Da Lane

EPIDEMIOLOGY OF COAGULATION FACTORS, INHIBITORS AND ACTIVATION MARKERS: THE THIRD GLASGOW MONICA SURVEY I. ILLUSTRATIVE REFERENCE RANGES BY AGE, SEX AND HORMONE USE

Coagulation factor activity (fibrinogen, VII, VIII and IX), coagulation inhibitor activity (antithrombin, protein C, protein S), and coagulation activation markers (prothrombin fragment F1,u2003u20032; thrombin–antithrombin complexes) were measured in 747 men and 817 women aged 25–74 years, randomly sampled from the north Glasgow population in the Third MONICA Survey. Significant effects of age, sex, menopause and hormone use were observed and specific reference ranges are presented to illustrate these effects. Significant correlations were observed between several coagulation factors and inhibitors. Increased levels of factors VII, VIII and IX and decreased levels of protein C were associated with increased coagulation activation. In general, increases in coagulation factors with age were greater than increases in coagulation inhibitors, especially in men; this imbalance may favour increased coagulation activation and hence increased thrombotic risk with age.

EPIDEMIOLOGY OF COAGULATION FACTORS, INHIBITORS AND ACTIVATION MARKERS: THE THIRD GLASGOW MONICA SURVEY II. RELATIONSHIPS TO CARDIOVASCULAR RISK FACTORS AND PREVALENT CARDIOVASCULAR DISEASE

Coagulation factor activity (fibrinogen, VII, VIII and IX), coagulation inhibitor activity (antithrombin, protein C, protein S), and coagulation activation markers (prothrombin fragment F1, 2; thrombin–antithrombin complexes) were measured in 746 men and 816 women aged 25–74 years, randomly sampled from the north Glasgow population in the Third MONICA Survey. After age‐adjustment, significant associations with cardiovascular risk factors were observed. Serum cholesterol and triglyceride were associated with increases in factors VII and IX, as well as antithrombin, protein C and protein S; and with increased fibrinogen and factor VIII in women. Apart from factor VIII (related to blood pressure in men, but not in women), similar associations were observed for blood pressure and body mass index. Smoking status and/or smoking markers were related to fibrinogen, factor IX, antithrombin and protein S. Alcohol intake was related to protein S, and inversely to fibrinogen and antithrombin in men. Low social class was associated with fibrinogen, factor VIII, factor IX, and with antithrombin, protein S, and low protein C in men. Serum vitamin C was associated inversely with coagulation factors and coagulation inhibitors. The only associations of activation markers were with low serum vitamin C, and with alcohol consumption and low social class in men. Prevalent cardiovascular disease was associated only with fibrinogen.

Role of factor XII in thrombin generation and fibrinolysis during cardiopulmonary bypass

During cardiopulmonary bypass, thrombin is generated, which is thought to be initiated by activation of factor XII on the surface of the bypass equipment. We present a patient with severe factor XII deficiency who underwent cardiac surgery. As much thrombin was formed during cardiopulmonary bypass (measured by the prothrombin activation fragment F1 + 2 and thrombin-antithrombin complexes) as in normal patients, showing that factor XII was not necessary for thrombin generation. Factor X, but not factor IX, was activated (as measured by their activation peptides), and this activation correlated with F1 + 2 and thrombin-antithrombin complexes, suggesting that the tissue-factor/factor-VIIa pathway is the trigger for thrombin formation.

Thrombogenic mechanisms in the human: fresh insights obtained by immunodiagnostic studies of coagulation markers

Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, meaningful in vivo studies of thrombogenic mechanisms have previously been hindered by the absence of suitable assays. This article reviews the recent development and/or contemporary clinical application of plasma-based immunoassays for coagulation markers (factor XIIa, factor IX activation peptide, prothrombin fragment F1 + 2, thrombin-antithrombin complex and fibrinopeptide A) and for the fibrinolytic marker, D-dimer, which have enabled a critical re-appraisal of some long-standing hypotheses. In chronic renal disease the intrinsic coagulation pathway was found to be activated before haemodialysis and increased end-stage coagulation activity was detected during dialysis when heparinization was limiting. No evidence was found to support the generally accepted hypothesis that thrombogenesis in dialysis is triggered by stimulation of the contact system following exposure of blood to the dialyser membrane. Instead, it is postulated that it is a failure of regulation of end-stage coagulation proteinases (owing to the absence of endothelium) which is responsible for increased thrombogenesis in the dialyser circuit. Excessive end-stage coagulation activity was observed during cardiopulmonary bypass (CPB) surgery and in patients undergoing general thoracic surgery. The data did not accord with the hypothesis that the contact system provides the major thrombogenic trigger in CPB surgery. It is proposed that, in general thoracic surgery, a powerful procoagulant stimulus is provided via the tissue factor-factor VIIa pathway and that the same mechanism is also primarily responsible for triggering thrombogenesis during CPB surgery. The established hypothesis of a prethrombotic state in hereditary AT III deficiency is challenged by the inability to detect increased coagulation activity in asymptomatic AT III deficient patients. It is concluded that the AT III concentration in deficient members is sufficient to enable regulation of the coagulation system in the basal state, whereas failure to regulate the coagulation system only occurs following a major procoagulant stimulus, which overwhelms the impaired inhibitory capacity and triggers thrombosis. These findings highlight the advantages of using plasma-based immunoassays to investigate thrombogenic mechanisms in hypercoagulable states and have important implications for the further study and treatment of blood-surface interactions and thrombotic disease.

Plasma concentrations of fibrinopeptide a, fibrinogen fragment bβ1-42 and β-thromboglobulin following total hip replacement

Abstract Plasma concentrations of thrombin sensitive peptide fibrinopeptide A (FpA), plasmin sensitive fibrinogen fragment Bβ1-42 and the platelet release product β-thromboglobulin (βTG) have been measured in 36 patients before and after total hip replacement. Statistically significant elevations of all three activation products were observed in the days following operation. There were small differences in plasma concentrations of FpA, Bβ1-42 and βTG in patients who did (n = 13) and did not (n = 23) develop post operative deep vein thrombosis, as assessed by ascending venography on post operative day 10, but these differences were not statistically significant. It is concluded that coagulation and fibrinolytic systems and also blood platelets are activated following total hip replacement operations. However, the formation of post operative deep vein thrombosis can not be effectively monitored by measurement of the activation products.

Antithrombin III Northwick Park: demonstration of an inactive high MW complex with increased affinity for heparin.

Summary. It has been shown previously that antithrombin III Northwick Park has reduced ability to inactivate thrombin and is characterized by an additional anodal component on crossed immunoelectrophoresis (Howarth et al. 1985). We have applied plasma from an affected family member to heparin‐Sepharose and eluted the antithrombin III with a salt gradient. Evidence is presented that a variant component has slightly higher affinity for heparin than normal antithrombin III. Furthermore, this variant component is present in plasma as an ∼ 120000 MW inactive antithrombin III complex that can be reduced with dithiothreitol to MW ∼ 60 000, indicating disulphide bridging. Using ionexchange chromatography, the inactive complex has been isolated and shown to migrate in the same position as the anodal peak on crossed immunoelectrophoresis.

Antithrombin III Glasgow: a variant with increased heparin affinity and reduced ability to inactivate thrombin, associated with familial thrombosis.

A functional antithrombin III (AT III) deficiency has been identified in two generations of a family with a high incidence of thrombosis. The deficiency presented as 50% reduction in heparin cofactor activity compared to its antigen concentration. No abnormality was detected by crossed immunoelectrophoresis in the presence or absence of heparin. Plasma from the propositus was precipitated with dextran sulphate, applied to heparin‐Sepharose and the AT III stepwise eluted with NaCl. The AT III had a reduced ability to inactivate thrombin, when this was monitored by substrate hydrolysis or by SDS polyacrylamide gel electrophoresis. Its mobility was normal by the latter technique using 10–20% gradient gels under reducing and non‐reducing conditions. AT III from the patient was reapplied to heparin‐Sepharose and eluted with a NaCl gradient. An active pool eluted in the same NaCl concentration range used to purify normal AT III, while predominantly inactive AT III eluted at higher NaCl concentrations. It is concluded that this variant, designated AT III Glasgow, has increased affinity for heparin but reduced ability to inactivate thrombin.

