It. J. Med. Sc. Sixth Series No. 491 pp. 493-496, November, 1966

IMMEDIATE EFFECT OF SUBCELLULAR COMPONENTS OF HUMAN CELLS ON BLOOD COAGULATION

By NOEL CLARKE Department of Pathology, University College, Dublin.

I T would appear that fibrin deposition in the body under " normal conditions " is much more common than has hitherto been thought. The occurrence of fibrin thrombi in many disease states is becoming

recognised and the very large number of diseases in which thrombosis occurs have been cited by McKay. 1

We .have been interested in the effect of human cells and their intra- cellular components on thrombosis, clotting and ,fibrinolysis. Since cell rupture or injury may often accompany thrombosis and haemorrhage, we have utilised the techniques of cell homogenisation to simulate cell injury. ,Chorion cells have been used as a preliminary model 2or studies on human cells.

Previous investigations on the subcellular components from chorion cells have established that the greatest coagulative activity in these cells is associated ~ith the microsomal fraction. 23

In the past ~ coagulative activity has been usually estimated on cell fractions which had been frozen. Since coagulation and thrombosis are rapid and dynamic phenomena it seemed important to determine the coagulative activity of the cell components immediately after cell rupture, rather ~than after introducing possible artefactual alteration of the subcellular particles by freezing at --20 ~ C.

'The purpose of this study is to determine the coagulative activity of human cell components as soon as possible after cell rupture and to hold the components at 2 ~ C. until incubar in order to preserve intracellular enzymic and metabolic activities.

Materials and Methods

A human placenta was collected at time of delivery and the chorion separated from the amnion. 4The chorion (vol. 20.0 ml.; wt. 20.0 g.) was cut into small pieces in two volumes of isotonic 0.25 M sucrose (pH 7.2), homogenised and filtered. Methods were as previously des- cribed unless otherwise indicated. 23 After removing an aliquot (10.0 ml.), the ho~nogenate (35.0 ml.) was subjected to differential centrifugation to obtain " nuclear ", " mitochondrial ", " microsomal " and soluble fractions. 'The pellets were washed with small quantities of sucrose and the final volume of the soluble fraction was 47.0 ml.

Dilution of fraction~ : The nuclear, mitochondrial and microsomal pellets were each made up separately to the volume of the original centrifuged homogenate, i.e. 35.0 ml., with cold unbuffered 0.85% saline.

Coagulation tests: The homogenate and cell fractions were further diluted with unbuffered 0.8'5% saline (to simulate intracellular pH as

493

494 IRISH JOURNAL OF MEDICAL SCIENCE

far as possible) and 0.1 ml. of the dilute cell fraction was added to 0.1 ml. plasma and 0.1 ml. M/40 calcium chloride at 37 ~ C.

Blood was collected from a normal donor into cold acid citrate-dextrose and the red cells and platclets removed. 3 'The fresh platelet-free plasma for the coagulation tests was placed at --20 ~ C. within 60 minutes of removal from the donor.

All procedures until incubation were carried out at 0-4 ~ C. Nitrogen assays were earried out on the ~ractions. 4

Results

The fractionation schedule and coagulation tests were completed within seven hours of the delivery of the placenta. The clotting times of the cell fractions, which had been diluted to the volume of the original homogenate, are shown on the right hand ordinate in Fig. 1. I t is seen that the fastest clotting time is shown by the homogenate, followed by the microsomal, nuclear, mitochondrial and soluble fractions in that order. It is recalled that, since each cell fraction has been diluted

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FIG. 1.--Clotting time of the saline-diluted fresh homogenate and cell fractions (0.1 ml.) when added to 0.1 ml. of previously frozen platelet-free plasma and 0.1 ml. calcium chloride (M/40) at 37~ T h e o r d i n a t e r e p r e s e n t s t h e clotting time in seconds on a logarithmic scale. The lower abscissa represents further dilutions of the homogenate a n d fractions on a logarithmic scale (2-cycle). The upper abscissa represents the fractions as a percentage of the original dilution. ~k----Soluble; o----Mitochondrial; X----Nuclear; I----Microsomal; O- -Homogena te ; D=Difco brain thrombo-plastin.

