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Interaction of High Molecular Weight Kininogen, Factor XII, and

Fibrinogen in Plasma at Interfaces

By L. Vroman, A. L. Adams, G. C. Fischer, and P. C. Munoz

Using ellipsometry. anodized tantalum interference color. and Coomassie blue staining in conjunction with immunologic
identification of proteins adsorbed at interfaces, we have previously found that fibrinogen is the main constituent
deposited by plasma onto many man-made surfaces. However, the fibrinogen deposited from normal plasma onto glass
and similar wettable materials is rapidly modified during contact activation until it can no longer be identified antigenically.
In earlier publications. we have called this modification of the fibrinogen layer “conversion,” to indicate a process of
unknown nature. Conversion of adsorbed fibrinogen by the plasma was not accompanied by marked change in film
thickness. so that we presumed that this fibrinogen was not covered but replaced by other protein. Conversion is now
shown to be markedly delayed in plasma lacking high molecular weight kininogen, slightly delayed in plasma lacking factor
XII. and normal in plasmas that lack factor XI or prekallikrein. We conclude that intact plasma will quickly replace the
fibrinogen it has deposited on glass-like surfaces by high molecular weight kininogen and, to a smaller extent, by factor XII.
Platelets adhere preferentially to fibrinogen-coated surfaces; human platelets adhere to hydrophobic nonactivating
surfaces, since on these, adsorbed fibrinogen is not exchanged by the plasma. The adsorbed fibrinogen will be replaced on
glass-like surfaces during surface activation of clotting, and platelets failing to find fibrinogen will not adhere.

T HE DEPOSITION of proteins by blood plasma factor-XII-deficient plasma. During conversion, the

onto flat surfaces of oxidized silicon crystal slices, thickness of the film did not change measurably. We
glass, and anodized tantalum-sputtered glass can be therefore presumed that the adsorbed fibrinogen loses
studied by means of ellipsometry, Coomassie blue its antigenicity as a result of replacement by some
staining, and observation of interference colors, other materials present in intact plasma and that the
respectively. After exposure to plasma, exposure to fibrinogen molecules are not merely distorted or
antisera will result in deposition of antibodies that masked.4 Fibrinogen conversion affects platelet adhe-
match the antigen substrate; the immune complexes sion: platelets adhere preferentially where fibrinogen
can be observed by the same methods and allow has been deposited and not where the film has been
identification of the proteins the plasma had depos- changed by the plasma.6’7 As a consequence, human
ited”2 without the need of labeled reagents. With these blood deposits markedly more platelets on hydro-
techniques we found earlier that normal intact (i.e., phobic surfaces (such as glass rubbed with ferric
not surface-activated) plasma, whether native, hepa- stearate) where fibrinogen remains present, than on
rinized, or citrated, deposits fibrinogen onto most hydrophilic glass-like surfaces where the platelets
surfaces we studied, including the wettable ones listed arrived too late to interact with unaltered fibrinogen.3
above, within a few seconds. While this fibrinogen Recently, we were able to study plasma from one
remains unaltered by the plasma when deposited on patient lacking high molecular weight kininogen
certain hydrophobic substrates,3 the fibrinogen depos- (HMK) and from another one lacking both HMK and
ited onto hydrophilic glass-like ones is altered by the low molecular weight kininogens. HMK is reported to
plasma. This “change” is evident by a loss of the film’s have a high affinity for clot-activating surfaces,8
ability to cause deposition of antibody from overlaid enabling it to carry factor XI to the interface9 where it
antisera to human fibrinogen.4’5’7 We did not know is activated by factor XII-the first factor to be
what process was involved in this loss of antigenicity, adsorbed and activated by the surface. On kaolin, the
and therefore, had called it “conversion.” At room interactions between HMI( and adsorbed factor XII
temperature in human blood, conversion occurs within now being studied’#{176}appear complex, but suggest to us
I mm on oxidized silicon crystals and within I 0 mm on that HMK may be one agent responsible for altering
glass or anodized tantalum. It is slightly delayed in preadsorbed fibrinogen.

