STUDIES ON THE CLOT-PROMOTING ACTIVITY
Eleazar Shafrir, Andre de Vries
J Clin Invest.
Find the latest version:
STUDIES ON THE CLOT-PROMOTING
By ELEAZAR SHAFRIR AND ANDRE Dz. VRIES 1
(From the Department of Clinical Research, Hebrew
(Submitted for publication February 21, 1956; accepted March 28, 1956)
glass has been
Quick maintained thatcontact
the precursor of
thromboplastin, possibly antihemophilic factor A
increases its prothrombin utilization.
They concluded that glass activates "plasma
Biggs, Douglas, and
developed in plasma by
with glass is
due to a
antihemophilic factor B).
Alexander, de Vries,
Goldstein (10) observed that contact with
during clotting of blood favors the evolution
SPCA (convertin, Factor VII).
Owren (11) showed that exposureto
markedly increases the activity of
of PTC. Other communications
(12, 13) attributed the
glass to removal of
first phase of
i.e., the evolution of
may be activated by contact with glass.
ofblood. Human venous blood was
col-lected by the two-syringe method using silicone coated needlesandglassware.
1Present Address: Department of
Hospital, Petah-Tikva, Israel.
2A 3 per cent solution in
SiliconeFluid, DowCorning Co., Midland, Mich., U. S. A.
Native "platelet free" (PF) plasma. Blood collected into a syringe chilled by an ice jacket was centrifuged at 4° C for 10 minutes at 14,000 g in a Servall angle
centrifuge. The supernatant plasma was separated and used immediately.
Oxalated "platelet free" (PF) plasma. One part of blood was mixed in a centrifuge tube with 19 parts of 0.2 M sodium oxalate. After chilling on ice for 5
min-utestheblood wascentrifuged at 4° C for 15 minutes at
14,000 g. The supernatant plasma was separated and used within an hour. Platelet counts performed on the undiluted supernatant by the method of Conley, Hart-mann, and Morse (5) showed that the number of platelets rarely exceeded 20 per mm.' (mean ± S.D. of 40plasmas was 14 ± 12, range 0to56 per mm.')
"Platelet free" (PF) BaSO,-plasma (prothrombin-,
proconvertin-, and PTC-free plasma). Finely powdered
BaSO,(Merck) was suspended in oxalated PF plasma, 50 mgm. per ml. The suspension was kept at20° C for 20 minutes and stirred from time to time; subsequently theBaSO, was sedimented bycentrifugationfor 10 min-utes'at 10,000 g and 4° C.
Isolation of platelets. One part of blood was mixed with 9 parts of a solution of 1 per cent EDTA8 and 1 percent Triton4in 0.7 per cent NaCl and chilledon ice. All subsequent procedures were carried out at 4° C. Platelet rich plasma was obtained by centrifugation of the blood at 500 g for 10 minutes and platelets were sedi-mented from this plasma by a second centrifugation for 5 minutes at 8,000 g. The sedimented platelets were rinsed and carefully resuspended in a washing fluid con-taining 0.05 per cent EDTA, 0.05 per cent Triton and 0.02 M sodium acetate in 0.7 per cent NaCI. The sus-pension was again centrifuged at 8,000 g for 5 minutes and the washing procedure repeated. The platelets were
finally resuspended in 0.9 per cent NaCl and counted by the method of Rees and Ecker (14). Phase microscopic examination showed the platelets to be morphologically intact
wathglass powder. Pieces of clean Pyrex glass were crushed in a mortar to fine powder. The specific surface area of the powder was 2,500 cm.' per gin
as determinedwith the methylene blue absorption method
(15). Variousplasmas were added to glass powder in a siliconed test tube as described in the results. The
mix-tures were kept at room temperature (20 to 220 C),
'EthyleneDiamine Tetra-Acetic Acid disodium salt
(Sequestrene), Alrose Chemical Co., Providence, N. J., U. S. A.
4Triton W. R. 1339, Rohm and Haas Co., Philadelphia,
Pa., U. S. A.
