[131

PROTHROMBIN

[13]

123

Prothrombin

B y KENNETH G. MANN

Prothrombin is the coagulation proenzyme present in highest concentration in blood (0.07-0.1 mg/ml) and was recognized very early 1 as a prime contributor to the blood coagulation process. The pioneering work of Smith e t al. 2 and Seegers 3-5 provided most of our present knowledge regarding the requirements for the maximum stimulation of thrombin production. The physiologically significant activator of prothrombin is thought to be a complex of two proteins--factor Xa (a proteolytic enzyme) and factor V (a cofactor protein)--phospholipid, and calciumY In addition, the proenzyme can be activated to its product by specific snake venoms s,' and by trypsin. TM During activation of the molecule to its product thrombin, there occurs a complex series of events that results in the deletion of about 42% of the proenzyme as "pro" fragments. 11 Figure 1 presents a conceptual image of the prothrombinase activator of prothrombin. One of the most intriguing and significant features of the prothrombinase catalyst is that the lipid provides a surface upon which the catalyst, factor Xa, and its activators, form the complex and perform their functions. Since plasma is an aqueous medium, the lipid matrix forms a separate phase from the surrounding medium, and in effect the activation of the prothrombin molecule takes place on an oil-water interface. In order to be utilized effectively, prothrombin must also bind to the interface; thus, prothrombin must be partitioned for its product, thrombin, to be generated at a rapid rate. Prothrombin is synthesized in the liver, 1~ and is one of the vitamin 1 p. Morawitz, Ergeb. Physiol. 4, 304 (1905). E. D. Warner, K. M. Brinkhous, and H. P. Smith, Am. J. Physiol. 125, 296 (1939). 3 A. G. Ware and W. H. Seegers, Am. J. Clin. Pathol. 19, 471 (1949). 4 W. H. Seegers, "Blood Clotting Enzymology." Academic Press, New York, 1967. 5 W. H. Seegers, "Prothrombin." Harvard Univ. Press, Cambridge, Massachusetts, 1962. ~D. Papahadjopoulos and D. J. Hanahan. Biochim. Biophys. Acta 90, 436 (1964). ~E. R. Cole, J. L. Koppel, and J. H. Olwin, Thromb. Diath. Haemorrh. 14, 431 (1965). s H. Pirkle, M. McIntosh, I. Theodore et al., Thromb. Res. 1, 559 (1972). 9 K. W. E. Denson, R. Borrett, and R. Biggs, Br. J. Haematol. 21, 219 (1971). 10F. Jobin and M. P. Esnouf, Nature (London) 211,873 (1966). 11K. G. Mann, C. M. Heldebrant, D. N. Fass et al., Thromb. Diath. Haemorrh., Suppl. 57, 179 (1974). 12M. I. Barnhart, Am. J. Physiol. 199, 360 (1960).

124

BLOOD CLOTTING ENZYMES

[13]

"Prothrombinase" a • • •

Factor X a Factor V Calcium Phospholipid /

Prothrombln

~

¢

~ - Thrombin

Snake Venom•

FIG. 1. Schematic prothrombin.

representation

of

the

"prothrombinase"

activation

of

K-dependent blood coagulation factors2 a Prothrombin synthesis in the absence of vitamin K or in the presence of vitamin K antagonists (in humans and cattle) results in the formation of an incomplete molecule, 14,15 which lacks certain calcium-binding sites 1~,16 that are vitamin K dependent. The incomplete protein is secreted into the plasma at normal levels, T M but is not subject to activation to thrombin by the physiological activator, presumably because the incomplete molecule which lacks the vitamin K-dependent calcium-binding sites is not capable of binding to the factor Xa, factor V, calcium, and phospholipid activator. The abnormal molecule produced in vitamin K deficiency states, however, is still susceptible to activation by snake venom activators.

Assay Two types of assays have been proposed to be used to evaluate the plasma level of prothrombin: the one-stage assay 19 and the two-stage assay. 3 In both assay systems, the process of prothrombin activation is initiated by adding a source of tissue factor, phospholipid, and calcium to the plasma sample. The tissue factor serves to activate factor VII in 2s C. A. Owen, Jr., Mayo Clin. Proc. 49, 912 (1974). 1, j . Stenflo and P.-O. Ganrot, J. Biol. Chem. 247, 8160 (1972). ~ G. L. Nelsestuen and J. W. Suttie, J. Biol. Chem. 247, 8176 (1972). ~aj . Stenflo and P.-O. Ganrot, Biochem. Biophys. Res. Commun. 50, 98, 1973. 17P.-O. Ganrot and J. E. Nil6hn, Scand. J. Clin. Lab. Invest. 22, 23 (1968). ~sF. Josso, J. M. Lavergne, M. Gouault, O. Prou-Wartell, and J. P. Soulier, Thromb. Diath. Haemorrh. 20, 88 (1968). ~gA. J. Quick, M. Stanley-Brown, and F. W. Bancroft, Am. J. Med. Sci. 190, 501 (1935).

[13]

PROTHROMBIN

125

the plasma, which subsequently activates the endogenous factor X to factor X~. The factor Xa produced combines with calcium, phospholipid, and factor V to form the catalyst that converts prothrombin to thrombin. In the one-stage assay, the amount of time required to produce a sufficient amount of thrombin to clot the fibrinogen present in the sample is recorded as a function of prothrombin concentration; in the two-stage type of assay, the thrombin produced is evaluated by a separate analysis, and thus both the rate and the amount of thrombin produced from the sample are recorded. In effect, a one-stage assay measures only the rate at which a given amount of thrombin is produced, and two-stage assays measure the quantity of product enzyme that can be produced when the zymogen is subjected to a quasi-physiological activation system. Thus, only the two-stage assay provides an adequate measure of the quantity of functional zymogen. The procedure our laboratory uses to assay prothrombin is basically a modification of the original two-stage procedure of Ware and Seegers2 Since purified prothrombin will not have present in it endogenous factor VII or factor X, ~° defibrinated plasma must be readded to the assay system as a source of these two factors. In addition, the assay of the product (thrombin) is accomplished by a modified N IH thrombin assay. 21 This provides for the evaluation of the concentration of the product directly in terms of the thrombin produced on a molar basis.

First Stage o] Assay Reagents Defibrinated plasma (factor VII and factor X source): 0.5 ml of citrated bovine plasma, 0.4 ml of 0.9% sodium chloride, and 0.1 ml of a 100 unit/ml solution of Parke-Davis Topical Thrombin in 50% glycerol are mixed, then incubated at 37 ° for 30 min. Prior to use, the plasma is expressed from the clot. Prothrombin to be measured: Protein concentration is evaluated speetrophotometrically using the ~~1~ 2 8 0 for bovine prothrombin 14.4 reported by Cox and Hanahan. 22 Protein concentrations are corrected for Rayleigh scattering using the equation: A2s0 = A2s0ob..... d -

1.706A320ob..... d

Factor V: 0.1 ml of AC-globulin (Difco), a barium-adsorbed serum preparation, is added to 15 ml of 0.9% sodium chloride. This 2oS. S. Shapiro and D. F. Waugh, Thromb. Diath. Haemorrh. 16, 469 (1966). 21R. L. Lundblad, H. S. Kingdon, and K. G. Mann, this volume [14].

126

BLOOD CLOTTING ENZYMES

[13]

solution is discarded after the completion of the assays. The freshly prepared solution is stored at 0 °. Tissue factor: Thromboplastin (Difco two-stage reagent) is prepared by reconstitution of dry reagent with 0.9% sodium chloride. Simplastin (General Diagnostics) may be substituted.

Assay Procedure. Two timers are required for the assay; one to measure the time of activation and one for the thrombin assay. To perform the assay, prepare an appropriate amount of NIH thrombin assay mixture 21 and the following solutions: solution A: 0.1 ml of defibrinated plasma and 2.4 ml of factor V reagent; solution B: 0.25 ml solution A and 0.75 ml of reconstituted thromboplastin (two-stage reagent) and incubate at 37 ° for 10 min; solution C: the prothrombin sample appropriately diluted with factor V reagent to a final concentration of between 0.25 and 2.0 mg/ml; solution D: mix 0.25 ml of solution C and 0.75 ml solution B, start the activation timer, and incubate the reaction mixture at 37 ° . Second Stage (Thrombin Assay) The reagents and procedures used for the modified NIH thrombin assay are detailed in this volume in Chapter [14] on thrombin. This assay is used for the second stage of the prothrombin assay. Remove aliquots for the NIH thrombin assay at various time intervals (~30 sec) and determine the thrombin activity present in the original sample at each time. Plot the thrombin activity generated in the first stage vs time. The point of maximum thrombin activity which can be generated from the prothrombin sample is the prothrombin activity of that sample. As an example, if a 10-~I sample of the activation mixture removed at the point of maximum activity has a clot time of 15 sec (1 unit/ml) in the NIH thrombin assay, and the original sample was diluted 1:3 (to prepare solution C) then the activity is 1 unit/ml × 310/10 × 4/1 X 3/1 = 372 units/ml Where 1 unit/ml is the thrombin activity (15 sec clot time in the thrombin assay) of the sample, determined from the clot time by reference to the standard plot, 21 310/10 is the dilution of the 10-~1 sample in the N I H thrombin assay, 4/1 is the dilution of solution C into the activation mixture (solution D), and 3/1 is the dilution of the original sample. This hypothetical sample, then, has an activity of 372 NIH thrombin units/ml in the modified two-stage assay. Bovine thrombin has a maximal specific activity of about 2700--3000

[13]

