THROMBOSIS RESEARCH 64; 455-476,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.

STUDIES ON FIBRIN NETWORK STRUCTURE IN HUMAN PLASMA. PART ONE: METHODS FOR CLINICAL APPLICATION

C.H.Nair, A.Azhar and D.P.Dhall Royal Canberra Research Unit, Thrombosis and Vascular Hospital (South),PO Box 11, Canberra, A.C.T., 2606, Australia.

(Received 28.1.1991; accepted in revised form 14.6.1991 by Editor J. Soria) (Received in final form by Executive Editorial Office 4.9.1991)

ABSTRACT on turbidity and permeability, for Methods based fibrin fibres ratio of measurement of mass-length solution, have been fibrinogen developed in pure to human of their applicability evaluated in respect plasma. Theoretical assumptions made in the calculation been critically mass-length ratios in plasma have of reproducibility examined. Methods of handling plasma, influence of age and sex have and the of technique used, as well as The anticoagulant been investigated. other factors such as time, venepuncture and effects of inhibitors are fibrinolytic fully calcium and suitable standardization the methods explored. With acceptable for application to clinical studies and are are reproducible.

LNTRODUCTION Although fibrin networks have been extensively investigated since the pioneering work of Ferry and Morrison in 1947 (l), much of the work has concentrated on the biophysics of network development. Investigations into the role of fibrin network structure in haemostatic disorders, in thrombosis and other clinical areas have been hampered on account of lack of suitable methods for study. Recently introduced methods allow characterization of fibrin networks through measurement of mass-length ratio from turbidity and permeability of fibrin clots. The mass-length ratio which is determined from turbidity is designated uT, while permeability of the network as a whole is designated by T; (2,3). Key words: Human Plasma.

Fibrinogen,

Fibrin

455

Fibres,

Mass-length

Ratio,

456

FIBRIN NETWORK STRUCTURE I

Vol. 64, No. 4

It is now known that in any given network, fibre thicknesses and fibres are divided into a major are not homogeneous network composed of thicker fibres and a minor network occupying the interstitial spaces between major network fibres Although mass-length ratio and permeability (4). determinations are said to be strictly applicable to networks developed in dilute fibrinogen solutions, it has been shown that both techniques are valid for comparative investigations in fibrinogen at concentrations similar to those in plasma (5). However, several consistent and pronounced differences have been shown between networks developed in fibrinogen solution and those in plasma (6). It is, thus, necessary to establish whether these methods are applicable to networks developed in plasma or whether they require modification for such an application. Theoretical validity for the application of these methods to investigations in plasma, recovery and reproducibility as well as the roles of anticoagulant, fibrinolytic inhibitors and calcium have been examined and the methods assessed for their applicability to clinical studies. MATRRlATaS AND METHODS Venous blood was collected using atraumatic venepuncture and mixed with 3.8% trisodium citrate in the ratio 9:1, and 35KIU/ml Trasylol (Bayer, Germany). The subjects had fasted overnight to avoid interference of turbidity measurement by abnormally elevated plasma lipids. The blood was centrifuged (2,400 x g) at room temperature for ten minutes to obtain platelet-poor plasma which was recentrifuged at 4,000 x g for five minutes to obtain essentially platelet-free plasma (PFP, platelet count < 3OOO/pl). The concentration of fibrinogen in plasma was determined according to Ratnoff and Menzies (7). The refractive index of plasma was measured with a Model 64420 Carl Zeiss refractometer (Germany). Networks were developed at 22" t 2°C by the addition of bovine in plasma pre-mixed with a e Davis, U.S.A.) thrombin (P The fibrinogen (Amersham, U.K.). trace of 1% I-labelled percentage of fibrinogen converted to fibrin was determined from the amounts of labelled fibrinogen in the perfusate (vide infra) or serum (6). Technique 0.9ml of platelet-free plasma was pipetted into cuvettes of O.lml of the appropriate thrombin was added, lcm path length. were and the solution stirred with a glass rod. Networks 22°C (22O ?r 2°C). develop at room temperature left to was adopted as the temperature for all experiments because preliminary studies showed that measurements made at 37°C were development of inaccurate on account of rendered often bubbles. Furthermore, it was found that in the range 22°C 37°C the fibre diameter was relatively temperature independent (8). Optical density at 8OOnm was recorded continuously (using unclotted plasma in the reference cell) with a Unicam SP 1800 spectrophotometer (Pye Unicam, U.K.), or a Hitachi U3200