ELEAZAR SHAFRIR AND ANDRE DE VRIES
Theeffectof contact of oxalated PF plasmawithvaryingquantitiesofglasspowder onthecloUingtime
Glass powder Clottingtime Serumprothromblnt Serumprothrombinz Serum convertin incontact (mi.)
with 0.4 ml.
plasma Glass Glass Glass Glaws Glass Glass Glass Glass
(mgm.) removed present removed presnt removed present removed present
0 14 78 60 17
2 8 7 80 70 62 40 100 123
10 8 6 75 62 64 38 120 110
s0 7 4 53 42 43 30 88 57
150 6 3 45 35 40 22 63 33
300 7 2 40 20 35 16 60 10
*Four-tenths ml. oxalated PF plasma treated with glass powder was
0.1ml. of 0.05 M
CaCI2,either after removal of the glass or in its presence.
Method of Rosenfield and Tuft (16). Method of Owren and Aas (17).
shaken gently by hand from time to time and after 10 minutes ofcontact centrifuged for 10 minutes at3,000g.
In the different experiments either the supernatant glass
activated plasma was transferred to another siliconed tube, ortheglass wasleft suspendedin the plasmainthe
Clotting times were measured at 37° C in siliconed
testtubes, internal diameter 11 mm.
Determination of clotting factors. Plasma and serum
prothrombin were determined by the one stage
proce-dure of Rosenfield and Tuft (16) and by the method of
Owren and Aas (17). SPCA (convertin, Factor VII)
in serum was assayed by the method of de Vries,
Alex-ander, and Goldstein (18). Determination of proaccelerin (plasma Ac-globulin, labile factor) wasperformed by the
method of Stefanini (19).
Quantity of glass;presence
The effect of varying quantitiesof
clotting timeof oxalated PF
is shown in Table I. Therewas a
glasswasremoved from the
before recalcificationor whether the
glass.In bothcases a
glass powder,if pres-ent
during clottingcaused additional
clot-accelera-tionbut if removed before
decrease in this factor. The
and convertin was
glass remained during
glass powder was removed from
plasma before recalcification
it was found that the
contact was not
increased by prolongation
the contact to 120
20, 200, 370
C. After 10
Clot promoting activity induced byglssin PFplasma: Effectofdecalkifyingagents
Clotting time* ofDPplasma (minutes) Without Glas Decalcifyingant glas treated
Sodiumoxalate,0.2Mt 16 8
Sodium fluoride, 0.4Mt 18 10
Sodiumcitrate,0.2Mt 16 9
EDTA,1%t 15 7
AmberliteIRC50§ 10 8
The amounts of CaCl2 applied were
optimalfor the differentlydecalcifiedPF
t Mixed with blood intheproportion of1 to19.
Four-tenths ml. of thederived PF plasmawasrecalcified with
0.1 ml. CaCI,0.05 M.
tEthylene diamine tetra-acetic acid disodium saltwas
added to the blood in the proportion of 1 to 9. Four-tenths ml. ofthe derived PF
plasmawasrecalcified with 0.1 ml.
IWhole bloodwasshaken for 10minuteswith
Xvolume oftheion exchange resin
Erlenmayerflask. Four-tenths ml. of the
plasmawas recalcified with0.1 ml. of
Clottingtimeand residual serum prothrombin*of oxalated PFpklsma recakiedinthe presence of
Whole Frozen andthawedi
Withoutglass Glasstreated: Withoutglass With glass Number of
plateletsper Serum Serum Serum Serum
mm.'of final Clotting pro- Clotting pro- Clotting pro- Clotting
pro-clotting time thromibin time thrombin time thrombin time thrombin
mixturet (mix.) (%) (mix.) (%) (mix.) (%) (mix.) (%)
160,000 16,000 1,600 400 100 25 0
*Determined by the method of Rosenfield and Tuft
(16).Valuesin excess of 100 percent obtained with this
method arediscussedby deVries, Herz,and Heiman-Hollander (20).
tOne-tenth ml. ofCaCi20.05 M addedtoamixture of 0.3 ml. oxalated PF
plasmaand 0.1 ml. of counted
tOne ml. ofsuspensionscontainingvaryingnumber ofplateletswereputinsiliconed tubes
(internaldiameter 20 mm.) containing200mgm. ofglass beads0.1 mm.indiameter and rotatedon adiscataslant of450at 20rpm. After
15 minutes of rotation theglass beadswere allowed tosettlefor 10minutes and thesupernatant platelet suspensions separated and used intheexperiment.
