active fibrinolytic agent if given in adequate dosage, but it was found that a small initial infusion gave rise to a paradoxical coagulant effect in the recipient. This effect was associated with the formation of‘cryofibrinogen’ and an increase in factor‐VIII activity. The coagulant effect was probably mediated by a low‐grade process of plasminogen activation rather than due to the preparation of urokinase itself. The preparation did have minor intrinsic thromboplastic activity similar to that described in other preparations of urokinase, but it is unlikely that this would produce a significant effect in vivo. The coagulant effect could be overcome by increasing the initial dose of urokinase to produce rapidly the haemostatic defect normally associated with fibrinolytic therapy.

Relationships between platelet function tests in normal and uraemic subjects

In tests of platelet function in normal subjects, the following relationships were found: the greater the platelet adhesiveness the less the ability to disaggregate after challenge with adenosine diphosphate (ADP), and the greater the disaggregation after ADP, the longer the clotting time in the test for platelet factor 3 availability. Such correlations were disturbed in uraemic patients.

Effect of normal and atheromatous aortic tissue on platelet aggregation in vitro

An investigation was made into the platelet-aggregating ability of human aortic tissue. It was found that potent aggregating substances are present in the vascular wall but that the degree and character of platelet aggregation produced was dependent on the method employed in the preparation of the tissue for study. The supernatants of aortic tissue homogenates were found to produce potent irreversible platelet aggregation that occurred after a latent period of one to two minutes. The activity was heat labile, destroyed by collagenase, sedimented by ultracentrifugation, and was considered to be identical or similar to collagen. An extract obtained by boiling aortic tissue with saline was found to cause less powerful reversible platelet aggregation that occurred within 15 to 30 seconds contact with platelets; the activity was not affected by heating, incubation with collagenase, or ultracentrifugation, and was considered to be due to adenosine diphosphate. Atheromatous aortic tissue when extracted by either method was found to contain less than one half of the potency of normal tissue in causing platelet aggregation.

Effect in vitro on platelet function of two compounds developed from the pyrimido-pyrimidines

VK 774 and VK 744, two new compounds developed from the pyrimido-pyrimidines, have been found to be powerful inhibitors of platelet function tested in vitro. They inhibit adenosine diphosphate (ADP)-induced platelet aggregation, and the release of platelet factor 3 by kaolin, and VK 774 also reduces platelet adhesiveness and inhibits platelet aggregation (`snowstorm effect) in the Chandler tube system. Although measured percentage whole blood clot retraction was uninfluenced by these drugs the clot produced with VK 774 was friable and soft. VK 774 appears to be the most powerful of these compounds reported so far, being active in some test systems at 10−6M, and, if the results of toxicity testing are satisfactory, it should be an important agent for therapeutic trial.

Plasminogen activation and the coagulation process.

C. R. M. PRENTICE, G. P. MCNICOL, AND A. S. DOUGLAS (University Department of Medicine, Glasgow Royal Infirmary) In the defibrination syndrome intravascular coagulation and the fibrinolytic process are often associated so that it is difficult to determine the primary cause of the process. The present studies were designed to examine some of the mechanisms by which intravascular coagulation is able to initiate, or is stimulated by, the fibrinolytic process. Several aspects of the problem have been well explored. Sherry, Fletcher, and Alkjaersig (1959) have suggested that a thrombus may selectively encourage local plasminogen activation as the inhibitors are excluded in the surrounding plasma. Kwaan and McFadzean (1957) have shown that the release of plasminogen activator is augmented by anoxiaof venous endothelium.A thrombus, by causing local endothelial ischaemia, could provide the stimulus for activator release. Niewiarowski and Prou-Wartelle (1959) and latridis, Wilson, Ferguson, and Rierson (1960) demonstrated that activation of the contact factors accelerates plasminogen activation. In the present studies, four situations in which coagulation and plasminogen activation were associated are described. Particular emphasis has been given to the linking mechanisms between these processes. The first studies were with urokinase. Using a proprietary preparation of urokinase a satisfactory fibrinolytic response was found in patients, but a transient coagulative effect was sometimes also seen. There was shortening of the recalcification time associated with a marked rise in factor VIII activity. Plasma obtained at this time formed a thrombin-clottable cryoprecipitate at 4°C. To determine if this effect was due to contamination of urokinase with thromboplastic substances or whether it was the direct result of plasminogen activation, volunteers were given two infusions on separate occasions. The infusions consisted of first, urokinase, and second, urokinase preceded by aminocaproic acid to inhibit plasminogen activation. The coagulative effect in the recipients plasma was, on the whole, greater when urokinase was administered alone than when it was given with aminocaproic acid. It is suggested that this coagulative effect was the result of plasminogen activation by urokinase. Possibly the patients fibrinogen may have been converted into a cold-precipitable form with consequent activation or release of factor VIII activity. The second studies were made following exertion. In volunteers, hard exertion produced the expected increase in plasma fibrinolytic activity and in factor VIII levels. Subjects who exercised following the administration of aminocaproic acid also showed a normal rise in factor VIII levels. It appears that the factor VIII increase is not dependent on plasminogen activation. Two splenectomized patients showed a normal rise of factor VIII activity following exertion. It is unlikely that factor VIII released by the spleen is responsible for the elevation of this factor seen after exercise. Platelet aggregation was increased and disaggregation impaired following exertion. The third series of studies were of three children from East Africa who developed thrombotic gangrene of the fingers, but they showed defective fibrinolysis associated with increased levels of fibrinolytic inhibitors. It is possible that this abnormality was, in part at least, responsible for their disease (Turpie, Forbes, and McNicol, 1967). The fourth series were of studies made during Caesarian section. Here, blood samples were taken simultaneously from peripheral and uterine veins. A progressive shortening of the whole blood clotting time, increase in factor VIII activity, and increased fibrinolysis were noted after placental separation. The changes were all greater in the uterine venous blood than in forearm vein blood. In some subjects raised levels of fibrinogen degradation products were found in the uterine venous blood, but not in the peripheral blood. It is apparent that both the coagulation and fibrinolytic processes are called into action following uterine incision and placental separation. Of practical importance is the observation that the uterine venous blood shows marked changes which are diminished by the time it reaches the periphery. This confirms the observation that the body has a great capacity to inhibit the active enzymes concerned with haemostasis. It also indicates that the peripheral venous blood may not give an accurate reflection of changes that occur locally in body tissues. These four situations, which reflect the close association between coagulation and fibrinolysis, illustrate that there are definite, but ill-defined, linking mechanisms between them. Further study in these areas is needed to increase our understanding of the physiology of haemostasis.