STUDIES ONTHE CLOT-PROMOTINGACTIVITY OF GLASS

By ELEAZARSHAFRIR ANDANDREDz. VRIES 1

(From the Department of Clinical Research, Hebrew University-Hadassah Medical School,Jerusalem, Israel)

(Submitted for publication February 21, 1956; accepted March 28, 1956)

Although the clot-promoting activity of foreignsurfaces has been the subject of many studies dur-ing almost a century (1) its nature is not yet un-derstood. While glass has been thought to acti-vate platelets (2), recent studies have mostly beenconcerned with the effect of glass on plasma clot-ting factors.

Quick maintained that contact with glass acti-vates the precursor of prothrombin, prothrombi-nogen (3) as well as the precursor of plasmathromboplastin, possibly antihemophilic factor A(4). Conley and his associates (5-8) have re-ported that contact with glass markedly shortensthe clotting time of silicone prepared "platelet free"plasma and increases its prothrombin utilization.They concluded that glass activates "plasmathromboplastin," a constituent residing in theglobulin fraction. Biggs, Douglas, and Macfar-lane (9) stated that the increased clotting activitydeveloped in plasma by contact with glass is prob-ably due to a change in Christmas Factor (PTC,antihemophilic factor B). Alexander, de Vries,and Goldstein (10) observed that contact withglass during clotting of blood favors the evolutionof SPCA (convertin, Factor VII). Rapaport,Aas, and Owren (11) showed that exposure toglass markedly increases the activity of procon-vertin as well as of PTC. Other communications(12, 13) attributed the clot-promoting activity of

glass to removal of lipid or protein inhibitors of thefirst phase of coagulation, i.e., the evolution ofthromboplastic activity.

In the present study it is shown that a plasmaclotting factor other than those mentioned abovemay be activated by contact with glass.

MATERIALS ANDMETHODS

Collection of blood. Human venous blood was col-lected by the two-syringe method using silicone coatedneedles and glassware.

1 Present Address: Department of Medicine, BeilinsonHospital, Petah-Tikva, Israel.

2A 3 per cent solution in CCIL of DC200 Silicone Fluid,Dow Corning Co., Midland, Mich., U. S. A.

Native "platelet free" (PF) plasma. Blood collectedinto a syringe chilled by an ice jacket was centrifugedat 4° C for 10 minutes at 14,000 g in a Servall anglecentrifuge. The supernatant plasma was separated andused immediately.

Oxalated "platelet free" (PF) plasma. One part ofblood was mixed in a centrifuge tube with 19 parts of0.2 M sodium oxalate. After chilling on ice for 5 min-utes the blood was centrifuged at 4° C for 15 minutes at14,000 g. The supernatant plasma was separated andused within an hour. Platelet counts performed on theundiluted supernatant by the method of Conley, Hart-mann, and Morse (5) showed that the number of plateletsrarely exceeded 20 per mm.' (mean ± S.D. of 40 plasmaswas 14 ± 12, range 0 to 56 per mm.')

"Platelet free" (PF) BaSO,-plasma (prothrombin-,proconvertin-, and PTC-free plasma). Finely powderedBaSO, (Merck) was suspended in oxalated PF plasma,50 mgm. per ml. The suspension was kept at 20° C for20 minutes and stirred from time to time; subsequentlythe BaSO, was sedimented by centrifugation for 10 min-utes'at 10,000 g and 4° C.

Isolation of platelets. One part of blood was mixedwith 9 parts of a solution of 1 per cent EDTA8 and 1per cent Triton 4 in 0.7 per cent NaCl and chilled on ice.All subsequent procedures were carried out at 4° C.Platelet rich plasma was obtained by centrifugation of theblood at 500 g for 10 minutes and platelets were sedi-mented from this plasma by a second centrifugation for5 minutes at 8,000 g. The sedimented platelets wererinsed and carefully resuspended in a washing fluid con-taining 0.05 per cent EDTA, 0.05 per cent Triton and0.02 M sodium acetate in 0.7 per cent NaCI. The sus-pension was again centrifuged at 8,000 g for 5 minutesand the washing procedure repeated. The platelets werefinally resuspended in 0.9 per cent NaCl and counted bythe method of Rees and Ecker (14). Phase microscopicexamination showed the platelets to be morphologicallyintact