§ Other platelet suspensions were frozen and thawed three times in siliconed tubes either in the presence of 200
mgm. ofglass beads0.1 mm.indiameter,orwithoutglassbeads. Supernatantswereseparatedasabove.
370 C. Therewere no
clotting time of plasma activated by
II. In PF
blood treated with the calcium
with blood, themselves shortened the
plasma. Contact of such plasma
glass or were frozen and thawed in
removal of the
the recalcification time
data in Table III
of contact of native or oxalated PF
Experiments in which glass acted upon three
kinds of PF
oxalated and oxalated
BaSO4-treated (prothrombin-, PTC-,
Table IV. In the
native plasma the
of the glass,
treated with glass.
activity developed by glass
and oxalated plasma was
demonstrated by shortening
appearance of serum
hand, the action
of glass on the oxalated
resulted in the development of
clot-pro-moting activity with
appearance of convertin in
The serum prothrombin measured by the
method of Owren and Aas (17) which gives
the variation in
serum convertin content, was slightly decreased
all three kinds of glass activated plasma. Serum
values obtained by the method of
Rosenfield and Tuft (16) express the combined
con-ELEAZAR SHAFRIR AND ANDRE DE VRIES
serumprotrombinand convertin content
Experi-ment No.* 1 2 3
Clotting time 4
(min.) 5 6 7 8 1 2 Serum 3 pro- 4 thrombin§ 5 (%) 6 7 8 1 2 Serum 3 pro- 4
(%)6 7 8 Serum convertin
(%)1 2 3 4 5 6 7 8
0.5ml. Without Glass glass treated 23 7 25 4 18 3 19 7 15 5 120
11S130 300 110 160 160 400
90 ' 115
90 60 100 90 100 75 85 S5 100 100 0 60 0 80 0 32 0 80 0 5
0.2 ml. 12 4 14 7 15 6 17 7 14 10 20 8 20 6 14 5 82 93 110 60 48 20 40 15 100 85 70 76 100 150 70 82 78 36 58 38 44 10 48 17 78 40 73 43 100 75 60 38 0 15 0 25 30 100 25 133 11 21 21 54 0 57 27 120
Oxal. PFBaSO4-plasma$ 0.2 ml. Without Glass
glass treated + Oxalated PF plasma
0.2 ml. 45 13 23 10 17 8 19 11 24 15 16 10 29 19 30 11 20 15 16 19 20 17 13 15 52 60 21 16 32 22 24 16 25 14 23 15 24 12 16 10 44 40 26 19 36 32 27 22 0 3 8 8 0 0 0 0 10 12 20 10 15 0 30 36
* Theclotting time,serumprothrombinandconvertin values of the
variousplasmasderivedfrom thesame blood
sample,areindicated bD the same
Clottingof 0.5 m?. of native PF
with theplasma during clotting.
tTwo-tenths ml. of glassactivated oxalated PF-oroxalated PF
wasadded to 0.2 ml. of oxalatedPF plasmaandrecalcifiedwith 0.1 ml. of 0.05 M
CaClI.For thecontrols,0.2 ml. of
E Method ofRosenfieldandTuft
(16).Methodof Owren and Aas
the variable results
ob-tained and the apparent increasein
pro-thrombin" in the
during contact with glass of a
A plasma rendered
The data in Table V show that
which shortens the clotting
mixture of such
proaccelerin, proconvertin, PTC and
A is present in the
the failure of
clotting time significantly
Caot-acceerating adtiviy deoped bycontact wit
glassinoxaktemd PF ImhiliaA plasmaredred
Oxalated PF plasma
0.5 ml. Without Glass
0.4 ml. Without Glass
0.2ml. Without Glass
gass treated Normal 0.2 ml.
0.2mil. Without Glass
glass treated Normal
0.2 ml. Without Glass
glass treated Normal
0.2 ml. Z. Z. >180 >180 >180 >26 12 30 17 35 18 18 10
F. M. >180 >180 >180 >180 50 16 84 18 65 30 28 14
N. L. 60 29 60 17 68 22 49 28
E. S. >180 80 20 12 45 18 18 12 34 22
Y.M. 68 30 45 19 120 64 180 72 54 32
1951 >180 >180 >180 >180 >180 >180 >180 >180 >180 >180 1954 >180 120 >180 48 180 38 >180 88
1951 >180 >180 >180 >180 >180 >180 >180 >180 >180 >180 >180 >180 1954 > 180 >180 >180 >180 180 24 >180 40 100 18 45 16
Clotting of 0.5 ml. of native PFhemophilicplasma withorwithout 50 mgm. glass powder; the glass powder re-mained in theplasma during clotting.
