Milica Brozović

Haemostatic function and ischaemic heart disease: principal results of the Northwick Park heart study

The Northwick Park Heart Study (NPHS) has investigated the thrombotic component of ischaemic heart disease (IHD) by the inclusion of measures of haemostatic function. Among 1511 white men aged between 40 and 64 at the time of recruitment, 109 subsequently experienced first major events of IHD. High levels of factor VII coagulant activity and of plasma fibrinogen were associated with increased risk, especially for events occurring within 5 years of recruitment. These associations seemed to be stronger than for cholesterol, elevations of one standard deviation in factor VII activity, fibrinogen, and cholesterol being associated with increases in the risk of an episode of IHD within 5 years of 62%, 84%, and 43% respectively. Multiple regression analyses indicated independent associations between each of the clotting factor measures and IHD but not between the blood cholesterol level and IHD incidence. The risk of IHD in those with high fibrinogen levels was greater in younger than in older men. Much of the association between smoking and IHD may be mediated through the plasma fibrinogen level. The biochemical disturbance leading to IHD may lie at least as much in the coagulation system as in the metabolism of cholesterol.

Factor VII in an Industrial Population

Factor‐VII levels were measured in members of an industrial population participating in a long‐term prospective study of arterial disease. A modified semi‐automated method with an artificially prepared factor‐VII deficient plasma, one batch of freeze‐dried thromboplastin and one freeze‐dried pooled reference plasma was used throughout. All calculations were carried out on a programmable desk calculator.

CHANGES IN HÆMOSTATIC SYSTEM AFTER APPLICATION OF A TOURNIQUET

In 35 patients undergoing routine orthopaedic operations in which occlusive tourniquets were used there was a pronounced rise in fibrinolytic activity in the systemic circulation which lasted for at least 15 minutes after the release of the tourniquet; this response was seen after operations on both arms and legs. In contrast there was no increase in fibrinolytic activity in the systemic circulation associated with venous occlusion. Neither the application of a tourniquet nor venous occlusion resulted in changes in factors V or VIII, fibrinogen, or platelet-count. The application of a completely occlusive tourniquet might be a simple form of prophylaxis against deep-vein thrombosis and would avoid the disadvantages of using heparin.

An epidemiological study of the haemostatic and other effects of oral contraceptives.

Summary. Factors V, VII and VIII (each determined by biological assay), fibrinogen, platelet count and adhesiveness, and fibrinolytic activity were measured in 234 white pre‐menopausal women, of whom 57 (24%) were on oral contraceptives and 177 (76%) were not. Cholesterol, triglyceride and blood pressure levels were also recorded. In 20 of the women on oral contraceptives, and in an age‐matched group of 20 who were not, prothrombin, factor X, antithrombin III and α2‐macroglobulin levels were determined, and factors VII and VIII were also measured immunologically. For the majority of the variables studied, the differences between those using and not using oral contraceptives were greater in younger than older women; in the case of factor VII (biological assay) and fibrinogen, the differences between the regression slopes on age were statistically significant, and mean values were substantially higher in those on oral contraceptives. There was also a significant difference between regression slopes on age for cholesterol. Mean levels of prothrombin, factors VII (immunological assay) and X, triglycerides and blood pressure were significantly higher, and mean levels of antithrombin III significantly lower, in those on oral contraceptives compared with those not. Overall, fibrinolytic activity was significantly higher in the women on oral contraceptives; this difference was, however, almost entirely due to the greatly increased fibrinolytic activity of the non‐smokers on oral contraceptives, activity in the smokers on oral contraceptives being similar to that of the women not on these preparations. There were no significant differences in mean platelet count or adhesiveness, or in haemoglobin, packed cell volume, uric acid and blood sugar levels. Among the women on oral contraceptives, there was a significant negative correlation between factor VIII and fibrinolytic activity; this was largely due to five women all of blood groups A and B, in whom, besides high factor‐VIII levels and poor fibrinolytic activity, other variables (e.g. fibrinogen) were raised in a direction that might be expected to favour thrombogenesis. It is possible that it is those women whose fibrinolytic activity does not increase in order to compensate for the effects of oral contraceptives on clotting factors, lipids and blood pressure, who are at special risk of thromboembolic episodes. The differential effects of oral contraceptives by age must be borne in mind in evaluating the effects of these preparations on the haemostatic and lipid systems.

Haemostatic changes following surgery.

Changes in factors V, VII and VIII and in fibrinogen were studied in 32 patients undergoing major abdominal surgery. Mean levels of factors V and VII were similar to population-based values preoperatively. Factor V fell following elective surgery and then rose above the mean pre-operative level before returning to it by the tenth post-operative day. Factor VII fell following both elective and emergency surgery and tended to remain depressed throughout the post-operative period. Pre-operative values of factor VIII and fibrinogen were higher than population-based values and higher in the emergency than in waiting-list patients. Both factor VIII and fibrinogen rose following elective surgery but no statistically significant change was seen following emergency surgery. The uncomplicated conditions leading to elective surgery, the acute complications leading to emergency surgery, and surgery itself may each have contributed to increases in factor VIII and fibrinogen levels, whereas the fall in factors V and VII was largely related to surgery itself. The findings may help in the interpretation of associations between clotting factors and thrombotic disease, particularly in the case of factor VII.