PROTHROMBIN

127

N I H units per milligram of protein; thus the theoretical specific activity for pure prothrombin is between 1500 and 1700 N I H thrombin units per milligram of prothrombin. In real assay conditions, however, this value will only be approached, since side reactions and degradation of the product eliminate the possibility of converting all the prothrombin present in the assay to thrombin. Operationally homogeneous bovine prothrombin will have a specific activity of about 1200 N I H thrombin units per milligram of prothrombin in the assay. Purification Purification of bovine prothrombin has been reported by a variety of laboratories. 22-3° In general, all purification schemes reported take advantage of a peculiar affinity of the vitamin K-dependent proenzymes for insoluble barium and magnesium salts. I t should be noted here t h a t those procedures involving insoluble barium and magnesium salts are suitable only for the normal vitamin K-dependent factors, as the proteins produced in the absence of vitamin K or in the presence of vitamin K antagonists are not adsorbable onto barium salts. In our early studies, our laboratory used a modification of the method of Ingwall and Scheraga 23 for the purification of prothrombin. In later studies, a procedure was developed that would provide for the isolation of both prothrombin and factor X from the same plasma sample, and this procedure will be detailed here. I s o l a t i o n of P r o t h r o m b i n f r o m B o v i n e P l a s m a

The isolation procedure for prothrombin and factor X is routinely carried out with 10-20 liters of plasma2 ° The purification cited for illustrative purposes was carried out with 14 liters of plasma. All steps were performed at 4 ° unless specified. Figure 2 shows the flow diagram of the steps involved in the purification. 52A. C. Cox and I). J. Hanahan. Biochim. Biophys. Acta 207, 49 (1970). ~3j. S. Ingwall and H. A. Scheraga, Biochemistry 8, 1960 (1969). ~ G. H. Tishkoff, L. C. Williamson, and D. M. Brown, J. Biol. Chem. 243, 4151 (1968) 25G. F. Lanchantin, J. A. Friedmann, and D. W. Hart, J. Biol. Chem. 240, 3276 (1965). ~"L. E. McCoy and W. H. Seegers, Thromb. Res. I, 461 (1972). ~ S. Magnusson, this series Vol. 19, p. 1957. ~8K. D. Miller, J. Biol. Chem. 231,987 (1958). ~ H. C. Moore, D. P. Malhotra, S. Bakerman and J. R. Carter, Biochim. Biophys. Acta 111,174 (1965). ~"S. P. Bajaj and K. G. Mann, J. Biol. Chem. 248, 7729 (1973).

128

[13]

BLOOD CLOTTING ENZYMES Citrated blood Flow separator Citrated plasma (1) Add BaCI 2 (2) Centrifuge

~

CeHs 4

Supe rnatant (source of factors V & VIII)

Barium citrate precipitate (1) Dissolve in citrate-saline Twice l 1(2) Add BaC12 (3) Centrifuge Supernatant 4 (discard)

Precipitate 4 (discard) Supernatant 9 (discard)

Barium citrate precipitate (1) Dialyze into EDTA (2! Dialyze into citrate-saline (3) Add saturated ammonium sulfate to 40~ saturation (4) Centrifuge Supernatant Add saturated ammonium sulfate to 60~ saturation Precipitate (1) DissolVepH 6, 10-3Min 0.025MDFpcitrate, (2) DialyzepH 6 into 0.025M citrate, (3) DEAE-ceUulose chromatography 0.1M chloride eluate (Factor H)

0.3M chloride eluate (Factor X, --~ 95% pure) DEAE -Sephadex chromatography

Contaminants ~ Factor X

FIG. 2. Flow diagram for prothrombin and factor X isolation. From S. P. Bajaj and K. G. Mann, J. Biol. Chem. 248, 7729 (1973).

Step 1. Collection o] Blood. The technology employed in the first two steps is t h a t of Moore et al. 29 Slaughterhouse blood was collected in 2.85% trisodium citrate (blood, 8 parts: anticoagulant, 1 part), and the cells and plasma were separated by the means of a continuous-flow separ a t o r ( D e L a v a l No. 518).31 This step was performed at room temperature. Step 2. Barium Citrate Adsorption and Elution. The v i t a m i n K - d e pendent factors were initially adsorbed onto barium citrate. The plasma was gently stirred and 1 M BaC12 (80 ml per liter of plasma, or 1120 ml of BaCl~ per 14 liters of plasma) was added dropwise. Stirring was continued for 10 min after all the BaC12 had been added. The suspension was then centrifuged for 30 min at 3600 g. We have used a Lourdes continuous-flow centrifuge ( B E T A - F U G E Model A-2, rotor 1010) for large preparations with good results. The supernatant from the barium citrate 31This is an antique hand-driven cream separator. These machines are relatively plentiful in the Midwest dairy regions. Other cream separators or continuous-flow centrifuges may be substituted.

[13]

PROTHROMBIN

129

adsorption may be used as a source of factors V and VIII. The barium citrate precipitate obtained was suspended in citrate-saline (9% NaC1 and 0.2 M trisodium citrate, 32 diluted 1:9 with distilled H~O) by means of a Waring blender at a low speed. The total volume of diluted citratesaline used was one-third of the volume of starting plasma (4670 ml for 14 liters of plasma). The resuspended protein was reprecipitated by addition of the same volume of 1 M BaC12 used in the initial precipitation step. Stirring was continued for 10 min after the addition of BaC12, and the suspension was covered and allowed to stand for 1 hr. (The suspension may be allowed to stand overnight, if desired.) The suspension was then centrifuged for 30 min at 3600 g, and the supernatant was discarded. The precipitate was resuspended in diluted citrate-saline as before, and the precipitation was repeated with same volume of 1 M BaC12. The barium citrate precipitate obtained at this stage may be stored indefinitely in the frozen state (--20°). The barium citrate precipitate was suspended in cold 0.2 M EDTA, pH 7.4 (120 ml per liter of plasma). A Waring blender at low speed was used to obtain a homogeneous suspension. The suspension was dialyzed for 40 rain vs 10 volumes of a mixture of 0.2 M EDTA, pH 7.4 (1 part), stock citrate-saline (0.2 M trisodium citrate, 0.9% NaC1, 1 part), and distilled water (8 parts). Dialysis was continued vs a similar volume of citrate-saline (1 part stock citrate-saline and 9 parts distilled water) for an additional 3 hr with the dialyzate changed every 30 rain and then continued overnight. The dialysis bags were mixed gently at each dialyzate change to ensure complete mixing. The dialysis step discussed above may be replaced by the following procedure, which is somewhat simpler and eliminates the need of dialyzing large quantities of particulate material. The barium citrate precipitate is suspended in 0.2 M EDTA pH 7.4 (120 ml of 0.2 M EDTA per liter of starting plasma). After suspension of the material, an equal volume of 0.026 M trisodium citrate, 0.9% NaC1 is added. Soybean trypsin inhibitor is then added to a final concentration equivalent to 3.5 mg per liter of starting plasma. After this addition, the suspension is made to 10% of saturation with cold saturated (NH,)_~SO~ (pH 7.0), and the suspension is allowed to stir overnight in the cold. After a minimum of 12 hr resuspension in this solvent system, the mixture is centrifuged at 6000 g for 15 min. Regardless of whether the resuspension of the barium citrate precipitate is carried out by the dialysis procedure alluded to previously or the direct redissolution procedure involving ammonium sulfate, step 3 is unchanged. 32Referred to as stock citrate-saline.

130

BLOOD CLOTTING ENZYMES

[13]

Step 3. Fractionation with Ammonium Sulfate. The viscous opaque suspension (EDTA eluate) obtained in step 2 was brought to 40% saturation with respect to ammonium sulfate. A saturated solution of ammonium sulfate, pH 7.0 (at 4 °, pH 7 adjusted with concentrated NH4OH), was added dropwise to the slowly stirring EDTA eluate. Stirring was continued for 15 min after all the ammonium sulfate solution had been added. The opaque suspension was centrifuged at 3600 g for 30 min and the precipitate was discarded. The supernatant was then brought to 60% saturation with respect to ammonium sulfate. Stirring was continued for an additional 10 min. Then the suspension was allowed to stand for 20 min, after which it was centrifuged at 3600 g for 30 min. The supernatant was discarded. Step 4. DEAE-Cellulose Chromatography. The precipitate obtained in step 3 was dissolved in a minimum volume of 0.025 M (sodium) citrate buffer, pH 6. The protein solution was made 1 mM in diisopropyl phosphorofluoridate (DFP) by the addition of 1 M DFP in isopropanol23 The DFP treated protein was dialyzed vs 2 liters of 0.025 M (sodium) citrate buffer, pH 6. The dialysis was continued overnight with two changes. Invariably after dialysis the solution had a small amount of insoluble protein; this was removed by centrifugation at 3600 g for 20 min. The supernatant was then applied to a DEAE-cellulose column (45 X 4 cm) equilibrated with the same buffer as used for the dialysis. The column was then washed with 0.025 M (sodium) citrate buffer, pH 6.0, until the absorbance of the effluent at 280 nm was less than 0.02. The buffer was then changed to 0.025 M (sodium) citrate, 0.1 M NaC1, pH 6. Prothrombin was eluted with this buffer. After the elution of prothrombin, the column was washed with the same buffer until the effluent had an absorbance of less than 0.01. At this point, a linear gradient of sodium chloride was applied to the column to elute the factor X. The gradient was formed by the presence of 1400 ml of 0.025 M (sodium) citrate, 0.5 M NaC1, pH 6, in the reservior and 1400 ml of 0.025 M (sodium) citrate, 0.1 M NaC1, pH 6, in the mixing chamber. The flow rate of the column was usually 80 ml/hr. A typical separation of bovine prothrombin and factor X effected by the DEAE-cellulose column is shown in Fig. 3. After application of the prothrombin-factor X containing sample to the column, washing the column with 0.025 M (sodium) citrate buffer, pH 6, results in the elution of peak A. Sodium dodecyl sulfate gel electrophoresis of the nonreduced and reduced samples from this peak revealed a major protein component (about 85%, by visual examination) composed of a single chain and 3sSoybean trypsin inhibitor (0.1 mg/ml) and benzamidine HC1 (0.01 M) may also be added in addition to the diisopropylphosphorofluoridate.

[13]

PROTHROMBIN o.IM NaCl

131

O.l-o.5M NaCI

120 3.0"

•

2.0-

0.5~ ~f

~o

0.3"~ ~ ]t

to.

¢

/-

-':=.:

80 0.t~

0.0

30

'

'

.

.