Activation wath glass powder. Pieces of clean Pyrexglass were crushed in a mortar to fine powder. Thespecific surface area of the powder was 2,500 cm.' per ginas determined with the methylene blue absorption method(15). Various plasmas were added to glass powder in asiliconed test tube as described in the results. The mix-tures were kept at room temperature (20 to 220 C),

'Ethylene Diamine Tetra-Acetic Acid disodium salt(Sequestrene), Alrose Chemical Co., Providence, N. J.,U. S. A.

4 Triton W. R. 1339, Rohmand Haas Co., Philadelphia,Pa., U. S. A.

1183

ELEAZAR SHAFRIR AND ANDREDE VRIES

TABLE I

The effect of contact of oxalated PF plasma with varying quantities of glass powder on the cloUing timeand serum prothrombin and convertin conten *

Glass powder Clotting time Serum prothromblnt Serum prothrombinz Serum convertinin contact (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 60 172 8 7 80 70 62 40 100 123

10 8 6 75 62 64 38 120 110s0 7 4 53 42 43 30 88 57

150 6 3 45 35 40 22 63 33300 7 2 40 20 35 16 60 10

* Four-tenths ml. oxalated PF plasma treated with glass powder was recalcified with 0.1 ml. of 0.05 MCaCI2, eitherafter 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 10minutes of contact centrifuged for 10 minutes at 3,000 g.

In the different experiments either the supernatant glassactivated plasma was transferred to another siliconedtube, or the glass was left suspended in the plasma in theoriginal tube.

Clotting times were measured at 37° C in siliconedtest tubes, 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 ofOwren 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) was performed by themethod of Stefanini (19).

RESULTS

1. Some factors influencing the clot-promoting ac-

tivity of glass on PF plasma

Quantity of glass; presence of glass during clot-ting. The effect of varying quantities of glasspowder on the clotting time of oxalated PF plasmais shown in Table I. There was a marked differ-ence in the clot-promoting effect of glass accordingto whether the glass was removed from the plasmabefore recalcification or whether the plasma was

recalcified in the presence of glass. In both cases

a small quantity of glass powder accelerated clot-ting. Larger quantities of glass powder, if pres-ent during clotting caused additional clot-accelera-tion but if removed before recalcification, had no

such effect. In addition, with increasing amountsof glass, serum prothrombin values decreased.While with both procedures serum convertin in-creased markedly, additional quantities of glass

powder caused a decrease in this factor. The de-crease in serum prothrombin and convertin wasmore pronounced when glass remained duringclotting.

Duration of contact with glass. In experimentsin which the glass powder was removed fromplasma before recalcification it was found that theclot-acceleration by a 2-minute contact was notincreased by prolongation of the contact to 120minutes.

Temperature during glass contact. OxalatedPF plasma was brought into contact with glasspowder at 20, 200, 370 and 420 C. After 10 min-

TABLE II

Clot promoting activity induced by glss in PFplasma:Effect of decalkifying agents

Clotting time* of DP plasma(minutes)

Without GlasDecalcifying ant glas treated

Sodium oxalate, 0.2 Mt 16 8Sodium fluoride, 0.4 Mt 18 10Sodium citrate, 0.2 Mt 16 9EDTA, 1%t 15 7Amberlite IRC 50§ 10 8Dowex 50§ 11 9

The amounts of CaCl2 applied were optimal for thedifferently decalcified PF plasmas.

t Mixed with blood in the proportion of 1 to 19. Four-tenths ml. of the derived PF plasma was recalcified with0.1 ml. CaCI, 0.05 M.

t Ethylene diamine tetra-acetic acid disodium salt wasadded to the blood in the proportion of 1 to 9. Four-tenths ml. of the derived PF plasma was recalcified with0.1 ml. CaClI 0.03 M.

