Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of factor VIII by activated protein C and protein S

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.     

Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.     

Reconstitution of human factor VIII from isolated subunits

Human factor VII heterodimers were fractionated into component heavy and light chains using an anti-light chain specific monoclonal antibody immunosorbant. Neither the light chain nor the heavy chain alone possessed activity. Factor VII activity was reconstituted by recombining the subunits in the presence of Mn2+ or Ca2+. Reconstitution of activity also showed ionic strength dependence suggesting the importance of hydrophobic and electrostatic interactions. All factor VIII heavy chains (93 to 210 kDa) recombined with the 83 kDa light chain as judged by retention of all reconstituted heterodimeric forms by the monoclonal immunosorbant. Maximum specific activity (3 units/micrograms) was obtained at a 1:1 molar ratio of light chain:heavy chain. The presence of von Willebrand factor enhanced the rate of factor VIII reconstitution as much as 5-fold. This effect was both ionic strength-dependent and dose-dependent up to a 25-fold weight excess of von Willebrand factor over factor VIII.     

Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II

Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.     

Purification and properties of an activating enzyme of blood clotting factor X from the venom of Cerastes cerastes

An activator of blood coagulation factor X was found in the venom of the horned viper Cerastes cerastes, and was purified by gel filtration, ion-exchange chromatography and chromatofocussing. The activator is a protein composed of a heavy and a light polypeptide chain linked by disulfide bonds. Two subforms of the activator were found. Both contained a heavy chain of Mr 58000 and are distinguished from each other by the presence of two different light chains of Mr 17700 and 15000. The activator appears to cleave the bond in the factor X molecule that is also cleaved by factor IXa. Factor X activation by the activator is strongly stimulated by Ca2+. The kinetic parameters for the activation reaction have been determined. A Km for factor X of 19.2 nM and a Vmax of 0.11 pmol of Xa/min per ng venom were found.     

Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function

We have recently demonstrated that human high molecular weight kininogen (HMWK) is a pro-cofactor that is cleaved by kallikrein to yield a two-chain cofactor (HMWKa) and the nanopeptide bradykinin. This proteolysis enhances its association with an activating surface, an event necessary for expression of its cofactor activity. We now report that factor XIa is capable of hydrolyzing HMWK and releasing bradykinin in a purified system as well as cleaving and inactivating HMWK in a plasma environment during the contact-activation process. The profile of proteolysis differs from that produced by kallikrein and by factor XIIa in that the first cleavage by factor XIa yields 75- and 45-kDa polypeptides, whereas both factor XIIa and kallikrein initially produce 65- and 56-kDa species. Further proteolysis by all three enzymes eventually produces similar heavy chains (Mr = 65,000) and light chains (Mr = 45,000). However, the amount of factor XIa generated in plasma during contact activation further degrades the light chain of HMWK, eventually destroying its coagulant activity. Furthermore, in a purified system, enhancement of the degradation of HMWK coagulant activity by factor XIa was achieved when kallikrein was included in the incubation mixture, suggesting that the preferred substrate for factor XIa is the active form of HMWK (HMWKa), and not the pro-cofactor. These data suggest that factor XIa has the potential to act as a regulator of contact-activated coagulation by virtue of its ability to destroy the cofactor function of HMWK after its generation by either kallikrein, factor XIIa, or to a lesser extent, factor XIa, itself.     

Blood coagulation induced by the venom of Bothrops atrox. 2. Identification, purification, and properties of two factor X activators

We have characterized and purified the two components of the venom of Bothrops atrox that activate the coagulation factor X. Activator 1 and activator 2 were separated by ion-exchange chromatography but otherwise presented similar characteristics. They consist of a heavy polypeptide of Mr 59,000 and either one or two light chains forming a doublet of Mr 14,000-15,000. They are inactive on synthetic substrates and on prothrombin or fibrinogen and thus appear to act specifically on factor X. They are not sensitive to inhibitors of serine proteases or thiol esterases. The activation of factor X is activated by Ca2+ ions with a Hill coefficient of 2.4 and is inhibited by Hg2+, Ba2+, and Cd2+. Its pH dependency suggests that the activity depends on the ionization of a group with an apparent pK of 6.9. We studied the cleavage of purified bovine factor X by B. atrox activators and compared it to that obtained with the factor X activator from Vipera russelli venom. Like the physiological activators, the venom's activators cleave the heavy chain of factor X, producing the activated factor Xa alpha. They produce however two other cleavages: one near the N-terminal end of the heavy chain of factor X, generating factor Xmu, and a second one located at one extremity of the heavy chain of factor Xa alpha, generating factor Xav.     

Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts.

Radiolabelled proteoheparan sulphates were isolated from confluent monolayers of fibroblasts and from their spent media. The cell-surface-associated proteoglycan (Mr 350 000) has a core protein of Mr 180 000 that is cleaved by reduction of disulphide bonds into polypeptides of Mr 90 000, both of which can bind transferrin [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661]. Thrombin digestion of the proteoglycan yielded two major fragments. The larger one contained the heparan sulphate chains and glycoprotein-type oligosaccharides, whereas the smaller one contained interchain disulphide bond(s) and had affinity for transferrin as well as for octyl-Sepharose. The larger thrombic fragment was cleaved by trypsin into fragments containing the heparan sulphate chains and the oligosaccharides respectively. The smaller proteoheparan sulphate derived from the culture medium (Mr 150 000) had a core protein of Mr 30 000, which contained heparan sulphate-attachment and oligosaccharide-attachment regions, but no domains for binding of transferrin or for hydrophobic interactions.     