1t0

.

.

.

'

'

'

'

'

' - " ~ "

190 270 350 Fraction number

~

430

490

0

Flu. 3. Chromatograph of the 60% (NI-I4)2804 precipitate on DEAE-cellulose. Absorbance at 280 rim, and factor X activity is plotted vs fraction number. From S. P. Bajaj and K. G. Mann, J. Biol. Chem. 248, 7729 (1973). having an apparent molecular weight of 55,000 ---+5000 (reduced sample). The ionic strength of the eluting buffer was then increased by the addition of NaC1 to 0.1 M and two partially resolved peaks (peaks B and C) were eluted. Both peaks contained prothrombin of identical specific activities (1300 _ 100 NIH thrombin units per milligram of protein). This elution pattern was characteristic of every preparation. However, occasionally the prothrombin peak C gave lower specific activity (about 1000 NIH thrombin units per milligram of protein). When proteins from peaks B and C were rechromatographed separately on a DEAE-cellulose column under the conditions described above, two partially resolved peaks were again eluted. This suggests that the partial resolution obtained is an artifact. These two prothrombin peaks, B and C, do not contain detectable levels of thrombin or factor VII, IX, or X. Application of the gradient results in the elution of peaks D and E. Peak E contains factor X. Electrophoretic analysis of the factor X obtained at this stage indicates that it is about 80% homogeneous. The factor X obtained may be purified to homogeneity by DEAE-Sephadex chromatography. 3° The yield of prothrombin is about 50%, based on the concentration of the protein in plasma.

Concentration and Storage of the Products The prothrombin and factor X purified by this procedure are concentrated by the addition of solid ammonium sulfate to 80% saturation to

132

BLOOD CLOTTING ENZYMES

[13]

the pooled fractions. The resulting precipitate is dissolved in 50% glycerol water (v/v) and stored at --20% Both proteins are stable for at least a year under these storage conditions. The prothrombin and factor X isolated by these procedures are electrophoretically and immunologically homogeneous, and effectively devoid of contaminating factor activities when assayed for presumed contamination by either prothrombin or factor X. Prothrombin from Other Sources

The procedure described here for bovine prothrombin has also been used for the isolation of equine prothrombin without modification of the procedure, and also for the isolation of human and canine prothrombin with slight modificationsY4 Human prothrombin has been isolated both from fresh human plasma anticoagulated with acid-citrate-dextrose A and from American Red Cross lyophilized factor IX concentrates. Isolations of human prothrombin from commercial prothrombin concentrates have not been successful in our hands, owing to the presence of activated material in these concentrates? 5 In the case of isolation from fresh human plasma anticoagulated with acid-citrate-dextrose A, the initial step is to adjust the pH of the starting plasma from its initial pH 6.6 to pH 8.6 with dilute sodium hydroxide. Succeeding steps of the purification procedure for human prothrombin are the same as for bovine prothrombin with the exception that the behavior of human prothrombin on the DEAE-column is somewhat different than from that of the bovine material. Human prothrombin is eluted from the DEAE-column by elution with 0.15 M sodium chloride present in the buffer, rather than with 0.1 M sodium chloride, as is the case with bovine prothrombin. Figure 4 presents a typical elution profile from DEAE-cellulose of a human prothrombin preparation when the starting material was fresh human plasma. The product obtained in human prothrombin isolation is electrophoretically homogeneous; however, unlike the bovine preparations it is contaminated with a small amount of factor X and factor VII (as detected by activity measurements). Canine prothrombin isolation is carried out in exactly the same way as the bovine material (in61uding anticoagulation) with the exception of the chromatographic steps. Like human prothrombin, canine prothrombin is eluted at a slightly higher ionic strength from the DEAE-cellulose column, being eluted at an ionic strength of 0.15 M sodium chloride. The ~4R. J. Butkowski, M.S. Thesis, Univ. of Minnesota, Minneapolis, 1974. 3~p. M. Blatt, R. L. Lundblad, H. S. Kingdon, G. McLean, and H. R. Roberts, Ann. Intern. Med. ~I, 766 (1974).

[13]

PROTHROMBIN

133

3.5-

3.0-

2.5-

¢

¢

¢

2,0-

t.5-

1.0-

0.5-

Aj

O-

0

20

'¢0

60

Fraction number

Fio. 4. Chromatograph of human prothrombin (60% (NH4):SO4 precipitate) on DEAE-cellulose. After the initial wash peak (eluted with 0.025 M sodium citrate pH 6.0), the buffer was changed to 0.025 M sodium citrate, 0.1 M NaC1, pH 6.0 (fraction 20). The buffer is then changed to 0.025 M Na citrate, 0,15 M NaC1, pH 6.0, to elute the prothrombin (fraction 40-50).

canine product is free of factor X, and in fact the canine factor X can be obtained by further elution of this column with the identical gradient used for the bovine factor X isolation. Canine prothrombin as isolated exists as two components that differ in molecular weight by about 2000. The amino acids deleted in the smaller canine prothrombin are from the carboxyl terminal24 The isolation of human prothrombin has been reported by Lanchantin et al26 and by Kisiel and Hanahan27 The latter authors made use of preparative electrophoresis to remove the contaminating vitamin K-dependent factors from human prothrombin. Moore et a129 and Zubairov et al. as have reported the isolation of canine prothrombin, and Li and Olson ~9 56G. F. Lanchantin, J. A. Friedmann, J. DeGroot, and J. W. Mehl, J. Biol. Chem. 238, 238 (1963). ~7W. Kisiel and D. J. Hanahan, Biochim. Biophys. Acta 304, 703 (1973). ~8D. M. Zubairov, V. N. Timerbaev, and V. M. Menshov, Biochemistry (USSR) 33, 5 (1968). 39L. F. Li and R. E. Olson, J. Biol. Chem. 242, 5611 (1967).

134

BLOOD CLOTTING ENZYMES

[13]

have reported the isolation of rat prothrombin. Horse prothrombin isolation and crystallization has been reported by Miller. 4° Useful P r o t h r o m b i n Derivatives

Two derivatives of prothrombin have been exploited by this laboratory to explore the process of prothrombin activation in complex biological systems. These derivatives have been used to demonstrate that the same four fragments that are produced in purified prothrombin activation systems are also produced in a complex biological system such as serum thromboplastin. 41,42

Fluorescein Isothiocyanate Labeling 4a Prothrombin may be labeled by a modification of the method of RinderknechtJ 3 The protein sample is brought to pH 8 by the addition of solid sodium bicarbonate and a small aliquot of fluorescein isothiocyanate, 10% on Celite (available from CalBiochem) is added. The reaction mixture is stirred for 10-15 min, and glycine is added to destroy the excess reagent. The protein sample is then centrifuged to remove the Celite, and freed from excess dye by gel filtration on a BiG-Gel P2 column, or by precipitation with ammonium sulfate at 80% saturation.

[~H-sialyl ] Prothrombin Prothrombin 42 may be labeled by the procedure of Van Lenten and Ashwell.44 Prothrombin to be labeled is dialyzed into 0.1 M sodium acetate, 0.15 M sodium chloride, pH 5.6, at 4 °. A 9- to 10-fold molar excess of 12 mM sodium metaperiodate with respect to the sialic acid is added to the sample in an ice bath. The reaction is allowed to proceed for 10 min at 0% At that time, a 100-fold molar excess of ethylene glycol is added to stop the reaction. The reaction mixture is then dialyzed against 0.05 M sodium phosphate, 0.15 M sodium chloride, pH 7.4, for 12 hr. After dialysis, an equimolar quantity (relative to prothrombin) of tritiated sodium borohydride (7.2 Ci/mmole) is added in 0.2 ml of 0.01 M sodium hydroxide. This solution is warmed to room temperature, and the reaction is allowed to proceed for 30 min. In order to ensure complete 4oK. D. Miller, this series Vol. 19, p. 140. 41D. N. Fass and K. G. Mann, 1. Biol. Chem. 248, 3280 (1973). 4zR. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 249, 6562 (1974). 43 H. Rinderknecht, Experienlia 16, 430 (1960). L. Van Lenten and G. Ashwell, J. Biol. Chem. 0.46, 1889 (1970).

[13]

PROTHROMBIN

135

reduction, a 3-fold molar excess of unlabeled sodium borohydride is then added, and the reaction is continued for an additional 30 min. Excess reagent is removed by dialysis against 0.1 M sodium acetate, 0.15 M sodium chloride, pH 5.6. The labeled prothrombin is recovered by precipitation with ammonium sulfate at 80% saturation and stored at --20 ° in 50% glycerol. The radiolabeled preparations possess 70-80% of the biological activity of the nonlabeled control. Roughly 90% of the label will be contained within the modified sialic acid, which has been identified as the 7-carbon analog of N-acetylneuraminic acid: [7-3H]5-acetimido 3,5-dideoxy-L-arabino-2-heptulosonic acid. 42

Congenitally Abnormal Prothrombins Five abnormal prothrombins have been described: prothrombin Cardeza, 45 prothrombin Barcelona, 46 prothrombin Brussels, 47 prothrombin San Juan, 4s and prothrombin Padua. 49 All five of the reported prothrombin variants appear to be different, the only common feature being a large discrepancy between the biological activity produced on prothrombin activation and the quantity of prothrombin zymogen as identified by immunochemical techniques. In the case of prothrombin Cardeza, an abnormal prothrombin fragment is produced during prothrombin activation without thrombin production. Prothrombin Padua and prothrombin San Juan also generate abnormal prothrombin activation fragments during activation. In the case of prothrombin Barcelona, the defect appears to be in the ability of the variant molecule to be activated by the prothrombinase complex rapidly, and in the case of prothrombin Brussels, a competitive inhibitor to normal prothrombin activation is produced. Physical and Chemical Properties Physical and chemical properties of prothrombin as isolated from all species are very similar with the exception of prothrombin isolated from the rat. 39 Values for the molecular weight of bovine and human prothrombin vary to some extent, partly owing to different values used for partial specific volume calculations (0.683 as to 0.72123 ml/g). Most values for the molecular weight, however, hover about numbers between 69,000 and 4~S. S. Shapiro, J. Martinez, and R. R. Holburn, I. Clin. Invest. 48, 2251 (1969). 4, F. Josso, J. Monasterio De Sanchez, J. M. Lavergne, D. M~nach~, and J. P. Soulier, Blood 38, 9 (1971). 47M. J. P. Kahn and A. Goverts, Thromb. Res. 5, 141 (1974). *~S. S. Shapiro, N. Maldonald, J. Fradesa, and S. McCord, J. Clin. Invest. 33, 6 (1974). 4~G. Girolami, A. Bareggi, A. Brunetti, and A. Stecche, J. Lab. Clin. Med. 84, 654 (1974).

