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Cerastotin, a thrombin-like enzyme from the venom of the desert viper Cerastes cerastes, has been purified by gel filtration on Sephadex G-75 and two ion-exchange chromatographies on Mono S columns. It is a neutral glycoprotein (PI = 6.6), present as a single polypeptide chain of 40 kDa. Its N-terminal sequence shows strong similarity with those of other thrombin-like enzymes from snake venoms. Cerastotin possesses esterase and amidolytic activities measured with W-tosyl-L-arginine methyl ester and the thrombin chromogenic substrate D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide, respectively. The amidolytic activity is inhibited by phenylmethylsulfonyl fluoride, W-tosyl-L-lysine chloromethane, N"-tosyl-L-phenylalanyl chloromethane, D-phenylalanyl-L-prolyl-L-arginyl chloromethane and benzamidine, suggesting that cerastotin is a serine protease. Cerastotin efficiently clots human plasma and cleaves preferentially the a chain of fibrinogen. Cerastotin did not induce aggregation of washed normal platelets, but did aggregate platelets in the presence of exogenous fibrinogen. A monoclonal antibody directed against glycoprotein (GPIb), which specifically inhibits induced agglutination by ristocetin also completely blocks platelet aggregation induced by cerastotin. However, another anti-GPIb monoclonal antibody, which specifically inhibits a-thrombin binding to GPlb, did not prevent this aggregation. Furthermore, platelets which were desensitised by a-thrombin still aggregate in the presence of cerastotin, but not a-thrombin. Similarly a monoclonal antibody, anti-GPIIb-IIIa, which blocks fibrinogen binding, did not inhibit cerastotin-induced platelet aggregation. This activity is abolished in the presence of 1 mM phenylmethylsulfonyl fluoride and/or 10 mM EDTA. Cerastotin also agglutinates formalin-fixed and washed platelets, only in the simultaneous presence of fibrinogen and of Von Willebrand factor.

A 55-year-old man with multiple myeloma developed sustained bleeding after bone marrow aspiration and cutaneous bleeding. Routine coagulation studies revealed a prolonged activated partial thromboplastin time and thrombin time (> 60 s) with a normal reptilase time. Further evaluation showed failure of the activated partial thromboplastin time to correct completely in a 1: 1 mixture with normal plasma. Treatment of the patient's plasma in vitro with protamine sulfate normalized the thrombin time. The presence of a heparin-like anticoagulant was suspected. The plasma heparin level was 0.73 IU/ml. Intravenous infusion of protamine sulfate appeared to neutralize the anticoagulant activity and stop the bleeding. The cancer cells themselves or the invasive nature of this type of cancer might result in a massive release of a heparinoid. Such coagulopathy appears to be a rare mechanism of bleeding and it is an important entity to consider since it is potentially reversible with protamine sulfate.