Search

The anticoagulant actions of the basic phospholipases A (PLA) from snake venoms were stronger than those of the neutral or acidic phospholipases A, and their enzymatic activities were not parallel with their anticoagulant activities. When the enzymatic activity of Trimeresurus mucrosquamatus snake venom phospholipase A (TMV-PLA) was almost completely (>99%) inhibited with p -bromophenacyl bromide, the anticoagulant activity was decreased markedly. The procoagulant activity of lysocephalin, derived from cephalin by the action of phospholipase A, was only about one hundredth of that of cephalin. When p -bromophenacyl bromide-modified TMV-PLA was added to the mixture of cephalin and lysocephalin, it still could prolong the Stypven clotting time. The anticoagulant activity of TMV-PLA may be due to both enzymatic and binding activities. The anticoagulant activity of Naja naja atra snake venom phospholipase A (NNAV-PLA) was very weak in the presence of plasma and cephalin. However, when NNAV-PLA was preincubated with cephalin at 37°C for 2 min and then plasma was added to this preincubation mixture, the anticoagulant effect of NNAV-PLA was more marked. This indicated that the weak anticoagulant activity of NNAV-PLA was due to interference of phospholipid binding to NNAV-PLA by plasma binding proteins. The phospholipid-binding activity of PLA is thought to be a prerequisite for its anticoagulant activity, with the enzymatic activity potentiating its anticoagulant activity. The anticoagulant activity of basic PLA was usually stronger than that of acidic PLA, possibly due to stronger binding activity.

Apparent Km and Vmax values for PC and PE hydrolysis were determined following exposure of HDL, LDL, and VLDL to a basic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. n. atra snake venom. Both enzymes hydrolyzed the lipoprotein phospholipids approximately as fast as they hydrolyzed pure phospholipids in mixed micelles, however, the N. nigricollis enzyme, which has a much stronger anticoagulant effect than the N. n. atra enzyme, had lower apparent Vmax values. These values were highest for phospholipids in VLDL and lowest for HDL, however, the differences between the lipoproteins were relatively small with the N. nigricollis enzyme while the differences were much larger with the N. n. atra enzyme. Fractions of the two enzymes in which varying numbers of lysines were carbamylated showed much larger differences in relative rates of phospholipid hydrolysis in HDL, LDL and VLDL. Triton X-100 eliminates these differences in rates of hydrolysis. These results are discussed in terms of the differences in the organized structure of the lipoprotein classes and in the penetration ability of the phospholipases.