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Adsorption of fibrinogen-125 I on Stellite 21
Adsorption of fibrinogen-"I on Stellite 21 Study with polished and Teflon-covered surfaces In this paper, we shall describe a means of determining the amount of fibrinogen adsorbed to foreign surfaces in a given amount of time. Polished Stellite 21, Teflon-coated Stellite 21, and polished glass are the surfaces that were tested. We developed a contact system which allowed fibrinogen, labeled with radioiodine-125, to be dropped at a constant rate onto these surfaces. Next, the test surfaces were evaluated by spectrometry and autoradiography. The reproducibility of our findings indicates that our contact system is a valid means of determining the degree of adsorption of fibrinogen to foreign surfaces.
P. C. P. Lemos, M.D., E. M. C. Tolosa, M.D., E. E. Camargo, M.D., J. G. Maksoud, M.D., B. Langer, M.D., N. A. G. Stolf, M.D., T. M. Barreto, M.D., A. Nasser,* and E. J. Zerbini,** Sao Paulo, Brazil
The liberation of thrombus fragments formed over the Stellite 21 surfaces of artificial heart valves is responsible for the large number of thromboembolic complications which occur in patients with those valves. The genesis of platelet thrombus over the foreign surfaces in contact with blood starts through the adsorption of a layer of plasma proteins, and the process is continued by the adhesion and aggregation of platelets. I, :l, 4 The physicochemical interactions which occur between the plasma proteins and the surface determine the activation of clotting factors with the consequent formation of fibrin.,." Fibrinogen has been identified as one of the constituents of this initial proteic phase, adsorption to foreign surfaces.": 1"-14 For this From the Heart Institute and Surgical Technic and Experimental Division of the University of Sao Paulo Medical School, Sao Paulo, Brazil. Received for publication March 13, 1974. • Professor of Surgical Technic. •• Professor of Surgery, Head.
reason, it is important to study the behavior of fibrinogen in adsorbing to the Stellite 21 surfaces used in the manufacture of heart valve prostheses. The purpose of this report is to standardize a simple methodology for such study. Methodology
SteIlite 21 rectangular plates with standard surfaces were polished (Fig. lA) or covered with a fine Teflon coat (Fig. IB) according to polishing specifications used for the cages of Starr-Edwards valves.'! Surfaces of polished glass with the same area and shape were used as a pattern for comparison (Fig. 1C) . Plasma with few platelets was obtained by centrifugation" of normal heparinized human blood. Then, 0.2 ml, of a saline solution of lyophilized fibrinogen (at 0.16 mg. per cent) with an 86.34 per cent index of coagulability, labeled with radioiodine-125,2 was added to 20 ml. of that plasma. Samples of 1 ml. of this plasma-fibrinogen-v-I (P-F-12f'I) solution were placed in 405
The Journal af
406
Lemos et al.
Thoracic and Cardiovascular Surgery
Fig. lA. Polished Stellite 21 test plate (P) and an enlargement of its surface (original magnification x30).
Fig. lB. Teflon-coated Stellite 21 test plate (T) and an enlargement of its surface (original magnification x30).
contact with the polished and Teflon-coated Stellite 21 surfaces and with polished glass surfaces. These were labeled P, T, and V test surfaces, respectively. A standardized system of plasma dropping was used for that purpose. The system (Fig. 2) was constructed so that 1 m!. of P_F_'25I solution was dropped through a standard pipet (Pp) over the test surface (S). The test surface was fixed to a support (G) at an incline of 30 degrees and was kept inside a container (L) under the lower end of the pipet. In order to regulate the dropping time, we employed a device to control the air intake through the upper end of the pipet. This device was composed of a rubber bulb (M) whose dome was transfixed by a needle (N) connected to a plastic cannula (0).
