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In the determination of plasma fibrinogen by electroimmunodiffusion, commercial sodium heparin, previously included in the agarose gel, by interacting with fibrinogen molecules, enhances their anodic mobility more strongly than after carbamylation with potassium cyanate. The proposed method provides an increase in sensitivity and specificity which practically eliminates the influence of fibrinogen degradation products.

Ten patients participated in this study which evaluated the effect of two heparin dose regimes, a high dose regime (mean dose 6750 IU) and a low dose regime (mean dose 3750 IU), on the thrombin activity, achieved during a 4-hour-dialysis. The thrombin activity was measured by use of the FPA assay. Heparin was administered as bolus dose followed by a constant rate infusion which was discontinued 1.5-2 hours prior to the end of the dialysis. Both dose regimes inhibited thrombin activity equally effectively as long as heparin was administered. In the high dose regime, the FPA levels remained unchanged until the end of the dialyses in all patients. In the low dose regime, six patients had the same FPA values at the end of the heparin infusion and at the end of the dialysis. In the remaining four patients much higher FPA levels were achieved at the end of the dialysis. No serious bleeding or clotting complications occurred, and all dialyses were uneventful.

Small modifications of the commercially available software of the I.L. Multistat Centrifugal Analyzer (MCA) enabled us to evaluate clot based prothrombin times (PT), activated partial thromboplastin times (APTT) and thrombin times (TT) simultaneously within one run. This imparted a great deal of flexibility to this procedure in that batches and/or panels of clot based and other types of turbidimetric tests can be performed concurrently in any combination. The PT and TT by this turbidimetric procedure correlated very well with those of the Fibrometer in normal specimens as well as in a wide variety of coagulation defects. The turbidimetric APTT procedure, however, often produced clotting times longer than those achieved with the Fibrometer. The discrepancy between the Fibrometer and turbidimetric APTT procedures was shown to be the consequence of greater sensitivity of the latter to detect low concentrations of heparin, elevated fibrinogen split products and mild factor deficiencies. The available clot based procedures are suitable for use in detecting specific factor deficiencies and the presence of coagulation inhibitors.

Incubation of plasma with granulocyte enzymes in the presence of kallikrein inhibitor resulted in a prolonged thrombin time of the plasma and in an increased level of fibrinogen fragments, indicating that fibrinogen was digested. In accordance with this, fibrinogen digestion products could be purified by affinity chromatography from plasma after incubation with granulocyte enzymes. The isolated products resembled early X-like fibrinogen fragments, which are produced by limited digestion of purified fibrinogen with elastase. On SDS gel electrophoresis both had no intact A alpha-chains, but apparently intact fibrinogen B beta- and gamma-chains. Also, both fragments isolated from plasma and the X-like fragments produced with purified elastase had a low anticoagulant activity. Although elastase, the main fibrinolytic enzyme of the granulocyte, was rapidly complexed with inhibitors, 10-20% of the elastase activity towards succinyl-trialanyl-paranitroanilide was detectable in plasma no matter if the mixture of granulocyte enzymes or purified elastase had been added. A possible role for the alpha 2-macroglobulin-granulocyte elastase complex in the production of the digestion products in plasma is discussed.

The influence of qualitative and quantitative changes in plasma fibrinogen upon the amount of heparin precipitable fraction (HPF), obtained during the course of acute disease states, were examined. Whereas fibrinogen in plasma includes 3 species of different molecular weights (HMW, LMW, LMW'), fibrinogen in HPF, from normal as well as from "acute phase" plasma, consisted almost exclusively of HMW-fibrinogen (3% SDS-PAGE). Sub-unit chain electrophoresis (10% PAGE-SDS and two-dimensional electrophoresis) did not disclose any signs of abnormalities in the fibrinogen precipitated in HPF. Studies on patients revealed that the composition of plasma fibrinogen changed (increased HMW) during the course of acute myocardial infarction and following hip operations, and indicated that the amount of HPF was governed by the relative amount of HMW-fibrinogen as well total fibrinogen. This assumption was strengthened by quantitating the amount of HPF obtained after addition of highly purified HMW and LMW fibrinogen to normal and "acute phase" plasma. It is concluded that fibrinogen quality (per cent HMW or HMW/LMW ratio) is of major importance for the amount of HPF, thereby explaining some of the HPF-variations seen during the course of acute diseases.

Experimental disseminated intravascular coagulation (DIC) can be induced by a 4-hr sustained infusion of endotoxin at a dose of 100 mg/kg in rats. This experimental model of DIC in rats was used to study the effects of defibrinogenation with batroxobin against DIC. One hour before the infusion of endotoxin, 200 batroxobin unit (BU)/kg of batroxobin was injected intraperitoneally. Immediately after the injection, fibrinogen level markedly decreased and fibrinogen and fibrin degradation products increased. Prothrombin time and partial thromboplastin time were also prolonged. Blood counts, platelet counts and hematocrit level only showed a slight decrease after the injection. The preventive effect against DIC was noted by the partial inhibition of the fall in the platelet counts and lessened number of renal glomeruli with fibrin thrombi, in the rats treated with 200 BU/kg of batroxobin 1 hr before the infusion of endotoxin (100 mg/kg/4 hr). From these results, it was shown that fibrinogen metabolism plays an important role in the DIC state.

Since thrombi continue to incorporate fibrin during lysis we tested the effect of pretreatment with ancrod, a defibrinating agent from Malaysian pit viper venom, on thrombolysis with urokinase and streptokinase. Thrombi were induced by copper-coils in the carotid arteries of the dogs, weighed after 1 hour and inserted into the femoral arteries of the same animals. They were then exposed for 15 min to iv boluses of streptokinase 10,000 U/kg, urokinase 10,000 U/kg and urokinase 25,000 U/kg with or without pretreatment with ancrod. Ancrod depleted fibrinogen within 5 min and enhanced the lytic effect of streptokinase from 25 +/- 8% to 59 +/- 13% (p less than .05), urokinase 10,000 U/kg from 16 +/- 11% to 66 +/- 18% (p less than .01) and urokinase 25,000 U/kg from 27 +/- 17% to 85 +/- 8% (p less than .001) of the initial thrombus weight. Ancrod itself did not activate plasminogen to plasmin. We conclude that ancrod enhances thrombolysis probably by depleting fibrinogen and preventing new fibrin incorporation into the thrombus during lysis.