Role of galactose in bovine factor V.

Using galactose oxidase as well as beta-galactosidase to produce modifications of the galactose units, the functional significance of these carbohydrate residues on the coagulant activity of bovine Factor V glycoprotein was evaluated. Incubation of native Factor V with galactose oxidase or hydrolysis of asialo-Factor V with beta-galactosidase results in a loss of Factor V activity. The inactivation of Factor V by oxidation of galactose moieties is partially reversible upon reduction of the newly formed aldehyde groups with sodium borohydride. The extent of reversal depends upon the degree of inactivation achieved. Thus, Factor V which retained 30% of the original activity following galactose oxidation returns to 75% of the original coagulant activity upon borohydride reduction; but, after destruction of 85% of the original activity treatment with borohydride returns to about 30%. In the initial stages of the inactivation of Factor V by treatment with galactose oxidase, the loss of Factor V coagulant activity is directly proportional to the moles of galactose oxidized. However, as the reaction progresses, the rate of galactose oxidation exceeds the rate of loss of Factor V activity. Moreover, galactose oxidation continues even after complete inactivation of Factor V. These results suggest that the galactose residues most susceptible to attack by galactose oxidase are those necessary for the activity of this coagulant protein. Only 15 galactose residues/mol of Factor V are susceptible to galactose oxidase prior to removal of sialic acid. In contrast, 37 galactose residues/mol of Factor V are found after acid hydrolysis. These results suggest that Factor V glycoprotein contains more than one type of sialyl-galactose linkages, the C2,3 or C2,4 linkages susceptible to oxidation in the native protein and the C2,6 linkage which is resistant. Native Factor V binds with diarachidonyl lecithin forming an active complex of lower buoyant density, while the Factor V oxidized with galactose oxidase does not. The Factor V-phospholipid complex is protected from inactivation by galactose oxidase. Moreover, lipid binding diminishes the extent of oxidation of galactose residues. Certain galactose groups are essential for coagulant activity probably because they are required for binding to phospholipid, a prerequisite to Factor V action.

Using galactose oxidase as well as /3-galactosidase to produce modifications of the galactose units, the functional significance of these carbohydrate residues on the coagulant activity of bovine Factor V glycoprotein was evaluated. Incubation of native Factor V with galactose oxidase or hydrolysis of asialo-Factor V with P-galactosidase results in a loss of Factor V activity. The inactivation of Factor V by oxidation of galactose moieties is partially reversible upon reduction of the newly formed aldehyde groups with sodium borohydride.
The extent of reversal depends upon the degree of inactivation achieved. Thus, Factor V which retained 30% of the original activity following galactose oxidation returns to 75% of the original coagulant activity upon borohydride reduction; but, after destruction of 85% of the original activity treatment with borohydride returns to about 30%. In the initial stages of the inactivation of Factor V by treatment with galactose oxidase, the loss of Factor V coagulant activity is directly proportional to the moles of galactose oxidized. However, as the reaction progresses, the rate of galactose oxidation exceeds the rate of loss of Factor V activity. Moreover, galactose oxidation continues even after complete inactivation of Factor V. These results suggest that the galactose residues most susceptible to attack by galactose oxidase are those necessary for the activity of this coagulant protein.
Only 15 galactose residues/m01 of Factor V are susceptible to galactose oxidase prior to removal of sialic acid. In contrast, 37 galactose residues/m01 of Factor V are found after acid hydrolysis. These results suggest that Factor V glycoprotein contains more than one type of sialyl-galactose linkages, the CZ,, or C 2,, linkages susceptible to oxidation in the native protein and the C 2, B linkage which is resistant.
Native Factor V binds with diarachidonyl lecithin forming an active complex of lower buoyant density, while the Factor V oxidized with galactose oxidase does not. The Factor V-phospholipid complex is protected from inactivation by galactose oxidase. Moreover, lipid binding diminishes the extent of oxidation of galactose residues. Certain galactose groups are essential for coagulant activity probably because they are required for binding to phospholipid, a prerequisite to Factor V action.