I Whole blood was shaken for 10 minutes with X volumeof the ion exchange resin (on the sodium cycle at pH 7.7)in a siliconed Erlenmayer flask. Four-tenths ml. of thederived PF plasma was recalcified with 0.1 ml. of CaC120.02 M.

I 1184

STUDIES ON THE CLOT-PROMOTINGACTIVITY OF GLASS

TABLE III

Clotting time and residual serum prothrombin * of oxalated PF pklsma recakied in the presence of glass-treated platelets

Platelets

Whole Frozen and thawediWithout glass Glass treated: Without glass With glass

Number ofplatelets per Serum Serum Serum Serummm.' of final Clotting pro- Clotting pro- Clotting pro- Clotting pro-

clotting time thromibin time thrombin time thrombin time thrombinmixturet (mix.) (%) (mix.) (%) (mix.) (%) (mix.) (%)160,000

16,0001,600

40010025

0

5 37 199 68

13 18414 19614 21014 200

4.5 37.5 18

10 8014 20015 20014 22015 206

6 38 16

11 6015 18016 18615 18216 190

7 39 20

10 7513 16016 20016 18015 194

* Determined by the method of Rosenfield and Tuft (16). Values in excess of 100 per cent obtained with thismethod are discussed by de Vries, Herz, and Heiman-Hollander (20).

t One-tenth ml. of CaCi2 0.05 Madded to a mixture of 0.3 ml. oxalated PF plasma and 0.1 ml. of counted plateletsuspension.

t One ml. of suspensions containing varying number of platelets were put in siliconed tubes (internal diameter 20mm.) containing 200 mgm. of glass beads 0.1 mm. in diameter and rotated on a disc at a slant of 450 at 20 rpm. After15 minutes of rotation the glass beads were allowed to settle for 10 minutes and the supernatant platelet suspensionsseparated and used in the experiment.

§ Other platelet suspensions were frozen and thawed three times in siliconed tubes either in the presence of 200mgm. of glass beads 0.1 mm. in diameter, or without glass beads. Supernatants were separated as above.

utes the glass was removed and the plasma recal-cified at 370 C. There were no significant differ-ences in the clotting time of plasma activated byglass at the different temperatures.

Method of decalcification of blood. Calcium wasremoved from blood with various decalcifyingagents as indicated in Table II. In PF plasma ob-tained from blood treated with the calcium pre-cipitating agents, sodium oxalate, sodium fluorideor with the chelating agents, EDTA or sodiumcitrate, clot-promoting activity developed on con-tact with glass. The decalcifying ion exchangeresins Amberlite IRC 50 or Dowex 50 in contactwith blood, themselves shortened the clotting timeof the derived PF plasma. Contact of such plasmawith glass did not result in further significant re-duction of clotting time.

2. Failure of glass to activate platelet clottingfactorsVarying quantities of platelets were treated with

glass or were frozen and thawed in the presence ofglass. After removal of the glass they were addedto oxalated PF plasma and the recalcification timeand residual serum prothrombin measured. Thedata in Table III show that the contact with glassdid not affect the activity of platelet clottingfactors.

3. Effect of contact of native or oxalated PFBaS04- plasma with glass on clotting time, se-rum prothrombin and serum convertin content

Experiments in which glass acted upon threekinds of PF plasma: native, oxalated and oxalatedBaSO4-treated (prothrombin-, PTC-, and pro-convertin-free) are recorded in Table IV. In thecase of native plasma the glass powder remainedduring clotting. With the oxalated plasmas, theclotting activity developed by contact with glasswas tested, after removal of the glass, on oxalatedPF plasma not treated with glass.

The clot-promoting activity developed by glassin PF native plasma and oxalated plasma wasdemonstrated by shortening of clotting time andappearance of serum convertin. On the otherhand, the action of glass on the oxalated BaSO.-plasma resulted in the development of clot-pro-moting activity with no appearance of convertin inthe clotting mixture.