136

BLOOD CLOTTING ENZYMES

[13]

74,000. ~'23,~°,51 I n contrast, rat prothrombin has a molecular weight of 90,00022 The sedimentation coefficient of prothrombin has been reported from a high of 5.3 S 53 to a low of 4.6 S. ~ E v a l u a t i o n of the sedimentation coefficient is made somewhat complex because the protein polymerizes in solvents of low ionic strength. ~ In addition, the choice of solvent in these sedimentation studies also complicates the picture, since it is clear t h a t the chromatographic behavior of prothrombin in citrate and phosphate buffers of equivalent ionic strength is different. 2~,~° Similarly, the question of the s y m m e t r y of the molecule in solution is complex because of its tendency to aggregate. Cox and H a n a h a n 2~ calculated a frictional ratio of 1.45 from both sedimentation and diffusion data t h a t would indicate the molecule to be assymetrical, whereas Ingwall and Scheraga 23 obtained a value for the intrinsic viscosity of prothrombin t h a t would indicate the molecule to be symmetrical (3.4 m l / g ) . A comparison of the amino acid compositions of bovine and h u m a n prothrombin obtained from several laboratories is presented in T a b l e I. I n general, there is reasonable agreement on the compositions of bovine and h u m a n prothrombin, and in addition, the compositions of these two proteins are not unexpectedly similar. Prothrombin is a glycoprotein which most likely contains between 8 and 10% carbohydrate. T a b l e I I provides a comparison of the carbohydrate data obtained in a v a r i e t y of laboratories. This carbohydrate appears to be distributed in three carbohydrate side chains. ~2,~ P r o t h r o m b i n Activation C o m p o n e n t N o m e n c l a t u r e Both h u m a n and bovine prothrombin activations have been studied by a v a r i e t y of laboratories, ~°-52,'~4a-~°~ and a relatively consistent picture C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 248, 7149 (1973). W. G. Owen, C. T. Esmon, and C. M. Jackson, J. Biol. Chem. 249, 594 (1974). ~2j. j. Morrissey and R. E. Olson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 3 (abstr.) 317 (1973). ~ W. H. Seegers, E. Marciniak, R. K. Kipfer, and K. Yasunaga, Arch. Biochem. Biophys. 121, 372 (1967). 51B. G. Hudson, C. M. Heldebrant, and K. G. Mann, Thromb. Res. 6, 215 (1975). ~" G. F. Lanchantin, J. A. Friedmann, and D. W. Hart J. Biol. Chem. 243, 476 (1968). ~ D. L. Aronson and D. M6nach6, Biochemistry 5, 2635 (1966). K. S. Stenn, E. R. Blout, Biochemistry 11, 45{)2 (1972). ~ H. Pirkle and I. Theodore, Thromb. Res. 5, 511 (1974). W. Kisiel and D. J. Hanahan, Biochim. Biophys. Acta 329, 221 (1973). 5gK. G. Mann, C. M. Heldebrant, and D. N. Fass, Fed. Proc., Fed. Am. Soc. Exp. Biol. 36, (abstr.) 539 (1971). K. G. Mann, C. M. Heldebrant, and D. N. Fass, I. Biol. Chem. 246, 6106 (1971). 6~aM. R. Downing, R. J. Butkowski, M. Clark, and K. G. Mann, J. Biol. Chem. 250, 8897 (1975).

[13]

PROTHROMBIN

137

TABLE I PROTHROMBIN AMINO ACID COMPOSITION Bovine prothrombin

Human prothrombin

Amino acid

a

b

c

d

e

f

g

Aspartic acid Threonine Serine Glutamic acid Proline Cysteine Glycine Alanine Valine Methionine Isoleuci~e Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

59 27 32 71 35 17 46 33 35 5 18 46 19 17 30 9 42 12

64 32 43 78 38 19 56 38 40 7 21 48 22 22 31 12 38 13

68 32 41 84 37 20 57 38 38 5 24 44 18 22 34 10 41 18

62 29 41 74 32 20 48 34 32 5 17 45 18 19 31 8 43 19

59 32 36 77 32 21 46 32 32 8 21 41 18 18 32 11 31 11

58 34 36 73 31 12 48 38 32 7 21 41 19 24 29 10 38 13

52 39 38 70 32 16 44 35 30 7 22 39 21 21 27 9 36 21

8.2%

(8.2%)

2.3%

9.6%

(8.2%)

(8.2%)

(8.2%)

%Carbohydrate

This laboratory. Based on C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 248, 7149 (1973), corrected to 8.2 % carbohydrate. r, W. H. Seegers, E. Marciniak, R. W. Kipfer, and K. Yasunaga, Arch. Biochem. Biophys. 121, 372 (1967), adjusted to a molecular weight of 70,000 and 8.2 % carbohydrate. c j. S. Ingwall and H. A. Scheraga, Biochemistry 8, 1960 (1969), molecular weight 74,000. a W. G. Owen, C. T. Esmon, and C. M. Jackson, J. Bwl. Chem. 249, 594 (1974), molecular weight 74,000. c This laboratory. Based on M. R. Downing, R. J. Butowski, Clark, M. and K. G. Mann, J. Biol. Chem. 250, 8897 (1975), adjusted to 8.2 % carbohydrate. / W. Kisiel and 1). J. Hanahan, Biochem. Biophys. Acta 304, 703 (1973), adjusted, to 8.2 % carbohydrate. a G. F. Lanchantin, J. A. Friedmann, J. DeGroot, and J. W. Mehl, J. Biol. Chem. 238, 238 (1963), adjusted to 8.2 % carboh);drate.

of t h e different f r a g m e n t s p r o d u c e d d u r i n g a c t i v a t i o n of t h e p r o t h r o m b i n m o l e c u l e has been a c c u m u l a t e d . I n contrast, h o w e v e r , the n o m e n c l a t u r e for p r o t h r o m b i n f r a g m e n t s has n o t been consistent. T h i s is p a r t l y o w i n g to t h e fact t h a t o n l y in a few cases h a v e p h y s i c a l and c h e m i c a l studies been car r i ed o u t to t h e degree t h a t d i r e c t a s s i g n m e n t s of p a r t i c u l a r c o m -

138

[13]

BLOOD CLOTTING ENZYMES

TABLE II CARBOHYDRATE COMPOSITION OF BOVINE PROTHROMBIN IN TERMS OF WEIGHT PERCENT

Component

a

b

c

d

e

Neutral sugar Glucosamine Galactosamine Sialic acid Total carbohydrate

2.9 2.5 0.1 2.7 8.2

3.6 2.5 0 3.2 9.3

3.0 1.8 0.2 4.2 9.2

3.8 3.3 -2.8 9.9

4.5 5.5 0 4.7 15.0

This laboratory: B. G. Hudson, C. M. Heldebrant, and K. G. Mann, Thromb. Res. 6, 215 (1975).

b W. G. Owen, C. T. Esmon, and C. M. Jackson, J. Biol. Chem. 249, 594 (1974). c S. Magnusson, A r k K e m i 23, 285 (1965). a G. H. Tishkoff, L. C. Williamson, and D. M. Brown, J. Biol. Chem. 243, 4151 (1968). " G. L. Nelsestuen and J. W. Suttie, J. Biol. Chem. 249, 6096 (1972).

ponents can be made. Our laboratory was one of the first ~9,G° to apply the sodium dodecyl sulfate (SDS) electrophoretic technique 61 to study the changes that occur during the activation of prothrombin. This technique has provided a primary tool for m a n y investigators for the identifications of prothrombin activation components. Thus, coidentities in component nomenclature can frequently be arrived at by inspection of the SDS gel electrophoretic data. Similarly, sequence data, when in agreement with published observations, have been used to establish the identities. Figure 5 provides an illustration of the fragmentation pattern of human prothrombin when it is subjected to factor Xa in the absence of thrombin inhibitors. It can be seen that four components are produced during the activation process when the analysis is conducted by SDS gel electrophoresis. The nomenclature of these different components as devised by different laboratories is presented in Table I I I . I n order to overcome the obvious difficulties of multiple nomenclatures for the same components of prothrombin, the question of prothrombin activation component nomenclature was placed under the scrutiny of a Task Force on Blood Clotting Zymogens and Zymogen Intermediates of the International Society on Thrombosis and Hemostasis, and the nomenclature question was examined at the Fifth Congress of this Society, held in Paris in July, 1975. As a consequence of this meeting, a tentative ~1K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).

