Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.
Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.
Qureshi
Shabir H
SH
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Yang
Likui
L
Manithody
Chandrashekhara
C
Rezaie
Alireza R
AR
D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005165:Factor V / Q000737:chemistry / Q000378:metabolism*; D015943:Factor Va / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D011516:Prothrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D017384:Sequence Deletion / Q000187:drug effects; D055672:Static Electricity; D013379:Substrate Specificity / Q000187:drug effects; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism
D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; C022475:prothrombinase complex; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D013925:Thromboplastin; C012007:prethrombins; D015951:Factor Xa