Compression of this cannula by a correctly adjusted forceps (Q) allowed the pipet to drop 1 ml. of the P_F_'25I solution over the test surface in 30 seconds. In this manner, the system for contact between the plasma and Stellite 21 surface was mounted and adjusted. All the material used in the collecting and handling of the plasma and, except for the test surfaces, all the surfaces of the contact system were coated with silicone. After 1 m!. of plasma was dropped over the test surface, the solution was withdrawn directly into a container (l) and removed from the system. Next, the excess plasma proteins which did not adsorb to the test surface during contact were washed out by the introduction of saline solution into the container (L) through the orifice (J) in its
Volume 68
Adsorption of fibrinogen-v -I on Stellite 21
Number 3
407
Sep tember, 1974
samples of P_F_l251 solution from the same individual were placed in contact with the eight test surfaces cv, V 2, Pi, P2' r., and T 2) to constitute a series of tests. Results
v Fig. Ie. Polished glass test plate (V) and an enlargement of its surface (original magnification x30) .
side wall. When the container was full, the saline was drained through the same orifice. By this means, the test surface was washed three times in succession. After being put in contact with the P-F-1251 solution and being washed, the test surface was transferred to a system of gamma spectrometry. Under conditions of standard geometry, the radiometry of the fibrinogen-v -I adsorbed to the surface was determined. Finally, the test surface was autoradiographed. By means of the contact system and the experimental dynamics described, eight samples of human plasma from different normal individuals were placed in contact with surfaces of polished glass, polished Stellite 21, and Teflon-coated Stellite 21 and were taken in duplicate: That is, eight 1 mI.
The radioactive values in impulses per minute of the gamma emissions of radioiodine-125 (tracer of the fibrinogen) are shown in Table 1. All the tested surfaces adsorbed the fibrinogen-v -I uniformly distributed among them, as shown by autoradiography. The statistical comparison between the averages (Student's t test- ") of the individual radioactive measurements of each test surface reveal the following (Fig. 3) : 1. The polished glass surfaces (Vi and V 2) adsorb less fibrinogen than the Tefloncoated Stellite 21 surfaces (T, and T z ) . 2. The polished Stellite 21 surfaces adsorb variable quantitiese of fibrinogen. At times the degree of adsorption is similar to that of polished glass (Pi = Vi = V z ) , whereas at other times it is similar to that of Teflon-coated Stellite 21 (V z = T, = T z ) (Table II). The reproducibility of the method for three polished glass surfaces is shown in Table III. Comments The reproducibility of the method for a given type of surface (glass) with a uniform polish indicates that our methodology is satisfactory for this type of study : that is, the comparative evaluation of the fibrinogen1201 concentrations adsorbed to foreign surfaces. The detection of labeled fibrinogen , adsorbed to all the tested surfaces, by both radioactive and autoradiographic methods demonstrates the constant presence of fibrinogen in the proteic layer at the initial phase of contact with plasma. Dutton and associates" suggested this fact in 1968 , while studying the formation of thrombi over glass surfaces by microscopy. Vroman and Adams' ? also noted that fibrinogen is consistently present in the proteic layer. Work-
408
The Journal of
Lemos et at.
Thoracic ond Cardiovascular Surgery
Pp
---
1 ",I
Fig. 2. Diagram of the experimental system used for the contact between the test surfaces and the plasma-fibrinogen-twl solution. Pp, Pipet. S, Test surface. G, Support. L, Container. M, Rubber bulb. N, Needle. 0, Plastic cannula. Q, Forceps. 1, Container. J, Orifice in side wall of container. For detailed description, see text.