Many of the proteins involved in blood coagulation are been probed by modifying the glycoprotein with galactose glycoproteins, but the functional role of the covalently linked oxidase and with P-galactosidase.
The results indicate that carbohydrates (l- 4) has not been defined. Recent observations galactose modification destroys Factor V activity and t,his that aggregation of platelets by collagen (5)(6)(7)  in Buffer A, and assayed for Factor V activity.
In control experiments, borohydride was shown to have no effect on the activity of native Factor V at the concentrations used in these experi-

Purification of Factor
V-The results of the purification procedure were essentially identical with those reported previously. Starting from the oxalated bovine plasma, the purification procedure involved five steps: adsorption on BaSO,, chromatography on TEAE-cellulose, ammonium sulfate precipitation, chromatography on cellulose phosphate, and finally gel filtration on Sepharose 4B. The increase in specific activity was nearly 2,000-fold from the starting plasma with a specific activity of 60 units/mg and with an over-all yield of 15% and the preparation was essentially free of other coagulation factors (11). Prior to gel filtration, larger aggregates of Factor V were ments.
seen on disc gel electrophoresis. Therefore, in the experiments reported subsequently only gel-filtered Factor V (K, = 0.60, M, 2 300,000 (24)) was used (Fig. 1, Fractions 60 to 80). After concentration polyacrylamide gel electrophoresis of gel-filtered Factor V gives two closely migrating protein bands both of which stain for carbohydrate.
Factor V activity is associated with each of the protein bands eluted from the disc gel. A small amount of protein representing less than 3% of the Factor V activity does not enter the gel (Fig. 2).
Effect of Galactose Oxidase on Factor V Activity-When Factor V was incubated with galactose oxidase there was a rapid loss of Factor V activity which was linear with respect to the galactose oxidase concentration (Fig. 3). Nearly 80% of Factor V activity was inactivated upon incubation with 200 units/ml of galactose oxidase for 10 min at 3'7'.
When the reaction was performed at different temperatures, it was found that the rate of inactivation was greater at higher temperatures.
The decrease in activity was first order with respect to Factor V activity with half-lives of 6, 9, and 11 min. (Fig. 4A) at 37, 25, and 15", respectively. An Arrhenius plot of the first order rate constant uersus reciprocal of the absolute temperature is shown in Fig. 48. The activation energy calculated from this curve was 4.3 kcal/mol. Acrylamide Electrophoresis-Using purified galactose oxidase, there was no change in the disc electrophoresis pattern in Factor V associated with galactose oxidase treatment (Fig.  5). When Factor V (500 pg) was incubated in the presence and absence of galactose oxidase (90 rg) at 37' for 30 min and subsequently precipitated with trichloroacetic acid; the resulting supernatant contained 2.5 to 2.9 pg/ml of peptide material (micro-Folin assay) in control as well as in the galactose oxidase-containing incubation mixtures. Reduction of Factor V with Sodium Borohydride-The loss of Factor V activity upon galactose oxidase treatment was partially reversible upon reduction with sodium borohydride ( Table I). The extent of reversibility depended largely on the extent of inactivation induced by galactose oxidase. When the residual activity was 30% of the original Factor V, the reduction with sodium borohydride resulted in a return to 75% of the original activity.
However, when the inactivation proceeded up to 85% the recovery upon sodium borohydride reduction was only up to 35%; sodium borohydride had no effect on control Factor V under the conditions of these experiments.