Saline clotting time---- 98 secs.; sucrose clotting time = 80 secs.

SUBCELLULAR COMPONENTS OF HUMAN CELLS ON" BLOOD COAGULATI01~ 495

to the volume of the original centrifuged homogena~e, the nuclear, mitochondrial and microsomal fractions are comparable from a dilution point of view, to each o'ther and the homogenate. The results of addi- tional dilutions ~)f the fractions have been plotted on log./log, graph paper in Fig. 1.

I f a horizontal s~tippled line is drawn (Fig. 1) to connect the homo- genate with the microsomal line, then the graph scale readings at each end of this line permit calculation 5 of the % thromboplastic activity of the microsomal fraction, taking the homogenate as 100%. The % thromboplastic activity of the fractions may be read as follows: nuclear----13.5%; mitochondrial=:6.5%; microsomal=78.0%; soluble =2.5% ; homogenate=100%.

The total nitrogen in each fraction is: nuclear----14,848~ (21.9%); mitoehondrial=3,248~ (4.8%); ,microsomal----l~,064~ (17.8%); soluble ----37,600v (55.5%); homogenate----~7,750~ (recovery=77.2%).

FIG. 2 . - -The fractions are represented along the abscissa in the order in which they are isolated. 1~ ----nuclear; M ~-mitochondrial; P ~microsomal; S ~-soluble.

Ord ina t e~Re la t ive "specific" a c t i v i t y ~ % thrornboplastic activity. % nitrogen.

Abscissa--f nitrogen content , % of total recovered nitrogen.

496 IRISH JOURNAL OF MEDICAL SCIENCE

The nitrogen content in 0.1 ml. of fraction at the level of the right hand ordinate is: nuclear----64.0~; mitochondrial=~14.0y microsomal= 52.0~; soluble---- 80.07, and homvgenate--~45.0~.

Fig. 2 shows correlation between the percentage thromboplastic activity and the percentage cell nitrogen. In the present state of knowl- edge regarding thromboplastic materiaP one cannot be certain that the use of nitrogen as a base line for calculating relar specific activity is a valid procedure. It is clear from fig. 2 that the microsomal fraction possesses the highest coagulativc activity when cell nitrogen is used as a basis.

D i s c u s s i o n a n d S u m m a r y

The preliminary evidence presented here indicates that Chese human cells possess, soon after cell rupture, high eoagulative activity associated with the microsomal fraction. Previous investigations have established that this activity is bound to a minute particle in the cell rather than the soluble material2

,Since the microsomal fraction is more active than the homogenate in some chorions, it is not always feasible using fresh material to plot ~he data as shown in Fig. 1; these and other results will be reported in detail later.

The association of coagulative activity with the microsomal fraction in these cells is interesting. Since cell injury is a constant factor in haemorrhage one might speculate 7 that injury to ~he cell membrane exposes the blood to intracellular endoplasmic reticulum and triggers off a " cascade " chain rea~ion in blood coagulation2 9 Again, particles of endoplasmic reticulum might be washed out from the injured cells by the streaming blood and provide a particulate nidus for coagulation, or " contact reaction ". A clinical indication of the powerful action of placental thromboplastins is manifes~ in the defibrination syndrome of pregnancy.

References

1. McKay, D. G. (1965). Disseminated Intravascular Coagulation, Harper and Row, London, p. 21.

2. Clarke, N. (1965). Nature (.Load.), 205, 608. 3. Clarke, ~ . and O'Meara, R. A. Q. (1966). Brit. J. Hacmat., 12, 536. 4, Johnson, M. (1941). J. Biol. Chem., 137, 575. 5. Biggs, 1~. and Maefarlane, 1~. G. (1962). Human Blood Coagulation and its Disorders,

Churchill, Oxford, 3rd. ed. p. 408. 6. Heeht , E. (1965). Lipids in Blood Clotting, Thomas, Illinois, p. 123. 7. Clarke, N. (1964). Ned. T. Geneesk., 108, 2374. 8. ]~Iacfarlane, R. G. (1964). Nature (Lond.), 202, 498. 9. Davie, E. W. and Ratnoff, O. D. (1964). Science, 145, 1310.