ing with plasma and antifibrinogen antisera, they were able to measure the width of the proteic layer adsorbed to foreign surfaces by using ellipsometry. The differences in the average radiometric values of the labeled fibrinogen adsorbed to polished Stellite 21 surfaces (Pi and P 2 ) can be related to the polishing technique. Because these surfaces are polished manually, as are the Starr-Edwards valves, the possibility of variation exists. However, on studying the radiometric values obtained from the uniformly polished glass surfaces (V1 and V 2 ) and the uniformly coated Stellite 21 surfaces, we find a
uniformity of adsorption of the "radiopharmac" (radioiodine): Vi = V 2 and T 1 = T 2 • Conclusions
1. The quantity of fibrinogen-P-I (from the P_F_125I solution) adsorbed by polished Stellite 21 surfaces in 30 seconds depends upon the polishing characteristics of the surfaces. 2. In 30 seconds, Teflon-coated Stellite 21 surfaces adsorb greater quantities of fibrinogen-v-I than do polished glass surfaces of the same size and shape. 3. The methodology used is reproducible and permits a comparative evaluation be-
Volume 68
Adsorption of fibrinogen-v-I on Stellite 21
Number 3
409
September, 1974
800
700 c-<~
';;
600
oM
~ §' oM ....,
c-<
!SOO
~
0
rl
~
H
Eo<
~Eo<
400
~
g 0
0
c-<~
0
«
300
!
r--<)M)-
~ ~
200
H
100
0
TEST SURFAOES Fig. 3. Diagram representing the average values and respective confidence intervals (p < 0.05) of the concentrations of radiopharmac (radioiodine) adsorbed to each test surface. VI and V" Polished glass. P, and P" Polished Stellite 21. T , and T" Teflon-coated Stellite 21.
Table I. Radiometric values of the tracer adsorbed to the test surfaces (measured in impulses per minute) Test surfaces
Series of experiments
VI
V,
PI
P,
TI
1 2 3 4 5 6 7 8
156 228 345 222 235 270 270 297
280 240
282 435 451 414
432 355 432 624 380 694 644 803
751
Legend: V, and
V"
363 252 300 246 330
Polished glass. P, and P"
267 190 260
951 619 659 559 435 513
T,
631 582 508 445 381 431
Polished Stellite 21. T, and T" Teflon-coated Stellite 21.
tween the concentrations of plasma proteins (fibrinogen) labeled with radioactive isotopes that are adsorbed to foreign surfaces. Summary
We have described a methodology for studying the adsorption of fibrinogen to both polished and Teflon-coated Stellite 21
surfaces at the initial phase of contact. The system used is composed of a standard pipet which can drop plasma with radiolabeled fibrinogen over the surface in a constant time (30 seconds). Radiometric values of gamma radiations of the fibrinogen-v-I adsorbed to the surfaces were compared among themselves. Values of
The Journal of
4 10
Lemos et al.
Thoracic and Cardiovascular Surgery
Table II. Comparison between the mean individual values of fibrinogen adsorption of the various test surfaces
materials in contact with blood at the initial phase of surface-induced thrombogenesis. REFERENCES
A verage values compared Unequal values
Equal values
V, V, V, P, p, T,
(V, =
= V, = P,
V, V, V, V, V, V, P, P, P,
= P, = T, = T, = T,
V,
=
P,)
<
=1= =1= =1= =1= =1= =1= =1= =1= =1=
P, T, T, p, T, T, p, T, T,
T,
(P2
=
2
3
T2 )
4
For legend, see Table I.
5
Table III. Verification of the reproducibility of the method: Test surface (glass) placed in contact with the same p_F_12.'[ solution
7
Test surface
V, Radioactive values (imp./min.) Mean ± S.D. Confidence interval (p < 0.05)
270
V, 246 264.0 ± 15.8
6
8
276
241 to 287
For legend, see Table I.