[131

PROTHROMBIN

139

I

FIG. 5. An illustration of the (human) prothrombin activation process when evaluated by the sodium dodecyl sulfate electrophoretic technique. The vertical column (II, 1, 2, 3, 4) refers to the activation component nomenclature previously used by this laboratory, and the letter designations on the gel correspond to the time of activation. Current nomenclature (which is used in subsequent sections of the text), refers to these components as prothrombin, prethrombin 1, prethrombin 2, prothrombin fragment 1, and prothrombin fragment 2, respectively. Reproduced from R. J. Butkowski, M. S. Thesis, Univ. of Minnesota, Minneapolis, 1974. standardized nomenclature system was devised that met with the approval of nearly all investigators who have published in the prothrombin field. This nomenclature system is also presented in Table III; it is the nomenclature system used throughout this manuscript in identifying the products of prothrombin activation. While the four components identified are those produced on prothrombin activation by factor X~ in any activation system in which a thrombin inhibitor is not present, the addition of thrombin inhibitors to the activation system results in the generation of a new fragment. The

140

BLOOD

~

00

~

CLOTTING

[13]

ENZYMES

~

oo

go

0

d°~ d •

~

ok 0

0 ,q

~

0

.~

~

"~-

o

0 0

8

0

0000

, IX-D~,-I

~ ' ~

~q

°0~ c~ Z

0000

m "N

~

•

•~._~

~

0

~ 0

° ~

'~

• .~0

'~

go

~r~1C~

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~.~ ~-~

:=~

~ 00 o~

o,~.~

J~NO io~O

00

13]

PROTHROMBIN

141

work of Stenn and Blout, 56 and Esmon et al., 6~ and Kisiel and Hanahan 63 have provided evidence that when thrombin inhibitors are present, a new fragment is produced which has been designated Fx~6 and fragment 1-2 ~,6~ by these investigators. This fragment has also been produced in our laboratory using DFP and hirudin as thrombin inhibitors; using our nomenclature system it would be denoted intermediate 3-42 °a Using the recently devised nomenclature system for prothrombin activation components from the Vth Congress of the International Society of Hemostasis and Thrombosis, this activation component would be designated prothrombin fragment 1.2. Prothrombin Activation Components B o v i n e P r e t h r o m b i n I and P r o t h r o m b i n F r a g m e n t 1

Prethrombin 1 and prothrombin fragment 1 are the sole products of bovine thrombin action on bovine prothrombin. Incubation of bovine prothr~mbin with a small amount of thrombin results only in the production of these two components (however, see section on Human Prothrombin). In a typical preparation of these two fragments, 200 mg of prothrombin in 30 ml was incubated with 200 units of thrombin in 0.144 M NaC1, 0.0168 M imidazole, pH 7.4. After 3 hr incubation, the reaction was terminated by the addition of DFP to a final concentration of 1 mM. The sample was then dialyzed vs 0.15 M sodium chloride, 0.02 M Tris, pH 7.4, and applied to a 2.5 X 59 cm DEAE-cellulose column equilibrated with the same buffer. Figure 6 presents the elution profile obtained for this chromatographic procedure. After application of the sample, the column was washed with starting buffer, which resulted in the elution of a large, protein containing peak. When no further protein was eluted with this buffer, the column was developed by means of a linear gradient formed by the presence of 500 ml of the initial buffer in the mixing flask, and 500 ml of 0.5 M sodium chloride, 0.6 M Tris, pH 7.4, in the reservoir. The first peak eluted from the column (fractions 10 to 40) corresponds to prethrombin 1. Two partially resolved components are eluted by the gradient. The shoulder corresponding to fractions 60-70 represents a small amount of prothrombin not converted to prethrombin 1 and prothrombin fragment 1, and the major peak (fractions 71 to 90) corresponds to prothrombin fragment 1. The prethrombin 1 and prothrombin fragment 1 protein pools obtained from the chromatographic C. T. Esmon, W. G. Owen, and C. M. Jackson, J. Biol. Chem. 249, 606 (1974). esW. Kisiel and D. J. Hanahan, Biochem. Biophys. Res. Commun. 59, 570 (1974).

142

[13:

BLOOD CLOTTING ENZYMES 2.5

2.0

1.5 ~o t.0

0.5

0

20

40

60

80

t00

Fraction number

FIG. 6. Chromatograph of bovine prethrombin 1 and prothrombin fragment 1 on DEAE-cellulose. Absorbance at 280 nm is plotted vs fraction number. From C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 248, 7149 (1973).

column were concentrated by precipitation with solid ammonium sulfate to 80% saturation. The individual proteins were then redissolved in 50% glycerol and stored at --20 ° . These two activation components are stable indefinitely when stored under these conditions.

Human Prethrombin 1 and Prothrombin Fragment 1

Human prethrombin 1 and prothrombin fragment 1 are not the sole products of thrombin treatment of human prothrombin. Prolonged treatment of human prothrombin with thrombin results in the slow production of two additional fragments which are similar when analyzed by electrophoresis in SDS to prethrombin 2 and prothrombin fragment 2. Therefore, it is essential that the reaction that is carried out with human prothrombin not be prolonged beyond 3 hr at a final concentration of 1 unit of thrombin per milligram of prothrombin. The separation of the human fragments, however, is entirely analogous to that used for bovine prothrombin fragment 1 and prethrombin 1. Bovine Prethrombin 2 and Prothrombin Fragment 2

Treatment of bovine prethrombin 1 with factor Xa results in the produetion initially of prothrombin fragment 2 and prethrombin 2, and subse-

[13]

PROTHROMBIN

143

quently the production of a-thrombin from the prethrombin 2. This second step (prethrombin 2 --->aIIa) proceeds at a rate which is roughly equivalent to the rate of production of prethrombin 2 from prethrombin 1 when factor X~ alone is used as a catalyst. In the presence of the complete prothrombinase catalyst (factor X~-factor V-calcium and phospholipid) the rate of prethrombin 2 conversion to a-thrombin is faster than the rate of prethrombin 2 production from prethrombin 1. In contrast, the activation of prethrombin 1 with factor Xa in the presence of 25% sodium citrate results in a rapid production of prethrombin 2 and prothrombin fragment 2, and a slow subsequent production of a-thrombin. This alteration in the rates of bond cleavage proves useful in the isolation of prethrombin 2 and prothrombin fragment 2. By using factor Xa in 25% sodium citrate as the activating system for prethrombin 1, one can obtain prethrombin 2 and prothrombin fragment 2 nearly quantitatively without the complication of contamination by thrombin and thrombin degradation products. To prepare prethrombin 2 and prothrombin fragment 2, prethrombin 1 (final concentration 1-10 mg/ml) in 25% trisodium citrate (w/v) is incubated with factor X~64 (final concentration 5-10 units/ml). During incubation, the production of thrombin by this system is evaluated by means of the standard NIH assay. 21 When 10 td of the reaction mixture will give a clotting time of approximately 15 sec in the NIH thrombin assay (about 30 min), DFP is added to the reaction mixture to a final concentration of 10-2 M. The sample is then dialyzed into 0.15 M sodium chloride, 0.02 M Tris, pH 7.4, in the cold and subjected to chromatography on DEAE-cellulose and equilibrated with the same buffer. Figure 7 shows the chromatographic separation of prethrombin 2 and prothrombin fragment 2. Prethrombin 2 is eluted in the wash peak on the column, while prothrombin fragment 2 is subsequently eluted with 0.5 M sodium chloride, 0.066 M Tris, pH 7.4. Frequently because of the noncovalent, association of prethrombin 2 and prothrombin fragment 2, prethrombin 2 obtained by this chromatographic procedure in the wash peak will be contaminated with prothrombin fragment 2. The second chromatographic separation is then required. The wash peak can be reehromatographed on sulfopropyl Sephadex C50-120. This chromatographic separation is essentially identical to the purification procedure used for a-thrombin. The wash peak is dissolved in or dialyzed vs 0.025 M sodium phosphate, pH 6.5, and applied to a 2.5 X 50 cm column of ~4We have used factor Xa(a) activated from factor X with trypsin insolubilized on polyacrylamide~°; (b) activated with insolubilized Russell's viper venom; (c) insolubilized factor X~ prepared by coupling factor X to Sepharose and activating the insoluble zymogen and Russell's viper venom.

144

BLOOD CLOTTING ENZYMES

[13]

0.3

0.2

0.t

%

i

2O

40

60

80

t 00

Fraction number

FIG. 7. Chromatograph of bovine prethrombin 2 and prothrombin fragment 2 on DEAE-cellulose. Absorbance at 280 nm is plotted vs fraction number. From C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 248, 7149 (1973). 2.8

A

B

2.0

t.2

0.4

"~

0

'~

20

40

"bO tt0 Fraction number

J

t30

t50

FIG. 8. Rechromatography of bovine prethrombin 2 and prothrombin fragment 2 on sulfopropyl-Sephadex C50-120. Absorbance at 280 nm is plotted against fraction number. Peak A corresponds to prothrombin fragment 2 and is eluted by the wash buffer, 0.025 M sodium phosphate, pit 6.5; peak B corresponds to prethrombin 2 and was eluted with 0.25 M sodium phosphate pH 6.5. From R. J. Butkowski, M. S. Thesis, Univ. of Minnesota, Minneapolis, 1974. SPC50-120 equilibrated with the same buffer. Prothrombin fragment 2 is eluted in the wash peak, while prethrombin 2 is eluted with 0.25 M sodium phosphate pH 6.5. This separation is presented in Fig. 8.

