Activation of protein C in vivo.

An endothelial cell-associated cofactor that greatly enhances the rate of protein C activation by thrombin has recently been described. The observation that the cofactor binds thrombin with unusually high affinity (K(d) = 0.5 nM) suggested that low level thrombin infusion into dogs might lead to the selective activation of protein C. Infusion of thrombin (1 U/min per kg body wt) into the jugular vein of dogs leads to the formation of a systemic anticoagulant activity within 5 min of starting the infusion. The plasma has a prolonged partial thromboplastin time and Factor X(a) clotting time, but there is no change in the thrombin clotting time. The systemic anticoagulant activity is identified as activated protein C for the following reasons: (a) anti-canine activated protein C IgG antibodies inhibit the anticoagulant activity; (b) the anticoagulant activity can be partially purified from the plasma of dogs infused with thrombin by barium citrate adsorption; (c) the anticoagulant has chromatographic properties on QAE Sephadex indistinguishable from those of activated protein C, and (d) the rate at which this anticoagulant is inhibited in citrated canine plasma is identical to that of canine activated protein C. The in vivo activation of protein C appears to be receptor mediated since it occurs at low thrombin concentration and since it can be progressively inhibited by simultaneous infusion of diisopropylphospho-thrombin with thrombin. The activation of protein C at low levels of thrombin is selective, since neither the platelet count nor the Factor V levels are altered. Thrombin infusion leads to an elevation in circulating plasminogen activator levels. This appears to be mediated through the activation of protein C since coinfusion of diisopropylphospho-thrombin with thrombin inhibits the increase in plasminogen activator levels. Pretreatment of dogs with dicumarol blocks both the formation of anticoagulant activity and the rise in plasminogen activator. When the dicumarol-treated dogs are supplemented with isolated protein C and thrombin is infused, the anticoagulant activity again appears and the circulating levels of plasminogen activator are again elevated. These studies illustrate that low levels of thrombin in vivo can activate protein C, which in turn can inhibit blood coagulation and initiate fibrinolysis by elevating circulating plasminogen activator levels.

A lysine-absorbable plasminogen activator is elevated in conditions associated with increased fibrinolytic activity.

A plasminogen activator, or class of activators, that absorbs to lysine-agarose is present in human plasma. We have developed a quantitative assay for this plasminogen activator. The assay involves removal of the activator from plasma with lysine-agarose affinity columns and subsequent measurement of the activity by the conversion of plasminogen to plasmin on standardized fibrin agar plates. Using this assay, we investigated three physiologic conditions that have in the past been associated with increased fibrinolytic activity to determine whether elevation of the LAPA was involved. Normal individuals undergoing strenuous physical exercise and others subjected to venous occlusion as well as patients with cirrhosis of the liver were examined. Treadmill exercise to maximal exertion produced up to 15-fold increases in the level of LAPA; venous occlusion produced similar elevation. Certain individuals did not show increase fibrinolytic activity in response to exercise or venous occlusion, as indicated by unchanged euglobulin lysis times. These fibrinolytic hyporesponders did not show an elevation of their LAPA levels. In the third group examined, patients with cirrhosis, 24 of 62 had elevated levels of LAPA. Supplementation of plasma from normal individuals with this plasminogen activator from exercised individuals and cirrhotics resulted in increased rates of clot lysis.

The dilute whole blood clot lysis assay: a screening method for identifying postoperative patients with a high incidence of deep venous thrombosis.

A dilute whole blood clot lysis assay was used to identify patients with a high incidence of DVT. Of 191 orthopedic and urologic patients who underwent surgery, the over-all incidence of DVT as determined by 125I-fibrinogen leg scan was 35% in the 92 individuals with abnormal assays and 1% in the 99 patients with normal assays. The likelihood that an individual patient might have developed DVT increased progressively with the number of abnormal assays (p less than 0.001). The incidence of DVT increased from 28% in patients who had one abnormal assay, to 35% in patients with two abnormal assays, to 56% in patients with three abnormal assays. These studies establish the clot lysis assay as a simple means to screen for patients with a high incidence of DVT. A normal assay can eliminate patients from consideration for more extensive studies (venography, fibrinogen scan, impedance plethysmography), whereas those patients with one or more abnormal assays should be seriously considered for these additional studies.