adsorption to polished glass surfaces were taken as a pattern. We have concluded that ( I) the adsorption of fibrinogen-v-I to polished Stellite 21 surfaces depends upon the polishing characteristics of those surfaces and (2) Teflon-coated Stellite 21 adsorbs more fibrinogen-v-I than do polished glass surfaces. Our method makes it possible to compare quantities of fibrinogen adsorbed from the plasma to foreign surfaces by means of radioactive tracers. We believe this same method should enable us to study the adsorption of other plasma proteins to foreign
9
10 11
12
13
14
15
Baier, R. E., and Dutton, R. C.: Initial Events in Interaction of Blood With a Foreign Surface, J. Biomed. Mater. Res. 3: 191, 1969. Barreto, T. M.: Marcacao de fibrinogenio humano com iodo radioativo (1 2 5 1). Estudo de sua coagulabilidade "in vivo," Tese, FMUSP, 1972. Dutton, R. c, Baier, R. E., Dedrick, R. L., and Bowman, R. L.: Initial Thrombus Formation on Foreign Surfaces, Trans. Am. Soc. Artif. Intern. Organs 14: 57, 1968. Dutton, R. c., Weber, A. J., Johnson, S. A., and Baier, R. E.: Microstructure of Initial Thrombus Formation on Foreign Materials, J. Biomed. Mater. Res. 3: 13, 1969. Gugler, E., and Liicher, E. F.: Platelet Function in Congenital Afibrinogenemia, Thromb. Diath. Haemorrh. 14: 361, 1965. Hellem, A. J.: Platelet Adhesiveness, Ser. Haematol. 1: 99, 1968. Salzman, E. W.: Nonthrombogenic Surfaces: Critical Review, Blood 38: 509, 1971. Salzman, E. W.: Role of Platelet Blood-Surface Interactons, Fed. Proc. 30: 1503, 1971. Scarborough, D. E.: The Pathogenesis of Thrombosis in Artificial Organs and Vessels, in Current Topics in Pathology, Berlin, 1971, Springer-Verlag, pp. 96-124. Snedecor, G. W., and Cochran, W. G.: Statistical Methods, ed. 6, Ames, Iowa, 1967, Iowa State University Press. Tsuzuki, S.: Valvula Cardiaca de Bola (fabricacao complicacoes tardias e sua aplicacao em 610 casos), Tese, FMUSP, 1969. Vroman, L., and Adams, A. L.: Possible Involvement of Fibrinogen and Proteolysis in Surface Activation, Thromb. Diath. Haemorrh. 18: 510, 1967. Vroman, L., and Adams, A. L.: Identification of Rapid Changes at Plasma-Solid Interfaces, J. Biomed. Mater. Res. 3: 43, 1969. Vroman, L., Adams, A. L., and Klings, M.: Interactions Among Human Blood Proteins at Interfaces, Fed. Proc. 30: 1494, 1971. Zucker, M., and Vroman, L.: Platelet Adhesion Induced by Fibrinogen Adsorbed Into Glass, Proc. Soc. Exp. BioI. Med. 131: 318, 1969.
P. C. P. Lemos, M.D., E. M. C. Tolosa, M.D., E. E. Camargo, M.D., J. G. Maksoud, M.D., B. Langer, M.D., N. A. G. Stolf, M.D., T. M. Barreto, M.D., A. Nasser,* and E. J. Zerbini,** Sao Paulo, Brazil
The liberation of thrombus fragments formed over the Stellite 21 surfaces of artificial heart valves is responsible for the large number of thromboembolic complications which occur in patients with those valves. The genesis of platelet thrombus over the foreign surfaces in contact with blood starts through the adsorption of a layer of plasma proteins, and the process is continued by the adhesion and aggregation of platelets. I, :l, 4 The physicochemical interactions which occur between the plasma proteins and the surface determine the activation of clotting factors with the consequent formation of fibrin.,." Fibrinogen has been identified as one of the constituents of this initial proteic phase, adsorption to foreign surfaces.": 1"-14 For this From the Heart Institute and Surgical Technic and Experimental Division of the University of Sao Paulo Medical School, Sao Paulo, Brazil. Received for publication March 13, 1974. • Professor of Surgical Technic. •• Professor of Surgery, Head.
reason, it is important to study the behavior of fibrinogen in adsorbing to the Stellite 21 surfaces used in the manufacture of heart valve prostheses. The purpose of this report is to standardize a simple methodology for such study. Methodology
SteIlite 21 rectangular plates with standard surfaces were polished (Fig. lA) or covered with a fine Teflon coat (Fig. IB) according to polishing specifications used for the cages of Starr-Edwards valves.'! Surfaces of polished glass with the same area and shape were used as a pattern for comparison (Fig. 1C) . Plasma with few platelets was obtained by centrifugation" of normal heparinized human blood. Then, 0.2 ml, of a saline solution of lyophilized fibrinogen (at 0.16 mg. per cent) with an 86.34 per cent index of coagulability, labeled with radioiodine-125,2 was added to 20 ml. of that plasma. Samples of 1 ml. of this plasma-fibrinogen-v-I (P-F-12f'I) solution were placed in 405
The Journal af
406
Lemos et al.
