Alan J. Johnson

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).

Methods for the production of clinically effective intermediate- and high-purity factor-VIII concentrates.

Summary. An intermediate‐ and a high‐purity factor‐VIII concentrate for clinical use have been prepared on a large scale by cryoethanol precipitation, extraction of the precipitate with tris buffer, and fractionation with polyethylene glycol. With bench‐scale fractionation, the intermediate material is 22‐fold purified on the average and the mean yield is 63%, while the high‐purity factor VIII is 274‐fold purified with a mean yield of 62%. With fractionation of 100 1. or more of fresh frozen plasma, the intermediate material shows a 30% yield and 14‐30‐fold purification; the high‐purity factor VIII shows an 18% yield and 125–350‐fold purification using 5.8 g/100 ml polyethylene glycol (PEG). A yield of nearly 30% should be possible with PEG, 4–5 g/100 ml. Both factor‐VIII preparations are stable for over a year in the lyophilized state at 4°C. Other plasma proteins can be fractionated from the residual plasma by routine Cohn procedures.

Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease

Summary. Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E‐5 (PE‐E5). The product was highly purified (3.5 u VIII: C/mg protein) compared to conventional concentrate (0.3 u VIII: C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII: C) to factor VIII related antigen (VIIIR: Ag) of 16:1.

The intravenous infusion of the streptococcal fibrinolytic principle (streptokinase) into patients.

The streptococcal fibrinolytic principle (streptokinase) has been demonstrated to be an effective agent, in vivo, in patients as a means of mediating the relatively rapid liquefaction of extravascular fibrin coagulums (1). Streptokinase (referred to in this article as SK) has also been shown to be useful as a therapeutic reagent, by which massive extravascular clots may be eradicated by liquefaction and aspiration (2). These findings have received extensive confirmation. (See Footnote 12 to the list of References.) It is the purpose of this article to describe the results of studies of the intravenous administration of SK into patients. The ultimate objective of the study is directed toward attempting to determine whether or not clots occurring within the vascular system as a result of disease may be subject to

Clinical Investigation of Intermediate- and High-Purity Antihaemophilic Factor (Factor VIII) Concentrates

Summary. Intermediate‐ and high‐purity antibaemophilic factor (factor‐VIII) concentrates developed by the American National Red Cross (ANRC) have undergone extensive clinical investigation. When administered to severely affected haemophiliacs, the recovery in vivo and the metabolic half‐disappearance time were similar to those achieved with plasma.

New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine☆

Abstract Methods for preparing biospecific adsorbents and immobilized enzymes utilizing Sepharose CL as a support and trichloro-s-triazine as the linking agent are described. The difficulties encountered during conventional aqueous and mixed aqueous-phase reactions of trichloro-s-triazine with insoluble polyols, particularly reagent hydrolysis, are avoided by performing the activation reactions in anhydrous organic phase and replacing the second chlorine on the triazine ring by an aromatic amine. Ligands can be coupled to the activated support in either aqueous or organic phase. The methods have been applied to the attachment of a number of different enzymes, proteins, and small-molecule ligands to Sepharose. The superiority of the triazine linkage to the cyanogen bromide linkage is demonstrated.

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.

Identification of an active site histidine in urokinase

Two forms of urokinase (EC 3.4.99.26) with apparent molecular weights of 33 400 and 47 000 purified by affinity chromatography have been modified specifically with newly synthesized peptide chloroketones by affinity labeline. Rapid inactivation of the enzyme preparations was observed with Ac-Gly-Lys-CH2 Cl and Nle-Gly-Lys-CH2 Cl which might be associated with a change in which a histidine residue is lost. After performic acid oxidation, an equivalent amount of 3-carboxymethyl histidine could be recovered, indicating alkylation at the N-3 of a histidine residue. In the case of the norleucine derivative, norleucine was concomitantly incorporated into the protein. It is thus likely that urokinase belongs in the class of enzymes utilizing the Asp..His..Ser triad for their catalytic action. The two active site residues so far identified, serine and histidine, were located in the heavy chain (33 100 mol. wt) of the 47 000 molecular weight form and in the 33 400 molecular weight form, the molecular weight of which remained constant.

Partial purification and properties of a plasminogen activator from human erythrocytes

The lysis time of euglobulin clots made with whole blood (plasma and red cells) was very much shorter than that of clots made with plasma alone, indicating a fibrinolytic component in red cells. A plasminogen activator was found in the stroma-free hemolysate, and proteolytic activity was found in the stromal fraction. The plasminogen activator, purified by using diethylaminoethyl-cellulose (DEAE-cellulose) in a batch procedure followed by column chromatography, was called erythrokinase (EK). On preliminary characterization, EK appears to activate human and bovine plasminogen in a manner similar to urokinase (UK), as determined by fibrinolytic and caseinolytic assays. The two enzymes can be separated by DEAE chromatography and acrylamide-gel electrophoresis, however, and they hydrolyze acetyl-L-lysine methyl ester and benzoyl arginine methyl ester at different rates.

An improved method for the purification of human fibrinogen

Abstract 1. 1. The isolation of human fibrinogen by differential polyethylene glycol precipitations of the fibrinogen contained in the cryoprecipitate of plasma is described. 2. 2. Purification was performed in the presence of 1 mM diisopropyl fluorophosphate and trasylol. 3. 3. The protein obtained is > 95% clottable. 4. 4. It contains no detectable prothrombim, thrombin, plasminogen or plasmin. 5. 5. It is highly soluble and apparently undegraded.