Intersubunit fluorescence energy transfer in human factor VIII

Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.     

Acetyl-coenzyme-A carboxylase from rat liver. Subunit structure and proteolytic modification.

The subunit structure of rat liver acetyl-coenzyme-A carboxylase has been studied by polyacrylamide gel electrophoresis in the presence of dodecylsulfate. A number of individual preparations of the enzyme purified by the same procedures exhibited three different types of electrophoretic patterns as follows: first, a single slow-moving protein bands (Mr 230000); secondly, two adjacent fast-moving protein band (M4 124000 and 118 000); finally, all three protein bands. With the use of the [14C]biotin-labelled enzyme, the biotinyl prosthetic group was shown to be associated with the polypeptide of 230000 Mr as well as with that of 124000 Mr, but not with the polypeptide of 118000 Mr. Studies were next made with the labelled enzyme to examine the possibility that the two light polypeptides might have been formed by proteolytic modification of the heavy polypeptide during the procedures used for the purification of the enzyme. Treatment of the enzyme with trypsin or chymotrypsin resulted in cleavage of the heavy polypeptide into two nonidentical polypeptides with molecular weights of approximately 120000. Incubation of the enzyme with proteases derived from rat liver converted the heavy polypeptide into lighter polypeptides of 80000-130000 Mr. Acetyl-CoA carboxylase isolated from crude rat liver extracts by means of immunoprecipitation with specific antibody invariably showed only the heavy polypeptide. The biotin content of the enzyme was found to be 1 mol per 237000 g protein. These results indicate that rat liver acetyl-CoA carboxylase, unlike bacterial and plant biotin enzymes, has only one kind of subunit, which has a molecular weight of 230000 and contains one molecular of biotin. Thus, the mammalian enzyme exhibits a highly integrated subunit structure.     

Purification and characterization of chicken liver cathepsin B

Cathepsin B was purified, 400-fold, to homogeneity from chicken liver. The enzyme comprised a mixture of two kinetically indistinguishable forms (approximately 1:1), which were separated on concanavalin A (Con A)-Sepharose; one consisting of Mr 25,500 and 5,000 polypeptide chains bound to Con A-Sepharose but the other, composed of Mr 24,500 and 5,000 polypeptide chains, did not. N-terminal amino acid sequence analyses of a mixture of the Mr 25,500 and 24,500 polypeptide chains, and of the Mr 5,000 polypeptide chain revealed single amino acid sequences, respectively. These amino acid sequences were homologous to those of the heavy and light chains of mammalian enzymes, respectively. The chicken liver and mammalian cathepsin B were similar in structure and properties.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.     

Chloroplast phosphoproteins. Phosphorylation of polypeptides of the light-harvesting chlorophyll protein complex.

When isolated, intact chloroplasts of pea (Pisum sativum) are incubated in the light with [32P]-orthophosphate, isotope is incorporated into several polypeptides. Among the most conspicuous phosphoproteins are two which form a very closely spaced doublet on dodecyl sulphate/polyacrylamide gels and co-electrophorese with the major polypeptide component of the light-harvesting chlorophyll a/b binding complex. Like the light-harvesting polypeptide, the phosphoprotein doublet is bound to thylakoids, sediments with the heavy particles released from thylakoids after digitonin treatment, is soluble in chloroform/methanol and has an apparent molecular weight of about 26 000. The doublet also appears in the highly purified light-harvesting chlorophyll a/b binding complex isolated from thylakoids by hydrosylapatite chromatography. I conclude that two polypeptide components of the complex are phosphorylated. One of these components may be the major light-harvesting chlorophyll a/b binding protein.     

Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.     

Biosynthesis, transport and processing of myeloperoxidase in the human leukaemic promyelocytic cell line HL-60 and normal marrow cells.

The processing and intracellular transport of myeloperoxidase were studied in the human promyelocytic leukaemia cell line HL-60 and in normal marrow cells labelled with [35S]methionine or [14C]leucine. Myeloperoxidase was precipitated with antimyeloperoxidase serum; the immunoprecipitates were subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and radiolabelled myeloperoxidase visualized by fluorography. During a 1 h pulse, myeloperoxidase was labelled in a chain of apparent Mr 90 000. With a subsequent chase, the Mr 90 000 polypeptide disappeared and was replaced by chains of Mr 62 000 and 12 400 corresponding roughly to the size of neutrophil myeloperoxidase subunits. The identification of the radioactive polypeptides as different forms of myeloperoxidase was established also by the similarity in patterns generated by partial proteolysis with V8 proteinase from Staphylococcus aureus. Processing of myeloperoxidase in HL-60 was slow; mature polypeptides were significantly increased only after 6 h. Another myeloperoxidase chain of apparent Mr 82 000 was an intermediate precursor or degradation form. Pulse-chase experiments in combination with sucrose-density-gradient separations of homogenates showed that the Mr 90 000 precursor was located in light density organelles only and not in granule fractions, whereas the Mr 82 000 precursor was located only in intermediate density organelles, suggesting that the latter is a product of the former. Processed mature myeloperoxidase was concentrated in the granule fraction, but some occurred in lower density organelles, which may indicate processing during intracellular transport. Only the Mr 90 000 polypeptide was secreted into the culture medium; this was also the only form found in the cytosol fraction.     

Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells

The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain.     

Light chains of sea urchin kinesin identified by immunoadsorption

Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartite tail [Scholey et al., 1989]. In the present, complementary study, we have used the monoclonal antikinesin, SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cytosol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitometry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equimolar quantities of heavy and light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.