The serum prothrombin measured by themethod of Owren and Aas (17) which gives pro-thrombin values independent of the variation inserum convertin content, was slightly decreasedin all three kinds of glass activated plasma. Serumprothrombin values obtained by the method ofRosenfield and Tuft (16) express the combinedactivity of residual serum prothrombin and con-

1185

ELEAZAR SHAFRIR AND ANDREDE VRIES

TABLB IV

Effect of contact of native or of oxalated BSOr-trsaed PF plasma with glass on dotting time,serum protrombin and convertin content

Experi-mentNo.*

123

Clotting time 4(min.) 5

678

12

Serum 3pro- 4

thrombin§ 5(%) 6

78

12

Serum 3pro- 4

thrombinal S(%) 6

78

Serumconvertin

(%)

12345678

Native PF plasmat0.5 ml.

Without Glassglass treated

23 725 418 319 715 5

120 11S130 300110 160160 40090 ' 115

90 60100 90100 7585 S5

100 100

0 600 800 320 800 5

Oxalated PF plasmat0.2 ml.

Without Glassglass treated

Oxalated PF plasma0.2 ml.

12 414 715 617 714 1020 820 614 5

82 93110 6048 2040 15

100 8570 76

100 15070 82

78 3658 3844 1048 1778 4073 43

100 7560 38

0 150 25

30 10025 13311 2121 540 57

27 120

Oxal. PF BaSO4-plasma$0.2 ml.

Without Glassglass treated

+Oxalated PF plasma

0.2 ml.

45 1323 1017 819 1124 1516 1029 1930 11

20 1516 1920 1713 1552 6021 1632 2224 16

25 1423 1524 1216 1044 4026 1936 3227 22

0 38 80 00 0

10 1220 1015 030 36

* The clotting time, serum prothrombin and convertin values of the various plasmas derived from the same bloodsample, are indicated bD the same experiment number.

t Clotting of 0.5 m?. of native PF plasma without or with 50 mgm. of glass powder; the powder remained in contactwith the plasma during clotting.

t Two-tenths ml. of glass activated oxalated PF- or oxalated PF BaSO4-plasma from which the glass was removed,was added to 0.2 ml. of oxalated PF plasma and recalcified with 0.1 ml. of 0.05 MCaClI. For the controls, 0.2 ml. ofnon-activated plasma was used.

E Method of Rosenfield and Tuft (16).Method of Owren and Aas (17).

vertin; this may explain the variable results ob-tained and the apparent increase in "serum pro-thrombin" in the experiments with native plasma.

4. Development during contact with glass of aclot-accelerating activity in oxalated PF hemo-philia A plasma rendered free of prothrombin,PTC, proconvertin, proaccelerin and fibrinogenThe data in Table V show that glass treatment

of oxalated PF hemophilia A plasma induces clot-promoting activity, which shortens the clotting

time of a mixture of such plasma with oxalatedPF normal plasma. For the development of theclot-promoting activity in hemophilic plasma,fibrinogen, proaccelerin, proconvertin, PTC andprothrombin are not required. This activity isnot well demonstrated unless antihemophilic fac-tor A is present in the clotting mixture. Thisfollows from the failure of glass to shorten theclotting time significantly in most of the hemo-philic PF plasma samples (native as well as oxa-lated) to which normal plasma had not been added.

1186

STUDIES ON THE CLOT-PROMOTINGACTIVITY OF GLASS

TABLE V

Caot-acceerating adtiviy deoped by contact wit glass in oxaktemd PF Imhilia A plasma redred free ofprohrombin, PTC, proconwri, proacerin and fibrinogen

Clotting time (min.)

Oxalated PF plasma

Native PFhemophilic

plasma*0.5 ml.

Without Glassglass treated

Hemophilict0.4 ml.

Without Glassglass treated

Hemophllct0.2 ml.

Without Glassglass treated

Normal0.2 ml.

BaSO4-hemophilIc$

0.2 ml.

Without Glassgass treated

Normal0.2 ml.

Stored BaSO-hemophilici

0.2 mil.

