Clarence Merskey

A Rapid, Simple, Sensitive Method for Measuring Fibrinolytic Split Products in Human Serum∗:

Summary and Conclusions A simplified procedure for recognizing and quantitating fibrinolytic split products has been described. It utilizes human group O cells which can be sensitized and stored at 4° for 3 weeks and at ‐20° for many months prior to use. It is very sensitive, detecting 1.0 μg/ml of fibrinogen or split products. Application of the test to the Microtiter and Autotiter systems permits assay of many samples at once. Only 1 hr is required to complete the test and read the results. The interpretation of the quantitative assay result should be carefully made since, while fibrin split products are not absorbed from the sera during the preparation of the sample and are relatively accurately measured, fibrinogen split products tend to be absorbed during sample preparation and are thus less accurately measured. The test would appear to be more useful with fibrin lysis than with fibrinogen lysis and to be especially applicable in the diagnosis of defibrination syndrome.

The Defibrination Syndrome: Clinical Features and Laboratory Diagnosis

THE development of incoagulable or hypocoagulable blood in a patient not previously suspected of having a blood-coagulation defect is a striking clinical phenomenon. On occasion, this is due to hypofibrinogenaemia which may result either from a primary lack, from decreased synthesis, from consumption by intravascular coagulation (defibrination), or from degradation by fibrinolysis. Clinical hypofibrinogenaemia is classically seen in diffuse hepatocellular disease, defibrination and so-called idiopathic primary pathological fibrinolysis. Whipple (1914) stated that it was well known that liver injury (due to chloroform or phosphorus) may be associated with a marked drop in plasma fibrinogen and that fibrinogen in whole blood of patients with severe liver disease may be digested within a few hours at 37’ C. Liver disease must be severe before hypofibrinogenaemia occurs (Ham and Curtis, 1938; Deutsch, 1965). Low levels of plasma fibrinogen have been found in a minority of patients with liver disease, for example, in only two of 20 patients with cirrhosis of the liver (Hallin and Nilsson, 1964). Hypofibrinogenaemia associated with cirrhosis is usually readily recognizable, as the diagnosis of severe hepatocellular disease is seldom in doubt. The term ‘defibrination’ denotes removal of fibrin, but clinical usage implies intravascular coagulation and depletion (consumption) of fibrinogen and other coagulation factors. ‘Consumption coagulopathy’ has been suggested as an alternate term (Rodriguez-Erdmann, 1965). Under optimal circumstances, objective pathological evidence of fibrin deposition or frank thrombosis may also be found. The subject has been extensively reviewed (Ratnoff, 1960; Hardisty and Ingram, 1965 ; Verstraete, Vermylen, Vermylen and Vandenbroucke, 1965; McKay, 1965; Hardaway, 1966). The syndrome varies markedly in intensity, thus it may be associated with catastrophic haemorrhage, symptoms of thrombus formation (with or without excessive bleeding) or it may even be occult (Jennison, 1959). It is found in many clinical conditions including carcinomatosis, leukaemia, giant haemangioma, purpura fulminans, drug reactions, haemorrhagic shock; also following snake bite or transfusion of mismatched blood and during extracorporeal shunts. A similar state may be induced experimentally by the infusion of thromboplastic material into the blood stream (Wooldridge, 1886; Gutmann, 1914; Mills, 1921) causing thrombus formation (Obata, 1919) and a concomitant fall in fibrinogen, prothrombin, Factors V and VIII, and blood platelets (Schneider and Engstrom, 1954; Penick, Roberts, Webster and Brinkhous, 1958). The injection of thrombin (Warner, Brinkhous, Seegers and Smith, 1939) or induction of the Shwartzman phenomenon has a similar effect (Cecil, 1935; Urbach, Goldburgh and Gottlieb, 1944).