Relation of Factor V Inactivation to Galactose Oxida- tion-The correlation of the rate of Factor V inactivation with quantity of galactose oxidized is shown in Fig. 6, A and B. Inactivation of Factor V by exposure to galactose oxidase was related directly to the number of galactose residues oxidized. As the time of incubation was increased, there was progressive decrease in Factor V activity and a gradual increase in the amount of galactose oxidized (Fig. 6A). In the initial stages of the reaction the loss of Factor V activity was directly proportional to the amount of galactose oxidized (Fig. 6B)   Thus, there are 37 galactose residues/mol of Factor V, assuming the molecular weight of Factor V to be 300,000. Incubation of native Factor V with galactose oxidase resulted in an oxidation of 15 galactose residues/m01 (8.5 pg/mg). Thus, 15 our of 37 galactose residues have the CH,OH group available for oxidation in native Factor V. When asialo-Factor V was incubated with galactose oxidase all of the 37 galactose residues were oxidized (Table II).
Binding of Factor V with Phospholipids-When Factor V was centrifuged in the sucrose density gradient, all the Factor V activity could be recovered in the first three to four fractions was subjected to sucrose density gradient centrifugation, two Factor V activity peaks were obtained. The first peak, which corresponds with the control Factor V, represents the concentration of this protein not bound to the lipid. The second peak, which has a lower buoyant density than the control Factor V, represents Factor V bound to the lipid (Fig. 7, top). The two Factor V activity peaks correspond with the two peaks obtained when the fractions were assayed for protein concentrations (Fig. 7,  bottom).
When the lipid concentration was increased for a constant amount of Factor V, there was a corresponding increase in the amount of Factor V bound to lipid (i.e. the second peak). This result thus indicates that the Factor V peak with lower buoyant density observed in the presence of lipid is due to the complex formations between the two constituents. The amount of Factor V binding to lipid approaches an optimum when the ratio of Factor V to lipid is 1:2 (~g/pg) (Fig. 8).
Effect of Galactose Oxidation on Binding of Factor V to Phospholipid-When Factor V which had been inactivated by the oxidation of galactose residues was subjected to sucrose density gradient centrifugation in the presence of phospholipid (Fig. 9), only one protein peak was obtained irrespective of whether phosphatidylcholine was added or not. This peak was in a position corresponding to that of control Factor V without phospholipid, indicating the absence of any lipid binding by the galactose-oxidized Factor V. The Factor V activity lost upon galactose oxidase treatment was not recovered upon the addition of lipids.  phospholipids could protect Factor V from inactivation by galactose oxidase.
Factor V which had been preincubated with phospholipid undergoes very little inactivation upon subsequent incubation with galactose oxidase (Fig. 10, top). Thus, while 90% of the activity is lost upon incubation of Factor V with galactose  I  I  I  I   0  2  4  6  6  IO  12  14  I6 FRACTION NUMBER 1 FIG. 9. Binding of galactose oxidase (GaLor)-treated Factor V with phospholipid. Factor V was incubated with galactose oxidase until the inactivation was about 90%. Sucrose density gradient centrifugation in the presence and absence of phosphatidylcholine (PC) was done as in the case of Fig. 7. @A, galactose-oxidized Factor V; x-x, galactose-oxidized Factor V + phospholipid; A---A, Factor V + phospholipid.
oxidase for 60 min, the loss of activity was only 20% when Factor V-lipid complex was incubated under similar conditions.
Oxidation of galactose residues in the Factor V-lipid complex by galactose oxidase proceeds at an initial rate almost identical with that of Factor V. In both cases about 3.7 pglmg of Factor V are oxidized in the first 40 min. Thus, these groups are equally accessible in the presence and absence of phospholipid. However, the galactose oxidation of the Factor V-phospholipid complex reaches a maximum by 40 min of incubation (Fig. 10, bottom). In contrast, the oxidation of uncomplexed Factor V continues over a much longer period. This further oxidation is best appreciated from the data in Table II where, in the absence of lipid, a plateau level after 3 hours of 8.5 rg of TIME ( MINI   FIG. 10. Effect of phospholipid on the galactose oxidase-induced inactivation of Factor V. Sonication of diarachidonyl lecithin was done as mentioned in the text. Factor V (400 pg) was incubated for 5 min at 37" with 400 pg of the sonicated lipid. The effect of galactose oxidase on the coagulant activity (Fig. 10, top) and on the oxidation of Factor V-linked galactose (Fig. 10, bottom) was assessed, using this preparation of lipid-bound Factor V. Experimental details are as given in the text. In experiments where galactose oxidation was measured in lipid-bound Factor V, the lipid was included in blank as well as in standards. Experiments with control Factor V without lipid were also performed simultaneously.