Relationship between ex vivo anti-proteinase (factor Xa and thrombin) assays and in vivo anticoagulant effect of very low molecular weight heparin, CY222

There is uncertainty as to which activities of unfractionated heparin (UFH) and low MW heparin are responsible for their anticoagulant and antithrombotic properties. We have sought to answer this question by examining plasma samples taken during a recently conducted dose‐finding study of the low MW heparin, CY222, in haemodialysis for chronic renal failure. In this study, in vivo anticoagulant effect was assessed by measurement of plasma FPA levels. UFH was administered as a dose of 5000 iu bolus + 1500 iu/h maintenance infusion, while the effects of three doses of CY222 were studied (10000, 15000 and 20000 Institute Choay anti‐factor Xa u bolus, all with 1500 Institute Choay anti‐factor Xa u/h maintenance infusion). Anti‐factor Xa levels were determined by chromogenic substrate assay. Anti‐thrombin levels were determined by chromogenic substrate assay and by quantitation of catalysed thrombin–inhibitor complexes (using autoradiography). Analysis of the results indicate that plasma fibrinopeptide A (FPA) levels correlate with anti‐factor Xa (r= 0.45) and anti‐thrombin (substrate) (r= ‐0.63) levels of UFH, but only with the anti‐factor Xa levels (r= ‐0.41) of CY222. These results suggest that the anti‐factor Xa assay is currently the most suitable assay for monitoring low MW heparins such as CY222 in humans.

Clearance of human desaminotyrosyl fibrinopeptide A from the rat circulation: role of kidney and proteolytic enzymes.

An investigation has been carried out of the elimination of fibrinopeptide A (FpA) from the rat circulation. 125I labelled desaminotyrosyl human FpA (125I-FpA) was injected into three groups of rats. These were (a) control rats (n = 9), (b) rats in which bilateral nephrectomy was performed (n = 10) and (c) rats which had acute bilateral ligation (n = 6). In the control rats elimination proceeded in a biphasic exponential manner. A fast initial clearance had a half life of less than 2 mins and this was followed by a slower component with an estimated half life of 51 mins. The fast component was caused by equilibration of the peptide throughout intra and extravascular spaces and the slow component reflected the action of catabolic processes once equilibration was attained. Nephrectomized rats had a similar initial fast clearance but the estimated half life of the slow component was 73 mins and also 10% less peptide was eliminated at a given time once equilibration had been achieved. The difference in half lifes of slow components of control and nephrectomized rats was not significant, while the difference in amount of excreted peptide was significant (p < 0.05) at each time point following equilibration. Rats with ligated ureters had almost identical clearance curves to nephrectomized rats (half life of slow component 73 mins), strongly suggesting that FpA is difectly filtered by the kidney. Blood samples withdrawn from control or nephrectomized rats at the completion of the experiments contained intact FpA and a major degradation fragment demonstrable by gel filtration on G10 Sephadex. A similar degradation could be elicited by in vitro incubation of 125I-FpA with fresh citrated blood. Urine also degraded 125I-FpA in vitro or in vivo to fragments, one of which was of similar molecular size to that produced in blood. It is concluded that proteolytic enzymes in blood degrade FpA which is then in part directly eliminated from the circulation by the kidneys: further degradation may then occur in the urine. The previously reported half life of FpA is a measure of its equilibration rather than of its catabolism.

Proteolysis of fibrinogen in healthy volunteers following major and minor in vivo plasminogen activation

Changes in immunoreactive fibrinopeptide A, fragment B beta 1-42 and fragment E were followed after major and minor in vivo plasminogen activation, after infusion of streptokinase and acylated streptokinase-plasminogen complex respectively, in healthy male volunteers. Major activation resulted in a dramatic rise in all three peptides, fragment E persisting in the circulation longest. Fragment B beta 1-42 was also markedly increased after minor systemic plasminogen activation despite apparently adequate levels of immediate antiplasmin.