Plasma Heparin Levels after Low Dose Subcutaneous Heparin in Patients Undergoing Hip Replacement

Plasma heparin levels were measured at 2 and 8 h after 5000 iu of heparin subcutaneously in 43 patients awaiting total hip replacement. At 2 h, 37 patients had plasma levels between 0.05 and 0.15 iu/ml, three patients had more than 0.2 iu/ml, and three had only trace levels. At 8 h, most patients had less than 0.1 iu/ml, and six patients had no detectable plasma heparin. There were no correlations between plasma heparin levels and age, sex, body weight or use of analgesics. Patients with high 2 h plasma heparin levels may bleed at operation, whereas those with very low plasma heparin concentrations may be at risk of post‐operative venous thrombosis; it may therefore sometimes be necessary to monitor plasma heparin levels in order to determine the optimal regime in individual cases.

ALTERED FACTOR VIII COMPLEXES IN SICKLE CELL DISEASE

The details of the patients are shown in Table I. All patients were studied on outpatient attendance while well and during the first day of admission for sickle cell crisis. All but one crisis were vasoocclusive; patient J.A. was admitted with chest infection due to M . pneumoniae. Factor VIII: C was measured using a two-stage assay (Denson, 1967); factor VIII: RAg was estimated the Laurell rocket immunoelect-rophoresis (Laurell, 1966); and factor VIII RiCoF was measured by a modification of the method of Evans & Austen (1977). Two-dimensional immunoelectrophoresis of factor VIII: RAg was carried out according to Laurell (1965) using a Behringwerke factor VIII antiserum. Cryoprecipitates were prepared by freezing 20 ml of plasma at -2OC, then thawing at 4°C for 10 h, followed by centrifugation at 3500 rpm at 4°C for 15 min. The precipitate was dissolved in 1 ml citrate saline, pH 7.4. Plasmas were also subjected to gel chromatography on Sepharose 6B. The eluting fluid was citrate saline. The results of factor VIII measurements are shown in Table I. Most patients had higher than normal values of factor VIII : C, RAg and RiCoF in both the steady state and during crisis. In four plasmas denoted by an asterisk, factor VII1:RAg showed two peaks on the twodimensional immunoelectrophoresis (Fig 1 b, c). When these plasmas were subjected to gel

Ristocetin-induced platelet agglutination in Afro-Caribbean and Caucasian people.

RISTOCETIN-INDUCED PLATELET AGGLUTINATION IN AFRO-CARIBBEAN AND CAUCASIAN PEOPLE A variety of haemostatic changes have been reported in patients with sickle-cell disease (SCD) including abnormalities of ristocetin-induced platelet agglutination (RIPA) (Sarji et al, 1 9 79; Leichtman & Brewer, 1977). While investigating haemostasis in a group of patients with SCD we confirmed the failure of platelets in platelet-rich plasma (PRP) to agglutinate to ristocetin, despite adequate plasma concentrations of factor VIII:C, VIII R:Ag and ristocetin cofactor (RiCoF). To our surprise, the PRP from healthy black controls also frequently failed to agglutinate to ristocetin; and the extent of aggregation to collagen and ADP was less than in age-matched Caucasian controls. Twelve healthy West Indian subjects, aged 15-41 (seven with Hb AA and five with Hb AS) were studied in parallel with eight non-Negro controls, aged 21-38. None were taking anti-platelet drugs. Platelet agglutination to ristocetin sulphate ( 1.2 mg/ml of PRP) was measured as the maximum change in light transmission and expressed as a percentage of chart paper units. Factor VII1:C was measured by a modified two-stage assay (Denson, 1967) using Diagen kits (Diagnostic Reagents Ltd, Thame, Oxon.). The assay was standardized with the sixth and seventh British Standards, 77/520 and 77/52 5 (NIBSC, Holly Hill, Hampstead). Factor VIII

ORAL ANTICOAGULANTS, VITAMIN K AND PROTHROMBIN COMPLEX FACTORS

Coumarins and indaiiediones are antagonists of vitamin K and are widely used for their anticoagulant effect. Considerable progress has recently been made in understanding their mode of action, mainly through elucidation of the role of vitamin K in the synthcsis of protlirombin and related coagulation factors. In 1963 Hemker and colleagues showed that anticoagulant treatment did not cause simple deficiency of vitamin K dependent factors; they demonstrated the presence of an abnormal protein in the plasma of patients on oral anticoagulaiit treatment and suggested that it was an inactive precursor of prothrombin. Hemker & Muller (1968) and Hemker et al(1968) named the abnormal protein PIVKA, abbreviated from protein, induced by vitamin K absence or antagonists. Later the presence of abnormal prothrombin, factor VII, IX and X (PIVKA 11, VII, IX and X) in the plasma of all patients on oral anticoagulants was confirmed by many investigators using immunological techniques (Ganrot & NilChn, 1968 ; Larrieu & Meyer, 1970; Denson, 1971). Studies in vitamin K deficient rats (Bell & Matschiner, 1969; Suttie, 1967, 1970) also indicated that a precursor protein is involved in the formation of prothrombin. When vitamin K was administered to vitamin K deficient rats, about half of the normal prothrombin concentration was restored quickly within the first hour, and this increase could not be blocked by cycloheximide, an inhibitor of protein synthesis. These observations suggested that vitamin K acts at a post-ribosomal site to convert an inactive precursor into a biologically active coagulation factor. Thus, the identification of the differences between normal vitamin K dependent factors and PIVKAs was obviously a key to the understanding of thc modc of action of vitamin K.