O----O, Factor V + phospholipid; x-x, Factor V-phospholipid. Acid hydrolysis of Factor V and estimations of galactose are described in the text. In the experiments with galactose oxidase, 1 mg of Factor V or asialo-Factor V was incubated with 150 units of galactose oxidase, 10 pg of peroxidase, and 0.5 ml of Chromogen in a total volume of 1 ml. At intervals shown, readings were recorded at 425 nm on the expanded chart of Gilford Spectrophotometer. In the parentheses the results are expressed as the number of galactose residues/m01 of Factor V. assuming the molecular weight of Factor V as 300,000. Experiments with &Galnctosidase-The importance of galactose in Factor V is illustrated further by the effect of &galactosidase upon asialo-Factor V. Although P-galactosi-dase had no effect on native Factor V and removal of sialic acid from Factor V resulted in an increase of activity (lo), incubation of asialo-Factor V with P-galactosidase resulted in rapid inactivation.
The inactivation was higher at higher temperature with half-lives of 7, 11.5, and 15 min at 37, 25, and 15", respectively. The plot of K versus l/T is linear and the first order rate constant calculated from the Arrhenius plot was 4.0 kcal/mol. a-Galactosidase from coffee beans had no effect on Factor V activity. DISCUSSION This investigation documents a significant role for the galactose residues of Factor V glycoprotein in the coagulant activity of this plasma protein. This conclusion has been arrived at from the various observations which link Factor V activity -to the presence of intact galactose units. Virtually complete loss of Factor V activity occurred upon treatment with galactose oxidase and this loss of activity was proportional to the amount of galactose oxidized in the initial stages of the reaction. The inactivation caused by the oxidation of galactose units was partially reversible by reduction with sodium borohydride, indicating the absence of any gross irreversible conformational or other related changes in the molecule. Finally the removal of galactose by fi-galactosidase from asialo-Factor V also resulted in loss of activity. The role of galactose in hemostasis has been reported by earlier workers. Thus, the ability of asialo-bovine von Willebrand's factor (6) to promote platelet aggregation has been shown to be destroyed by incubation with galactose oxidase. In a similar way, the binding of collagen to platelets, the primary event in hemostasis, has been shown to be destroyed by galactose oxidase treatment and reversed by borohydride reduction (7). For quantitative conclusions about the number and role of galactose groups in Factor V, enzymatic modification of the coagulant protein was used as the primary tool. This approach demands purity of both the substrate and the enzyme. Although the cellulose phosphate eluate contains only Factor V protein as previously shown by activity and immunological studies (25), the preparations show considerable molecular weight heterogeneity including the presence of aggregates as well as active fragments derived from in vivo proteolysis. These were eliminated by using a single peak of gel-filtered Factor V with an estimated molecular weight of 300,000 probably representing Factor V monomer. When the preparation was analyzed by alkaline acrylamide disc gel electrophoresis two protein bands both of which have Factor V activity were evident. They grossly appeared to have similar carbohydrate as measured by periodic acid-Schiff staining, but heterogeneity of carbohydrate composition, particularly sialic acid, might still account for the slightly different migration of the two bands of Factor V. In evaluating the purity of the enzyme, proteolytic activity was found in the galactose oxidase preparation (26). Purification of galactose oxidase eliminated any proteolytic activity toward Factor V and thus the inactivation is not due to peptide bond cleavage.
