Factors affecting plasma clot absorbance and fibrin mass

CLINICA CHIMICA ACTA

FACTORS

AFFECTING

I

PLASMA

CLOT

ABSORBANCE

AND

FIBRIN

MASS

LOUIS KOSENFELD

DeQartmentof Pathology, New

York

Uniwvsity

(Revised manuscript

New York University School of Medicine, Medical Center, New York, N. Y. 1oor6 (U.S.A.)

received June rqth, 1967)

SUMMARY I. The reactions

of thrombin

and plasma were studied.

in physical and chemical parameters on clot absorbance mined. 2. Variations in pH, ionic strength, temperature

The effect of variations

and fibrin mass were deterand protein

concentration

affect the spectrophotometric measure of absorbance of a thrombin-plasma clot, but not the mass of fibrin formed. 3. Citrate ion at pH 6.0 greatly retards the development of both absorbance and fibrin formation. 4. Absorbance depends on thrombin concentration and is maximal at 1.0 unit thrombin per milliliter reaction mixture over the pH range 6-8. 5. One unit of thrombin will clot completely (> 95%) 1.17 mg plasma nogen (3.4 nmoles) in 1.0 ml reaction solution at pH 7.35, 38” and ionic strength

fibri0.16.

6. At protein concentrations below 5%, transition from a fine to a coarse clot occurs over the pH range 8.6-6.0. 7. The presence of lipoproteins and other non-clottable globulins affects clot absorbance and measurable fibrin.

ISTRODUCTION In affect the strength, thrombin. were due

a recent report variations of physical and chemical factors were shown to absorbance of a plasma clot’. Among the parameters studied were pH, ionic temperature, and concentration of citrate ion, protein, fibrinogen and It was not apparent from absorbance measurements whether differences to altered fibrin structure or incompleted fibrinogen-fibrin conversion.

Morrison2 achieved complete clotting of concentrated fibrinogen fractions at pH 6.3 with a thrombin (units per ml) to fibrinogen (mg per ml) ratio of I to IO allowing 4 to 5 h for the reaction at room temperature. Saifer and Newhouse obtained complete yields from plasma with a ratio of 5 to I for I h at room temperature. Jacobsson reported complete yields with purified fibrinogen fractions using a ratio of IO to I at room temperature for z h. Others reported ratios of 35 to I for IO min5, and 7 to I for I he and for 2 min’. In this study, a ratio of about I to I is effective in Clin. Chim.

Acta,

18 (1967)

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ROSENFELD

2

min at 38” for test reaction mixtures representing plasma with up to 0.60% fibrinogen. This is similar to that found effective by Shinowara and Rostnfeld* for fibrinogen separated as Fraction I from titrated plasma. This study reports the striking effects of pH on clot absorbance and fibrin mass, when protein, citrate and thrombin concentrations are varied. The influence of lipoproteins and other globulin protein fractions on clot absorbance and measurable fibrin are also described. MATERIALS

AND

METHODS

The technics

for collection

of blood, the chemical

determination

of fibrinogen

and the spectrophotometric measurement of clot absorbance were described viouslyl. The preparation of citrate-phosphate buffer (pH 7.35, ionic strength and standard

fibrinogen

and thrombin

solution

were also described.

prc0.16),

Other reagents

are 0.08 M acetate buffer (pH 4.0), and 0.07 M phosphate buffer, pH 7.35 and 7.90. Human plasma was used throughout. Fibrin clots were analyzed by a micro-modification of the Folin-Ciocalteug reaction for tyrosine and are reported as clottable protein. The chromogenic factor for tyrosine in fibrin is taken as 11.0, and was determined gravimetrically. Absorbance measurements are usually made in IO min at room temperature (24’-26’), after mixing 0.8 ml of substrate with 0.2 ml of thrombin solution of specified strength in citrate-phosphate buffer’. Fibrin clots for tyrosine analysis for 15 min at 38” except when absorbance measurements are made. All experimental

are developed

pH values given are for the substrate.