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FIBRIN NETWORK STRUCTURE I

457

spectrophotometer (Hitachi, Japan). After optical density had plateaued, turbidity (2.303 x optical density) was measured at wavelengths between 600 and 8OOnm. on Te&n.lque cut from 2ml glass Fibrin networks were formed in glass tubes in 1% which had been pre-etched by immersion pipettes, hydrofluoric acid for 48 hours. Each tube was 1OOmm in length internal individual internal the and 3mm in diameter: Clots were dimensions of each tube were carefully measured. formed in tubes with bottom end covered with 2 layers of Parafilm (American Can Co., Greenwich, U.S.A.). Networks were allowed to develop for a time corresponding to that required for turbidity to plateau in similar networks (see above) by adding 89ul of the appropriate thrombin to 0.8ml of plateletfree plasma. The tube containing the clot was then attached, using plastic tubing, to a horizontally mounted lml pipette on The network was perfused with Tris HCl a retort stand. saline buffer of pH 7.35, ionic strength 0.153, at a pressure Care was taken to head of less than or equal to 150mm water. increase the pressure head gradually in steps of three minutes by 50mm water. The transit time of an accurately measured volume of perfusate was then recorded. The initial 0.05ml of perfusate was collected to allow calculation of the fibrin content of the network. The conversion was also measured by crushing clots with a wooden spatulum in the spectrophotometer and measuring the radioactivity in 0.05ml of microcuvettes This was expressed as a percent ratio of the expelled serum. initial plasma radioactivity. Networks developed in plasma are much more permeable than those developed in fibrinogen (6). These networks collapse in permeation tubes even at low pressures (100 - 150mm H20). the inside of the This can by precoating be overcome permeation tube with a thin layer of fibrin, as follows: O.l5u/ml thrombin was added to a solution of fibrinogen at a concentration of 2.5mg/ml, ionic strength 0.153, pH 7.35. The mixture was then flushed through the permeation tubes, and the tubes left to stand for one hour at room temperature to ensure maximal conversion of fibrinogen to fibrin. From an initial volume of 0.80m1, only 0.03ml of solution was left inside the tube (n = 9, SEM = 0.003). The resulting thin adhesive layer of fibrin gel does not significantly alter the permeability of the network. Furthermore, the total amount of thrombin (0.0005 units) in the tube is an insignificant fraction of that normally used to clot plasma (l.OOu/ml) and may be ignored. In each experiment at least four replicate measurements were made with the permeability technique and duplicate measurements with the turbidity technique.

Compaction is based on the method of Dhall et al (9). Fibrin networks were formed in 1.5ml Eppendorf microcentrifuge tubes, In-e-sprayed with a lecithin-based aerosol (Spray and Cook,

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Woolworths) to render the surface non-adhering. The clots were centrifuged at 8,000 x g for 45 seconds in the Eppendorf Zentrifuge, model 3200 (Germany), after they had been allowed to clot for a period of time to correspond to that required to reach plateau in the turbidity curve. The volume of fluid expelled from the network was measured with a lml syringe, and expressed as a percentage of initial volume. ERCGVRRY,

RFPRGDUCIBSJ~JTY AND HANDJlUJG

Replicate measurements, six in turbidity studies, eight in compaction technique and ten in permeation method, were made immediately in each of plasma samples obtained from five separate donors. Effect of Calcium. 1.

Conversion

of fibrinogen to fibrin.

An appropriate number of samples of 0.45ml of platelet-free plasma was clotted with 0.05ml of appropriate thrombin in lml Eppendorf centrifuge tubes. Conversion of fibrinogen tp fibrin was measured by counting radioactivity in fluid expelled from the networks squeezed with a wooden spatulum at 5, 10, 15, 30 and 60 minute intervals. Each measurement was performed in duplicate. Networks were formed w$$h either 0.25 or 1.5u/ml thrombin and with or without 25mM Ca . ii.

Fibrin Network Characteristics.

Conversion

of

fibrinogen

to

fibrin, concentration of fibrin compaction were determined in %%k?to;:$'w:& and without 25mM Ca2+. Thrombin concentration used was lu/ml. This final concentration was used so that sufficient time was available to allow proper mixing of thrombin and plasma to develop a homogeneous network. ‘c and

and Dav To Davm

.

.

(with Calcium) .

60ml of blood collected from each of thirteen healthy donors (aged 15 to 47 years) was divided into two aliquots in 50ml test tubes. The first 30ml was centrifuged to obtain plateletpermeability and plasma as described above. VT, poor tube determined. The second test compaction were then left to room containing titrated blood was stand at the same procedure was for one hour before temperature Blood was then collected from the same donors 24 repeated. hours later and the whole procedure repeated to investigate day to day variations. of Plasma ande

Blood

Pools of platelet-free plasma obtained from 40 healthy donors were stored in 30ml aliquots in six 50ml test tubes (3 test Measurements of uT# pool of plasma). for each tubes

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FIBRIN NETWORK STRUCTURE I

459

permeability and compaction were made on fresh pooled plasma then after storage at -70°C for 24 hours. Plasma and and whole blood collected in sodium citrate were stored at 4°C for 24 hours and the appropriate indices measured. .

.

.

Effect of F-tic

.

Inh;Lbitor

To investigate the effect of fibrinolytic inhibitor, 35~1 of Trasylol (Aprotinin) (Bayer, Germany) was added to lOm1 blood, at a final concentration of 35 KIU/ml. In control experiments 35~1 normal saline was added in place of Trasylol. Platelet free plasma was then prepared from both the test and the control samples of blood and uT determined.

Effect of varying citrate concentration was investigated by collecting blood in a 9:l ratio in O.lOM, O.l3M, and 0.16M Platelet-free plasma was then prepared trisodium citrate. blood and from each of the three samples of and uT permeability determined. These studies were conducted without the addition of calcium. ct of Aae and Sex Forty-nine healthy subjects (32 males and 17 females) were investigated for the effect of age and sex on uT, r and of the blood donors informed consent compaction. From each was obtained for these investigations. . . Coefficients of Correlation and Derivations.