Clottingof 0.4 ml. ofoxalated PF
hemophilic plasma,recalcifiedwith 0.1 ml. of 0.05 M
wasremoved from the activatedsample beforerecalcification.
t Two-tenths ml. of thevarious glass-activated oxalated PF hemophilic plasmas were added after removal ofthe
glass to 0.2 ml. of oxalatedPFnormal plasma and the mixtures recalcified with 0.1 ml. of 0.05MCaCl2. For the con-trols, 0.2ml. of non-activatedsampleswereused.
§ Oxalated PFhemophilicplasma was stored inasiliconed tube at 4° C for 18 days. (One stage prothrombintime more than 50seconds.) Theplasma was then treated withBaSO4.
II PF BaSO4-hemophilicplasmawasheated for10min. at 56° C. and the supernatant separated by centrifugation. The supernatant was fibrinogen-free as tested with thrombin and proaccelerin free as tested with the procedure of
Y.S.wereexaminedtwice. In 1951 bothwere
"refractory" hemophiliacsand in 1954
both had again become responsive to normal plasma, evidenced by correction of clotting time and prothrombin con-sumption.
Noteworthy is the difference in the results
ob-tained with the plasma of patients R. F. and Y. S.
1951 both patients were
"refractory hemophiliacs"; the prolonged clotting
time and the disturbed prothrombin utilization of
blood could not be corrected by the addition
At that time glass treatment
did not induce clot-promoting activity in their
plasmas (21). In 1954 the
become responsive to normal plasma and
paralleling this, glass treatment resulted in the
appearance of the clot-promoting activity.
to show adsorption of a clotting
elute a clotting
possibly adsorbed on glass which had been in
contact with normal or hemophilic plasma.
powder, after contact with a considerable
was extracted with
quan-tities of 0.9 per cent NaCl or
normal plasma, while organic solvent extracts
0.9 per cent NaCl before the test.
experiments the eluates
di-luted brain thromboplastin extract for 10 minutes
of the mixture
determined by the
stage prothrombin test
case, however, could a clot
detected in these eluates.
experiment (Table VI)
powder sample was brought
oxalated PF normal or
ELEAZAR SHAFRIR AND ANDRE DE VRIES
Effect ofcontact of glasspowderwithsuccessive
time andon the
Clottingtime*of Proteint onthe Esterifiedlipidston plasma
aftercontact ghsspowdersample the glasspowder
with theglass after contact sampleafter con-powdersample withplasma tact withplasma
glass powder (mis.) (mgm.) (mgm.)
withplasma Hemo- Hemo(
Hemo-aliquots Normal philic Normal philic Normal philic
Nocontact 15 80
Firstcontact 6 19 0.28 0.30 0.17 0.19
Secondcontact 5 16 0.30 0.35 0.16 0.17
Third contact 5 21 0.27 0.32 0.14 0.16
Fourthcontact 6 24 0.32 0.30 0.15 0.18
Fifthcontact 5 25 0.33 0.37 0.16 0.16
*Two-ml. aliquots ofoxalated PF plasma in 10 test tubeswereactivated each with 500 mgm. ofglass powder.
Aftercentrifugationthe supernatants wereseparated,pooledand a0.4-mi. samplerecalcifiedwith0.1ml.of 0.05 MCaC12. Theglass powderfrom twotubeswasprocessedasdescribed below. To theglasspowder ineachof theremainingeight tubes a new2-ml. aliquot of theoriginal non-activated plasma was added and the aboveprocedurewasrepeatedeach
time withtwotubes less.
glass powderfromonetubewaswashed threetimeswith5 ml.of0.9 percent
NaCifollowing which 1 ml.of2.5 NNaOH wasadded andthemixtureheated on aboilingwaterbath for20minutes. Thetyrosine-likeactivityofthe supernatantwasdetermined bythe method ofHerriott (22). As determined with this
method theinitialproteincontentof thenormalplasmawas7.4gm.per 100ml.,of thehemophilicplasma 7.9 gm.per 100ml.