RESULTS

PH, ionic strength and temperature The general effects of pH, ionic strength and temperature on the clotting process1F10-14 are to produce fibrin clots ranging between two extremes of opacity. Clots so formed were analyzed to determine whether variations in absorbance were due to altered fibrin structure or incompleted fibrin formation. Oxalated plasma was diluted with phosphate buffer (pH 7.9) and the pH varied by addition of dilutions of 0.08 M acetate buffer (pH 4.0). Four plasma dilutions ranging in pH from 7.85 to 6.05 were thus obtained. The clottable protein was unchanged. To vary ionic strength, 1.0 ml of oxalated plasma was diluted to 5.0 ml with phosphate buffer (pH 7.35) and water. Five different ionic strengths ranging from 0.056 to 0.158 were obtained. In another experiment, plasma was diluted withalbumin* solutions of o, I, 4 and 7% (w/v) protein concentration and clotted attemperatures of 5”, 15”, 25” and 38” (ref. I). There was no variation clottable protein. Eflects of pH and protein concentration The dependence of absorbance on pH, as protein concentration is varied is shown in Fig. I. Dilutions of titrated plasma with albumin solutions, and adjustments of pH, were made as previously described’. At pH 6.0 there is a marked increase in * Dilutions were made from 257; (w/v) normal buffer

(PH

7.35).

Clin. Chim. .4cta, 18 (1967)

I--IO

human

serum

albumin

with

0.07 JI phosphate

PLASMA

CI.OT ABSORBAKCE

% PROTEIN

Fig. I. Effect of protein concentration and of pH on clot absorbance (560 m,u). The numbers of the curves above refer to the substrate pH. At pH 6.0, the r-h reading is indicated by A and the Io-min reading by a. Additional conditions were 0.064% fibrinogen, and thrombin, 1.0 N.I.H. unit/ml reaction mixture. The ho-min readings at pH 6.8 to 8.6 are virtually identical with their respective Io-min values, and are not shown separately.

absorbance at protein concentrations of absorbance is slowed considerably, 60 min. Morrison2 reported a clotting described the transition from a coarse

below 5% protein. However, the development and maximal absorbance is not reached until delay at lower pH, and Ferry and Morrisonr” to a fine clot in the pH range from 6.2 to 7.2.

Despite the variations in absorbance, the clottable protein after all protein concentrations at each pH respectively is unchanged. Effect

IO

and 60 min for

of$H ami citrate Dilutions of oxalated

plasma with sodium citrate solution of various concentrations (o-100/0 (w/v)) and at diff erent pH were made and clotted as previously described’. In Fig. z are shown the effects of pH on the variable relationship between citrate concentration and clot absorbance for IO and 60 min of reaction. Analysis of clottable protein showed no effect by citrate on fibrin mass at either time interval for pH 7.37 and 8.37 despite some retardation of fibrin polymerization. Data for pH 6.05 is shown in Table I. The mass of clottable protein formed at pH 6.0 was also measured at the end of IO min, 60 min and 4 h of substrate reaction with 1.0,z.o and 5.0 units of added thrombin (Table II). At pH 6.0 there is a marked difference in absorbance and fibrin mass at IO and TABLE EFFECT

citvate

1 OF CITRATE ON ABSORBANCE

24bsorbance

(560

AND

MASS

OF CLOTTABLE

~____

mp)

Clottable

pvotein

PROTEIN

(mg/ml)

AT

pH 6.05

Clottable protein in supernatant (mg/ml)

M IO min

I h

4h-

IO milz

I h

4h

IO min

I h

4h

0.210

0.000

0.102

0.000

0.299

0.449

0.790

0.196

0.080

0.127

0.000

0.064 0.116

0.086 0.045 0.014 0.003

0.020 0. I99 0.274 0.274

0.192 0.256 0.269 0.289

0.158 0.207 0.256 0.269 0.289

0.0*9 0.354 0.814 0.922 0.939

0.572 0.810 0.939 0.977 0.981

0.675 0.834 0.955 0.957 0.961 ~-~

0.730 0.438 0.229 0.013 0.000

0.081 0.018 0.000 0.000 0.000

0.042 0.013 0.000 0.000 0.000

___

Clin. Chim.