Between Various Measurements

and fibrinogen T;r 1-1Tr compaction, network fibrin content (CN) measured in a group of 49 concentration were derived or healthy subjects. Coefficients of correlations were calculated for the various permutations of relationships amongst uT, t, and fibrinogen conversion. Unpaired t-tests compaction, CN were used to investigate differences in means of different populations. Statistical analysis was performed using NSW Statpak (Northwest Analytical, Inc., Portland, Oregon, U.S.A.) THEORETICAL .

CONSIDERATIONS

.

iditv Technique According to Carr and Hermans (2), the turbidity of clots developed in dilute fibrinogen solutions ( I 1.00 mg/ml ) is given by the equation : T = (44/15)mKcXuT/n

(1)

where c is the fibrin content, A is the wavelength, n is the refractive index of the solution and uT is the mass-length ratio of the fibres. The constant K is given by: K = 2rt2n(dn/dc)2/Nah4

(2)

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FIBRIN NETWORK STRUCTURE I

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where dn/dc is the specific refractive index increment of the solute in the solvent and N is Avogadro's number. quations 1 and 2 imply that the turgidity should vary as l/x3 if the slight wavelength dependence of n and of dn/dc is ignored. Indeed, networks developed in dilute fibrinogen solutions under certain conditions do satisfy this relationship (2). In this event, pT can be computed at any given wavelength by use of equation 1. When the clotting conditions are altered to favour the formation of very turbid networks, the above-mentioned rel tionship is not satisfied. Plots of turbidity against 3 for networks developed in fibrinogen solution containing l/A 1.50 to 5.40 mg/ml of fibrinogen (final thrombin concentration was 1.50 u/ml; pH 7.35; ionic strength 0.153) have been previously shown by Shah et al (5). As fibrinogen concentration is increased, the relationship becomes increasingly curvilinear. However, at a fibrinogen concentration of 1.50 mg/ml the curve extrapolates to the origin so that u can be obtained by using the following relationship (2): (44/15)nKcX/nT

= (1+184n2p2n2/77A2

. ..)/u

(3)

Thus, in plots of c/TA3 as a function of l/h2 the intercept, A, can be used to calculate uT (1) according to the equation: uT=

10 x 1012 daltons/cm 1.48A

(4)

Unless otherwise stated, turbidity is measured at a range of wavelengths between 600 and 800 nm. uT is derived from equations 3 and 4. The higher wavelengths are recommended (as against wavelengths between 350 and 600 nm) because the errors due to non-compliance with theory are minimised (10). the turbidity versus In the event that l/h3 curves do not extrapolate to the origin, computation of pT from equations 3 Nonetheless, it has been shown that such and 4 is not valid. derivations of uT may be used as highly acceptable values (6). 1 shows the envelope of plots of turbidity versus l/A3 Figure bank donors, developed in plasma of blood for networks preoperative laboratory workers and healthy apparently patients admitted for elective abdominal surgery. These curves failure to their and similar in curvilinearity are extrapolate to the origin - to those observed for networks solutions with comparable in purified fibrinogen developed fibrinogen concentrations (5). The mean refractive index of plasma in 17 samples was 1.3450 ( 1SD = .0030 ) and was 1.3370 in fibrinogen solution containing 2.50 mg/ml of fibrinogen. Thus, the error associated with using the refractive index of fibrinogen solution instead of plasma, and that associated with variation between plasmas is less than 1% and may be ignored. Hence, the derivation of uT using equations 3 and 4 appears to be as valid in plasma as it is in fibrinogen

Vol. 64, No. 4

FIBRIN NETWORK STRUCTURE I

461

solution.

FIGURE 1 for networks Envelope of plots of turbidity against 1/X3 developed in human plasma (n = 17). Curves in these plots are similar in their curvilinearity and relationship to the origin purified fibrinogen networks developed in for those as solutions with comparable fibrinogen concentrations (5).

Permeation

Technique

In networks developed in fibrinogen solution the permeability or Darcy constant is calculated from the equation: r = Qlh/Ftp

(5)

where Q is the volume flow in time t; 1 is the viscosity of the liquid; h is the length of the clot, F its cross-section and p the applied pressure (3). pp or mass-length ratio from permeability may be derived from permeability according to Carr and Hermans (3). However, this derivation is based on a packing constant of 10 which introduces and error in the computation of uP. This aspect will be discussed in another paper. Compaction

Techniaue

Theoretical background and quantative aspects of this technique will be separately described (12). It is generally

FIBRIN NETWORK STRUCTURE I

462

Vol. 64. No. 4

accepted (11) that basic determinants of compaction are (its time, centrifugal distance related to centrifugal field and force) on the one hand and particle characteristics on the other (surface area, volume ratio, buoyancy and density). In a constant centrifugal field compaction is dependent mainly upon particle characteristics. Major perturbations from this are caused by the quality and degree of crosslinking within a fibrin network. Previous experimental studies (9) have shown compaction correlates with that inversely tensile fibrin characteristics of Young's modulus of such as elasticity and strength at break (Figure 21, both measured using an Instron. However, this method of examining tensile characteristics requires large volumes of plasma and access to sophisticated equipment. With compaction, precise calculation of Young's modulus of strength at break - accepted indices for tensile strength of a variety of materials - is not possible. Nonetheless, since the correlation is close compaction gives an overall index of tensile behaviour in fibrin network. This in all probability is a index in more appropriate relationship to the network capacity of compression or stress in the vascular system modulus of elasticity, or strength at break.

to withstand than Young's

S T

\

25 -

*

2I

1,5' 1

I

I

t / /IIll

I

/

/ IIII

10 % -

YOUNG’S

,2 100

COMPACTION

MODULUS

*

STRENGTH

AT BREAK

FIGURE 2 Correlation of compaction against measured Young's modulus of Compaction is elasticity and against final strength at break. inversely proportional to the Young's modulus of elasticity of the network and to the final strength at break, determined from load elongation curves of fibrin strips.