$After each contact-experiment the glasspowder from thesecondtube waswashed asaboveand extractedwith 3ml.ofalcohol-ether mixture3: 1. Esterifiedlipidsweredetermined in the extract by the method of Stern and Shapiro (23). The plasmavalues obtained with this method were: normal 340 mgm. per 100
ml.,hemophilic 370 mgm. per
not lose its
the experiment recorded
oxalated PF normalor
with successivealiquots ofglass powderon
Contacts*of (misn.) (
successivealiquots Hemo- Hemo-ofglass powder Normal philic Normal philic
Nocontact 18 80 100 100
First contact 7 23 96 94
Secondcontact 7 19 94 94
Thirdcontact 9 26 85 90
Fourth contact 11 35 75 82
Fifthcontact 15 70 65 80
*Oneplasmasamplewasactivated five times with fresh
aliquots ofglass powder, 250 mgm. per ml. Theinitial amount of plasma was 10 ml. After each contact with glass 2 ml. were removed for
tFour-tenths ml. of plasma were recalcified (after
re-movalofglass)with 0.1 ml.of 0.05 MCaCIt.
bythemethodofOwren and Aas
aliquots of glass powder. The shortening of the
clotting time after the first contact did
plasma prothrombin accompanying successive
glass, does not seem
the prolongation of the
time. However, evidence has
that the application of excessive
plasma proconvertin activity (24).
other clotting factors such
is not known.
the development of
is not due to removal of a
described above itwas
On the other
STUDIES ON THE CLOT-PROMOTING ACTIVITY OF GLASS
of "platelet free" (PF) plasma with
study, the clot-accelerating
variously treated oxalated
meas-ured by the ability of such plasmas
recalcification time of mixtures of these
activity is developed by glass in
rendered free of prothrombin, PTC,
proaccelerin and fibrinogen. The
clot-ting factor activated by glass in such plasma has
identified. It is unlikely that the factor
concerned is the "fourth thromboplastic factor"
Spaet, Aggeler, and Kinsell (26)
factors would have
destroyed by the
fibrinogen from the
other factors recently
evolution of blood thromboplastic
the "Hageman Factor" (28) might be
These observations do
reported action of glass
com-patible (Table IV) with the
Aas, and Owren
by glass. Considerable
activity developed in native
oxalated PF normal
plasma, on contact with glass.
plasma (proconvertin free),
after removal of glass,
(proconvertin containing), and the mixture
resulted in shortening of clotting time of the
of detectable serum
thus seems to act on
Whether the greater
glass present during clotting of oxalated PF
plasma, as compared with the effect of glass when
before recalcification (Table I), is due
or on other plasma
that the clotting of
accelerated in the
fibrinogen before the
the formation of
The observations that the residual high level of
prothrombin remaining after clotting of
with glass before recalcification
those of Dick, Jackson, and Conley
attribute the decrease
1. The effect of glass
glass, of the duration of
of the method of
Contact of glass with isolated platelets
platelet factors liberated by freezing
3. Contact with glass induceda
platelets, prothrombin, PTC, proconvertin,
plasma clotting inhibitor
to remove some
1. Lister, J., On the coagulation of blood. Proc. Roy.
Soc., 1862-1863, 12, 580; other authors cited by Dicketal. (8).
2. Morawitz, P., Die Chemie der Blutgerinnung. Ergebn. d.
Physiol., 1905,4, 307.
3. Quick A. J., The interrelationship of prothrombin,
prothrombinogen, and labile factor. Conference on
ELEAZAR SHAFRIR AND ANDRE DE VRIES
Blood Clotting and Allied Problems, 1950, vol. 3, p. 182.
4. Quick, A. J., and Epstein, E., Thromboplastic activity in human blood. J. Applied Physiol., 1952, 4, 840.
5. Conley, C. L., Hartmann, R. C., and Morse, W. I., II, The clotting behavior of human "platelet free" plasma: Evidence for the existence of a "plasma thromboplastin." J. Clin. Invest., 1949, 28, 340. 6. Ratnoff, 0.D.,andConley,C. L, The r6le of surface
and of calcium in the coagulation of aglobulin
frac-tionof platelet-deficient plasma. Bull. Johns Hop-kins Hosp., 1951, 89, 245.