Acta,

18 (1967)

I--IO

4

ROSENFELD

60 min, indicating a greatly slowed reaction. After 4 h, less than 50% ot the available fibrinogen has clotted in the presence of the high concentration of citrate ion. TABLE

II

THE EFFECT AT pH 6.06

OF

CITRATE

AND

THROMBIN

CONCENTRATION

ON

THE

MASS

Time

Citrate M

I

2

5

IO min

O.LIO

0.013

0.000

0.088

0.127 0.086

O.I2I

0.301

0.570 0.981 1.036 1.058

0.585 0.968

0.493 0.733 I.1222

0.585 0.785 0.992 1.089

0.748 0.895 1.008 I .056

I.135 I.133 0.873 0.990 1.067 1.104

I.078

I.102

I.102

I.082

I.133

I.124

0.210 0.127

0.086

I_

0.045 0.014 0.003

.-.

I.144 I.140

Effect of thrombin concentration and $H on clot absorbance Maximum plasma clot absorbance depends on thrombin results with 1.0 N.I.H. unit thrombin per ml reaction mini. To test whether the maximum is dependent on pH, fibrinogen concentration and varying pH were clotted of thrombin. Fig. 3 shows that maximum absorbance thrombin

CLOTT.IBLE

PROTEIN

Clottable $wotein (mg/ml) Thrombin (units/ml)

0.045 0.014 0.003 60 min

OF

over the entire pH range tested

concentration

and

mixture at pH 7.35 in ten plasma dilutions of constant by different concentrations was achieved with 1.0 unit

(6.12-7.86).

E$ect of thyombin concentration on formation of fibrin mass Volumes of titrated plasma at pH 7.35 were diluted to 0.8 ml with citratephosphate buffer and clotted with 0.2 to 5.0 N.I.H. units thrombin. The data in Table III point up the limitations to the enzymatic nature of the thrombin-plasma reaction. The action is not always complete and depends on the relative concentrations of thrombin enzyme and fibrinogen substrate. For each thrombin concentration tested, there is an upper limit of fibrinogen above which complete (> 95%) conversion to fibrin does not occur. In 1.0 ml of reaction mixture, one N.I.H. unit completely clotted about 1.17 mg (3.4 nmoles). The molecular weight for human fibrinogen

is taken

as 340000

(ref. 15).

Effects of albumin and other sewm proteins on apparent jibrin mass Plasma fibrinogen was clotted in the presence of known amounts of added proteins, and their effect on fibrin clot analysis and clot absorbance was measured. Two-hundred and fifty mg each of human protein Cohn Fractionsla, II (y), III-o (P-lipoprotein), IV-I (a-globulin), IV-4 (a- and /I-globulins) and V (albumin, recrystallized), purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio, were dissolved in 5.0 ml of 0.07 M phosphate buffer (pH 7.35). These were dialyzed overnight at 5” against the phosphate buffer, then centrifuged to remove trace amounts of insoluble protein. Only Fractions II and V contained no other electrophoretic components. Clottable protein was absent from all fractions. Clip.

Chim.

Acta,

18

(1967)

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PLASMA CLOT ABSORBANCE TABLE

5

III

EFFECT OF

THROMBIN

CONCENTRATION

ON

YIELD

OF

FIBRIN

MASS

Initial concentration per ml

Clotted protein

Thrombin 1V.I.H. units

mpmoleslml

o/O of substrate

I.59

102

0.2

0.5

1.0

Fibrinogen

mpmoles 1.56 2.33 2.63 3.13 3.90 4.62 0.54 I.09 1.64 2.17 2.41 2.96 3.62 4.08 4.83 6.04 7.24 8.12 I.09

