Lipid mobility in the assembly and expression of the activity of the prothrombinase complex.

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.

Section: Resultssupporting

confidence: 90%

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

Stefansson¹,

Kini²,

Evans³

1990

“…Our studies confirm the observations of Higgins et al (1985) on the occurrence of lag phases in thrombin generation when prothrombin activation is started with factor Xa or factor Va on phospholipid membranes in the gel state. Such a presteady-state interval indicates that membrane fluidity affects the time required for the assembly of the membrane-bound factor Xa-Va complex.…”

Section: Discussionsupporting

confidence: 91%

“…This demonstrates that the time courses shown in Figure 1C represent steady-state rates of prothrombin activation. In contrast to Higgins et al (1985), we observed that membrane fluidity affected the steady-state rate of prothrombin activation. On membranes in the gel phase, the steady-state rate of prothrombin activation was about 7-fold slower than on membranes in the liquid-crystalline state (Figure 1C).…”

Section: Resultscontrasting

confidence: 81%

“…This was confirmed by subsequent studies with vesicles composed of synthetic phospholipids in which it was shown that the phase transition from the gel state to the liquid-crystalline state is accompanied by a sharp increase of the clot-promoting activity of the vesicles (Tans et al, 1979). In a recent study, Higgins et al (1985) investigated the effect of membrane fluidity on the catalytic properties of the prothrombinase complex. They observed a considerable lag in the time required for the assembly of the prothrombinase complex on membrane surfaces in the gel state and concluded that lipid fluidity affects the rate of assembly of the factor Xa-Va complex at the membrane surface.…”

mentioning

confidence: 71%

See 1 more Smart Citation

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles

Govers‐Riemslag¹,

Janssen²,

Zwaal³

et al. 1992

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

“…1994 American Chemical Society its interaction with the cofactor (Nesheim et al, 1984). This suggestion is based on the data available for other analogous enzyme complexes of coagulation and is primarily supported by spectral changes in a fluorescent reporter group covalently incorporated into the active site of factor Xa (Higgins et al, 1985;Husten et al, 1987). However, measurements of the reaction of a fluorescent peptidyl chloromethyl ketone with factor Xa in the presence or absence of other constituents of prothrombinase failed to yield evidence for detectable alterations in the catalytic residues of factor Xa (Walker & Krishnaswamy, 1993).…”

mentioning

confidence: 97%

Assembly of the Prothrombinase Complex Enhances the Inhibition of Bovine Factor Xa by Tick Anticoagulant Peptide

Krishnaswamy¹,

Vlasuk²,

Bergum³

1994

The interaction of factor Xa with factor Va on a membrane surface results in the assembly of the prothrombinase complex. The highly specific and multistep interaction between recombinant tick anticoagulant peptide (rTAP) and factor Xa was used to probe perturbations in the macromolecular interaction sites of factor Xa that accompany prothrombinase assembly. Steady-state kinetic studies indicated that the incorporation of factor Xa into prothrombinase resulted in a modest 3-fold increase in the rate constant for inhibition by rTAP. However, the overall dissociation constant for the enzyme-inhibitor interaction (Ki*) was decreased approximately 30-fold to 5.3 pM. This finding was verified by fluorescence stopped-flow studies of the multistep reaction between rTAP and solution-phase factor Xa or prothrombinase by using 4-aminobenzamidine. The second-order rate constant for the binding or rTAP to the protease (k + 1 = 3.35 x 10(6) M-1.s-1) was increased approximately 2-fold (k + 1 = 6.6 x 10(6) M-1.s-1) following the assembly of prothrombinase, while the rate constant for the subsequent slow displacement of the fluorophore from the active site of factor Xa was decreased by 20-fold. Therefore, factor Va alters macromolecular interaction sites on factor Xa; which leads to the stabilization of intermediates in the reaction of the protease with rTAP and an increased overall affinity for the inhibition of factor Xa. Fluorescence measurements of prothrombinase assembly using factor Xa modified with dansylglutamylglycinylarginine chloromethyl ketone (DEGR-Xa) indicated that the preformed rTAP-Xa binary complex bound to factor Va more tightly (Kd = 30.7 +/- 6.2 pM) than factor Xa alone (Kd = 1.25 +/- 0.29 nM). The 30-fold higher affinity of the rTAP-Xa complex for factor Va can completely account for the increased affinity of rTAP for prothrombinase and implies adequate thermodynamic description of the reactions involved. Collectively, the data suggest that the interaction of factor Xa with factor Va on a membrane surface alters macromolecular recognition sites on factor Xa involved in binding rTAP. As a result of this conformational change, the inhibition of factor Xa by rTAP is thermodynamically favored when the enzyme is assembled in the prothrombinase complex.