Thoracic and Cardiovascular Surgery
Fig. lA. Polished Stellite 21 test plate (P) and an enlargement of its surface (original magnification x30).
Fig. lB. Teflon-coated Stellite 21 test plate (T) and an enlargement of its surface (original magnification x30).
contact with the polished and Teflon-coated Stellite 21 surfaces and with polished glass surfaces. These were labeled P, T, and V test surfaces, respectively. A standardized system of plasma dropping was used for that purpose. The system (Fig. 2) was constructed so that 1 m!. of P_F_'25I solution was dropped through a standard pipet (Pp) over the test surface (S). The test surface was fixed to a support (G) at an incline of 30 degrees and was kept inside a container (L) under the lower end of the pipet. In order to regulate the dropping time, we employed a device to control the air intake through the upper end of the pipet. This device was composed of a rubber bulb (M) whose dome was transfixed by a needle (N) connected to a plastic cannula (0).
Compression of this cannula by a correctly adjusted forceps (Q) allowed the pipet to drop 1 ml. of the P_F_'25I solution over the test surface in 30 seconds. In this manner, the system for contact between the plasma and Stellite 21 surface was mounted and adjusted. All the material used in the collecting and handling of the plasma and, except for the test surfaces, all the surfaces of the contact system were coated with silicone. After 1 m!. of plasma was dropped over the test surface, the solution was withdrawn directly into a container (l) and removed from the system. Next, the excess plasma proteins which did not adsorb to the test surface during contact were washed out by the introduction of saline solution into the container (L) through the orifice (J) in its
Volume 68
Adsorption of fibrinogen-v -I on Stellite 21
Number 3
407
Sep tember, 1974
samples of P_F_l251 solution from the same individual were placed in contact with the eight test surfaces cv, V 2, Pi, P2' r., and T 2) to constitute a series of tests. Results
v Fig. Ie. Polished glass test plate (V) and an enlargement of its surface (original magnification x30) .
side wall. When the container was full, the saline was drained through the same orifice. By this means, the test surface was washed three times in succession. After being put in contact with the P-F-1251 solution and being washed, the test surface was transferred to a system of gamma spectrometry. Under conditions of standard geometry, the radiometry of the fibrinogen-v -I adsorbed to the surface was determined. Finally, the test surface was autoradiographed. By means of the contact system and the experimental dynamics described, eight samples of human plasma from different normal individuals were placed in contact with surfaces of polished glass, polished Stellite 21, and Teflon-coated Stellite 21 and were taken in duplicate: That is, eight 1 mI.
The radioactive values in impulses per minute of the gamma emissions of radioiodine-125 (tracer of the fibrinogen) are shown in Table 1. All the tested surfaces adsorbed the fibrinogen-v -I uniformly distributed among them, as shown by autoradiography. The statistical comparison between the averages (Student's t test- ") of the individual radioactive measurements of each test surface reveal the following (Fig. 3) : 1. The polished glass surfaces (Vi and V 2) adsorb less fibrinogen than the Tefloncoated Stellite 21 surfaces (T, and T z ) . 2. The polished Stellite 21 surfaces adsorb variable quantitiese of fibrinogen. At times the degree of adsorption is similar to that of polished glass (Pi = Vi = V z ) , whereas at other times it is similar to that of Teflon-coated Stellite 21 (V z = T, = T z ) (Table II). The reproducibility of the method for three polished glass surfaces is shown in Table III. Comments The reproducibility of the method for a given type of surface (glass) with a uniform polish indicates that our methodology is satisfactory for this type of study : that is, the comparative evaluation of the fibrinogen1201 concentrations adsorbed to foreign surfaces. The detection of labeled fibrinogen , adsorbed to all the tested surfaces, by both radioactive and autoradiographic methods demonstrates the constant presence of fibrinogen in the proteic layer at the initial phase of contact with plasma. Dutton and associates" suggested this fact in 1968 , while studying the formation of thrombi over glass surfaces by microscopy. Vroman and Adams' ? also noted that fibrinogen is consistently present in the proteic layer. Work-
408
The Journal of
Lemos et at.