1.64 I.93 2.04 2.42 2.72 2.93 3.40 3.61 3.89 4.85 6.04 7.25 8.19 2.0

1.09 1.62 2.43 3.64 4.09 4.84 6.05 7.25 8.10

5.0

0.54 1.08 1.64 2.03 2.43 2.72 3.00 4.06 4.82 6.11 7.25 8.14

2.10

2.26 2.41 2.69 3.77 0.55 1.09 1.62 2.17 2.46 2.72

90 86 77 69 60 IOI 100

99 100 102

3.33 3.51 3.86

92 92 86 80

4.41 4.56 4.86

73 63 60

1.09 1.62

100

I.93 2.04 2.42 2.72 2.87 3.23 3.43 3.62

99 IO0 IO0 100 100 98 95 95 z 80

4.27 4.83 5.44 5.73

75 70

1.09

100

I.59 2.38

98 98 98 98 91 81

3.57 4.01 4.40 4.90 5.44 5.83 0.56 1.07 I.57 I.97 2.38 2.67 3.06 3.86 4.43 4.95 5.14 5.78

75 72 IO3 99 96 97 98 98 102 95 92 81 75 71

Clin. Chim. Acta, 18 (1967) I-IO

6

737 786

I

I

0

0.05

0.10

M CITRATE

0.15

IN REACTION

0.20

MIXTURE

I

_~_L_

c

10

UNITS

20

THROMBIN

72

io

PER ML REACTION

5’0 MIXTURE

Fig. L. Effect of citrate concentration and of pH on clot absorbance (560 m,/~).The numbers of the curves above refer to the substrate pH. All reaction mixtures contained thrornbin, 1.0 N.1.H. unit/ml. Absorbance readings were made under the following conditions: pH 0.04, O.IOL”~, fibrinogen; pH 7.37, 0.087qa fibrinogen; pH 8.37, o.rozq/, fibrinogen. l IO min; 00 min. Fig. 3. Effect ot thrombin concentration and of pH on clot absorbance (560 m/l, IO min). The numbers of the curves above refer to the substrate pH. -411 reaction solutions contain4 o. ICY”,, fibrinogen.

One volume of titrated plasma adjusted to pH 7.35 was mixed with two volumes of each reconstituted fraction: undiluted, ~1~diluted and 11~ diluted with the phosphate buffer. The mixtures were clotted with 1.0 N.I.H. unit thrombin and the absorbance was read. The clots were removed, washed, and analyzed for fibrin’. The protein fractions and the plasma were analyzed for total protein by the biuret reaction. Correction was made for the dilution of the thrombin solution, but not for the negligible protein content of the thrombin. The control reference consisting of one volume plasma and two volumes phosphate buffer was similarly treated and the apparent increase in clot analysis was determined. The percentage of occluded protein responsible for the fibrin error was calculated by dividing the percentage increase in apparent fibrin by the ratio of added protein to fibrin. This data appears in Table IV. The amount of occluded protein varies widely with the nature of the protein added but is proportional to the amount added. Fractions IV-4 and V are not occluded at all, and Fraction II only at moderately high concentration. Although a greater range of added protein was tested in this study, the findings are in general agreement with Morrison2 who clotted concentrated fibrinogen fractions.

of

globulins on clot absorbance Absorbance measurements of plasma clots were found to be markedly affected by the nature and concentration of the added protein fraction, as shown in Fig. 4. Effect

Clin.

Chzm. Acta,

18 (1967)

I-IO

PLASMA

CLOT ABSORBANCE

ThBLE

IV

THE

OCCLUSION

OF

PLASMA

7

PROTEIN

FRACTIONS

Protein concentration

FVaCtiOn

II

111.0

IV-1

IV-4

1.

BY

(“/b)

Increase in apparent fibrin f 96)

.4 dded

Total*

2.73

4.26

12.5

1.82 0.91

3.36 2.45

6.1 -o.2

1.19 0.79 0.40 1.89

2.72

1.26 0.63 2.11 1.4’ 0.70

2.32 I.93 3.42 2.79 2.16 3.64 2.94 2.24

2.19 1.46 0.73

3.72 2.99 2.26

in reaction

solution

1

0

I %

=

Plasma

I

I

2

3

protein

Ratio of added protein to fibrin

Occluded protein (%)

29 20 IO

0.4 a.3 0.0

16.6 II.4 5.8 43.3 26.5 16.6 0.1 -o.2 -0.7

13 8 4 20

I.3 I.4 1.5 2.2

14 7 23 15 8

1.5 - I.0 - 1.9

24 16 8

I.9 2.4 0.0 0.0 -o.1 0. I -o.1 -o.2

The constant conditions of each of the 15 solutions unit/ml; pH 7.35; 24’-26’; ionic strength 0.15. * Total

FIBRIN

were 0.093%

~~ Added

fibrinogen;

thrombin,

1.0 N.I.H.

fraction.