H u m a n Prothrombin Fragment 2 and Prethrombin 2 H u m a n prethrombin 2 and prothrombin fragment 2 can be prepared in the same w a y as the bovine activation components with one primary exception: human thrombin, when it is produced, cleaves at the amino terminal of human prethrombin 2, deleting the first 13 residues2 °a Thus,

[13]

PROTHROMBIN

145

precautions must be taken to block the production of thrombin during the preparation of this component. We have included DFP (10 -3 M) or hirudin at a concentration of 100 t~g/ml to block the back reaction of thrombin on human prethrombin 2. If the human prethrombin 1 conversion is carried out so that thrombin activity is generated, the product isolated will not be prethrombin 2, but rather prethrombin 2', from which the first 13 residues have been deleted. This component is designated in the current nomenclature system as prethrombin 2'(d~ R~-R~3). Human prothrombin fragment 1-2 can be prepared from prothrombin analogously with human prothrombin, using hirudin (2-fold molar excess) as the thrombin inhibitor. The solvent in this case is 0.02 M Tris, 0.15 M NaC1, pH 7.4. I m m u n o l o g i c a l Properties of Prothrombin Activation C o m p o n e n t s

The first successful antibody preparations prepared against prothrombin activation components were reported by Shapiro. 65 We have recently prepared antibodies to prothrombin and the activation fragments, as well as thrombin, by emulsifying the antigen in Freund's adjuvant containing killed tubercule bacilli. Final concentrations of the antigen in each case was 0.1 mg/ml and of the bacilli was 5 mg/ml. We have used rabbits, chickens, and burros to produce the antisera, and the animals in each case received weekly subcutaneous injections of 0.2 ml of these preparations. The antibodies have been evaluated by immunodiffusion66,67 as well as hemagglutination inhibition, and more recently by radioimmunoassays for prethrombin 1 and prothrombin fragment 1. In our hands, 66 the appropriate immunological recognition between the various prothrombin fragments has been observed in all assays. Antisera prepared against prethrombin 1 recognizes prothrombin, prethrombin 1, prothrombin fragment 2, and prethrombin 2, and antisera directed against prothrombin fragment 1 recognize only prothrombin fragment 1 and prothrombin. Similarly, antisera prepared against prothrombin fragment 2 recognize prethrombin 1, prothrombin, and prothrombin fragment 2, but not prethrombin 2 nor prothrombin fragment 1. Antisera prepared against prethrombin 2 recognize prothrombin and prethrombin 1, but not prothrombin fragment 2 or prothrombin fragment 1. A recent brief communication by Hewett-Emmett et al. 6s has reported immunological 65S. Shapiro, Science 162, 127 (1968). ~6A. H. Auernheimer and F. O. Atchley, Am. J. Clin. Pathol. 38, 548 (1962). 67C. Taswell, F. C. McDuffie,and K. G. Mann. Immunochemistry 12, 339 (1975). 6~D. t{ewett-Emmett, L. E. McCoy, H. I. Hassoura, J. Reuterby, D. A. Walz, and W. H. Seegers, Thromb. Res. 5, 421 (1974).

146

BLOOD CLOTTING :ENZYMES

[13]

TABLE IV PHYSICAL PROPERTIES OF BOVINE PROTHROMBIN AND ITS ACTIVATION COMPONENTS

Prothrombin Prethrombin 1 Prethrombin 2

Prothrombin fragment 1

Prothrombin fragment 2

23,000 24, O00

13,000 12,900

O. 682 0. 692

O. 704 0. 705

10.5 10.1

12.5 13.8

Molecular weights

70,000 74,000

51, OOO~ 49,700/

41,000 37,000 Partial specific volumes

O. 708 0.711

O. 719 0. 720

O. 728 -Extinction coe~cient (E ~ )

14.4 c 14.4

16.4 16.1

19.5 d 15.8

a This laboratory: C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 9.48, 7149 (1973). b W. G. Owen, C. T. Esmon, and C. M. Jackson, J. Biol. Chem. 249, 594 (1974). c A. C. Cox and D. J. Hanahan, Biochim. Biophys. Acta 207, 49 (1970). d D. J. Winzor and H. A. Scheraga, J. Phys. Chem. 68, 338 (1964). 60,300-65,000 on sodium dodecyl sulfate electrophoresis. I 61,000 on sodium dodecyl sulfate electrophoresis. cross-reactivity between prothrombin fragment 1 and prothrombin fragment 2. However, no primary data were presented.

Physical and Chemical Properties of Bovine P r o t h r o m b i n Activation Components The physical properties of the bovine prothrombin activation components in terms of molecular weight, partial specific volume, and extinction coefficients are presented in Table IV. R e m a r k a b l y good agreement is obtained by the two laboratories ~°,~1 that have provided properties for these fragments. Table V presents the amino acid compositions of bovine prethrombin 1 and prothrombin fragment 1, and preliminary composition data for human prethrombin fragment 1, and Table VI presents similar data for bovine prethrombin 2 (a-thrombin) and prothrombin fragment 2, and human prethrombin 2 and prothrombin fragment 2. Quantitative amino-terminal analysis 5° and amino terminal sequence data 69 from this laboratory initially allowed the orientation of the bovine prothrombin activation components within the prothrombin primary C. M. Heldebrant, C. Noyes, H. S. Kingdon, and K. G. Mann, Biochem. Biophys. Res. Commun. 54, 155 (1974).

[13]

PROTHROMBIN

147

TABLE V PRETHROMBIN 1 AND PROTttROMBIN FRAGMENT 1 AMINO ACID COMPOSITION Prethrombin 1 Amino acid a Aspartic acid Threoninc Serine Glutamic acid Proline Cysteinc Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan % Carbohydrate

Bovine b

Prothrombin fragment 1

c

Human d

Bovine a b

Human d

46 17 22 48 24 9 35 24 25 4 14 35 15 15 22 5 29 8

46 17 27 49 22 12 38 24 23 5 14 36 14 16 25 6 29 15

48 21 26 53 28 11 38 26 28 5 15 36 17 17 26 8 30 13

47 18 24 58 20 14 39 25 25 7 17 34 15 14 26 8 28 9

16 9 l0 24 10 10 12 11 10 1 4 11 6 5 6 2 12 4

16 10 14 23 10 9 12 10 9 1 4 10 4 4 5 2 14 5

16 15 11 25 9 7 12 13 9 2 4 9 5 5 6 3 12 2

3.75

4.5

(3.75)

(3.75)

16.6

20

(16.6)

a This laboratory: C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 248, 7149 (1973). b W. G. Owen, C. T. Esmon, and C. M. Jackson, J. Biol. Chem. 249, 594 (1974). c W. H. Seegers, E. Marciniak, R. K. Kipfer, and K. Yasunaga, Arch. Biochem. Biophys. 121, 372 (1967). Calculated assuming 3.75 % carbohydrate and a molecular weight of 51,000. This laboratory: M. R. Downing, R. J. Butkowski, M. Clark and K. G. Mann, J. Biol. Chem. 250, 8897 (1975). Calculated assuming the same carbohydrate composition as the bovine material. structure. P r o t h r o m b i n f r a g m e n t 1 corresponds to t h e a m i n o t e r m i n a l s e g m e n t of p r o t h r o m b i n , w h il e p r e t h r o m b i n 1 r e p r e s e n t s t h e c a r b o x y l t e r m i n a l p o r t i o n of the p r o t h r o m b i n molecule. P r o t h r o m b i n f r a g m e n t 2 r e p r e s e n t s t h e a m i n o t e r m i n a l end of p r e t h r o m b i n 1, while p r e t h r o m b i n 2 represents t h e c a r b o x y l - t e r m i n a l end of p r e t h r o m b i n 1. P r e t h r o m b i n 2 a m i n o - t e r m i n a l sequence corresponds to t h e a m i n o t e r m i n a l sequence of b o v i n e a - t h r o m b i n A - c h a i n as p r e s e n t e d by M a g n u s s o n . ~° T h e a m i n o t e r m i n a l sequences of p r o t h r o m b i n , t h r o m b i n , and t h e a c t i v a t i o n corn~°S. Magnusson, Thromb. Diath. Haemorrh., Suppl. 38, 97 (1970).

148

[13]

BLOOD CLOTTING ENZYMES

TABLE VI PRETHROMBIN 2 (o~-THROMBIN) AND PROTHROMBIN FRAGMENT 2 AMINO ACID COMPOSITIONS

Prethrombin 2 (a-thrombin) Amino acid

Aspartic acid Threonine Serine Glutamic acid Proline Cysteine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan % Carbohydrate

Bovine

Prothrombin fragment 2

a

b

Human c

a

Bovine b

d

Human c

35(30) 16(14) 17(14) 40(40) 20(19) 6(6) 30(27) 17(15) 25(21) 5(5) 15(13) 32(28) 13(11) 15(16) 25(22) 7(11) 25(20) 8(8)

32(30) 13(13) 15(18) 32(34) 16(15) --(8) 26(25) 15(15) 18(20) 4(4) 12(14) 27(26) 9(10) 13(12) 22(23) 6(7) 21(20) --(11)

34 15 19 41 18 9 29 17 18 7 17 29 12 11 23 6 25 8

18 4 8 17 10 3 12 10 5 0 1 9 3 3 3 1 7 --

18 5 10 15 10 5 12 l0 5 0 1 9 4 3 2 0 8 4

17 5 9 14 8 4 10 10 4 0 1 9 4 3 2 0 8 2

14 4 9 18 6 5 15 10 9 0 1 9 3 3 4 2 5 2

4.67 %

--(6.3)

--

0

O

0

0

a This laboratory: C. M. Heldebrant, R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem. 248, 7149 (1973). b W. G. Owen, C. T. Esmon, and C. M. Jackson, J. Biol. Chem. 249, 594 (1974). This laboratory: M. R. Downing, R. J. Butkowski, M. Clark and K. G. Mann, J. Biol. Chem. 250, 8897 (1975). d Calculated from the sequence data of J. Reuterby, D. A. Walz, L. E. McCoy, and W. H. Seegers, Thromb. Res. 4, 885 (1974). p o n e n t s are p r e s e n t e d in T a b l e V I I . A t e n t a t i v e c o m p l e t e s e q u e n c e for p r o t h r o m b i n f r a g m e n t 2 has been r e p o r t e d by R e u t e r b y e t al. ~1 an d a t e n t a t i v e c o m p l e t e s e q u e n c e of p r o t h r o m b i n has been r e p o r t e d b y Magnusson.~2, 7~ 71j. Reuterby, D. A. Walz, L. E. McCoy, and W. It. Seegers, Thromb. Res. 4, 885 (1974). ~S. Magnusson, L. Sottrup-Jensen, T.-E. Peterson, and H. Claeys, "Boerhaave Course on Synthesis of Prothrombin and Related Coagulation Factors," Leiden MP4, 1974. ~S. Magnusson, L. Sottrup-Jensen, T.-E. Petersen, and H. Claeys, Abstr. Cold Spring Harbor M e e t . Proteases Biol. Control, Sept. 10, 1974, p. 7.