Increase in Fibrinogen and Fibrin-Related Antigen in Human Serum Due to In Vitro Lysis of Fibrin by Thrombin

In vitro lysis of fibrin, as indicated by increased fibrinogen-fibrin-related antigen (FR-antigen) in serum is usually seen when whole blood, or plasma, or highly purified fibrinogen prepared by several different procedures is clotted and kept at temperatures above 0 degrees C. This increase is both time and temperature dependent, occurs despite the addition of various plasmin and cathepsin inhibitors, and is probably caused by thrombin evolved during clotting and/or added in vitro. In these experiments, the FR-antigen was measured by a sensitive, reproducible hemagglutination inhibition immunoassay adapted to the AutoAnalyzer. Serum from whole blood contained more than serum from plasma, and fibrin rather than fibrinogen proved to be essential for the in vitro lysis. The phenomenon was also caused by Arvin or Reptilase, suggesting that splitting of one or more arginine or lysine bonds in fibrin may be at least partially responsible. To obtain minimal levels of FR-antigen (< 0.5 mug/ml), plasma is clotted for 4 hr at 0 degrees C with 1.0-5.0 U/ml thrombin, CaCl(2) (0.0125 mole/liter), and epsilon aminocaproic acid (0.05 mole/liter). Slightly higher levels, probably adequate for clinical diagnosis, are obtained by 10-30 min clotting at room temperature. Since endogenous and/or exogenous thrombin is essential for the collection of serum FR-antigen, all the FR-antigen found in normal serum probably results from an irreducible amount of in vitro lysis rather than from continuous intravascular clotting and fibrinolysis.

Defibrination in Normal and Abnormal Parturition

Summary Studies of blood coagulation and fibrinolysis in women undergoing uncomplicated single delivery showed a significant fall in plasma fibrinogen and factor VIII. The levels were lowest 1–4 hr postpartum and returned to pre‐existing or higher levels within 24 hr. A significant fall in fibrinogen also occurred in women with twin delivery or caesarean section without labour. Similar changes were noted in factors V and VIII. Fibrinolytic activity was markedly decreased during delivery but increased rapidly thereafter in women with a single birth or caesarean section without labour. In women with twin delivery, it remained low. No consistent changes in the level of fibrinolytic inhibitors or plasminogen were noted. Mean levels of fibrinolytic degradation products (FDP) were in the upper level of the normal range and increased in the first 1–4 hr postpartum. These changes were even more marked in twin delivery. In women with abruptio placentae or prolonged intrauterine death (IUD), levels of fibrinogen, factors V and VIII and blood platelets were markedly reduced and generally dropped even lower during the first 4 hr after delivery, returning within 24 hr to the levels found in nonpregnant women. Levels of plasminogen and factor X were also reduced. High levels of FDP were found during labour, returning within 24 or 48 hr to the range observed in women 24 hr after normal delivery. Immunodiffusion and immunoelectrophoresis of serum in agar gel disclosed abnormal double or split precipitin lines. Sequential changes in these precipitin lines pointed to a continuing process which started before delivery, reached a maximum shortly thereafter, disappearing within the next 10–24 hr. The changes found in early (vs. late) labour, or during uterine surgery not associated with pregnancy, did not uphold the supposition that similar changes observed during normal or abnormal parturition might be due to the stresses of labour or surgery. The findings reported herein suggest that a minor degree of physiological defibrination develops during normal labour which is qualitatively similar to, but of much lesser magnitude than, the pathological defibrination syndrome (with local lysis of fibrin) commonly associated with abruptio placentae or prolonged intrauterine death.

DIAGNOSIS AND TREATMENT OF INTRAVASCULAR COAGULATION

Intravascular coagulation is considered by some to be a continuing, balanced dynamic equilibrium in which the fibrin deposited on vascular intima is removed by active fibrinolysis (Astrup, 1958). Although this interesting concept is not yet proved, florid forms of intravascular coagulation undoubtedly do occur frequently, giving rise to a clinical syndrome called defibrination (Ratnoff, 1960; Hardisty and Ingram, 1965 ; Rodriguez-Erdmann, 1965 ; Verstraete, Vermylen, Vermylen and Vandenbroucke, 1965 ; Merskey, Johnson, Kleiner and Wohl, 1967). The term ‘defibrination’ denotes removal of fibrin. In clinical usage, however, it implies intravascular coagulation as well as depletion (consumption) of fibrinogen and other coagulation factors ; ‘consumption coagulopathy’ has been suggested as an alternative term (Rodriguez-Erdmann, 1965).