Without Glassglass treated

Normal0.2 ml.

Heated BaSO4-hemophilici

0.2 ml.

Without Glassglass treated

Normal0.2 ml.

Z. Z. > 180 >180 >180 >26 12 30 17 35 18 18 10F. M. >180 >180 >180 >180 50 16 84 18 65 30 28 14N. L. 60 29 60 17 68 22 49 28E. S. >180 80 20 12 45 18 18 12 34 22Y. M. 68 30 45 19 120 64 180 72 54 32R. F.¶

1951 >180 >180 >180 >180 >180 >180 >180 >180 >180 >1801954 >180 120 >180 48 180 38 >180 88

Y. S.¶1951 >180 >180 >180 >180 >180 >180 >180 >180 >180 >180 >180 >1801954 > 180 > 180 >180 > 180 180 24 > 180 40 100 18 45 16

Clotting of 0.5 ml. of native PF hemophilic plasma with or without 50 mgm. glass powder; the glass powder re-mained in the plasma during clotting.

t Clotting of 0.4 ml. of oxalated PF hemophilic plasma, recalcified with 0.1 ml. of 0.05 MCaCi2. The glass powderwas removed from the activated sample before recalcification.

t Two-tenths ml. of the various glass-activated oxalated PF hemophilic plasmas were added after removal of theglass to 0.2 ml. of oxalated PF normal plasma and the mixtures recalcified with 0.1 ml. of 0.05 MCaCl2. For the con-trols, 0.2 ml. of non-activated samples were used.

§ Oxalated PF hemophilic plasma was stored in a siliconed tube at 4° C for 18 days. (One stage prothrombin timemore than 50 seconds.) The plasma was then treated with BaSO4.

II PF BaSO4-hemophilic plasma was heated for 10 min. 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 ofStefanini (19).

1 Both patients R. F. and Y. S. were examined twice. In 1951 both were "refractory" hemophiliacs and in 1954both 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.in 1951 and 1954. In 1951 both patients were"refractory hemophiliacs"; the prolonged clottingtime and the disturbed prothrombin utilization oftheir blood could not be corrected by the additionof normal plasma. At that time glass treatmentdid not induce clot-promoting activity in theirplasmas (21). In 1954 the blood of these patientshad become responsive to normal plasma andparalleling this, glass treatment resulted in theappearance of the clot-promoting activity.

5. Attempts to show adsorption of a clotting in-hibitor from plama on glass powder

Attempts were made to elute a clotting inhibi-tor, possibly adsorbed on glass which had been incontact with normal or hemophilic plasma. Theglass powder, after contact with a considerableamount of plasma, was extracted with small quan-

tities of 0.9 per cent NaCl or 0.1 MCaCl2 of dif-ferent pH (range 4 to 12) or with ether, alcohol-ether 3: 1, acetone or chloroform. The NaCl andCaCl2 eluates were tested directly on oxalated PFnormal plasma, while organic solvent extractswere evaporated and the residues resuspended in0.9 per cent NaCl before the test. In additionalexperiments the eluates were incubated with di-luted brain thromboplastin extract for 10 minutesat 370 C and the clotting potency of the mixturewas determined by the one stage prothrombin testsystem. In no case, however, could a clot re-tarding activity be detected in these eluates.

In another experiment (Table VI) one glasspowder sample was brought into contact with suc-cessive aliquots of oxalated PF normal or hemo-philic plasma and the clotting time (after removalof glass) and the protein and lipid adsorbed bythe glass were determined. Although the glasspowder during the successive contacts with new

1187

ELEAZAR SHAFRIR AND ANDREDE VRIES

TABLE VI

Effect of contact of glass powder with successive aliquots of oxlated PF normal or hemophilc plasma on thedcotting time and on the amount of protein and lipid adsorbed

Clotting time* of Proteint on the Esterified lipidst onplasma after contact ghss powder sample the glass powder

with the glass after contact sample after con-powder sample with plasma tact with plasma

