During the tenth biennial examination of the Framingham Study, 1315 participants who were free of cardiovascular disease had fibrinogen levels measured. During the ensuing 12 years, cardiovascular disease developed in 165 men and 147 women. For both sexes, the risk of cardiovascular disease was correlated positively to antecedent fibrinogen values higher than the 1.3 to 7.0 g/L (126 to 696 mg/dL) range. The magnitude of the risk diminished with advancing age in women but not in men. Risk for coronary heart disease also was significantly related to fibrinogen level. Here, the magnitude of risk displayed diminishing impact with age, again only in women. Risk of stroke increased progressively with fibrinogen level in men but not in women. The impact of fibrinogen value, considered as a separate variable, on cardiovascular disease was comparable with the major risk factors, such as blood pressure, hematocrit, adiposity, cigarette smoking, and diabetes. Fibrinogen values were also significantly related to these risk factors. Taking all these into account in a multivariate analysis, fibrinogen level was still significantly related to the incidence of cardiovascular disease in men and marginally significant in women. For coronary heart disease, the fibrinogen level was significant for both men and women. Elevated fibrinogen level is a predictor of cardiovascular disease that should be added to the cardiovascular risk factor profile. ( JAMA 1987;258:1183-1186)

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Fibrinogen and risk of cardiovascular disease. The Framingham Study.

Since thrombin cleaves fibrinopeptides A (FPA) and B from the NH(2)-terminal end of the fibrinogen molecule, measurement of fibrinopeptide levels in plasma may provide a direct index of thrombin action. Recently a radioimmunoassay for FPA has been developed, and in the present paper, we describe the application of this assay to the measurement of FPA levels in clinical blood samples. Since fibrinogen cross-reacts with antibodies to FPA, dialysis was used to extract the peptide from plasma. In vitro generation of FPA was prevented by removing the fibrinogen from the plasma by precipitation with ethanol before dialysis. The processing technique permitted recovery of 75% of FPA added to blood in vitro. Evidence that the immunoreactivity measured in plasma is due to FPA was provided by the results of experiments in which two antisera to FPA with different specificities showed comparable results and addition of thrombin caused no change in immunoreactivity. In contrast, extracts of streptokinasetreated plasma showed a five-fold increase in activity when treated with thrombin and markedly different immunoreactivity with the two antisera. Plasma FPA levels in 30 normal men were below 2 ng/ml, with a mean of 0.5 ng/ml. FPA levels in 12 patients with reduced fibrinogen levels or reduced platelet counts or both ranged between 4 and 289 ng/ml. FPA levels in 13 patients with normal or elevated fibrinogen levels, including 6 patients with clinical evidence of venous thrombosis or pulmonary embolism or both, ranged between 5 and 23 ng/ml. FPA and fibrinogen degradation product levels did not correlate, and in several patients, elevated FPA levels were found in the presence of normal fibrinogen degradation product levels. After infusion of FPA-containing solutions in four normal individuals, FPA showed a disappearance rate from the plasma consistent with a t((1/2)) of 3-5 min. Heparin infusions in six patients with venous thrombosis or pulmonary embolism or both and elevated FPA levels were followed by a prompt decline in FPA level at a mean rate equivalent to a 3-5 min t((1/2)).

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Measurement of Fibrinopeptide A in Human Blood

Normal human and animal sera contain a glob-ulin, plasminogen, which in the presence of acti-vators is rapidly converted to plasmin, a pro-teolytic enzyme active at neutral hydrogen ion concentrations. Plasmin is an enzyme of wide specificity and will attack such varied substrates as gelatin, casein, certain synthetic esters, accelerator globulin, complement, fibrinogen and most importantly fibrin. Plasminogen activation occurs spontaneously (1), or as a result of contact with activators of tissue (2), body fluid (3, 4), or bacterial origin (5). The conversion of plas-minogen to plasmin involves the loss of a peptide moiety and there is evidence to suggest that the plasmin obtained by different modes of activation may vary in composition (6). Biochemically, considerable species differences exist not only between the plasminogen system of man and animals, but more particularly between the systems of various animals; this variability is most extreme with regard to the differential effectiveness of streptokinase. Rigid kinetic studies (6) reveal that the activation of human plasmino-gen under the influence of streptokinase (an ex-tracellular product of hemolytic streptococcal metabolism), trypsin and urokinase (prepared from human urine) results from a first order enzymatic reaction. Since physiological fibrinolytic phenomena result from activity of the plasminogen system, attempts to use plasmin or plasminogen activators to effect therapeutic thrombolysis have been numerous. Animal experiment, despite the difficulties and confusion of species variability, has yielded striking findings (7, 8, 9). Though the demonstration of experimental thrombolytic action has been of an unequivocal nature, the precise mechanism of its production has hitherto been obscure. experiments and in vivo observations bearing upon thrombolytic' mechanisms in man. The results indicate that since plasminogen is found both in plasma and also as a constituent of thrombi, clot lysis occurs by a dual mechanism. The chief and primary mechanism of thrombolysis involves the diffusion or adsorption of plasminogen acti-vator to the thrombus, activation of intrinsic clot plasminogen and thrombolysis. The secondary mechanism involving digestion of the thrombus by extrinsic plasmin action appears to be of little quantitative importance. MATERIALS AND METHODS Streptokinase2 was a highly purified preparation, bio-physically though not immunochemically homogenous; the specific activity was 600 to 700 streptokinase units per lug. nitrogen. Human plasminogen (1) contained 100 to 150 casein units per mg. tyrosine. Plasmin was prepared by autocatalytic activation of plasminogen in glycerol (1) and contained 95 casein units per mg. tyrosine (see Casein assay). Epsilon amino caproic acid2 (6-amino hexanoic acid) had …

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The mechanism of clot dissolution by plasmin

ANTITHROMBIN III (heparin cofactor) is known to inhibit thrombin1 and Factor Xa1–3 (activated Factor X). We have purified antithrombin4 from human plasma by a series of chromatographic and electrophoretic separation techniques. The homogeneity of the final product is demonstrated by disc gel electrophoresis, SDS gel electrophoresis and immunoelectrophoresis4. Using this preparation, we have shown that antithrombin forms a 1 : 1 stoichiometric complex with thrombin which cannot be dissociated with denaturing and reducing agents. Addition of heparin dramatically accelerates the rate of formation of this complex. Complex formation is completely dependent on an interaction between the serine active centre of thrombin and an arginine reactive site of antithrombin. Furthermore, e-amino lysyl groups of antithrombin serve as binding sites for the highly negatively charged heparin. Based on these data and other evidence, we have proposed that heparin acts to accelerate inhibitor function by binding to antithrombin and inducing an allosteric modification in it, which renders the arginine in its reactive site more accessible to the serine in the active centre of thrombin4.

C Menzie

Papers

A new method for the determination of fibrinogen in small samples of plasma.