Thoracic ond Cardiovascular Surgery
Pp
---
1 ",I
Fig. 2. Diagram of the experimental system used for the contact between the test surfaces and the plasma-fibrinogen-twl solution. Pp, Pipet. S, Test surface. G, Support. L, Container. M, Rubber bulb. N, Needle. 0, Plastic cannula. Q, Forceps. 1, Container. J, Orifice in side wall of container. For detailed description, see text.
ing with plasma and antifibrinogen antisera, they were able to measure the width of the proteic layer adsorbed to foreign surfaces by using ellipsometry. The differences in the average radiometric values of the labeled fibrinogen adsorbed to polished Stellite 21 surfaces (Pi and P 2 ) can be related to the polishing technique. Because these surfaces are polished manually, as are the Starr-Edwards valves, the possibility of variation exists. However, on studying the radiometric values obtained from the uniformly polished glass surfaces (V1 and V 2 ) and the uniformly coated Stellite 21 surfaces, we find a
uniformity of adsorption of the "radiopharmac" (radioiodine): Vi = V 2 and T 1 = T 2 • Conclusions
1. The quantity of fibrinogen-P-I (from the P_F_125I solution) adsorbed by polished Stellite 21 surfaces in 30 seconds depends upon the polishing characteristics of the surfaces. 2. In 30 seconds, Teflon-coated Stellite 21 surfaces adsorb greater quantities of fibrinogen-v-I than do polished glass surfaces of the same size and shape. 3. The methodology used is reproducible and permits a comparative evaluation be-
Volume 68
Adsorption of fibrinogen-v-I on Stellite 21
Number 3
409
September, 1974
800
700 c-<~
';;
600
oM
~ §' oM ....,
c-<
!SOO
~
0
rl
~
H
Eo<
~Eo<
400
~
g 0
0
c-<~
0
«
300
!
r--<)M)-
~ ~
200
H
100
0
TEST SURFAOES Fig. 3. Diagram representing the average values and respective confidence intervals (p < 0.05) of the concentrations of radiopharmac (radioiodine) adsorbed to each test surface. VI and V" Polished glass. P, and P" Polished Stellite 21. T , and T" Teflon-coated Stellite 21.
Table I. Radiometric values of the tracer adsorbed to the test surfaces (measured in impulses per minute) Test surfaces
Series of experiments
VI
V,
PI
P,
TI
1 2 3 4 5 6 7 8
156 228 345 222 235 270 270 297
280 240
282 435 451 414
432 355 432 624 380 694 644 803
751
Legend: V, and
V"
363 252 300 246 330
Polished glass. P, and P"
267 190 260
951 619 659 559 435 513
T,
631 582 508 445 381 431
Polished Stellite 21. T, and T" Teflon-coated Stellite 21.
tween the concentrations of plasma proteins (fibrinogen) labeled with radioactive isotopes that are adsorbed to foreign surfaces. Summary
We have described a methodology for studying the adsorption of fibrinogen to both polished and Teflon-coated Stellite 21
surfaces at the initial phase of contact. The system used is composed of a standard pipet which can drop plasma with radiolabeled fibrinogen over the surface in a constant time (30 seconds). Radiometric values of gamma radiations of the fibrinogen-v-I adsorbed to the surfaces were compared among themselves. Values of
The Journal of
4 10
Lemos et al.