Contacts* ofglass powder (mis.) (mgm.) (mgm.)with plasma Hemo- Hemo( Hemo-

aliquots Normal philic Normal philic Normal philic

No contact 15 80First contact 6 19 0.28 0.30 0.17 0.19Second contact 5 16 0.30 0.35 0.16 0.17Third contact 5 21 0.27 0.32 0.14 0.16Fourth contact 6 24 0.32 0.30 0.15 0.18Fifth contact 5 25 0.33 0.37 0.16 0.16

* Two-ml. aliquots of oxalated PF plasma in 10 test tubes were activated each with 500 mgm. of glass powder.After centrifugation the supernatants were separated, pooled and a 0.4-mi. sample recalcified with 0.1 ml. of 0.05 MCaC12.The glass powder from two tubes was processed as described below. To the glass powder in each of the remaining eighttubes a new 2-ml. aliquot of the original non-activated plasma was added and the above procedure was repeated eachtime with two tubes less.

t After each contact experiment the glass powder from one tube was washed three times with 5 ml. of 0.9 per centNaCi following which 1 ml. of 2.5 N NaOHwas added and the mixture heated on a boiling water bath for 20 minutes.The tyrosine-like activity of the supernatant was determined by the method of Herriott (22). As determined with thismethod the initial protein content of the normal plasma was 7.4 gm. per 100 ml., of the hemophilic plasma 7.9 gm. per100 ml.

$ After each contact-experiment the glass powder from the second tube was washed as above and extracted with3 ml. of alcohol-ether mixture 3: 1. Esterified lipids were determined in the extract by the method of Stern and Shapiro(23). The plasma values obtained with this method were: normal 340 mgm. per 100 ml., hemophilic 370 mgm. per100 ml.

plasma aliquots did not lose its ability to induceclot-promoting activity, the amount of proteinand lipid adsorbed by the glass did not increaseabove that adsorbed during the first contact.

In the experiment recorded in Table VII, onesample of oxalated PF normal or hemophilicplasma was brought into contact with successive

TABLE ViI

Effect of contact of a PFnormal or hemophilic plasma samplewith successive aliquots of glass powder on clotting

time and plasma prothrombin content

Clotting timet Plasma prothrom-after contact with bint after contact

glass powder with glass powderContacts* of (misn.) (plasma with

successive aliquots Hemo- Hemo-of glass powder Normal philic Normal philic

No contact 18 80 100 100First contact 7 23 96 94Second contact 7 19 94 94Third contact 9 26 85 90Fourth contact 11 35 75 82Fifth contact 15 70 65 80

* One plasma sample was activated five times with freshaliquots of glass powder, 250 mgm. per ml. The initialamount of plasma was 10 ml. After each contact withglass 2 ml. were removed for clotting and prothrombintests.

t Four-tenths ml. of plasma were recalcified (after re-moval of glass) with 0.1 ml. of 0.05 MCaCIt.

t Determined by the method of Owren and Aas (17).

aliquots of glass powder. The shortening of theplasma clotting time after the first contact didnot become more pronounced on repeated con-tacts with new glass; on the contrary, the clottingtime again increased. The observed decrease inplasma prothrombin accompanying successive con-tacts of the plasma with glass, does not seem suffi-cient to explain the prolongation of the clottingtime. However, evidence has also been obtainedthat the application of excessive amounts of glassto oxalated PF plasma results in diminution ofplasma proconvertin activity (24). Whether theconcentration of other clotting factors such asPTC, Factor X (25), etc. is also decreased by thesuccessive contacts with glass, is not known.

These results indicate that the development ofclot-promoting activity in plasma in contact withglass is not due to removal of a clotting inhibitorby adsorption onto the glass surface.

DISCUSSION

In the experiments described above it was ob-served that the clotting activity of isolated plate-lets, or of platelet clotting factors, liberated byfreezing and thawing of isolated platelets, wasnot enhanced by contact with glass. On the other

1188

STUDIES ON THE CLOT-PROMOTINGACTIVITY OF GLASS

hand contact of "platelet free" (PF) plasma withglass induced a clot-promoting activity.