FIBRIN NETWORK STRUCTURE I

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Recovery

463

Wad&s_

Recoveries of uT, r and compaction are shown in Table 1. Coefficient of variation is acceptably small in turbidity measurements and there seems little merit in making more than two measurements for each test. Permeability of networks on the other hand is subject to a larger variation. To minimize were made in quadruplicates. Although error, measurements in compaction measurements had a smaller recoveries of variation than permeability, coefficient at least four observer were made in each test to minimize measurements error. TABLE 1 RECOVERIES

IN PLASMA: VT, r AND COMPACTION

1-1T

t (x1011cm2)

Compaction (%I

(Za~~Zs/cm) 14.08

417

21

n=3

n=5

n=5

Coefficients of Variation (%) 3

6.5

1.8

Table 1 shows recoveries in plasma of mass length ratio from turbidity (uT), permeability (r) and compaction. As shown, coefficient of variation in recovery measurements are small in turbidity and compaction, and comparatively larger in the permeability. These experiments were performed without calcium. Effect of CalConversion of fib inogen to fibrin in networks developed in the presence of Ca?2+ was approximately 92% irrespective of the thrombin concentration used (Figure 3). Without calcium, however, while conversion was dependent on the concentration of thrombin even with 1.5 u/ml thrombin total convers'on after 1 hour was only around 75%. In the presence of Ca $+ however, the conversion was increased and became relatively thrombin-concentration independent.

FIBRIN NETWORK STRUCTURE I

Vol. 64, No. 4

100% I,~,_,_, _ __ __ __ _.__..__._.._ Q._____.-._._.

~;~l_I-_8=_:~,sit,~,~~~,~~~~~~~,~,~,~

_._._-.-.Q

_‘__._‘._.~_“_._“_.._’

,_,_,___.___._._,_._

90 -

80 -

E

”

”

”

K

._

f

70-

2 s

60-

--_c-------_--_c-_--__-_-____-_-

8 50-

J

40

1 0

I 5

1 10

I 15

I

I

I 30

t

I

I

I

1

I 60

Minutes

FIGURE 3 Conversion of fibrinogen to fibrin 'n networks developed with (+.-.-•) and without (+-s-*) 25mM Ca ++ with 0.25u/ml thrombin, and with (o-. ._o) and without (B-PC ) 25mM Ca2+ with 1.5u/ml thrombin. Conversion of fibrinogen to fibrin is maximal concentration when networks are irrespective of thrombin thrombin-concentration with calcium, but is developed dependent in its absence. While uT (Table 2) was higher in networks developed with Ca2+, variation for invariant. Coefficients of 'c remains permeability and convession were lower in networks developed It was also observed that collapse in the presence 0fCa . after which can occasionally occur even networks, of precoating of permeability tubes with a thin layer of fibrin, was more or less eliminated in the network developed with 25mM Collapse was more of a problem with lower calcium or calcium. higher citrate concentration (vide infra).

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FIBRIN NETWORK STRUCTURE I

465

TABLE 2 Effect of Calcium on Fibrin Network Characteristics. Without Indices PT

19.90

r

452

C.V. (%)

Indices

(7.00)

26.70

(0.40)

1.49

n

With 25mM Calcium

(14)

8 Conv. 75 CN

Calcium

(0.01)

C.V. (%) (6.00)

453

(7)

90

(0)

1.76

(0.02)

6

6

Permeability (r) and mass-length ratio (uT) show and higher small coefficients of variation (C.V.) conversion when networks are developed with 25mM Final thrombin concentration in both cases calcium. was 1.0 u/ml. Note, when calcium is added coefficient of permeability is halved.

of variation

uT is expressed in x l$)t" d3ltons/cm. r is expressed in x 10 cm . Network fibrin content (CN) is in mg/ml.

.

.

R-roduubLa&ay

.

.

.

to Day Varlatlon.

measured within 20 minutes were not ‘c and compaction significantly different from the values obtained from plasma prepared from blood which is stored for one hour on a bench at room temperature after venepuncture (Table 3). These values were also very similar to those obtained in plasma made from freshly obtained blood from the same subject by venepuncture a day after the initial investigation. Fibrinogen concentration and fibrin conversion also did not change significantly. In contrast to the values obtained without calcium addition (Table 2), co 2yersion values were near maximal (91%) in the presence of Ca . I-y-I

466

FIBRIN NETWORK STRUCTURE I

TABLE 3 Effect of a Delay of One Hour in Blood Processing Day to Day Variation with 25mM Calcium.

