Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Staphylocoagulase-binding region in human prothrombin

A staphylocoagulase-binding region in human prothrombin was studied by utilizing several fragments prepared from prothrombin by limited proteolysis. Bovine prothrombin, prethrombin 1, prethrombin 2, and human diisopropylphosphorylated alpha-thrombin strongly inhibited formation of the complex ("staphylothrombin") between human prothrombin and staphylocoagulase, but bovine prothrombin fragment 1 and fragment 2 had no effect on the complex formation, indicating that the binding region of human prothrombin for staphylocoagulase is located in the prethrombin 2 molecule. To identify further the staphylocoagulase-binding region, human alpha-thrombin was cleaved into the NH2-terminal large fragment (Mr = 26,000) and the COOH-terminal fragment (Mr = 16,000) by porcine pancreatic elastase. Of these fragments, the COOH-terminal fragment, which includes Asn-200 to the COOH-terminal end of the alpha-thrombin molecule, partially inhibited the complex formation between staphylocoagulase and human prothrombin. In contrast, the NH2-terminal large fragment did not show any inhibitory effect on the staphylothrombin formation. These results suggest that the staphylocoagulase interacts with human prothrombin through the COOH-terminal region of alpha-thrombin B chain. Other plasma proteins, factor X, factor IX, protein C, protein S, protein Z, all of which are structurally similar to prothrombin, did not inhibit the staphylothrombin formation at all, indicating that a specific interaction site with staphylocoagulase is contained only in the prothrombin molecule.     

Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum.

Exposure of sarcoplasmic reticulum to trypsin in the presence of 1 M sucrose results in degradation of the Mr = 102,000 ATPase enzyme to two fragments of Mr = 55,000 and 45,000 with subsequent appearance of fragments of Mr = 30,000 and 20,000. These fragments were purified by column chromatography in sodium dodecyl sulfate. Antibodies were raised against the ATPase and the Mr = 55,000, 45,000, and 20,000 fragments. There was no antigenic cross-reactivity between the Mr = 55,000 and 45,000 fragments, indicating that they were derived from a single linear cleavage of the larger enzyme. There was antigenic cross-reactivity between the Mr = 20,000 and 55,000 fragments, indicating an origin of the Mr = 20,000 fragment in the Mr = 55,000 fragment. None of the antibodies inhibited (Ca2+ + Mg2+)-dependent ATPase or Ca2+ transport. The Mr = 20,000 fragment and the Mr = 55,000 fragment were active in Ca2+ ionophore assays. The active site of ATP hydrolysis was labeled with [gamma-32P]ATP and the site of ATP binding was labeled with tritiated N-ethylmaleimide. In both cases radioactivity was found in the intact ATPase and in the Mr = 55,000 and 30,000 fragments, indicating that the Mr = 30,000 fragment was also derived from the Mr = 55,000 fragment. Amino acid composition data showed that the Mr = 45,000 fragment contained about 60% nonpolar and 40% polar amino acids, while the Mr = 55,000 fragment and the Mr = 20,0000 fragment contained about equal amounts of polar and nonpolar amino acids. Studies of the reaction of various antibodies at the external surface of sarcoplasmic reticulum vesicles showed that the ATPase was exposed, whereas calsequestrin and the high affinity Ca2+-binding protein were not. The use of antibodies against the various fragments indicated that the Mr = 55,000 fragment was in large part exposed, whereas the Mr = 20,000 and the 45,000 fragments were only poorly exposed. It is probable that the site of ATP hydrolysis in the Mr = 55,000 fragment is external, whereas the ionophore site is only partially exposed and the Mr = 45,000 fragment is largely buried within the membrane.     

Activation of key proenzymes of the blood coagulation system by the fibrin E fragment

Plasminogen and prothrombin are key proenzymes of fibrinolytic and clotting system. It is known that they can be activated by indirect activators streptokinase and staphylocoagulase. In this paper it is shown that fibrin E fragment purified from DD-E complex induced catalytic activity in plasminogen and clotting activity in prothrombin. Streptokinase increased 2 times the rate of catalytic activity induction by E fragment in prothrombin. It's possibly that process is one of the factors providing for rethrombosis after thrombolytic therapy of myocardial infarction.     

Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin

Staphylocoagulase with a molecular weight of 64,000 and subspecies ranging in molecular weight from 36,000 to 64,000 were purified by affinity column chromatography on bovine prothrombin-Sepharose 4B from the culture filtrates of the Staphylococcus aureus strains, st-213 and 104. The samples containing all molecular species from both strains had the same NH2-terminal sequence, Ile-Val-Thr-Lys-Asp-Tyr-Ser-Lys-Glu-, implying that the molecular heterogeneity was due to proteolytic degradation to some extent of the COOH-terminal portion during cultivation or purification. Staphylocoagulase (Mr = 64,000) from strain st-213 formed an active complex, "staphylothrombin," with human prothrombin in a molar ratio of 1 to 1.1. Staphylothrombin was unstable at 37 degrees C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmentation of prothrombin into prethrombin 1 and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent fluorography for the products of prothrombin activation by staphylocoagulase in the presence of [3H]diisopropylphosphofluoridate (DFP) demonstrated the formation of a DFP-sensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of alpha-thrombin was found. The enzymatic properties including the pH-dependency of the activity, substrate specificity and behavior towards thrombin inhibitors of staphylothrombin differed from those of alpha-thrombin, although the active site titration of staphylothrombin with p-nitrophenyl-p'-guanidinobenzoate showed 0.95 +/- 0.2 mol of active site/mol of enzyme.     