In the present study, the clot-accelerating ac-tivity induced by transient contact with glass invariously treated oxalated PF normal and hemo-philic A plasmas was estimated. This was meas-ured by the ability of such plasmas to shorten therecalcification time of mixtures of these plasmaswith oxalated PF normal plasma. With thismethod it was possible to show that clot-promotingactivity is developed by glass in PF hemophilia Aplasma, rendered free of prothrombin, PTC, pro-convertin, proaccelerin and fibrinogen. The clot-ting factor activated by glass in such plasma hasnot been identified. It is unlikely that the factorconcerned is the "fourth thromboplastic factor"of Spaet, Aggeler, and Kinsell (26) or the Clot-ting Factor X (25), as these factors would havebeen destroyed by the heating to 560 C employedto remove fibrinogen from the test plasma. How-ever, among other factors recently postulated tocontribute to the evolution of blood thromboplasticactivity, PTA (plasma thromboplastin antecedent,27) and the "Hageman Factor" (28) might bepresent during activation by glass of the plasmasystem used here.

These observations do not disagree with thereported action of glass on proconvertin andPTC (9-11). Indeed some of them are com-patible (Table IV) with the conclusions of Rapa-port, Aas, and Owren (11) as to proconvertinactivation by glass. Considerable serum convertinactivity developed in native or oxalated PF normalplasma, on contact with glass. However whenglass was applied to oxalated BaSO4-treated PFnormal plasma (proconvertin free), no serumconvertin developed when such plasma was addedafter removal of glass, to oxalated PF plasma(proconvertin containing), and the mixture re-calcified. In this experiment, the action of glassresulted in shortening of clotting time of the mix-ture without development of detectable serumconvertin. Glass thus seems to act on procon-vertin as well as on other, yet unidentifiedfactor(s).

Whether the greater clot-accelerating effect ofglass present during clotting of oxalated PFplasma, as compared with the effect of glass whenremoved before recalcification (Table I), is dueto an action on proconvertin or on other plasma

factors, has not been elucidated. It is pertinentthat the clotting of purified fibrinogen-thrombinmixture is accelerated in the presence of suspendedglass powder, while removal of the glass powderafter contact with the fibrinogen before the addi-tion of thrombin does not result in shortened clot-ting time (24). It is possible that the finely di-vided glass particles present during clotting maypromote the formation of primary fibrin strands.

The observations that the residual high level ofserum prothrombin remaining after clotting of PFplasma was decreased when the plasma was treatedwith glass before recalcification are in agreementwith those of Dick, Jackson, and Conley (8).However, as in the present experiments (TableVII) it was shown that after contact with a largearea of glass prothrombin may be partly removedfrom plasma, we hesitate to attribute the decreasein serum prothrombin after contact with glass, toenhanced prothrombin utilization.

SUMMARY

1. The effect of glass on the evolution of clot-promoting activity in "platelet free" plasma wasexamined. Data are presented on the influence ofthe amount of glass, of the duration of contact, ofthe temperature and of the method of decalcifica-tion of the plasma on this process.

2. Contact of glass with isolated platelets orwith platelet factors liberated by freezing andthawing, did not result in increase of their clottingactivity.

3. Contact with glass induced a clot-promotingactivity in oxalated hemophilia A plasma, free ofplatelets, prothrombin, PTC, proconvertin, pro-accelerin and fibrinogen.

4. Attempts to demonstrate the adsorption of aplasma clotting inhibitor onto glass were unsuc-cessful. Contact with large area of glass wasshown to remove some prothrombin from oxa-lated plasma.

REFERENCES

1. Lister, J., On the coagulation of blood. Proc. Roy.Soc., 1862-1863, 12, 580; other authors cited byDick et al. (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

1189

ELEAZAR SHAFRIR AND ANDREDE VRIES

Blood Clotting and Allied Problems, 1950, vol. 3,p. 182.

4. Quick, A. J., and Epstein, E., Thromboplastic activityin 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 "plasmathromboplastin." J. Clin. Invest., 1949, 28, 340.

