A membrane-mediated catalytic event in prothrombin activation.

C Kung,E Hayes,K G Mann

doi:10.1016/s0021-9258(18)47323-1

Abstract

Prothrombinase assembly takes place on the surface of unsaturated phosphatidylcholine (PC), phosphatidylserine (PS) membranes in the presence of Ca2+, through the rapid association of membrane-bound factor Va and factor Xa. The present study uses saturated PCPS (75:25, w/w) vesicles to study prothrombinase assembly and catalytic properties in order to differentiate the influences of the membrane upon catalyst assembly, substrate delivery, and peptide bond cleavage. In contrast to studies using unsaturated phospholipid, prothrombin activation studies using saturated PCPS (75:25, w/w) (C14:0, C16:0, and C18:0) revealed up to a 20-fold decrease in prothrombinase activity. C18:0 membranes support at least 50% of the prothrombinase binding capacity (KdVa-Xa = 1 nM and nVa-Xa = 1.1) of C18:1 PCPS (75:25, w/w). Thus, the 95% loss in activity cannot be explained by gross alterations in catalyst concentration or assembly. Stopped-flow studies with saturated lipids demonstrate that factor Va, factor Xa, and prothrombin have decreased kon values. Compensatory changes in koff leave the Kd values for these protein-lipid interactions almost unchanged relative to unsaturated PCPS. The profoundly decreased activation rate on saturated phospholipid membranes as compared to unsaturated phospholipids is in part due to slowed substrate/enzyme delivery caused by the saturated lipids. However, studies using prethrombin-1 and C18:0 PCPS (75:25, w/w) also revealed a 15-fold decrease in activity for preassembled prothrombinase. Although there was a slight change in Km (+2-fold), the major cause of the decrease is an 18-fold decrease in kcat. Similar differences for Km and kcat values were obtained for prothrombin. Substrate delivery is thus only partially responsible for the diminished prothrombinase activity observed with saturated phospholipids. Since the activity of prothrombinase is decreased for both prothrombin and prethrombin-1 principally by reducing kcat, it appears that catalyst formation on saturated phospholipids somehow compromises the proteolytic activity of the enzyme complex. This implies that the phospholipid bilayer serves not merely as a surface for condensing the proteins but also as a functional element of the prothrombinase enzyme.

Highlights

Prothrombinase assembly takes place on the surface 1981,1987, 1990a, 1990b)
Pholipid membranesas compared to unsaturated phos- Both natural and synthetic lipids have been used in studies pholipidsis in part due to slowedsubstrate/enzyme of a-thrombin generationby prothrombinase
There was a slight change in K, (+2-fold)t,he major causeof the decrease is an 18-fold decrease kincat.Similar differencefsor K,and kcatvalues wereobtained for prothrombin

Summary

A Membrane-mediated Catalytic Evenitn Prothrombin Activation*

The bound factor Va and factor Xa rapidly associate to create the catalyticcomplex In this reaction scheme, the rate-determining steps for the assembly of prothrombinase are theassociation of factor Xa and factorVa with the lipid membrane surface. Prothrombinactivationrequires the Kd values forthese protein-lipid interactions almost phospholipid binding for optimal delivery and proper presenunchanged relative to unsaturated PCPs.Theprotation of prothrombin to the active siotef the enzyme complex foundly decreased activation rate on saturated phos- (Husten et al, 1987). E.g. phosphatidic acid and phosphatidylglycerol, have been shownprtoomote prothrombin activation (Higgins et al, 1985; Jones etdl., 1985; Gerads etal., 1990), pointing to electrostatic contributions to protein-phos-. This implies that the phospholipid bilayer serves not pholipid interactions.

Presentaddress

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Methods