Biomedical subjects

Per-Ola Freskgård

Publications and source records attributed to Per-Ola Freskgård.

4 recordsLinked to original sources

PubMed2005

Sequential coagulation factor VIIa domain binding to tissue factor.

Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the gamma-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.

Factor VIIa↗

PubMed2005

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway.

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.

Blood Coagulation↗

PubMed2003

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains.

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.

Calcium↗

PubMed2003

Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

Amino Acid Substitution↗