Biomedical subjects

Helen Philippou

Publications and source records attributed to Helen Philippou.

3 recordsLinked to original sources

PubMed2005

Directing thrombin.

Following initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of the prothrombinase complex. Thrombin then has multiple roles. The way in which thrombin interacts with its many substrates has been carefully scrutinized in the past decades, but until recently there has been little consideration of how its many functions are coordinated or directed. Any understanding of how it is directed requires knowledge of its structure, how it interacts with its substrates, and the role of any cofactors for its interaction with substrates. Recently, many of the interactions of thrombin have been clarified by crystal structure and site-directed mutagenesis analyses. These analyses have revealed common residues used for recognition of some substrates and overlapping surface exosites used for recognition by cofactors. As many of its downstream reactions are cofactor driven, competition between cofactors for exosites must be a dominant mechanism that determines the fate of thrombin. This review draws together much recent work that has helped clarify structure function relationships of thrombin. It then attempts to provide a cogent proposal to explain how thrombin activity is directed during the hemostatic response.

Allosteric Regulation↗

PubMed2005

Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation.

Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 x 10(5) m(-1).s(-1), a dissociation rate constant of 7.61 x 10(-2) s(-1) and equilibrium binding constant (K(D)) of 147 nm. It was activated by thrombin over endothelial cells with a K(m) of 213 nm and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K(m) (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.

Antigens, CD↗

PubMed2003

Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate.

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.

Binding, Competitive↗