Vol. 64, No. 4

and of

BLOOD PROCESSED Without Delay

After 1 Hour

Day 1

T

383 (23)

368 (23)

381 (29)

u'T

21.54 (1.48)

21.39 (1.24)

Compaction

$6)

31 (0.6)

CN

2.12 (0.14)

2.17 (0.13)

20.46 (1.11) 31 (0.6) 2.17 (0.13)

SEM is shown in parenthesis.

The measurement of permeability (r), masslength ratio from turbidity (uT), compaction fibrinogen and conversion are not altered significantly in thirteen subjects when anticoagulated blood is left for one hour at room prior to temperature preparing platelet-poor plasma by centrifugation. zlasma was clotted with the addition of 25mM Ca + and lu/ml thrombin. Permeability, mass-length ratio compaction and CN when measured (VT), next day are in the subject the same constant reproducible showing relatively characteristics from one day to the individual fibrinogen conversion is and next, the maximal (91%) in contr%s+t to < 75% in nearly experiments performed without Ca (Table 2).

Network Fibrin content

. . ect of Flbrlnolvtlc

.

(cN)

is in mg/ml.

.

Table 4A shows the effect of Trasylol on mass-length ratio (uT) and permeability (T). The presence of 35KIU/ml of Trasylol does not affect mass-length ratio and permeability Thus, blood was always collected into citrate significantly. containing this concentration of Trasylol to avoid spontaneous Table 4B shows the effect of Trasylol on fibrinolysis. fibrinogen conversion and fibrin content of the network (CN).

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FIBRIN NETWORK STRUCTURE I

467

Trasylol does not affect the conversion of fibrin or the amount of fibrin in the network.

fibrinogen

to

TABLE 4A Effect of Trasylol on uT and Permeability

(r) 'I:

Expt.No. daltons/cm) Control

(xlOllcm2)

Test

Control

Test

1.

15.79 (0.11)

16.56 (0.3)

332 (57)

356 (21)

2.

17.31 (0.31)

16.73 (0.25)

417 (38)

429 (42)

3.

20.53 (0.43)

19.59 (0.29)

330 (29)

345 (24)

SEM (n = 5 for each experiment) is shown in Differences were not significant parenthesis. according to the paired t test. TABLE 4B Effect of Trasylol on Fibrinogen Conversion

and CN

% Conv.

CN (mg/ml)

Control

81 (1)

1.87 (0.04)

Test

82 (.06)

1.90 (0.008)

Significant differences were not observed when fibrin developing Trasylol is added to networks were developed in networks. These the absence of calcium. Means (SEM) of three experimants are shown.

Effect of Anticoagulants Preliminary experiments showed that EDTA and Heparin were not suitable anticoagulants; networks developed in plasma in which these were used as anticoagulants consistently collapsed during permeation. In addition, when Heparin was used as the anticoagulant conversion of fibrinogen to fibrin varied widely

FIBRIN NETWORK STRUCTURE I

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presumably depending from one plasma to the next, antithrombin III concentration and other plasma factors.

upon

Fffect of Citrate Concentration The effect of varying concentration of citrate on t and uT is shown in Table 5. r and uT both decrease with increase in larger coefficient of citrate concentration. A generally with O.lOM citrate. Thus, variation was observed lower citrate concentrations were avoided. With the use of 0.13M and uT was acceptably small and (3.8%) citrate, variation in 'I: citrate in this concentration is suggested as the optimal further Increasing citrate anticoagulant for work. also increased conversion of fibrinogen to fibrin. However, maximal conversion could be achieved only when 25mM calcium was added. TABLE 5 Effect of Citrate Concentration

on uT and t.

Expt. No.

-c (x 10:: daltons/cm) O.lOM

0.13M

0.16M

1.

30.48 (4.53) % Conv 67 CN 1.3

25.93 24.56 (2.58) (3.66) 75 81 1.46 1.57

2.

30.67 (10.40) % Conv 64 CN 1.44

26.48 (3.02) 66 1.49

22.78 (1.36) 76 1.71

3.

15.31 (2.02) 82 1.92

14.20 (2.04) 82 1.96

17.01* (1.94) 8 Conv 75 CN 1.76

(x1011cm2) O.lOM

0.13M 0.16M

390 (26)

312 (12)

527 (18)

466 (12)

263 (12)

483 (11)

359 (13)

314 (7)

324 (8)

in plasma using O.lmM, uT and 'c measured O.l3mM, and 0.16mM Trisodium citrate as the separate experiments. three anticoagulant in in the absence of Networks were developed shown in is calcium. Coefficient of Variation * significant denotes parenthesis. for 0.13M from measurements difference of and 0.16M; p < 0.05. Concentration found to influence uT and r and citrate was of Variation was larger at the Coefficient lower citrate concentrations. CN is expressed in mg/ml.

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FIBRIN NETWORK STRUCTURE I

Effect of Storage

of

Plasmaand

469

Blood.

