"Human factor VII is a single chain, vitamin K-dependent, plasma glycoprotein which is synthesized in the liver. Prior to secretion into the blood, post translational modification by a vitamin K-dependent carboxylase produces ten-carboxyglutamic acid (gla) residues located in the NH2-terminal portion of the molecule, which facilitate cell membrane binding. Factor VII is proteolytically activated to the serine protease, factor VIIa, during coagulation. Factor VII can be activated by thrombin, factor IXa, factor Xa or factor XIIa. The activation results in cleavage of the single chain molecule on the COOH-terminal side of arginine-152, to produce an NH2-terminal derived light chain (Mr=20,000) and a COOH-terminal derived heavy chain (Mr=30,000) which remain covalently associated by a single disulfide bond. The light chain region contains the gla domain, as well as two growth factor domains which are homologous to human epidermal growth factor (EGF). A single β-hydroxyaspartic acid identified in factor VII is also located in the light chain region. The heavy chain region of factor VIIa contains the catalytic domain. Factor VIIa and the cofactor, tissue factor, may combine on negatively charged cell surfaces in a calcium dependent manner to form the extrinsic factor Xase enzyme complex. This enzyme complex catalyzes the conversion of both factor IX to factor IXa and factor X to factor Xa. The cDNA for factor VII has been isolated and the nucleotide sequence determined. Factor VII shares extensive sequence homology with other serine proteases including factor IX, factor X and protein C.
Human factor VII is purified using a combination of conventional techniques and immunoaffinity chromatography. The purified protein is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20°C. Purity is determined by SDS-PAGE analysis and activity is measured in a factor VII clotting assay."
"The zymogen factor IX is a single chain vitamin K-dependent glycoprotein which is synthesized in the liver. The domain structure of factor IX is similar to that of the other vitamin K dependent coagulation factors. The NH2-terminal region contains 12 γ-carboxyglutamic acid (gla) residues which facilitate the calcium dependent binding of factor IX to negatively charged phospholipid surfaces. Two domains which are homologous to epidermal growth factor (EGF) span the region between the NH2-terminal gla domain and the activation peptide (Ala-146 to Arg-180).
Factor IX is activated by either factor XIa or the factor VIIa/tissue factor/phospholipid complex. Cleavage at site A (see figure) yields the intermediate IXa which is subsequently converted to the fully active form IXaβ by cleavage at site B. The NH2-terminal light chain (GLA and EGF domains) remains covalently attached to the COOH-terminal heavy chain by a disulfide bond. The serine protease catalytic triad (Ser-365, His 221, Asp-269) is located in the heavy chain. Factor IXaβ is the catalytic component of the ""intrinsic factor Xase complex"" (factor VIIIa/IXa/Ca2+/phospholipid) which proteolytically activates factor X to factor Xa.
Human factor IX is prepared from fresh frozen plasma by a combination of conventional procedures and immunoaffinity chromatography. Bovine factor IX is prepared from fresh citrated bovine plasma by a modification of the method described by Fujikawa et al. The purified proteins are supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20°C. Purity is determined by SDS-PAGE analysis and activity is measured using a factor IX clotting assay."
"Factor X is a vitamin K-dependent protein zymogen which is synthesized in the liver and circulates in plasma as a two chain molecule linked by a disulfide bond. Prior to secretion into plasma, post-translational modifications produce 11 gamma-carboxyglutamic acid (gla) residues and a single b-hydroxyaspartic acid residue, which are located within the NH2-terminal light chain. The light chain also contains two epidermal growth factor (EGF) homology domains. The COOH-terminal heavy chain of factor X contains most of the carbohydrate moieties, as well as the latent serine protease domain. The activation of factor X is catalyzed by either the intrinsic factor Xase complex (factor IXa, factor VIIIa, cellular surface and calcium ions) or the extrinsic factor Xase complex (factor VIIa, tissue factor, cellular surface and calcium ions). Activation of human factor X by either complex results in cleavage at Arg52-Ile53 of the COOH-terminal heavy chain and subsequent release of a 52 amino acid activation glycopeptide. Factor Xa then serves as the enzyme component of the prothrombinase complex which is responsible for the rapid conversion of prothrombin to thrombin. The gla residues enable factor X/Xa to bind phospholipid (i.e. cell surfaces) in a calcium dependent manner; a requirement for assembly of the prothrombinase complex. The first EGF homology domain contains a Ca2+ binding site which acts as a hinge to fold the EGF and GLA domains towards each other. This region of the molecule is involved in the recognition of cellular binding domains.
Human factor X is isolated from fresh frozen human plasma by a combination of conventional techniques and immunoaffinity chromatography. In addition to the standard human factor X preparation, Gla-domainless human factor X is also available. Bovine factor X is isolated from fresh bovine plasma using a modification of the procedure reported by Bajaj et al. The purified zymogen is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20°C. Purity is determined by SDS-PAGE analysis and activity is measured in a factor X clotting assay."
"Factor XII (XII) (Hageman Factor) is a single chain (Mr=78,000) glycoprotein zymogen that circulates in plasma at a concentration of 40 μg/ml. Reciprical activation of XII to the active serine protease factor XIIa (XIIa) by kallikrein is central to initiation of the intrinsic coagulation pathway. Surface bound α-XIIa in turn activates factor XI to XIa. Secondary cleavage of α-XIIa by kallikrein yields β-XIIa, which catalyzes solution phase activation of kallikrein, factor VII and the classical complement cascade.