6. Ratnoff, 0. D., and Conley, C. L, The r6le of surfaceand of calcium in the coagulation of a globulin frac-tion of platelet-deficient plasma. Bull. Johns Hop-kins Hosp., 1951, 89, 245.

7. Hartmann, R. C., and Conley, C. L., Studies on theinitiation of blood coagulation. III. The clottingproperties 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., Theinitial stages of blood coagulation. J. Physiol.,1953, 122, 538.

10. Alexander, B., de Vries, A., and Goldstein, R., Afactor in serum which accelerates the conversion ofprothrombin to thrombin. II. Its evolution withspecial reference to the influence of conditions whichaffect blood coagulation. Blood, 1949, 4, 739.

11. Rapaport, S. I., Aas, K., and Owren, P. A., The effectof glass upon the activity of the various plasmaclotting factors. J. Clin. Invest., 1955, 34, 9.

12. Tocantins, L. M., Relation of contacting surface andanticephalin activity to the maintenance of thefluidity and coagulability of blood. Blood, 1946,1, 156.

13. Fiala, S., On the role of a protein inhibitor in thefirst stage of blood coagulation. Arch. internat. dephysiol., 1951, 58, 386.

14. Rees, H. M., and Ecker, E. E., An improved methodfor counting blood platelets. J.A.M.A., 1923, 80,621.

15. Nevo, A., de Vries, A., and Katchalsky, A., Inter-action of basic polyamino acids with the red bloodcell. I. Combination of polylysine with singlecells. Bioch. Biophys. Acta, 1955, 17, 536.

16. Rosenfield, R. E., and Tuft, H. S., Estimation of pro-thrombin level from prothrombin time. Am. J.Clin. Path., 1947, 17, 405.

17. Owre.n, P. A., and Aas, K, The control of dicumaroltherapy and the quantitative determination of pro-thrombin and proconvertin. Scandinav. J. Clin. &Lab. Invest., 1951, 3, 201.

18. de Vries, A., Alexander, B., and Goldstein, R., A fac-tor in serum which acceerates the conversion ofprothrombin to thrombin. I. Its determination andsome physiologic and biochemical properties.Blood, 1949, 4, 247.

19. Stefanini, M., New one-stage procedures for thequantitative determination of prothrombin andlabile factor. Am. J. Clin. Path., 1950, 20, 233.

20. de Vries, A., Herz, N., and Heiman-Hollander, E.,Observations on prothrombin consumption duringclotting of normal blood in glass. Acta med.Scandinav., 1950, 138, 219.

21. de Vries, A., and Shafrir, E., Methode d'evaluation del'activit6 thromboplastique du plasma. Applica-tions cliniques et physiologiques. Rev. d'hemat.,1952, 7, 481.

22. Herriott, R. M., Reaction of Folin's reagent withproteins and biuret compounds in presence of cu-pric ion. Proc. Soc. Exper. Biol. & Med., 1941,46, 642.

23. Stem, I., and Shapiro, B., A rapid and simple methodfor the determination of esterified fatty acids andfor total fatty acids in blood. J. Clin. Path., 1953,6, 158.

24. Unpublished observations.25. Duckert, F., Fluicldger, P., Matter, M., and Koller, F.,

Clotting Factor X. Physiologic and 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 plasma thromboplastinantecedent (PTA) in relation to blood coagulation.J. Lab. & Clin. Med., 1955, 45, 123.

28. Ratnoff, 0. D., and Colopy, J. E., A familial hemor-rhagic trait associated with a deficiency of a clot-promoting fraction of plasma. J. Clin. Invest.,1955, 34, 602.

29. Lanchantin, G. F., and Ware, A. G., Identification ofa thromboplastin inhibitor in serum and in plasma.J. Clin. Invest, 1953, 32, 381.

30. Jiirgens, J., Factor VII-inhibitor. A new physiologi-cal serum accelerator inactivation principle. ActaHaemat, 1955, 14, 57.

1190