Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase,

Hirbawi, Jamila; Vaughn, John; Bukys, Michael A.; Vos, Hans L.; Kalafatis, Michael

doi:10.1021/bi101097t

Cited by 14 publications

(35 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The investigation of the activation rates of plasmaderived and of all rFII molecules, cleavage and activation by fXa alone or prothrombinase was performed according to a protocol previously described by our laboratory using plasma-derived FII or rFII (47,49,69). The calculation of the rates of all FII molecules consumption by fXa alone or by prothrombinase were performed as described previously with the software Prizm (GraphPad) (47,49,69).…”

Section: Studies Of the Pathway For Fii Activation By Gel Electrophormentioning

confidence: 99%

“…The calculation of the rates of all FII molecules consumption by fXa alone or by prothrombinase were performed as described previously with the software Prizm (GraphPad) (47,49,69).…”

Section: Studies Of the Pathway For Fii Activation By Gel Electrophormentioning

confidence: 99%

“…Kinetic Titrations of Prothrombinase-To investigate the kinetic constants (K m and k cat ) of prothrombinase, assays with a set amount of plasma-derived fVa and fXa (as described in the legend to the figures) were executed as described by our laboratory in many instances (47,49,69,70). The initial rate of IIa generation was analyzed with the software Prizm (GraphPad) according to the Michaelis-Menten equation, and all final numbers reported are derived directly from the graphs.…”

Section: Studies Of the Pathway For Fii Activation By Gel Electrophormentioning

confidence: 99%

“…Earlier investigations have shown the existence of binding sites on FII for fVa in each of the kringle domains (39 -41) and within the Gla domain (42). Furthermore, significant protein-protein interactions between the acidic COOH-terminal region of fVa and a region rich in basic amino acids of FII have been inferred and characterized indirectly by employing molecular techniques involving specific hirudin-like ligands and the anion-binding (pro)exosite I (ABE I) of FII derivatives, as well as directly using a specific acidic peptide derived from the COOH-terminal region of the fVa heavy chain and recombinant fVa molecules (43)(44)(45)(46)(47)(48)(49). Site-directed mutagenesis of the basic residues in the proenzyme generated a recombinant FII molecule impaired in its ability to be activated by fully assembled prothrombinase (50).…”

mentioning

confidence: 99%

“…Although a crystal structure and a model of fVa have been available for some time now (51,52), the crucial interaction of the acidic hirudin-like COOH-terminal portion of the heavy chain of the cofactor with FII required for efficient IIa formation was initially ignored because it was missing from the crystal structure of the cofactor (51). This interaction was further discounted without providing any solid evidence (53) despite initial findings by Guinto and Esmon (54) and more recent original findings from our laboratory (47)(48)(49). A very recent model of prothrombinase using as a template the crystal structure of prothrombinase from the snake venom of Pseudonaja textilis verified and established the critical role of the acidic COOHterminal region of fVa heavy chain for optimal rates of FII cleavage at two spatially distinct sites by prothrombinase resulting in timely IIa formation at the place of vascular injury (27,55).…”