MATERIALS AND METHODS

From the Interface Laboratory, Veterans Administration Medi-
High Molecular Weight
cal Center. Brooklyn. N.Y.
Kininogen-Deficient Plasmas
Supported in part by National Heart, Lung and Blood Institute
Grant I ROl HL2389901. Human plasma lacking in only this factor” was obtained from
Submitted March 30. 1979; accepted September 28. 1979. George King Biochemical Inc., Overland Park, Kans. Plasma defi-
Address reprint requests to Leo Vroman. Ph.D.. Interface Labor- cient in both high and low molecular weight kininogen (Williams
atory. Veterans Administration Medical (‘enter, 800 Pol; Place. trait)’2 was generously provided by Dr. Robert W. Colman, Phila-
Brooklyn. N.Y. 11209. delphia, Pa. These and other plasmas were collected into I part of
(c) / 980 by Grune if Stratton, Inc. 3.8% sodium citrate/9 parts blood and stored at about -60#{176}C.
0006-4971/80/5501-0026$0l .00/0 Purified human high molecular weight kininogen was generously

156 Blood, Vol. 55, No. 1 (January), 1980

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KININOGEN AND FIBRINOGEN AT INTERFACES 157

provided by Dr. 0. D. Ratnofi, Cleveland, Ohio (9.2 U/mg). Glass Table 1 . Alteration in Dc posited Fibrinog en in Vario us Plasmas
slides were obtained from A. H. Thomas, Philadelphia, Pa., Cat. Anodized
6685-Q-20. Tantalum-sputtered glass slides were kindly provided by Plasma Tantalum Glass
Dr. N. Schwartz, Bell Telephone Labs., Murray Hill, N.J., and
Normal 4 444 4 4444 4
were anodized at about 20 V in 0.01% HNO3 Polyvinylchloride 4 444 3 4443 3
slides (PVC) came from A. H. Thomas, Cat. 6686.V15. Sparkleen
was obtained from Fisher Sci. Co., Springfield, N.J., and Kimwipes Prekallikrein 4 4 4 3 3 4 4 3 3 2

from Kimberly-Clark Corp., Neenah, Wisc. For details regarding deficient 3 2 2

veronal buffer in saline (VS), see refs. 1 and 3; for those regarding FactxXl 4 444 4433 3
Coomassie brilliant blue R, see ref. 2, however, the following is used deficient
without further dilution: 250 mg of the dye is dissolved into 50 ml
methanol and 10 ml glacial acetic acid; bring volume to 100 ml with FactorXll 3 333 3 222 1 1
deficient
distilled water. Antisera to human fibrinogen came from Behring
Diagnostics, Somerville, N.J., Cat. 14-513-301, and Schwarz- Fitzgerald 0 0
Mann, Division of Becton, Dickinson & Co., Orangeburg, N.Y.,
Cat. 904764. Williams 1 00 1 10

Williams and HMKt 4 3

Treatment of Surfaces Each entry, ranging from 4 to 0, represents ranking of a single
experiment, with 4 indicating complete alteration and 0 indicating none
All slides were cleaned by brushing with concentrated Sparkleen.
at all (see text).
using Kimwipes wrapped around applicators; the slides were then
rinsed with large amounts of distilled water, air-dried, and quickly to. 1 ml Williams plasma premixed with 0.02 ml (about 0.02 ) of
purified high molecular weight kininogen.
passed over a colorless bunsen burner flame. They were used when
cooled, within about I hr. To avoid plasma/air/solid interfaces.
drops were deposited onto slides as follows: 0. 1 ml of VS was placed
on the center of a slide and spread to an approximately I x I inch RESULTS
area; 0.1 ml of plasma was placed into the VS and mixed without
Normal Plasma
disturbing the solid/liquid interface. The slide was placed in a moist
chamber and removed after 10 mm. Then, 0.1 ml VS was placed On the area (See Fig. 1 ) where plasma resided for
near the free end of the slide and allowed to spread over the 10 sec only, antiserum to human fibrinogen placed
remaining I x I inch area of the surface. After merging with the
onto area ‘b’ deposited additional material: area ‘b’ is
area containing plasma, the 2 deposits were allowed to mix for about
darker than area ‘d.’ In contrast, where the normal
10 sec, following which the entire slide was rinsed with VS. followed
by water, and air-dried. Next, 0.1 ml VS was spread across part of plasma had resided for 10 mm, the antiserum placed
both areas (the one where plasma had resided for 10 mm and where on area ‘a’ left only traces of material: areas ‘c’ and ‘a’
it had stayed for about 10 sec only), and into this VS. antiserum to are about equally light. All samples of normal plasma
human fibrinogen was placed and mixed. This was rinsed off with
tested on glass and on anodized tantalum showed this
VS and water 3 mm later. The glass slides were stained with
marked effect on the deposited fibrinogen (see
Coomassie blue for 3 mm and rinsed with water; all slides were
air-dried. Table 1).