Isolation of Fibrinogen–Fibrin Related Antigen from Human Plasma by Immunoaffinity Chromatography: its Characterization in Normal Subjects and in Defibrinating Patients with Abruptio Placentae and Disseminated Cancer

Summary. Highly purified, fibrinogen–fibrin related antigen (FR‐antigen) was isolated with good recovery from 1.0–2.0 ml of human plasma, by immunoaffinity chromatography with antibody specific for fibrinogen and fibrin, and plasmin degradation products X, Y, D and D‐D dimer. In FR‐antigen from defibrinating patients there was evidence for thrombin activity alone (mainly disseminated cancer) or both plasmin and thrombin (mainly abruptio placentae). Thus, the molar ratio of N‐terminal Gly – Tyr in the FR‐antigen of 18 of 20 patients strongly suggested thrombin activity (95th percentile). In addition, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) on unreduced samples frequently showed bands similar in mol wt to fragments X, Y and D, and in the reduced samples A α and Bβ chain degradation, both indicating plasmin activity. N‐terminal β chain Ala was elevated in the antigen of four of 20 patients, also suggesting plasmin activity (99th percentile). Combined thrombin, plasmin and factor‐XIII activity, as shown by bands in similar position to D‐D and γ chain dimers, was commonly associated with high levels of serum FR‐antigen (> 10 mg/dl). In some defibrinating patients, especially those with disseminated cancer, heterogeneity of unreduced FR‐antigen and Aα chain degradation, both indicators of mild plasmin‐like activity which are commonly seen in normals, were absent.

PATHOGENESIS AND TREATMENT OF ALTERED BLOOD COAGULABILITY IN PATIENTS WITH MALIGNANT TUMORS

It must be admitted that we have little sound information about the pathogenesis of either the thrombotic or the bleeding syndrome that may occur with malignant tumors. For many years, we have attempted to place the blame on a hypercoagulable state, because levels of many blood coagulation factors, especially fibrinogen, factors V and VIII, and blood platelets, are known to be elevated in patients with cancer.’.’ We cannot conclude that such elevated levels cause thrombosis, however. At the other end of the spectrum are patients with so-called “disseminated intravascular coagulation” (DIC) ; the release of thromboplastic substances into their bloodstream is widely believed to trigger the formation of thrombin. Clotting is then initiated inside the blood vessels with “consumption,” or a reduction in the levels, of some of the factors that normally disappear during blood clotting, for example, platelets, fibrinogen, and factors V and VIII. When the fibrinolytic system is activated, local deposits of fibrin are lysed, thus resulting in the accumulation of fibrinogen-fibrin digestion products (FDP) in the circulaAlthough this concept is attractive and may sometimes be valid, it remains to be shown that thromlbin is evolved during the process. Perhaps more frequently encountered are patients with apparently similar diseases who have normal or even moderately elevated levels of these clotting factors. A possible explanation is that although these levels are considered to be in the normal range, they actually represent a precipitous fall in the patient in question. In other instances, one-stage assays reveal apparently normal or even elevated values (perhaps due to the presence of aotivated intermediate clotting factors), whereas two-stage assays show reduced values.4-5 Autopsy examination of persons with DIC might be expected to reveal many thrombi, yet intensive search is sometimes necessary to identify even a few minute bnes6 This discrepancy usually is attributed to local lysis of previously deposited thrombi. Also, a similar mechanism is generally assumed to be responsible for FDP, despite little or no evidence of increased systemic fibrinolytic activity; the euglobulin clot lysis time is usually prolonged and fibrin plate lysis is commonly less than normal. The level of a protein in the circulation represents, at least to some extent, a balance between its production and its destruction. Thus, when synthesis exceeds destruction, levels might be elevated; conversely, reduced levels might represent excessive destruction, whereas normal levels might represent a rate of destruction in balance with the rate of production. In this way, any plasma level could be associated with accelerated destruction. The presence of both accelerated fibrinogen turnover and reduced platelet survival in patients with cancer has been considered to show that DIC has occurred.7 Similarly, shortened plasminogen

In vitro Production of Coagulation Factors VII-X by Liver Slices from Rats Fed Atherogenic Diets.

Conclusion and summary Coagulation Factor VII-X production by liver slices was studied because it was found that plasma Factor VII-X levels were significantly elevated in rats fed atherogenic diets. Although unexplained variations occurred, in 15 out of 18 paired experiments the production of Factor VII-X during incubation was greater in flasks containing liver slices from test animals than from controls. Expressed as a ratio (test:control) the median value of Factor VII-X production per mg protein in the flask was 1.9. Thus there appeared to be increased synthesis of Factor VII-X in flasks from test rats though the possibility of delayed degradation was not excluded.