1

4

PROTEIN

Fig. 4. Effect of protein fractions on clot absorbance (560 mp, IO min). The numbers of the curves above refer to the Cohn Fraction added. Additional conditions were: o.103~/0 fibrinogen, and thrombin, r.o N.I.H. unit/ml reaction mixture.

The graphs radiate from a focal point representing zero concentration of added protein fraction. Since we can correlate increased clot absorbance with decreased clotting timelo, the data of Fig. 4 agrees with Shinowaral’, who reported the effects on clotting time of similar protein fractions. Occlusion of plasma non-clottable proteins To determine the extent of this occlusion, 0.2 ml of oxalated plasma was diluted to 0.8 ml with varying volumes of normal serum and citrate-phosphate buffer, and C&n. Chim. Acta,

18 (1967)

I-IO

S

ROSENFELD

then clotted with 1.0 N.I.H. unit thrombin. The percentage increment to the fibrin analysis ranged from O.OOO~/~ to 0.024% in three experiments with plasma fibrinogen levels ranging from 0.328% to 0.540%, and were not proportional to the amounts of serum added. This narrow range probably reflects the analytical error of a constant occlusion. Occlusion was not dependent on the fibrinogen concentration either, an observation also made by others 2- 4. The average increment for the three experiments was o.orro/,, representing an error of 2.00/, 2.80/, and 3.4% respectively. This compares favorably with the r.g”/b reported by Jacobsson *. Errors due to occlusion of serum proteins have been reported by others as 5% (ref. 5), 157,; (ref. 2), and 2096 (ref. 3). DISCUSSIOK

As pH decreases toward the isoelectric point repulsion between fibrinogen molecules is sufficiently

of fibrinogen, the electrostatic decreased to permit formation

of larger aggregates resulting from side-to-side alignment. At pH 6.0, this type of grouping forms more readily at low than at high protein concentrations probably owing to the physical impediment of the protein molecule. While pH is the most important factor influencing fibrin clot structure, it has no influence on formation of fibrin mass. Increases in fibrin yield at higher pH values reported by Morrison2 and by Saifer and Newhouses were attributed to the probable occlusion of non-clottable protein. Although variations in ionic strength, temperature, pH and protein concentration affect absorbance, fibrin formation is complete. In mixtures with added citrate ion at pH 7.37 and 8.37 changes in absorbance suggest polymer growth and continuing realignment of insoluble fibrin polymer taking place after IO min rather than formation of new insoluble protein, and that the citrate interacts primarily with fibrinogen. The inhibitory effect of citrate ion in diminishing absorbance is due to binding by the fibrin monomer and the resulting steric blocking of the polymerization sites as described13yls for neutral polyhydric alcohols. Formation of an opaque clot should be enhanced by conditions which favor lateral aggregation, such as slow rate of formation and low pH (ref. IO). Both these conditions are in effect in the retarding action of citrate at pH 6.0. The citrate ion delays but does not inhibit clotting, and thus ma? be classed a “retarder” according to Shulman la. At pH 6.0 there is a greater degree of dissociation of citrate ions and the resultant increase of ionic strength enhances the binding effect of the increasing citrate concentration. The effects of thrombin and pH on fibrinogen showed that what favored occlusion also favored formation of a “fine” clot, thus indicating that physical entrapment of large and asymmetric molecules in the smaller interstices of the fine clot was largely responsible for occlusion 2. The percentage of occluded protein reported here for plasma fractions and serum are considerably less than that found by Morrison at pH 7.2. However, Morrison estimated fibrin gravimetrically, and included lipid material. From the data presented here, occlusion would not be a serious problem except in lipemic specimens4 or plasma with high lipoprotein content2. The technic for syneresis and washing the fibrin clot free of serum proteins may be a critical step in the analysis of fibrin. Morrison2 and other+13 allow the liquid Clin. Chim. Acta,

18 (1967) I--IO

PLASMA CLOT ABSORBANCE

of the clot to be passively or pressure

contact

9 absorbed.