[131

149

PROTHROMBIN T A B L E VII NH2-TERMINAL SEQUENCE Prothrombin and prothrombin fragment 1

Bovine a .b.c

Ala Ash Lys Gly Phe Leu Glu* Glu* Val Arg Lys Gly Asn Leu Glu Arg Glu* Cys Leu H u m a n d.e

Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu . . . (Glu* = -~,-carboxyglutamic acid) Prethrombin 1 and prothrombin fragment 2 Bovine ~,b,]

Ser Gly Gly Ser Thr Thr Ser Gln Ser Pro Leu Leu Glu Thr Cys Val Pro Asp Arg Human a

Ser Glu Gly Ser Ser Val Asn Leu Ser Pro Pro Leu Glu Gln Cys Val Pro Asp Arg Prethrombin 2 and q-thromb~n A chain Bovine a .b .o

Thr Ser Glu Ash His Phe Glu Pro Phe Phe Asn Glu Lys Thr Phe Gly Ala Gly Glu H u m a n d,h,i

Thr Ala Thr Ser Glu Tyr Gln Thr Phe Phe Asn Pro Arg ],Thr Phe Gly Ser Gly Glu !

J a This laboratory: C. M. Heldebrant, C. Noyes, H. S. Kingdon, and K. G. Mann, Biochem. Biophys. Res. Commun. 54, 155 (1973). t, Magnusson et al. [S. Magnusson, L. Sottrup-Jensen, T. E. Petersen, and H. Claeys, "Boerhaave Course on Synthesis of Prothrombin and Related Coagulation Factors, Leiden, MP4, 1974; Abstr. Cold Spring Harbor Meet. Proteases Biol. Control, Sept. 10, 1974, p. 7; S. Magnusson, L. Sottrup-Jensen, and T. E. Petersen, F E B S Lctt. 44, 189 (1974)] identified GluT, Glus, Glu~7 as ~,-carboxyglutamic acid. c Stenflo Identified Glut Glu8 as ~-carboxyglutamic acid: J. Stenflo, J. Biol. Chem. 249, 5527 (1974). a This laboratory: M. R. Downing, R. J. Butkowski, IV[. Clark and K. G. Mann, J . Biol. Chem. 250, 8897 (1975). H. Pirkle and I. Theodore, Thromb. Res. 2, 461 (1973): residues 1-5. s j. Reuterby, D. A. Walz, L. E. McCoy, and W. H. Seegers, Thromb. Res. 4j 885 (1974). S. Magnusson; this series, Vol. 19, Page 1957. h A. R. Thompson, L. H. Ericsson, and D. L. Enfield, Circulation 50, 292 (abstr.), 1119 (1974): human thrombin A-chain. i D. A. Walz and W. H. Seegers, B~ochem. Biophys. Res. Commun. 60, 717 (1974): human thrombin A-chain. / P o i n t of thrombin cleavage in human intermediate 2. IV[. R. Downing, R. J. Butkowski, M. Clark, and K. G. Mann, J. Biol. Chem. 250, 8897 (1975).

150

BLOOD CLOTTING ENZYMES

[13]

It has been mentioned previously that human prothrombin cleavage by thrombin produces, in addition to prethrombin 1 and prothrombin fragment 1 (the initial products), two additional products that are electrophoretically similar to prethrombin 2 and prothrombin fragment 2. These products arise as a result of the cleavage of the prethrombin 1 produced by thrombin at a position 13 residues removed from the aminoterminal of the factor X~-produced human prethrombin 2. Studies by Thompson et a l Y 4 and by Walz and Seegers ~ on human thrombin A-chain indicate that the amino terminal sequence of human thrombin A-chain is homologous with bovine thrombin beginning with residue 14. Human prethrombin 2', as isolated in this laboratory from factor Xa activation of prethrombin 1 in the absence of thrombin inhibitors has the same sequence as that reported for human a-thrombin A-chain. However, if human prethrombin 1 is isolated from short-term thrombin treatment of human prothrombin and the resulting prethrombin 1 is subsequently factor X~ activated to prethrombin 2 and prothrombin fragment 2 in the presence of DFP or hirudin, a new fragment is isolated which begins with residues homologous to the bovine prethrombin 2 and a-thrombin A-chain. These data suggest that there are two sites of cleavage of human prothrombin by thrombin: one position analogous to the Arg-Ser bond in bovine prothrombin, and another, cleavage 13 residues removed from the amino terminal of the factor X~, produced human prethrombin 2. A summary of the sites of cleavage of human and bovine prothrombin during activation in a noninhibited system are presented in Fig. 9. The cleavage (b') indicated by the dotted line occurs only in human prothrombin. The carboxyl terminals of all the bonds cleaved are arginine, 11,72,73,76 and recent reports by Magnusson et al. ~2,~ indicate that cleavages b and c occur after the sequence Ile-Gly-Glu-Arg. Studies by Stenn and Blout ~6 suggested that the fragments produced from prothrombin during activation by factor Xa or by thrombin were different. These authors proposed that bovine prothrombin can be activated by two pathways; one pathway, catalyzed by factor Xa, took place in two steps, while the second pathway was initiated by thrombin and took place in three steps. This proposal was confirmed by Esmon et a l 2 ~" who, repeating the studies of Stenn and Blout, '~6 showed that, in the presence of DFP factor X,~ produced fragments consistent with cleavage only at positions b and c. These authors were able to show the isolation of the entire "pro" end of the molecule (prothrombin fragment 1 cova,4A. R. Thompson, L. H. Ericsson, and D. L. Enfield, Circulation 50, 292 (abstr) 1119 (1974). ,5D. A. Walz and W. H. Seegers, Biochem. Biophys. Res. Commun. 60, 717 (1974). '~H. Pirkle and I. Theodore, Thromb. Res. 5, 511 (1974).

[13]

PROTHRO3IBIN

Prothrombin ALA 14

CiHOClHO

o

ISER

b

Prothrombin fragment 1

Prothrombin

6c

~THR~ I

LE

CH JO

Prethrombin 1

, Prothrombin, _ '-'fragment 2"-' I"

151

fragment 1.2

Prethrombin 2 ......

SER ~=1

=',

=J

a-Thrombin

Fzo. 9. Schematic representation of the orientation of prothrombin activation components within the prothrombin molecule. CHO represents a carbohydrate side chain. The letter designations a, b, and c represent the sites of cleavage of bovine and human prothrombin. An additional cleavage, b', occurs in human prothrombin. Currently accepted nomenclature for the pieces produced during prothrombin activation are also designated in the figure. lently linked to prothrombin fragment 2) and prethrombin 2 (a-thrombin) as the products of the reaction. This study has recently been extended to human prothrombin by Kisiel and Hanahan, 63 who provided electrophoretie data which indicate that the same process occurs in human prothrombin activation. The studies with hirudin and human prothrombin have been confirmed by this laboratory.

Calcium and V i t a m i n K The vitamin K-dependent blood coagulation factors (factor IX, factor VII, factor X, and prothrombin) all required calcium and phospholipid surfaces for either their activation to enzymes or their suSsequent enzymic functions. With respect to prothrombin activation, Papahadjahopoulos and H a n a h a n 6 demonstrated that calcium was essential for maintaining the integrity of the prothrombin-activating complex. These authors showed that factor V and phospholipid form a complex in the absence of calcium, but that the binding of factor Xa to the complex is dependent upon the presence of calcium. Cole and associates 7 demonstrated that the complexing of factor Xa to phospholipid could occur in the absence of factor V, but not in the absence of calcium. Ganrot and NilShn 17 and Josso et al. 18 in 1968 demonstrated by immunochemical techniques that individuals treated with the vitamin K

152

BLOOD CLOTTING ENZYMES

[13]

antagonist drugs (eoumarins) had in their plasma an abnormal prothrombin fraction. Suttie et al. 77 have isolated a prothrombin liver precursor in the wa.rfarin-treated rat. These studies were extended by Stenflo and Ganrot 14 a n d Nelsestuen and Suttie, 15 who demonstrated that prothrombin binds calcium and that the abnormal prothrombin antigen, which is induced with vitamin K antagonists, does not bind calcium. Equilibrium dialysis studies conducted by Stenflo and Ganrot TM and in our laboratory indicate that prothrombin has 10 or 11 calcium binding sites. Work in our laboratory 7s-s° indicates t h a t these sites are distributed in the two components t h a t are not on the pathway to thrombin, namely, prothrombin fragment 1 and prothrombin fragment 2. Prothrombin fragment 1 has five or six sites, with a log K association of 3.7, and prethrombin 1 has four or five sites t h a t are weaker, having a log K association of about 2.5. The calcium-binding sites in prethrombin 1 are contained in the amino-terminal prothrombin fragment 2 segment of the molecule. Prethrombin 2, the carboxyl-terminal segment of the prothrombin molecule, and the immediate precursor of thrombin has no affinity for calcium. The question of cooperativity between these sites in prothrombin, as well as the precise number of sites, cannot be adequately determined at the present time because of the relatively low affinities of these molecules for calcium. The nature of the vitamin K-dependent alteration in the prothrombin molecule that confers upon it the tight calcium-binding sites has recently been elucidated by a number of laboratories. Stenflo and associates 8~,s2 and Nelsestuen and co-workers s3-8~ have reported the isolation of peptides which contain the strong calcium-binding sites in prothrombin. The peptide isolated by Stenflo contained the Glu-Glu sequence in position 7 and 8 of the amino terminal of bovine prothrombin. Both Nelsestuen and Stenflo et al. have identified a unique amino acid present in pro"J. W. Suttie, G. A. Grant, C. T. Esmon, and D. V. Shah, Mayo Clin. Proc. 49, 933 (1974). ,8 S. P. Bajaj, R. J. Butkowski, and I(. G. Mann, Fed. Proc. Fed. Am. Soc. Exp. Biol. 33, (abstr.) 1473, (1974). ~K. G. Mann and D. N. Fass, Mayo Clin. Proc. 49, 929 (1974). 8oS. P. Bajaj, R. J. Butkowski, and K. G. Mann, J. Biol. Chem. 250, 2150 (1975). J. Stenflo, Y. Biol. Chem. f~49, 5527 (1974). 8sj. Stenflo, P. Fernlund, W. Egan, et al., Proc. Natl. Acad. Sci. U.S.A. 71, 2730 (1974). ~J. B. Howard and G. L. Nelsestuen, Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, (abstr.) 1473 (1974). G. L. Nelsestuen, T. Zytkovicz, and J. B. Howard, Y. Biol. Chem. 249, 6347 (1974). u G. L. Nelsestuen, T. H. Zytkovicz, and J. B. Bryant, Mayo Clin. Proc. 49, 941 (1974).