The ability of a variety of negatively charged substances, both physiological and nonphysiological to promote XII activation and, thus, initiation of the intrinsic pathway has led to the psuedonym ""contact activation"". Binding to anionic surfaces induces a conformational change, making the XII zymogen more susceptible to cleavage by a variety of proteases. It is unlikely that binding to negatively charged surfaces alone is sufficient to activate XII, since highly purified preparations of XII and plasma deficient in prekallikrein and high molecular weight kininogen do not undergo this ""autocatalysis"".
A single cleavage by kallikrein at R353-Val354 of XII yields α-XIIa, a 2 chain protease (Mr=80,000) held together by disulfide bonds. The COOH-terminal light chain (Mr=28,000) contains the catalytic triad (His-40, Asp-89, Ser-191), while the NH2-terminal heavy chain (Mr=52,000) conatins the anionic surface binding portion of the molecule. A secondary cleavage of α-XIIa by kallikrein outside the disulfide bond yields β-XIIa (XIIf, BHFa, HFf, hageman factor fragments) (Mr=28,000), which no longer binds anionic surfaces. β-XIIa can activate prekallikrein, but has little procoagulant activity. Several other minor intermediate forms of XIIa are indicated in the figure above.
Inhibitors of XIIa include C1-INH, α2-antiplasmin, α2-macroglobulin and antithrombin III. At physiological concentrations, the relative effectiveness of these inhibitors is 91 : 4.5 : 3 : 1.5, respectively. The ratio of C1-INH to XII has been implicated in the ""cold activation"" of factor VII and the conversion of prorenin to renin on storage of plasma.
Human factor XII is prepared from fresh frozen plasma by immunoaffinity chromatography and supplied in 50% glycerol for storage at -20°C."
"Plasminogen is a single chain glycoprotein zymogen which is synthesized in the liver and circulates in plasma at a concentration of approximately 2.4 μM. The plasminogen molecule contains 790 amino acids, 24 disulfide bridges, no free sulfhydryls and 5 regions of internal sequence homology, known as kringles, between Lys77 and Arg560. These five triple-looped, three disulfide bridged, kringle regions are homologous to the kringle domains in t-PA, u-PA and prothrombin. Plasminogen contains one high affinity (Kd=9x10-6M) and four low affinity (Kd=5x10-3M) lysine binding sites. The high affinity binding site resides within the first kringle region of plasminogen. The interaction of plasminogen with fibrin and α2-antiplasmin is mediated by these lysine binding sites. Native glu-plasminogen (Mr=88,000) is readily converted to Lys-77-plasminogen (Mr=83,000) by plasmin hydrolysis of the Lys76-Lys77 peptide bond. Elastase catalyzed cleavage of the Val441-Val442 peptide bond of glu-plasminogen yields a functionally active zymogen termed Val-442 plasminogen or mini-plasminogen.
The conversion of plasminogen to plasmin occurs by a variety of mechanisms, but all result in hydrolysis of the Arg560-Val561 peptide bond of plasminogen, yielding two chains which remain covalently associated by a disulfide bond.
Native glu-plasminogen is prepared from fresh frozen human plasma by a modification of the procedure of Castellino, utilizing gel filtration and affinity chromatography. The two carbohydrate variants of glu-plasminogen (CHOI and CHOII) are isolated by gradient elution from lysine-Sepharose using the lysine analog, e-aminocaproic acid. The plasminogen is supplied in 50% (vol/vol) glycerol/H2O for storage at -20°C. Purity is determined by SDS-PAGE analysis."
The vitamin K-dependent zymogen, protein C, is synthesized in the liver as a single chain polypeptide and is subsequently converted to a disulfide linked heterodimer, by removal of a dipeptide (Lys-146 and Arg-147) from the precursor molecule.
Prothrombin is a vitamin K-dependent plasma protein which is synthesized in the liver. Prior to secretion into plasma, prothrombin undergoes post-translational modification by a vitamin K-dependent carboxylase which converts ten specific glutamic acid residues to y-carboxyglutamic acid (gla).
Purified from fresh frozen human plasma. Human Prekallikrein is a single chain gamma globulin glycoprotein that participates in the early phase of contract activation, kinin formation and fibrinolysis. Prekallikrein purity is determined by SDS-PAGE and shows no reduction upon incubation with 2-mercaptoethanol. Activity is determined via clotting assay.
High Molecular Weight Kininogen (HK) is a non-enzymatic cofactor of the contact activation system. HK is thought to have two functions in the contact activation system. First, HK links Prekallikrein to a negatively charged surface thereby allowing activation of Kallikrein by surface bound Factor a-XIIa. HK also forms a complex with Factor XI and accelerates its activation to XIa by a-XIIa. Additionally HK serves as a source of Bradykinin, a potent vasoactive peptide important in hypotension studies. The protein purity is determined by SDS-PAGE.
ERL offers the two chain Kinin-free form of Kininogen. This is prepared by Kallikrein digestion of Kininogen which is then repurified to remove traces of Kallikrein.