Kininogen-Deficient Plasmas
Evaluation of Results
A representative slide (see Fig. 1, Williams) is
The deep bronze first-order interference color of the anodized
shown. Where plasma lacking high molecular weight
tantalum was shifted to red by adsorption of plasma proteins; where
antibody to fibrinogen had been deposited on top of this film, the kininogen, or both high and low molecular weight
color shifted to purple or from purple to violet. On the glass slides, kininogens, had resided on glass for 10 mm, fibrinogen
sites ofdeposited antibody stained a deeper blue than did the protein remained: area ‘a’ left by the antiserum to fibrinogen
film, where it had not adsorbed antibody. Quantitative data could be
is markedly darker than the background color ‘c’ left
obtained in several ways: (I) by measuring azimuth and ellipticity
by the plasma after 10 mm. In the sample shown, area
of light reflected by the film-bearing anodized tantalum surfaces in
the ellipsometer; (2) by measuring the intensity of light transmitted ‘a’ is only slightly lighter than area ‘b,’ indicating that
by the treated glass slides after staining; and (3) simply by ranking during the 10 mm of contact, the plasma converted
colors by eye. All appeared reliable, but ranking was preferred. Data only a small amount of the deposited fibrinogen. Both
are listed (Table I ) as ranked differences in color between areas ‘a’
Williams and Fitzgerald plasma samples were defi-
and ‘b’ (Fig. I ): site of antiserum to human fibrinogen on area
previously exposed for 10 mm (area ‘a’) versus 10 sec (area ‘b’) to
cient in this activity (see Table 1). Plasma lacking
plasma. Values from 0 to 4 were assigned for each slide as follows: factor XII was somewhat deficient, while plasmas
(0) color identical for the two areas (no conversion); ( I ) area ‘a’ lacking factor XI or prekallikrein did convert their
slightly lighter than area ‘b’; (2) area ‘a’ lighter than area ‘b’ and fibrinogen deposits as did normal plasma.
clearly distinguishable from background protein film color ‘c’ (Fig.
1); (3) area ‘a’ much lighter than ‘b’ and faintly distinguishable DISCUSSION
from ‘C’; (4) area ‘a’ much lighter than ‘b’ and indistinguishable
from background color ‘c’ (complete conversion-no antibody to Before we had begun to identify the proteins depos-
fibrinogen deposited). ited by plasma, we had discovered that on a clot-
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158 VROMAN ET AL.

Fig. 1 . Williams and normal citrated plasma were applied into buffer (see text) as follows: on areas a and c. plasmas resided for 10
mm, on areas b and d. for 10 sec. After being rinsed with buffer and water. and air-dried. the slides were covered with rabbit antiserum to
human fibrinogen on areas a and b. rinsed off 2 mm later with buffer and water, and stained with Coomassie brilliant blue R while wet.
Comparison of areas a and b shows complete alteration of fibrinogen by normal plasma (no deposition of antifibrinogen antibody on a. and
only a trace of alteration by Williams plasma (a nearly equals b) Note also that areas c stained lighter than areas d, indicating less matter
was left by plasmas in 10 mm than had been deposited in 10 sec. This process of removal46 requires intact factor XII.