mentioning

confidence: 99%

See 4 more Smart Citations

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Wiencek

Hirbawi

Yee

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Prothrombin (FII) is activated to ␣-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa ( In the presence of a procoagulant membrane surface and divalent metal ions, factor Va (fVa) 3 binds factor Xa (fXa) to form prothrombinase. Prothrombinase is the two-subunit enzymatic complex where the non-enzymatic regulatory subunit (fVa) controls the rate and directs cleavage of prothrombin (FII) by the catalytic subunit (fXa) at two spatially distinct sites resulting in timely ␣-thrombin (IIa) formation at the place of vascular injury (1-3). Cleavage at Arg 271 and Arg 320 of FII is required to form the active serine protease IIa. The essential IIa molecule bears strong homology with other serine protease enzymes, such as activated protein C (APC), chymotrypsin, and fXa. Several different numberings of IIa residues appear in the literature based on either the chymotrypsin numbering (4) or IIa numbering (5, 6) or the entire FII sequence (7). The latter nomenclature is used herein with the appropriate chymotrypsin numbering in parentheses when required for comparison with the existing data in the literature.Historically, it has been shown that in the absence of fVa, initial cleavage at Arg 271 of FII results in the generation of the inactive intermediate prethrombin-2 and fragment 1⅐2. Further cleavage of prethrombin-2 at Arg 320 generates IIa (prethrombin-2 pathway) (8 -21). Concurrent with the appearance of excess fVa during clotting and in the presence of a procoagulant surface, the order of cleavages is reversed, and initial cleavage at Arg 320 generates a transient enzymatically active intermediate, meizothrombin, that has much higher catalytic efficiency than IIa toward chromogenic substrates usually employed to assess IIa activity (18,(22)(23)(24). Meizothrombin is next cleaved at Arg 271 resulting in the generation of IIa and fragment 1⅐2 (meizothrombin pathway). Although efficient cleavage at each site requires the presence of phospholipids, initial cleavage at Arg 320 is entirely fVa-dependent. In the absence of fVa, the two activation cleavage sites are not readily available, and FII is activated at a slow non-physiological rate by membrane-bound fXa alone. Interactions between fXa and FII are known to exist in the presence and absence of fVa; however, the enhanced activity of fXa within prothrombinase toward both activating cleavage sites is controlled solely by the membrane-bound non-enzymatic cofactor (3,16,17,25,26 3 The abbreviations used are: fVa, factor Va; fV, factor V; FII, prothrombin; fXa, factor Xa; IIa, ␣-thrombin; r, recombinant; PC, L-␣-phosphatidylcholine; PCPS, small unilamellar phospholipids vesicles composed of 75% PC and 25% L-␣-phosphatidylserine (w/w); rFII WT , recombinant...

show abstract

Section: Studies Of the Pathway For Fii Activation By Gel Electrophormentioning

confidence: 99%

“…The calculation of the rates of all FII molecules consumption by fXa alone or by prothrombinase were performed as described previously with the software Prizm (GraphPad) (47,49,69).…”

Section: Studies Of the Pathway For Fii Activation By Gel Electrophormentioning

confidence: 99%

Section: Studies Of the Pathway For Fii Activation By Gel Electrophormentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Wiencek

Hirbawi

Yee

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Cryo‐EM structures of coagulation factors

Mohammed

Pelc

et al. 2022

Research and Practice in Thrombosis and Haemostasis

View full text Add to dashboard Cite

A State of the Art lecture titled “Cryo‐EM structures of coagulation factors” was presented at the ISTH Congress in 2022. Cryogenic electron microscopy (cryo‐EM) is a revolutionary technique capable of solving the structure of high molecular weight proteins and their complexes, unlike nuclear magnetic resonance (NMR), and under conditions not biased by crystal contacts, unlike X‐ray crystallography. These features are particularly relevant to the analysis of coagulation factors that are too big for NMR and often recalcitrant to X‐ray investigation. Using cryo‐EM, we have solved the structures of coagulation factors V and Va, prothrombinase on nanodiscs, and the prothrombin‐prothrombinase complex. These structures have advanced basic knowledge in the field of thrombosis and hemostasis, especially on the function of factor V and the molecular mechanism for prothrombin activation, and set the stage for exciting new lines of investigation. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

show abstract

Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function

Hirbawi

Kalafatis

2017

ACS Omega

Self Cite

View full text Add to dashboard Cite

Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg701 of hfVa for human prothrombin (hPro) activation by prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by prothrombinase. The difference between the two cofactor molecules was also found within the Asn700–Arg701 dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp–Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution 700NR701→DE. rhfVNR→DE together with the wild-type molecule (rhfVWT) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the Kd of rhfVaNR→DE for human fXa as well as the kcat and Km of prothrombinase made with rhfVaNR→DE for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVaWT. Remarkably, sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of hPro activation time courses demonstrated that the rate of cleavage of hPro by prothrombinase reconstituted with rhfVaNR→DE was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVaWT. These unanticipated results provide significant insights on the role of the carboxyl-terminal end of the heavy chain of hfVa for hPro cleavage and activation by prothrombinase and show that residues 700NR701 regulate at least in part the enzyme–substrate/product interaction during fibrin clot formation.

show abstract

Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase,

Cited by 14 publications

References 55 publications

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Cryo‐EM structures of coagulation factors

Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function

Contact Info

Product

Resources

About