Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651567 ◽

1993 ◽

Vol 69 (02) ◽

pp. 124-129 ◽

Cited By ~ 2

Author(s):

Susan Solymoss ◽

Kim Thi Phu Nguyen

Keyword(s):

Western Blotting ◽

Protein C ◽

Thrombin Generation ◽

Blood Platelets ◽

Activated Protein C ◽

Phospholipid Vesicles ◽

Factor V ◽

Two Stage ◽

One Stage ◽

Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

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Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615223 ◽

1998 ◽

Vol 80 (09) ◽

pp. 418-422 ◽

Cited By ~ 31

Author(s):

Kirsty Lapan ◽

Philip Fay

Keyword(s):

Serine Protease ◽

Heavy Chain ◽

Solid Phase ◽

Activated Protein C ◽

Cross Linking ◽

Factor X ◽

Serine Protease Domain ◽

C Terminus ◽

Factor Viiia ◽

Solid Phase Binding

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

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Expression and purification of recombinant serine protease domain of human coagulation factor XII in Pichia pastoris

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2019.1621151 ◽

2019 ◽

Vol 83 (10) ◽

pp. 1815-1821 ◽

Cited By ~ 1

Author(s):

Bangya Peng ◽

Guangpu Xue ◽

Dongfang Xu ◽

Zanjie Feng ◽

Jing Chen ◽

...

Keyword(s):

Pichia Pastoris ◽

Serine Protease ◽

Coagulation Factor ◽

Factor Xii ◽

Protease Domain ◽

Expression And Purification ◽

Serine Protease Domain

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Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

Scientific Reports ◽

10.1038/s41598-019-55531-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Nis V. Nielsen ◽

Elfie Roedel ◽

Dipankar Manna ◽

Michael Etscheid ◽

Jens Preben Morth ◽

...

Keyword(s):

Enzymatic Activity ◽

Serine Protease ◽

Active Site ◽

Factor Vii ◽

Protease Domain ◽

Functional Changes ◽

E Coli ◽

Catalytic Activation ◽

Serine Protease Domain ◽

Tissue Factor Pathway

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

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