promoting surface, intact plasma deposits material inized or citrated blood will deposit a dense layer of
and then removes some of it again in a process requir- platelets within 10 mm, but surface coagulation
ing intact factor XII.’3 Later, we discovered that much factors will not be activated, and the fibrinogen depos-
of the material deposited initially is fibrinoge& and ited by plasma will not be converted.’5 The present
that intact plasma destroys the antigenicity of this study indicates that conversion is related to surface
deposit in a temperature- and concentration-depen- activation of clotting as follows. (A) Plasma deposits
dent reaction.’4 We called this process “conversion.” fibrinogen on the glass or glass-like surface. (B)
Factor XII seemed less essential for conversion than Factor XII may displace some of the fibrinogen, but
for subsequent removal of material. Since removal most is replaced by high molecular weight kinino-
followed, and was not part of the conversion process,4 gen. (C) Removal of some product of activation
we assumed that fibrinogen deposits were replaced by follows. (D) Platelets will only adhere where fibrin-
other material so that film thickness remained ogen remains on the surface.6
constant. Conversion and removal are both clearly One consequence of this apparent antagonism
evident in this study (see Fig. I and legend). Neither between the effects of surface properties on the activa-
of these events takes place on hydrophobic substrates, tion ofclotting versus the “stickiness” to platelets may
such as polyvinylchloride or glass that had been very well be that a material that prevents both clotting
polished with ferric stearate. On these surfaces, hepar- and platelet adhesion will be hard to find.

REFERENCES
I . Vroman L: Effects of hydrophobic surfaces upon coagulation. ways to detect antibody--antigen complex on flat surfaces. J Immu-
Thromb Diath Haemorrh 10:455, 1964 nol Meth 3:227, 1973
2. Adams AL, Klings M, Fischer GC, Vroman L: Three simple 3. Vroman L, Adams AL, Klings M, Fischer GC: Blood and air.
 From www.bloodjournal.org by guest on August 23, 2019. For personal use only.

KININOGEN AND FIBRINOGEN AT INTERFACES 159

Blood bubble interaction in decompression sickness, in Acles KN ofcoagulation factor Xl and prekallikrein. Proc NatI Acad Sci USA
(ed): DCIEM Conference Proceedings, 73-CP-960 Defence and 74:4636, 1977
Civil Institute on Environmental Medicine. Ontario, Canada, 1973, 10. RatnoffOD: Personal communication, 1979
p49
11. Saito H, Ratnoff OD, Waldmann R, Abraham JP: Defi-
4. Vroman L, Adams AL: Findings with the recording ellipsom-
ciency of a hitherto unrecognized agent, Fitzgerald factor, partici-
eter suggesting rapid exchange of specific plasma proteins at pating in surface-mediated reactions of clotting, fibrinolysis, gener-
liquid/solid interfaces. Surface Sci I 6:438, 1969 ation of Kinins, and the property of diluted plasma enhancing
5. Vroman L, Adams AL: Possible involvement offibrinogen and
vascular permeability (PF/DIL). J Clin Invest 55:1082, 1975
proteolysis in surface activation. Thromb Diath Haemorrh I 8:510,
I 2. Colman RW, Bagdasarian A, Talamo RC, Scott CF. Seavey
I967
M, Guimaraes JA, Pierce JV, Kaplan AP: Williams trait. Human
6. Zucker MB, Vroman L: Platelet adhesion induced by Ii-
kininogen deficiency with diminished levels of plasminogen proacti-
brinogen adsorbed onto glass. Proc Soc Exp Biol Med I 31:318,
vator and prekallikrein associated with abnormalities of the Hage-
I 969
man factor-dependent pathways. J Clin Invest 56:1650, 1975
7. Vroman L, Adams AL, Klings M, Fischer GC, Munoz PC,
Solenzky RP: Reactions of formed elements of blood with plasma
13. Vroman L, Lukosevicius A: Ellipsometer recordings of
changes in optical thickness of adsorbed films associated with
proteins at interfaces. Ann NY Acad Sci 283:65, 1977
surface activation of blood clotting. Nature 204:701 , I 964
8. RatnoffOD, Saito H: Evidence that Fitzgerald factor counter-
acts inhibition by kaolin or ellagic acid of the amidolytic properties 14. Vroman L. Adams AL: Identification of rapid changes at
ofa plasma kallikrein. Blood 47:243, 1976 plasma-solid interfaces. J Biomed Mater Res 3:43, 1969
9. Wiggins RC, Bouma BN, Cochrane CG, Griffin JH: Role of I 5. Vroman L: What factors determine thrombogenicity? Bull
high-molecular-weight kininogen in surface-binding and activation NY Acad Med 48:302, 1972
 From www.bloodjournal.org by guest on August 23, 2019. For personal use only.

1980 55: 156-159

Interaction of high molecular weight kininogen, factor XII, and fibrinogen in

plasma at interfaces
L Vroman, AL Adams, GC Fischer and PC Munoz

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