Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

Yegneswaran, Subramanian; Kojima, Yumi; Nguyen, Phuong Mai; Gale, Andrew J.; Heeb, Mary J.; Griffin, John H.

doi:10.1074/jbc.m704316200

Cited by 12 publications

(10 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…More experimental data are needed to confirm this hypothesis. Our data are in agreement with a reported global involvement of the area around Ile 311 -Phe 325 in the FVa-APC interaction as was recently shown by the inhibitory activities of a peptide, comprising residues 311-325 of FVa, in the inactivation of FVa by APC (38). One might expect that the inactivation rate of the other FVa variants carrying mutations at Lys 320 , Arg 321 , and Arg 400 would also be increased, but this was not the case.…”

Section: Dsupporting

confidence: 82%

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Segers

Dahlbäck

Rosing

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the downThe protein C pathway provides a major anticoagulant mechanism for the regulation of hemostasis by nullification of the procoagulant activities of FVIIIa and FVa, the cofactors in the tenase and prothrombinase complexes, respectively. Protein C is a vitamin K-dependent protein composed of a heavy chain and a light chain that are connected through a single disulfide bridge (1). The light chain is composed of an N-terminal ␥-carboxyglutamic acid-rich domain and two epidermal growth factor-like domains. The heavy chain contains a short (12 amino acid) signal peptide and the serine protease (peptidase) domain.Previously, we and others have demonstrated that three surface loops (37, 70, and 148, chymotrypsin numbering) in the serine protease domain of APC 2 form an extended FVabinding site (2-4). Furthermore, a cluster of basic residues located on loops 37, 60, and 70 form a positively charged exosite on the surface of APC important for its interaction with thrombomodulin, protein C inhibitor, ␣1-antitrypsin, and heparin (2, 5-7).APC is able to cleave FVa, the nonenzymatic cofactor of FXa in the prothrombinase complex, at least at five peptide bonds, of which those at Arg 306 and Arg 506 are considered most important for the inactivation of FVa (8 -10). FVa is a multidomain protein that consists of a 71/74-kDa light chain (A3, C1, and C2 domains) and a 105-kDa heavy chain (A1 and A2 domains) that are noncovalently associated. All APC cleavage sites are located in the FVa heavy chain (8). In the presence of negatively charged phospholipids, the cleavages at Arg 506 and Arg 306 are primarily responsible for inactivation of FVa (11). Cleavage of Arg 506 is kinetically favored over the Arg 306 cleavage (up to 20-fold rate difference) and results in an FVa inactivation intermediate with partial cofactor activity, whereas cleavage at Arg 306 fully inactivates FVa (11,12). In the presence of its cofactor protein S, however, APC cleaves the Arg 306 peptide bond with a rate that is approximately similar to that of cleavage at Arg 506 (13). Heparin is a naturally occurring sulfated glycosaminoglycan that can be found in several organs and tissues of the human body, notably on the surface of endothelial cells that form the inner lining of our vasculature. Unfractionated heparin (UFH) and low molecular weight heparin are widely used as effective pharmacotherapeutics for the prevention and treatment of thrombosis. The anticoagulant functions of heparin are diverse and include stimulation of the inhibition of various coagulation factors (thrombin, FIXa, FXa, FXIa, and FXIIa) by antithrombin as well as inhibition of platelet functions (14 -17). Apart from its anticoagulant functions, heparin also has anti-inflammatory properties as it modulates the production and release of proinflammatory factors (18).* This work was supported by VIDI Grant 916-046-330 from the Dutch Organization for Scientific Research (to G. A. F. N.). The costs of publica...

show abstract

Section: Dsupporting

confidence: 82%

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Segers

Dahlbäck

Rosing

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The modifying effects that FXa, protein S, and prothrombin have on the expression of the anticoagulant properties of APC(S360A) were not studied, however, and all effects were attributed to FVa. In a more recent publication Yegneswaran et al (11) propose that the mode of action of APC(S360A) is through competition between APC and FXa, which is in agreement with our observations. It was also observed previously that APC(S360A) has much less effect on the clotting (APTT) of plasma of a homozygous carrier of the FV Leiden mutation (37).…”

Section: Discussionsupporting

confidence: 83%

“…Protein C numbering is used throughout the text, followed by chymotrypsinogen numbering in parentheses. Several epitopes on the surface of FVa contribute to the interaction with APC at the Arg 506 and the Arg 306 cleavage sites (10,11). Mutagenesis studies suggest the presence of an extended exosite on FVa near Arg 506 , which is much less prominent around the Arg 306 cleavage site (10).…”