[13]

PROTHROMBIN

153

thrombin as a result of vitamin K action. This unique amino acid corresponds to a ~,-carboxyglutamic acid. This unusual residue has also recently been identified by Magnusson et al., s6 who identified it in 11 of the first 42 positions in the bovine prothrombin amino-terminal sequence, namely positions 7, 8, 15, 17, 20, 21, 26, 27, 30, 33. The human prothrombin sequence reported by our laboratory shows a Glu-Glu sequence at positions 6 and 7 homologous to the ~,-COOH Glu, ),-COOH Glu sequence at positions 7 and 8 in the bovine prothrombin sequence identified by both Stenflo s2 and Magnusson. s6 The significance of the calcium-binding sites in prothrombin can be seen in studies of the kinetics of activation of prothrombin and the two thrombin precursors, prethrombin 1 and prethrombin 2. The addition of calcium to the factor Xa-catalyzed activation of prothrombin increases the rate of prothrombin activation by about 40-fold. The addition of phospholipid to this activation mixture increases the rate by another 2.5-fold, or about 100 times the rate of prothrombin activation in the presence of factor X~ alone. In contrast, the activation of prethrombin 1 and prethrombin 2 in the presence of calcium and/or calcium and phospholipid results in no enhancement of the rate. Thus, the deletion of the prothrombin fragment 1 segment of the molecule eliminates the calcium accelerating effect, and subsequently that of phospholipid in enhancing the rate of prothrombin activation. Calcium-dependent phospholipid binding to prothrombin and its fragments has been reported by Gitel et al. s7 These authors showed that prothrombin fragment 1 is the calcium-dependent phospholipid-binding segment of the prothrombin molecule. The significance of the weak calcium-binding sites present in the prothrombin fragment 2 segment of the prothrombin molecule can be seen in studies of complete prothrombinase activation of prethrombin 1 and prethrombin 2. Although the addition of calcium and phospholipid to factor X~ as the catalyst for prethrombin 1 activation has no effect on the rate of thrombin production from prethrombin 1, factor V addition results in a dramatic increase in the thrombin production from prethrombin 1.79,8°,s8 The stimulatory influence of factor V on the rate of prethrombin 1 activation is not observed in the absence of calcium. 79,s° Similarly, the rate of activation of prethrombin 2 is not enhanced by the addition of calcium, calcium-phospholipid, or calcium-phospholipid-factor V. 8~S. Magnusson, L. Sottrup-Jensen, and T.-E. Petersen, F E B S Lett. 44, 189 (1974). sTS. N. Gitel, W. G. Owen, C. T. Esmon, et al., Proc. Natl. Acad. Sci. U.S.A. 70, 1344 (1973). C. M. Jackson, W. G. Owen, S. N. Gitel, and C. T. Esmon, Thromb. Diath. Haemorrh., Suppl. 57, 273 (1974).

154

BLOOD CLOTTING ENZYMES

[13]

Thus, the sensitivity to factor V acceleration is lost when the prothrombin fragment 2 segment of the molecule, which contains the weak calciumbinding sites, is deleted. When unactivated factor V is added, the acceleration of prethrombin 1 activation is preceded by a definite lag. When activated factor V (factor Va) is used, the lag is substantially decreased, or nonexistent. These observation suggest that activated factor V (factor V~) can accelerate the activation of prethrombin 1. Esmon et al. ~'' have provided evidence that factor V.~, but not factor V, binds to prothrombin in the presence of calcium. Prothrombin fragment 2 noncovalently associates with both bovine a-thrombin 9°-92 and with prethrombin 2. 92 The addition of prothrombin fragment 2 to the factor X~-factor V-calcium-phospholipid activation of prethrombin 2 restores the factor V.~ acceleration of thrombin production. Jackson et al. 93 have recently reported that the addition of the whole "pro" piece, prothrombin fragment 1.2 to prethrombin 2 in the presence of factor X~, factor V~, calcium and phospholipid results in a rate of prothrombin production faster than the rate of prothrombin conversion to thrombin. Two conclusions can be drawn from the studies of the calcium binding and kinetics of activation of prothrombin and the thrombin-producing intermediates: (1) the prothrombin fragment 1 segment of the prothrombin molecule which contains the strong calcium binding site is responsible for the calcium-dependent phospholipid binding of prothrombin to the prothrombinase complex; (2) the prothrombin fragment 2 segment of the molecule, which contains the weak calcium binding sites, is implicated in the factor V~ dependent association of the prothrombin molecule with the prothrombinase complex.

Prothrombin Activation

An overall picture of prothrombin activation is beginning to emerge which is consistent with the data provided by all laboratories. An illustration of the hypothetical pathway for prothrombin generation from prothrombin is presented in Fig. 10. Presumably, the initial event in ~ C. T. Esmon, W. G. Owen, D. L. Duiguid, and C. M. Jackson, Biochim. Biophys. Acta 310, 289 (1973). C. M. Heldebrant and K. G. Mann, J. Biol. Chem. 248, 3642 (1973). D1K.G. Mann, S. P. Bajaj, C. M. Heldebrant et al., Set. Haematol. 6, 479 (1973). 95K. It. Myrmel, R. L. Lundblad, and K. G. Mann, Circulation 50, 292 (abstr.) 118 (1974). 93C. M. Jackson, C. T. Esmon, and W. G. Owcn, Abstr. Cold Spring Harbor Meet. Proteases Biol. Control, Sept. 10, 1974,p. 7.

[13]

PROTHROMBIN HUMAN

"IT

ACTIVATION

"r CHO

CHO

~Ia

h

i

d

CHO

THR

b.d

~:~::~::.:~:'::::-:. ~,~-,::i:i:!:i:!:!:!:'~' CHO ii b,d.a

155

CHO

GLV

-V-~._~..v.a a

ARGISERI m

b

d

CHO

ARG1 THR

GLU

a,b.d CHO

iii

c

THRIARG

GLU CHO

c

S--

:FIG. 10. Schematic representation of the events that occur during human prothrombin activation. For the nomenclature of prothrombin pieces, see :Fig. 9. The letters a, b, c, and d represent cleavage sites in the human prothrombin molecule that occur during activation; the orders of these cleavages are presented by the roman numerals i, ii, and iii. P. L. designates phospholipid, and the A on the prothrombin fragment 1 s~ment of the molecule represents the 7-carboxyglutamie acids. prothrombin activation is the formation of factor Xa by either the intrinsic or extrinsic pathway. The factor Xa produced forms a complex with calcium, phospholipid, and probably with unactivated factor V. The binding of the factor V to the complex would be most likely by virtue of lipid-protein interaction rather than direct interaction of prothrombin with factor V, since the work of Esmon e t al. s9 indicates that unactivated factor V does not bind to prothrombin. In the initial event of activation, probably only factor Xa, calcium, and phospholipid serve as the catalyst, and bind the prothrombin through the prothrombin fragment 1 segment of the molecule, which contains the vitamin K-dependent calcium-binding sites. Cleavage of the prothrombin by the complex at position b (see Fig. 10) produces the prothrombin fragment 1-2 plus prethrombin 2. Prethrombin 2 is subsequently cleaved at position d to produce a-thrombin. Once a-thrombin is produced, it can serve two functions. It can activate factor V to factor Va, which results in factor V becoming a calcium-dependent prothrombin and prethrombin 1 binding protein. At this point, the rate of prothrombin activation would be greatly accelerated, and would probably proceed by the two routes originally suggested by Stenn and Blout, 56 one route being catalyzed only by factor X~ cleavage at positions b and d, the other route being catalyzed by a

156

BLOOD CLOTTING ENZYMES

[14]

combination of thrombin cleavage at position a and factor Xa cleavage at positions b and d. An additional side effect of thrombin production, at least in the case of human prothrombin, is the cleavage of a small 13-residue peptide from the amino-terminal end of both prethrombin 2 and the A-chain of thrombin. Prothrombin fragment 2, the factor Va binding segment of the prothrombin molecule, not only binds to factor Va and to prethrombin 2, but also binds a-thrombin itself. Therefore, it is conceivable that in the final prothrombinase activator formed, thrombin itself may be a constituent part, bound to the complex noncovalently through prothrombin fragment 2, which binds to the complex via factor V~ and calcium.

[14]

Thrombin

By ROGER L. L~NDBLAD, HENRY S. KINGDON, and KENNETH G. MANN

Thrombin is the enzyme involved in the final step in the coagulation of mammalian blood. This step involves the conversion of fibrinogen to fibrin through the cleavage of four arginyl-glycyl peptide bonds. 1 Thrombin therefore is a highly specific proteolytic enzyme. Thrombin also possesses the ability to hydrolyze a variety of ester and amide substrates, but, at least in the case of ester substrates, these synthetic substrates do not effectively measure changes in biological (fibrinogen-clotting) activity. 2,3 In addition, thrombin is not specific for either ester or amide substrates. Therefore it is preferable to use fibrinogen clotting as the specific assay for thrombin. Assay The procedure used in our laboratory is a modification of the NIH thrombin assay. 4 The principal difference between this assay and the standard NIH assay procedure is that all reagents are pre-prepared separately in large quantities, and then aliquoted into approximately 2-ml volumes into polyethylene vials (flip-top polyethylene vials Model B, 2-dram capacity, obtainable from Laboratory Supplies Company, Inc., I K. Laki and J. A. Gladner, Phy~oL Rev. 44, 127 (1964). 2G. F. Lanchantin, C. A. Presant, D. W. Hart, and J. A. Friedmann, Thromb. Diath. Haemorrh. 14, 159 (1965). *R. L. Lundblad, K. G. Mann, and J. H. Harrison, Biochemistry 12, 409 (1973). "Minimum Requirement for Dried Thrombin," 2nd Revision, Division of Biologic Standards, National Institutes of Health, Bethesda, Maryland, 1946.