A more forceful expressing

of a glass rod496 or applicator

of fluid with weight%7

stick1 is probably

more effective

in minimizing occlusion or retention of non-clottable protein. Jacobsson stresses the importance of complete syneresis rather than prolonged washing to minimize occlusion. Small amounts of thrombin will initiate clotting of large amounts of fibrinogen, however, this enzymatic action goes to completion only when added thrombin and plasma fibrinogen concentrations are within certain limits. Above these limits, greater amounts but not all the fibrinogen will be clotted. This agrees with the theory of the action of thrombin to release available sites for subsequent polymerizationls and clotting of fibrin monomer. If too great a concentration of fibrinogen is present, the molecules are so closely packed that even high concentrations of thrombin cannot have access to the available bonds. In addition, molecules of fibrin monomer that are formed cannot approach one another for cross-linking. Morrison2 used 0.05 to 0.20 unit thrombin for complete clotting of purified fibrinogen solutions. However, because of the presence of antithrombic activity in plasma the thrombin concentration cannot be precisely defined and a greater amount of thrombin is needed. The data of Table III shows there is no effective increase in the mass of fibrin formed beyond a thrombin concentration of 2.0 units per ml. This is in close agreement with the 3 units reported by Saifer and Newhouse and 4 units reported by Jacobsson4. Although 1.0 N.I.H. unit thrombin is optimal for maximum absorbance, its effectiveness for completely clotting fibrinogen has an upper limit of fibrinogen concentration. Attempts to quantitate the sol-gel transformation of fibrinogen to fibrin by absorbance measurement should take advantage of this optimum, but be cognizant of its limitationsZO. The results of this and previous report’ show that many physical and chemical parameters are in operation during the thrombin-plasma reaction to affect the estimation of two end-of-reaction products: clot absorbance and insoluble fibrin polymer. ACKNOWLEDGEMENT

The author wishes to express his appreciation to Mrs. Gloria Feig for her technical assistance, and to Professor George Y. Shinowara for his helpful suggestions, valuable criticisms, and the general facilities of his laboratory. This investigation was supported in part by the Health Research Council of the City of New York, Contract No. I-146 and by Grant No. H-4843 from the National Institutes of Health, Bethesda, Md.

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IO 7 H. 0. BANG, Scand. J. Clin. Lab. Invest., g (1957) 205. 8 G. Y. SHINOWARA AND L. ROSENFELD, .I. Lab. Clin. Med., 37 (1951) 30.3 g 0. FOLIN AND V. CIOCALTEU, J. Biol. Chem., 73 (1927) 627. IO J. D. FERRY AND P. R. MORRISON, J. Am. Chem. Sot., 6g (1947) 388. II S. SHULMAN AND J. D. FERRY, J. Phys. Colloid Chem., 54 (1950) 66. 12 J. D. FERRY AND S. SHULMAN, J. Am. Chem. Sot., 71 (1949) 3198. 13 J. T. EDSALL AND W. F. LEVER, J, Biol. Chem., 191 (1951) 735. 14 M. BURSTEIN AND J. LOEB, Rev. He’matol., 7 (1952) 385. 15 E. A. CASPARY AND R. A. KECKWICK, Biochem. J., 56 (1954) xxxv.

16 E. J. COHN, L. E. STRONG, W. L. HUGHES, Jr., D. J. MULFORD, J. N. ASH!I.ORTH, 41. MIXIN AND H. L. TAYLOR, J. Am. Chem. SW., 68 (1946) 459. 17 G. Y. SHINOWARA, J. Lab. Clin. Med., 34 (1949) 477. 18 S. SHULMAN, Discussions Faraday SOL., 13 (1953) Iog. 19 H. A. SCHER~ZGA,in N. 0. KAPLAN (Ed.), MolecularBiology, Vol. I, Academic I’ress, New York, 1961, p. 129. 20 L. ROSENFELD, J. Lab. Clin. Med., accepted for publication (1967). Clin.

Chim.

Acta, 18 (1967) I-IO