The basic phospholipase A2 from Naja nigricollis venom inhibits the prothrombinase complex by a novel nonenzymic mechanism

Abstract

The three phospholipase A2 isoenzymes from Naja nigricollis venom inhibit blood coagulation with different potencies. The strongly anticoagulant basic isoenzyme CM-IV inhibits the prothrombinase complex, whereas the weakly anticoagulant isoenzymes CM-I and CM-II do not. To determine the role of enzymatic activity of the phospholipases in the inhibition of prothrombinase, we varied the time of incubation of each of these isoenzymes with the prothrombinase complex. The inhibition by CM-IV did not increase with time of incubation. CM-I and CM-II failed to inhibit the complex, even with complete hydrolysis of phospholipids in the assay mixture. After alkylation of its active-site histidine, CM-IV lost 97% of its enzymatic activity but retained 60% of its inhibitory potency on prothrombinase. CM-IV also inhibited prothrombinase activity in the absence of phospholipids, whereas CM-I and CM-II did not. The inhibition of the prothrombinase complex by CM-IV is thus not due to its binding to or hydrolysis of phospholipids. The kinetics of CM-IV inhibition of the prothrombinase complex in both the presence and absence of phospholipids was noncompetitive. This inhibition can be explained by binding of CM-IV to either factor Va or Xa, or both, to inhibit the complex. CM-IV differs from previously described nonenzymatic anticoagulants that are proteinase inhibitors or that inhibit the coagulation complexes by interfering with the binding of clotting factors to phospholipids. We conclude that the basic enzyme, CM-IV, inhibits the prothrombinase complex by a novel mechanism independent of enzymatic activity.

Total chemical synthesis of enzymatically active human type II secretory phospholipase A 2

“…sPLA 2 Anticoagulant Activity. PLA 2 is an anticoagulant and inhibits the prothrombinase complex activity of blood coagulation (8)(9)(10)(11)(12). To compare synthetic sPLA 2 with recombinant sPLA 2 , each protein was preincubated with factor Xa at 37°C for 15 min before adding the other components of prothrombinase, i.e., factor V, prothrombin, Ca 2ϩ , and phospholipid vesicles.…”

“…Some snake-venom sPLA 2 molecules express anticoagulant activity by inhibiting the prothrombinase complex activity and͞or the extrinsic factor X-activating complex (7). It was proposed that in addition to the lipolytic activity due to the enzyme molecule, basic residues located in a variable surface loop (residues 51-77) were involved in the anticoagulant action of venom sPLA 2 (8)(9)(10). Human sPLA 2 exerts anticoagulant activity in plasma (11) and inhibits prothrombinase activity independent of its lipolytic activity (12).…”

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

“…For example, CM-IV, a strongly anticoagulant phospholipase A 2 from Naja nigricollis venom, prolongs coagulation by inhibiting two successive steps in the coagulation cascade. It inhibits the TF⅐FVIIa complex by both enzymatic and nonenzymatic mechanisms (63), whereas it inhibits the prothrombinase complex by only a nonenzymatic mechanism (64,65). Hemextin A and its synergistic complex are the first reported specific inhibitors of FVIIa isolated from snake venom.…”

Inhibition of Prothrombinase by Human Secretory Phospholipase A2 Involves Binding to Factor Xa

“…This inhibitory effect does not require the enzymatic activity of the enzyme, indicating that hsPLA 2 may interact with non-phospholipid targets (24). It was suggested that some venom sPLA 2 s are able to affect blood coagulation by mechanisms that do not involve their catalytic activity, although the targets of the venom enzymes were not identified (41)(42)(43). We investigated which plasma coagulation factors may be affected by hsPLA 2 .…”