7. Hartmann, R. C., and Conley, C. L., Studies on the initiation of blood coagulation. III. The clotting properties of canine platelet-free plasma. J. Clin. Invest., 1952, 31,685.
8. Dick, F. W., Jackson, D. P., and Conley, C. L., Sur-face as a quantitative factor in prothrombin utili-zation. J. Clin. Invest., 1954, 33, 1423.
9. Biggs, R., Douglas, A. S., and Macfarlane, R. G., The initial stages of blood coagulation. J. Physiol.,
10. Alexander, B., de Vries, A., and Goldstein, R., A factor in serumwhich accelerates the conversion of prothrombin to thrombin. II. Its evolution with special reference to the influence of conditions which affect blood coagulation. Blood, 1949, 4, 739. 11. Rapaport, S. I., Aas, K., and Owren, P. A., The effect
of glass upon the activity of the various plasma clotting factors. J. Clin. Invest., 1955, 34, 9. 12. Tocantins, L. M., Relation of contacting surface and
anticephalin activity to the maintenance of the fluidity and coagulability of blood. Blood, 1946, 1, 156.
13. Fiala, S., On the role of a protein inhibitor in the first stage of blood coagulation. Arch. internat. de physiol., 1951, 58,386.
14. Rees, H. M., and Ecker, E. E., An improved method for counting blood platelets. J.A.M.A., 1923, 80,
15. Nevo, A., de Vries, A., and Katchalsky, A., Inter-action of basic polyamino acids with the red blood cell. I. Combination of polylysine with single
cells. Bioch.Biophys. Acta, 1955, 17, 536. 16. Rosenfield, R. E., and Tuft, H. S., Estimation of
pro-thrombin level from propro-thrombin time. Am. J. Clin. Path., 1947, 17, 405.
Owre.n,P. A.,andAas, K, The control of dicumarol therapy and the quantitative determination of pro-thrombin andproconvertin. Scandinav. J. Clin. & Lab. Invest., 1951, 3, 201.
18. deVries, A.,Alexander,B., andGoldstein, R., A fac-tor in serum which acceerates the conversion of
prothrombinto thrombin. I. Itsdetermination and some physiologic and biochemical properties.
19. Stefanini, M., New one-stage procedures for the
quantitative determination of prothrombin and labilefactor. Am. J. Clin. Path., 1950,20, 233. 20. de Vries, A., Herz, N., and Heiman-Hollander, E.,
Observations on prothrombin consumption during clotting of normal blood in glass. Acta med.
Scandinav., 1950, 138, 219.
21. deVries,A., and Shafrir, E., Methode d'evaluation de
l'activit6 thromboplastique du plasma. Applica-tions cliniques et physiologiques. Rev. d'hemat.,
1952, 7, 481.
22. Herriott, R. M., Reaction of Folin's reagent with proteins and biuret compounds in presence of
cu-pric ion. Proc. Soc. Exper. Biol. & Med., 1941,
23. Stem,I., and
Shapiro,B., Arapidand simplemethod for the determination of esterified fatty acids and for total fatty acids in blood. J. Clin. Path.,
24. Unpublished observations.
25. Duckert, F.,Fluicldger, P., Matter, M., and Koller, F.,
ClottingFactor X. Physiologicand
physico-chemi-cal properties. Proc. Soc. Exper. Biol. & Med., 1955, 90, 17.
26. Spaet, T. H., Aggeler, P. M., and Kinsell, B. G., A possible fourth plasma thromboplastin compo-nent J. Clin. Invest., 1954, 33, 1095.
27. Rosenthal, R.L., Properties of plasmathromboplastin
antecedent (PTA) inrelationtobloodcoagulation.
J. Lab.& Clin. Med., 1955,45, 123.
0.D., and Colopy, J. E., A familial
hemor-rhagictrait associated with a deficiencyof a
clot-promoting fraction of plasma. J. Clin. Invest.,
Lanchantin,G. F., andWare, A. G., Identification of
a thromboplastininhibitor in serum and in
J. Clin. Invest, 1953, 32, 381.
Jiirgens, J.,FactorVII-inhibitor. A new physiologi-cal serum accelerator inactivation principle. Acta