Section: Human Activated Protein C (Apc)mentioning

confidence: 99%

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Nicolaes

Bock

Segers

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Human activated protein C (APC)2 is formed after activation of zymogen (protein C) on endothelial cells by the thrombin⅐ thrombomodulin (TM) complex. APC is a serine protease that, together with its cofactor, protein S, down-regulates thrombin formation by inactivating the activated forms of the coagulation factors V and VIII (FVa and FVIIIa) via limited proteolysis. The protein C pathway is vital to normal hemostasis, as is indicated by the severe thrombotic events observed in individuals with homozygous or compound homozygous protein C deficiency (1, 2). Heterozygous individuals have an increased risk for thromboembolic events (3, 4). Another commonly observed (ϳ5% of Caucasians) impairment of the anticoagulant properties of protein C is associated with the factor V Leiden missense mutation (FV(R506Q)), causing the ablation of one of two APC cleavage sites, the other being at Arg 306 . Carriership of the FV Leiden mutation is the most common hereditary risk factor of thromboembolic disease.Interactions between APC and its substrates (FVa and FVIIIa) are largely dependent on three exposed surface loops (the 37-loop: Lys 191(37) ; the 60-loop: Lys 217(62) and Lys 218(63) ; and the 70 -80-loop: Arg 229(74) , Arg 230(75) , and Lys 233(78) ) that together form an electropositive exosite on APC (5-9). Protein C numbering is used throughout the text, followed by chymotrypsinogen numbering in parentheses. Several epitopes on the surface of FVa contribute to the interaction with APC at the Arg 506 and the Arg 306 cleavage sites (10, 11). Mutagenesis studies suggest the presence of an extended exosite on FVa near Arg 506 , which is much less prominent around the Arg 306 cleavage site (10). These data are in line with three-dimensional molecular models for the complexes between FVa and APC (at Arg 506 and Arg 306 ), which suggest that exosite-mediated contacts are more important for docking of APC at Arg 506 than for docking at Arg 306 , by virtue of the extended character of the surface loop in which Arg 306 is located (12). Much less information is available about the interaction between APC and FVIIIa. Manithody et al. (13), however, have reported that the same basic residues in the 37-loop and the calcium-binding 70 -80-loop of APC are also critical for recognition and inactivation of factor VIIIa.The affinity of APC for FVa has not been measured directly but can be inferred from the apparent K m for the proteolysis at Arg 506 in native FVa of between 2 and 20 nM and for cleavage at

show abstract

“…FVa binds aPC through both the light (19) and heavy chains (20,21). FXa also binds FVa through both the heavy (22,23) and light chains (23), and FXa protects FVa from aPC-mediated proteolysis (24,25).…”

Section: Introductionmentioning

confidence: 99%

Down regulation of prothrombinase by activated protein C during prothrombin activation

Kim

Nesheim²

2010

Thromb Haemost

View full text Add to dashboard Cite

SUMMARY Activated protein C (aPC) proteolytically inactivates factor Va (FVa) and thereby down regulates prothrombinase. Although FVa inactivation by aPC has been studied extensively, the inactivation of prothrombinase during prothrombin activation has not. Therefore, prothrombin activation initiated both without and with aPC (5.0, 7.5 or 10.0nM) was monitored over time by fluorescence. The experiments were performed with 0.075nM FVa and 1.0nM FXa, and with these concentrations reversed. The time courses of the residual prothrombinase activity with aPC, determined from the slopes of fluorescence over time, were pseudo first order with both limiting and excess FVa. With FVa limiting or in excess, the second rate constants for inactivation of prothrombinase were 1.98±0.09 × 105 M−1s−1 and 2.54±0.13 × 105 M−1s−1, respectively. The former value is 101-fold smaller than that for FVa inactivation by aPC alone. Since with limiting FVa the second order rate constants for prothrombinsase inactivation and FVa inactivation are equal, FVa is protected 101-fold, presumably by both FXa and prothrombin. In contrast, with excess FVa, the calculated rate constant for FVa inactivation exceeds that for prothrombinase inactivation 17.3-fold, which reflects a loss of protection by FXa. Since the protective effects of the two proteins are theoretically multiplicative, FXa protected 17.3-fold and prothrombin protected 5.8-fold. With 150nM protein S and limiting FVa, prothrombinase inactivation was two-fold faster, yet it was still protected 91-fold. These studies show that FVa is down-regulated by aPC during prothrombin activation, but FXa and prothrombin together are highly protective, both with and without protein S.

show abstract

Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

Cited by 12 publications

References 36 publications

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Down regulation of prothrombinase by activated protein C during prothrombin activation

Contact Info

Product

Resources

About