Thoracic and Cardiovascular Surgery
Table II. Comparison between the mean individual values of fibrinogen adsorption of the various test surfaces
materials in contact with blood at the initial phase of surface-induced thrombogenesis. REFERENCES
A verage values compared Unequal values
Equal values
V, V, V, P, p, T,
(V, =
= V, = P,
V, V, V, V, V, V, P, P, P,
= P, = T, = T, = T,
V,
=
P,)
<
=1= =1= =1= =1= =1= =1= =1= =1= =1=
P, T, T, p, T, T, p, T, T,
T,
(P2
=
2
3
T2 )
4
For legend, see Table I.
5
Table III. Verification of the reproducibility of the method: Test surface (glass) placed in contact with the same p_F_12.'[ solution
7
Test surface
V, Radioactive values (imp./min.) Mean ± S.D. Confidence interval (p < 0.05)
270
V, 246 264.0 ± 15.8
6
8
276
241 to 287
For legend, see Table I.
adsorption to polished glass surfaces were taken as a pattern. We have concluded that ( I) the adsorption of fibrinogen-v-I to polished Stellite 21 surfaces depends upon the polishing characteristics of those surfaces and (2) Teflon-coated Stellite 21 adsorbs more fibrinogen-v-I than do polished glass surfaces. Our method makes it possible to compare quantities of fibrinogen adsorbed from the plasma to foreign surfaces by means of radioactive tracers. We believe this same method should enable us to study the adsorption of other plasma proteins to foreign
9
10 11
12
13
14
15
Baier, R. E., and Dutton, R. C.: Initial Events in Interaction of Blood With a Foreign Surface, J. Biomed. Mater. Res. 3: 191, 1969. Barreto, T. M.: Marcacao de fibrinogenio humano com iodo radioativo (1 2 5 1). Estudo de sua coagulabilidade "in vivo," Tese, FMUSP, 1972. Dutton, R. c, Baier, R. E., Dedrick, R. L., and Bowman, R. L.: Initial Thrombus Formation on Foreign Surfaces, Trans. Am. Soc. Artif. Intern. Organs 14: 57, 1968. Dutton, R. c., Weber, A. J., Johnson, S. A., and Baier, R. E.: Microstructure of Initial Thrombus Formation on Foreign Materials, J. Biomed. Mater. Res. 3: 13, 1969. Gugler, E., and Liicher, E. F.: Platelet Function in Congenital Afibrinogenemia, Thromb. Diath. Haemorrh. 14: 361, 1965. Hellem, A. J.: Platelet Adhesiveness, Ser. Haematol. 1: 99, 1968. Salzman, E. W.: Nonthrombogenic Surfaces: Critical Review, Blood 38: 509, 1971. Salzman, E. W.: Role of Platelet Blood-Surface Interactons, Fed. Proc. 30: 1503, 1971. Scarborough, D. E.: The Pathogenesis of Thrombosis in Artificial Organs and Vessels, in Current Topics in Pathology, Berlin, 1971, Springer-Verlag, pp. 96-124. Snedecor, G. W., and Cochran, W. G.: Statistical Methods, ed. 6, Ames, Iowa, 1967, Iowa State University Press. Tsuzuki, S.: Valvula Cardiaca de Bola (fabricacao complicacoes tardias e sua aplicacao em 610 casos), Tese, FMUSP, 1969. Vroman, L., and Adams, A. L.: Possible Involvement of Fibrinogen and Proteolysis in Surface Activation, Thromb. Diath. Haemorrh. 18: 510, 1967. Vroman, L., and Adams, A. L.: Identification of Rapid Changes at Plasma-Solid Interfaces, J. Biomed. Mater. Res. 3: 43, 1969. Vroman, L., Adams, A. L., and Klings, M.: Interactions Among Human Blood Proteins at Interfaces, Fed. Proc. 30: 1494, 1971. Zucker, M., and Vroman, L.: Platelet Adhesion Induced by Fibrinogen Adsorbed Into Glass, Proc. Soc. Exp. BioI. Med. 131: 318, 1969.