Only a small fraction of the total galactose units are involved in Factor V activity. Thus, complete loss of activity resulted from the oxidation of about 15% of the total galactose content of the Factor V protein. Assuming the molecular weight of Factor V to be 300,000 (21) the oxidation of only 7 to 8 galactose residues out of 37 residues/m01 of Factor V, leads to the complete inactivation. Galactose units which are important for Factor V activity are probably in 2.3. or 2.4glycosidic linkage with the terminal sialic acid since inactivation by galactose oxidase occurred without prior removal of sialic acid.
Oxidation of galactose by galactose oxidase takes place specifically at the hydroxyl group at carbon 6 (27). The observation that only 15 galactose residues of 37lmol of Factor V are available to the enzyme prior to hydrolysis of sialic acid indicates that these sugar residues are in 2,3 or 2,4 linkages with adjacent sialic acid residues. Since removal of sialic acid by neuraminidase results in the oxidation of all the galactose residues by galactose oxidase, this finding suggests the existence of 2,3-or 2,4-as well as 2,6-sialyl galactose linkages in Factor V, as in the case of aI-glycoprotein (28). However, the increased availability of galactose for oxidation in asialo-Factor V might also be due to the minor conformational changes in protein resuiting from the removal of negatively charged sialic acid residues (29,30).
While reduction with sodium borohydride restored the activity of galactose-oxidized Factor V, the recovery of the activity was never complete.
Since no loss of activity occurred when Factor V was incubated with borohydride alone in the absence of galactose oxidase, the reason for lack of complete reversibility is unclear. Perhaps this is due to a conformational change such as that documented by Harper et al. (31) after galactose oxidation of collagen.
The binding of Factor V and Factor Xa. the components of the prothrombinase complex, to phospholipids and the importance of such a binding in the optimal activity of prothrombinase complex has been reported (32234). Our studies imply that Factor V can bind in a stoichiometric fashion with the highly unsaturated synthetic phospholipid-diarachidonyl phosphatidylcholine to alter its buoyant density. Interaction of Factor V with phospholipids has been demonstrated previously by gel filtration (32) and by precipitation methods (34). We chose density gradient centrifugation since this approach allows not only the separation of free Factor V from phospholipid-bound Factor V but also the determination of whether the complex is heavier than native Factor V or lighter. No protein-phospholipid interaction as demonstrated by sucrose density gradient ultracentrifugation was observed when Factor V previously inactivated by galactose oxidase was incubated with phospholipid. The hypothesis that galactose oxidation prevents the binding of Factor V to phospholipid, thus leading to a loss of coagulant activity, is further substantiated by the observation that phospholipid protects Factor V from galactose oxidase-induced inactivation and also make some of the galactose residues unavailable for oxidation by galactose oxidase. In view of the fact that only a fraction of the total oxidizable galactose residues are involved directly in Factor V activity, it seems probable that the galactose residues which are oxidized in Factor V-phospholipid complex are those which are not involved in Factor V activity. Alternatively, the possibility exists that binding with phospholipid has caused conformational changes in the Factor V molecule (35,36), which restricts the number of available galactose residues for oxidation.
The role of galactose in facilitating the formation of Factor V-phospholipid may be analogous to the attachment of glycoproteins with plasma membranes. It has been suggested (37-39) that the association of glycoproteins with membranes involves the attachment of lipophilic groups of the protein molecule to the lipoidal membrane with the peptide region containing.
the carbohydrate chain extending outside the membrane brarrier. According to the theory of Spiro (40), the membrane glycoproteins in the externally located hydrophilic area serve as a probe for the noncovalent attachment of proteins to the lipoidal membrane, By analogy with the binding of glycoproteins to membranes, it seems logical to postulate that the carbohydrates of Factor V glycoprotein play a role in regulating the binding to platelet membrane with the concomitant binding of the other participants of the prothrombinase complex.