Various properties and kinetics of interaction of high and low molecular weight human kininogens with human plasma kallikrein

A relatively simple procedure for isolation and purification of human blood plasma kallikrein (HPK) by QAE-Sephadex A-50 SP-Sephadex C-50 and affinity chromatography on Sepharose 4B with immobilized soybean trypsin inhibitor with the activity yield of about 40% has been developed. The method allows for simultaneous isolation of low (LMW) and high molecular weight (HMW) kininogens from the same HPK sample. HPK preparations are homogeneous upon 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% SDS; its Mr is 90,000. After treatment with beta-mercaptoethanol, HPK dissociates into two fragments with Mr of 43,000 and 37,000. HPK preparations have high specific activities of esterase (31 microM/min), amidase (78 microM/min), and kininogenase (420 micrograms equiv. bradikinin/min). The high degree of protein purification was demonstrated by titration of active centers with 4-methylumbelliferylguanidine benzoate. The values of equilibrium dissociation constants for the HPK complex with aprotinin (Ki) equal to 1 X 10(-8) M (ethyl ester of N-alpha-benzoyl-L-arginine) and 1,5 X 10(-9) M (HMW) were determined. The kinetics of HPK-induced liberation of bradikinin from purified preparations of HMW and LMW was studied. The kinetic parameters (Km, kcat and kcat/Km) of this reaction suggest a high affinity of HPK for HMW, but not for LMW. LMW does not compete with HMW for the enzyme active center. It is assumed that LMW is not a physiological substrate for HPK.     

Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin

Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu-Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity.     

Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.     

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.     

Evidence that binding to the carboxyl-terminal heparin-binding domain (Hep II) dominates the interaction between plasma fibronectin and heparin

We assessed the participation of the three known heparin-binding domains of PFn (Hep I, Hep II, Hep III) in their interaction with heparin by making a quantitative comparison of the fluid-phase heparin affinities of PFn and PFn fragments under physiologic pH and ionic strength conditions. Using a fluorescence polarization binding assay that employed a PFn affinity-purified fluorescein-labeled heparin preparation, we found that greater than 98% of the total PFn heparin-binding sites exhibit a Kd in the 118-217 nM range. We also identified a minor (less than 2%) class of binding sites exhibiting very high affinity (Kd approximately 1 nM) in PFn and the carboxyl-terminal 190/170 and 150/136 kDa PFn fragments. This latter activity probably reflects multivalent inter- or intramolecular heparin-binding activity. Amino-terminal PFn fragments containing Hep I (72 and 29 kDa) exhibited low affinity for heparin under physiologic buffer conditions (Kd approximately 30,000 mM). PFn fragments (190/170 and 150/136 kDa) containing both the carboxyl-terminal Hep II and central Hep III domains retained most of the heparin-binding activity of native PFn (Kd = 278-492 nM). The isolated Hep II domain (33-kDa fragment) exhibited appreciable, but somewhat lower (2-5-fold), heparin affinity compared to the 190/170-kDa PFn fragment. Heparin binding to the 100-kDa PFn fragment containing Hep III was barely detectable (Kd greater than 30,000 nM). From these observations, we conclude that PFn contains only one major functional heparin-binding site per subunit, Hep II, that dominates the interaction between heparin and PFn.     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

Characterization of the calmodulin-binding sites of muscle phosphofructokinase and comparison with known calmodulin-binding domains

Calmodulin has been shown to interact with high affinity with muscle phosphofructokinase (Mayr, G. W. (1984) Eur. J. Biochem. 143, 513-520, 521-529). In this study, direct binding measurements indicated that each of the two subunits of dimeric phosphofructokinase bound two calmodulins with Kd values of about 3 nM and 1 microM, respectively, in a strictly Ca2+-dependent way. To get more detailed information about this interaction, calmodulin-binding fragments were isolated from a CNBr digest of phosphofructokinase using affinity chromatography on calmodulin-agarose. Two fragments, M11 (Mr 3080) and M22 (Mr 8060), formed a 1:1 stoichiometric complex with Ca2+-calmodulin. The amino acid sequences of these fragments were determined, and their positions in the three-dimensional structure-model of phosphofructokinase are proposed. Fragment M11, which binds to calmodulin with the higher affinity (Kd 11.4 nM), is located in a region of the subunit where two dimers have been proposed to make contacts if associating to active tetrameric enzyme. A stabilization of the dimeric form of the enzyme by binding of calmodulin supports this location of M11. The weaker binding fragment M22 (Kd 198 nM) corresponds to the C-terminal part of the polypeptide and contains the site which is phosphorylated by cAMP-dependent protein kinase. Both fragments have structural properties in common with the isolated calmodulin-binding domains of myosin light chain kinase: two cationic segments rich in hydrophobic residues, one constantly possessing a tryptophan, and the other exhibiting an amino acid sequence resembling sites phosphorylated by cAMP-dependent protein kinase.     