When essentially platelet-free plasma was stored at 4°C for 24 decreases in uT, 'c and compaction were hours, significant fibrin observed. Network content also decreased (cN) significantly (Table 6), and this was due to a significant fibrinogen in the plasma stored sample. decrease in were Significant changes in values of VT, CN and compaction when plasma was stored at -70°C for 24 hours. observed When whole However, 'c was decreased, but not significantly. blood was stored at 4°C and plasma prepared freshly for tests the variation in the various indices with the exception of compaction was less marked than when platelet-free plasma was stored. These alterations in network structure highlighted the possible perform the tests as soon as after need to venepuncture. TABLE 6 Storage of Plasma and Whole Blood for 24 Hours. A VT

'c

20.73 (0.39)

C

B 19.13 ** (0.31)

D

23.23 * (0.95) 334 (19)

20.86 (0.71) 322 (IO)

374 (31)

285 * (26)

Compaction (%) 31 (2)

26 * (2)

25 * (1)

26 * (1)

2.29 ** (0.05)

2.28 ** (0.07)

2.31 (0.06)

CN

2.45 (0.05)

Conversion (%) $35) *

* (:26)

** (09:)

(09;)

P < 0.05 ** P < 0.005 Columns A (before storage), B (plasma stored for 24 hours at 4"C), C (plasma stored for 24 hours at -70°C) and D (whole blood stored for 24 hours at 4°C) represent mean (SEM) of five experiments. Significant decreases in all measurements were observed when plasma was stored at 4°C for 24 hours. Changes in all observations except permeability (T) were apparent when plasma was stored at - 70°C for 24 hours. Interestingly, when whole blood was stored at 4°C for 24 hours significant alterations in only compaction were observed. n = 4. uT is expressed in x 1pJ2c;qltons/cm. 'I: is expressed in x 10 . Network fibrin content (C,) is in mg/ml.

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FIBRIN NETWORK STRUCTURE I

470

d

.

Aae and

Tables 7A, 7B and 8 are data from statistical analyses of values obtained from 49 healthy adults. The mean u this population was 23.11 * 1.01, while the me?&' ",""a:: compaction + SEM was 407 f 19 and 26 f 0.7 respectively. Males found to have a significantly higher uT, 'I:and compaction were than females (Table 7B). TABLE 7A uT# t and Compaction as Measured in a Population of 49 Normals (32 males and 17 females) Between the Ages of 13-61. u'T

T

Compaction

X

23.11

407

26

2.25

SEM

1.01

19

0.7

0.12

CN

uT is expressed in x 1nt” d3ltons/cm. 'c is expressed in x 10 cm . Network fibrin content (C,) is in mg/ml.

Population

TABLE 7B I: and Compaction Means of p ark?'17 Females.

uT

Compaction (%) Fibrinogen Concentration CN

Age

Males

Females

24.75 (1.30)

20.25 (1.33)

P < 0.025

443 (25)

341 (21)

P < 0.005

Significance

P < 0.05 $8) 2.09 (0.13)

3.02 (0.19)

P < 0.0005

1.92 (0.11)

2.77 (0.17)

P < 0.0005

(?OO)

30 (2.51)

(yrs)

Age range

in 32 Males

(15-47)

(13-49)

Differences in means were tested using unpaired T statistics. Figures in brackets are of the mean.uT is exprlfssed2in x .Network fibrin content is expressed in x 10 concentration are in (C ) and plasma fibrino:n mg rml.

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FIBRIN NETWORK STRUCTURE I

471

However, females showed a higher plasma fibrinogen (and therefore higher fibrin content, CN) than males.

content

Although a weak but significant negative correlation was found between permeability of the network (T) and age (P < O.Ol), no significant correlation with age was found with compaction and uT and permeability (t) showed a significant uT (Table 8). correlation (P < 0.0001) positive which from this analysis would seem to arise from network fibrin content (CN): uT is affected by CN which is one variable in the mathematical derivation of PT. r also is determined by CN, i.e. fibrin content is one determinant of permeability of the network.

Correlations

TABLE 8 Between Measurements of Fibrin Network Characteristics. Correlation 0.4733

uT vs =

Significance P < 0.0001

uT vs compaction

-0.0152

NS

uT vs cN

-0.7165

P < 0.0001

uT vs Age

-0.1274

NS

uT vs Sex

-0.3208

P < 0.01

0.2782

P < 0.05

'I; vs Compaction r vs CN

-0.6386

P < 0.0001

'I: vs Age

-0.3176

P < 0.01

Compaction

vs Age

0.1518

NS

Compaction

vs CN

-0.1856

NS

Fibrinogen

vs Age

0.2640

P < 0.033

Correlations between various measurements of fibrin network characteristics were obtained from data from 49 normal subjects (32 males and 17 females). uT and COmpaCtiOn, however, do not correlate, showing these two measurements are distinct and independent. Interestingly, permeability ‘c and compaction correlate significantly (P < 0.05). Compaction or collapsibility of the network depends strongly on the degree of crosslinking and of branch point density of the fibres in the minor network. Such structural reinforcement not only makes the network more resistant to

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collapse but also less permeable. It is interesting that compaction, unlike permeation (r) and mass-length ratio from turbidity (u ), is relatively independent of fibrin content of network (C,T. It thus seems to be a measure of primary crosslinks and branching in network. It has the been shown (11) that when the number of primary separately crosslinks and branch points are reduced, fibrin fibres become thick and compaction increases proportionately. fibrinogen Plasma concentration had a positive correlation with age. (P < 0.05) the other hand, negatively correlates with age (P < O:bOOy). Thus, network permeability (t) decreases with age because of the increased amount of fibrin within the network.