The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies

The receptor for human interferon-gamma (IFN-gamma) was purified from foreskin fibroblasts. Triton X-100 extracts obtained from either intact cells or membrane preparations were passed through an immobilized interferon-gamma column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of eluted fractions revealed a major band of Mr = 95,000 and minor bands of Mr = 80,000 and 60,000. Further purification was obtained by steric exclusion and by lectin chromatography. The purified receptor retained the ability to bind 125I-IFN-gamma with a Kd of 2.2 X 10(-10) M, a value close to that obtained with intact fibroblasts (5 X 10(-10) M). A complex of Mr = 105,000-125,000 was visualized by immunoprecipitation of 125I-IFN-gamma cross-linked to the purified receptor followed by SDS-PAGE and autoradiography. A similar complex was obtained when 125I-IFN-gamma was cross-linked to intact cells. Immunization of mice with the excised SDS-PAGE band of Mr = 95,000 elicited antibodies that blocked the antiviral activity of IFN-gamma and immunoprecipitated the cross-linked complex of 125I-IFN-gamma and its receptor.     

Interaction of human thrombin I fragment, prethrombin I, and alpha-thrombin with tissue thromboplastin]

The binding of 125I-labeled prothrombin fragment I. prethrombin I and alpha-thrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 of EDTA was studied. The experimental curves plotted in the Scatchard coordinates testify to the presence in thromboplastin of two types of fragment I binding sites: those with a high (Kd = 7.6 x 10(-6) M) and moderate (Kd = 1.3 x 10(-8) M) binding affinity. The parameters of fragment I binding and their changes reproduced, for the most part, the mode of prothrombin binding observed in previous studies. The experimental results provide indirect evidence in favour of a hydrophobic role of Ca(2+)-dependent binding of prothrombin fragment I to thromboplastin. The binding of prethrombin I was nonspecific and Ca(2+)-independent, whereas alpha-thrombin showed a relatively high level of nonspecific electrostatic binding which was competitively inhibited by Ca2+. Thromboplastin proteins interacted (both directly and in a Ca(2+)-independent fashion) with all the prothrombin derivatives under study.     

Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen (plasmin fragments Y)

The present study was undertaken as a step to delineate further the localization of the calcium-binding sites in fibrinogen and to assess the anticlotting properties of fibrinogen degradation products. To this purpose, fragments Y were prepared by plasmin digestion of human fibrinogen in the presence of added Ca2+, and purified. We found that, on a molar basis, fragments Y exhibit twice as much anticlotting activity as fragments X. They possess two calcium-binding sites with Kd = 1.9 . 10(-5) M. Their predominant amino-terminal amino acids are alanine and tyrosine. It is known that one binding site in fragment Y is related to its D moiety. We conclude that the other calcium-binding site may be located in the central domain of the molecule.     

Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of Sarcoplasmic reticulum.

The (Ca2+ + Mg2+)-dependent ATPase of sarcoplasmic reticulum has been shown to ast as a Ca2+-dependent and selective ionophore in artificial lipid bilayers. Four fragments of 55,000, 45,000, 30,000, and 20,000 daltons have been purified from tryptic digests of the enzyme and it has been shown that the 55,000- and 45,000-dalton fragments are obtained from a single cleavage of the 100,000-dalton ATPase, while the 30,000- and 20,000-dalton fragments are obtained subsequently by a cleavage of the 55,000-dalton fragment. The 55,000- and 20,000-dalton fragments have ionophore activity inhibited by ruthenium red and by mercuric chloride but not by methylmercuric chloride, an inhibitor of the hydrolytic site of the enzyme. Under standard conditions the 45,000-dalton fragment was not active as an ionophore, while the 30,000-dalton fragment acted as a nonselective ionophore. The 55,000- and 30,000-dalton fragments have been shown to contain the site of phosphorylation and of N-ethyl [2-3H]-maleimide binding indicative of the hydrolytic site in the enzyme, and this site is absent from the 20,000-dalton fragment. Therefore, the ionophoric and hydrolytic sites are localized in separate regions of the ATPase molecule and they have now been physically separated. The 20,000-dalton fragment was degraded with cyanogen bromide and fragments were separated by molecular sieving. Ionophore activity was found in fragments of molecular mass less than 2,000 daltons.     

The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells

We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I-labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase.     

Structural requirements for expression of factor Va activity

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.