Previous studies on fibrin network structure have concentrated on the mechanisms of fibrin fibre growth and the biophysics of network development. Physiological relevance of network structure and its role in disease remains an unexplored area. Often conclusions have been drawn from studies based on unphysiological concentrations of fibrinogen manipulated under highly artificial conditions. However, studies on networks developed in fibrinogen solution and human plasma (6) have shown important differences in the two systems even when the amount of fibrinogen converted to fibrin is similar in both. It has thus become necessary to establish methods for the study of fibrin networks in plasma so as to investigate their roles in physiological haemostasis as well as in clinical disorders such as thrombosis, malignancy and diabetes. The investigations in this study show that methods used to characterize networks developed in fibrinogen can, with minor modifications, be used with equal validity in plasma. One of fundamental assumptions in is a linear the the method relationship between turbidity and the wavelength of incident light (1). In networks developed in plasma the relationship between turbidity and l/h3 is not linear, a situation not dissimilar to that in fibrinogen solution in which thrombin concentration is lowered or fibrinogen concentration is increased (1). In the latter case it is possible to derive a rom an intercept obtained measurement of mass- ength ratio from a plot of C/TX 4 against l/A 5 . Investigations described here have shown that similarly in plasma networks plots of may be also used to derive a reliable versus l/h2 C/TA3 measure of PT. For the measurement of permeability (T) of a network developed in plasma the theoretical basis of the method requires little modification and may be used confidently, in a fashion similar to that with fibrinogen solution. However, the networks in plasma are more permeable and tend to collapse more readily. For this reason, permeation tubes require to be coated with a thin layer of fibrin so that the network adheres to the perfusion tube without collapsing.

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from ratio acknowledged that mass-length Whilst it is permeability (up) can be derived from 'I;, it must be appreciated that the packing constant used in this derivation is based on that for longitudinal fibres (3). In the original derivation this constant was calculated to be 10 which is the packing constant for cotton fibres. Thus, computation of uP using this packing constant introduces serious errors. up, thus, is not a satisfactory description of mean fibrin fibre size. Compaction technique describes the tensile behaviour of fibrin fibres. The inverse correlation between compaction and Young's modulus, and between compaction and strength at break, shows on the number that the simple method of compaction depends and strength of the primary crosslinks and branch points in the network. It is interesting that compaction is not affected by fibrin content of the network in the way that permeability is. The method is highly reproducible, inexpensive and can be in used in any clinical laboratory. The use of compaction addition t0 permeability and uT for characterization of fibrin networks in plasma provide three independent methods. Recovery experiments (Table 1) have shown all three methods are highly reliable when used to examine fibrin networks in Turbidity measurements showed little variation and it plasma. was considered adequate to make observations in duplicates. degree of subject to a greater however, is Permeation, variation and as a rule at least four permeation tubes were used in each experimental condition. Compaction showed very However, little variation within a set of measurements. because the expelled fluid was measured with the aid of a syringe it was considered open to a greater degree of observer compaction error. To minimize observer error at least four tubes were used. of 25mM calcium to the system ensures that The addition to fibrin is near maximal. The conversion of fibrinogen which develops into a strong structure attaches network of the permeation tubes securely to the inside without collapse when these tubes are lined with fibrin. There is no leakage of perfusion buffer along the side of the clots when tested with coloured buffer. Lower calcium concentrations or higher citrate concentrations are to be avoided because these factors modify network adherence and integrity. The effect of citrate may be due to both the influence of ionic strength and a direct effect of citrate ions (8). Further investigations are required to dissect out each contribution. Calcium seems to overcome any inhibition of conversion of fibrinogen. Using calcium the derivation of u is based on near maximal conversion of fibrinogen to fi% rin in the network. It is suggested that 25mM calcium is always added to plasma prior to the addition of thrombin. Furthermore, coefficients of variation in each measurement or derivation is smaller when calcium is used, and thus it improves sensitivity of the techniques. On the other hand (Table 2), when calcium is not added or is added in an inadequate amount, the

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.conversion of ,.fibrinogen to fibrin is not near maximal in many subjects and the degree of inhibition o thrombin varies The role of Ca &+ is perhaps of from subject to subject. theoretical interest in studies with purified fibrinogen, but in any modification of these techniques for clinical studies it is important to standardize measurements of fibrin network structure with near maximal conversions of fibrinogen by the addition of calcium. Otherwise the variation in the network characteristics would be more from variation in fibrinogen conversion than fr m clinical the condition under investigation. 25mM CaS+ was the concentration of choice as flooding of the plasma system with an abundance of the divalent cation was the aim. Calcium is bound to many plasma proteins as well as the sodium citrate, which was used as the anticoagulant. It was found that blood need not be immediately processed after venepuncture and could be left to stand for one hour without significant change arising in the structure of the network subsequently developed (Table 3). However it is recommended that plasma not be frozen at -70°C or stored at 4°C over night (Table 6) for reliable measurement of network characteristics. free plasma Thus, platelet should be processed and used for studies on fibrin network no later than one hour after venepuncture. A significant variation was not found in any given subject in network characteristics, plasma fibrinogen or network protein content from one day to the next (Table 3). These results validate the use of these techniques in clinical situations, and show that under physiological conditions basal values of compaction, uT and t remain stable Furthermore, these studies show that if blood and unchanged. is not processed for up to one hour after venepuncture the results are still reproducible and reliable. The type of anticoagulant used was found to influence network structure in found fashion and some anticoagulants were a dramatic Citrate was a reliable anticoagulant but its unsuitable. concentration was found to be of critical importance. On the basis of these studies, 3.8% sodium citrate in a ratio of 1:9 consistent and reliable values for in blood was found to give 'c, uT and compaction. Since fibrinolytic activity in plasma could influence the in patients characterization of fibrin network, especially with enhanced fibrinolytic activity, Trasylol (Aprotinin) was It has added to blood to inhibit spontaneous fibrinolysis. been shown that Trasylol itself does not influence mass-length ratio and the very small volume of the additive (0.35% of the total volume) ensures that the plasma itself is not diluted to any great extent. statistically significant correlation was found between A sex of the subject. Males were network characteristics and found to have a higher uT, t and compaction than females. Interestingly, females had a higher fibrinogen and therefore males (Table 6B). than the network fibrin content of Permeability (r) was inversely related to age in the less were networks Thus, tested (Table 8). population

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permeable with an increase in age. In the population tested, mean uT was 23.11 f 1.01 (SEM), and T was 407 * 19. Compaction was 26 + 0.7. There is no previous physiological data for human fibrin networks and in this regard these values are of great interest. These measurements may be considered physiologically normal and may be used to compare data from with clinically disordered patients network structure. since it is shown that it is now possible to Furthermore, fibrin networks in disease and investigate the role of pathophysiological states (13), it is to be expected that a new area of investigation will lead to better understanding of haemostasis/thrombosis. significant correlations found Statistically were between fibrin fibres network mass-length ratio of and (uT) permeability (r;) and permeability (T), and also between Network fibrin compaction, but not between uT and compaction. significant correlations with both fibre content (C ) showed thickness r uT) and permeability (r). It is to be noted that consistently ar und 92% of plasma conversion was the fibrinogen concentration when Ca ?+ was prelsyIt End e;=;;;, Studies in which fibrinogen content using measured using Ratnoff and Menzies' content of networks technique, showed that a fibrinogen conversion calculated from in the serum was not simply radio active labelled fibrinogen the clotability of the label. Fibre thickness a measure of (u ) and permeability (T) are both dependent on the network fi-8rin content and reinforce the concept that the structural integrity and network characteristics are strongly related to the fibrin content of the networks. The lack of correlation between compaction and uT indicates that collapsibility of the the major fibres network is dependent not on the size of which make up the network, nor on the organization of the minor network, but on the strength and degree of crosslinking of the network as a whole. ACKNOWLEDGEMENTS The Authors acknowledge the experiments concerned with anticoagulants.

assistance of Dr.G.A.Shah in citrate concentration and

REFERENCES 1. FERRY J.D. and MORRISON P.R. Preparation and properties of serum and plasma proteins. VIII. The conversion of human fibrinogen to fibrin undr various conditions. J Am Chem Sot, 69, 388-400. 1947. 2. CARR M.E Jr. and HERMANS J. Size and density of fibres from turbidity. Macromolecules,ll, 46-50. 1978

fibrin

3. CARR M.E Jr., SHEN L.L. and HERMAS J. Mass-length ratio of fibrin fibres from gel permeation and light scattering. Biopolymers, 16,1-15. 1977.

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4. SHAH G.A., FERGUSON I.A., DHALL T.Z. and DHALL D.P. diameter of fibres in fibrin networks: Polydispersion in the the measurement of mass-length ratio by consequence permeability a:: turbidity. Biopolymers, 21, 1037-1047. 1982. 5. SHAH G.A., NAIR C.H. DHALL D.P. and Physiological studies on fibrin network structure. !fhromb Res, 40,181-188. 1985. 6. SHAH G.A., NAIR C.H. and DHALL D.P. Comparison of fibrin networks in plasma and fibrinogen solution. Thromb Res,45, 257-264. 1987. RATNOFF O.D. and MENZIES C. 7. A new method for the determination of fibrinogen in small samples of plasma. J Lab Clin Med. 37, 316-320. 1951. 8. NAIR C.H.,SHAH G.A. and DHALL D.P. Effect of temperature, and composition on fibrin network PH and ionic strength structure and its development. !Chromb.Res, 42, 809-816. 1986. DHALL T.Z., BRYCE W.A.J. 9. and DHALL D.P. Effects dextran on the molecular structure and tensile behaviour human fibrinogen. !Chromb & Haemos, 35, 737-745. 1976.

of of

10. CARR M.E. and GABRIEL D.A. The effect of dextran 70 on the structure of plasma-derived fibrin gels. J Lab Clin Med, 96, 985-993. 1980. 11. DHALL D.P., and NAIR C.H. Functional adaptation of fibrin network structure and its regulation. In: Protein Structure Zaidi, Smith Karachi:TWEL Function, Abassi, (Eds.) Publishers,l990, pp. 27-40. 12. DHALL, D.P. and fibre diameter and compaction technique.

NAIR C.H. Network regulation: its In preparation.

crosslinking Observations

fibrin into

13. NAIR, C.H., AZHAR, A., WILSON, J.D. and DHALL, D.P. Studies on fibrin network structure in human plasma part II Clinical application: Diabetes and antidiabetic drugs. !Chromb.Res. 1991. In Press