THROMBOSIS RESEARCH Printed in the United
vol. States
7, pp. 471-480, Pergamon Press,
1975 Inc.
EFFECTS OF COLLAGEN AND ARTIFICIAL SURFACES ON PLATELETS THAT INFLUENCE BLOOD COAGULATION
R.G. Mason and KS. Read Department of Pathology University of North Carolina Chapel Hill, N. C. 27514
in revised (Received 8.6.1975; Accepted by Editor A.L.
form 4.8.1975. Copley)
ABSTRACT Human platelets were isolated by albumin density gradient technique. Isolated platelets were exposed to collagen or particles of polymeric and other materials and then assayed for the effects that this exposure might have upon the clotting time when the various mixtures were added to platelet-poor plasmas. Normal plasma and plasmas from patients deficient in factors VII, IX, or XII were tested. Normal platelets and platelets from patients deficient in factors XI or XII were used in these studies. Collagen, Silastic, and silicone shortened clotting times of platelet-poor plasmas primarily through their effect on factor XI present on the surface of test platelets. Cuprophane, glass, kaolin, polyethylene, polyvinyl chloride, and Teflon produced shortening of clotting times of platelet-poor plasma by diverse means. These means included probable activation of factor XI on the platelet surface, probable activation of plasma factor XII, activation of platelet factor 3 and activation or unmasking of a tissue factor-like activity in the platelet preparation. These findings indicate the complexity of the inter action of artificial surfaces with blood and point out that a truly blood compatible artificial surface may be nearly impossible to fabricate.
INTRODUCTION The ability of collagen preparations and of kaolin to activate factor XI or a similar factor present on the outer surfaces of the platelet plasma membrane has been reported recently (1,2). Collagen and kaolin are known also to increase the amount of platelet factor 3 "available" on platelets (3,4). In addition, both collagen and kaolin activate factor XII present in plasma (5,6). Hence, collagen and kaolin, two "foreign" surfaces not normally in contact with blood, can influence the blood coagulation mechanisms at at least three different points. The increased use of numerous artificial materials with diverse surface
471
472
CGLUGET,PLATELETS
AND
CLOTTING
Vo1.7,No.3
properties in cardiovascular prostheses and in various extracorporeal devices that contact flowing blood is well documented (7,8). Most, if not all, artificial surfaces in contact with native blood activate the intrinsic blood clotting system. The mechanisms by which such activation takes place are only partially understood at present. Certain effects of a number of different artificial or "foreign" surfaces on the blood clotting system were investigated in order to gain a better understanding of blood-artificial surface interactions. Surfaces studied included collagen and kaolin as well as a number of materials commonly used in cardiovascular prostheses and in other devices that contact blood ex vivo. Effects of these foreign surfaces on activation of factor XII in plasma, activation of factor XI on platelets, activation of platelet factor 3, and possible activation of tissue factor on platelets was quantitated.
MATERIALS AND METHODS Platelets in Plasma Platelets were prepared by the albumin density gradient procedure of Walsh (9) from blood of normal subjects and patients with congenital bloo$+ coagulation defects or hemostatic disorders and finally resuspended in Ca free Tyrode's solution. Platelet-poor plasma (PPP) was prepared from whole blood by centrifugation at 39,000 g for 30 minutes at 4'C; only the middle two quarters of the PPP column were collected. All blood was anticoagulated with acid-citrate-dextrose (ACD) (7.5 ml ACD to 42.5 ml of blood). Washed or isolated platelets and plasmas were kept in silicone-coated glass tubes at 28'C for not longer than one hour before use. Test Materials Collagen was prepared from human Achilles tendon by the method of Hovig (10). Cuprophane PT 150 was obtained from Gulf South Research Institute, New Orleans, La. Glass beads measuring 0.25 to 0.30 mm in diameter were obtained from B. Braun Co., &lsungen, Germany. Kaolin was obtained from Fisher Scientific Co., Raleigh, N.C. and used in the form obtained. Polyethylene (PE) low density, polyvinyl chloride (PVC), and Teflon TFE were obtained from Commercial Plastics, Raleigh, N.C. Silastic (Dow Corning Co., Midland, Mich.) was prepared by mixing 10 ml of MDX4-4092 and 6 drops of catalyst. Siliconecoated glass beads and laboratory glassware were prepared by expsing glass to a 10% solution of Dri-film SC-87 (Pierce Chemical Co., Rockford, Ill.) in toluene for 15 min. followed by rinsing in 0.001 N NaOH solution, then in tap water, and finally in distilled water. All test materials were washed in a dilute solution of detergent (FL-70, Fisher Scientific Co., Raleigh, N.C.) and rinsed 5 times in tap water and twice in demineralized water. Washed materials were dried in air. All polymeric materials were ground in a Wiley mill with a 0.3 mm mesh screen. Only polymer particles which would pass through a 297 micron sieve but not through a 210 micron sieve were used. Surface area values for certain test materials as determined by gas adsorption techniques (11) (Nu lear Materials and Rqui ment Corp., Appollo, Penn.) were: 4 glass beads, 0.77 m5 /gm; and Teflon, 0.08 m /gm. Blood was obtained from donors congenitally deficient in factors IX, q or XII. PPP from a patient congenitally deficient in factor VII was obtained commercially (Medical Sciences International, Stoneham, Mass.). All patients except the one deficient in factor VII have been examined repeatedly in the Clinical Coagulation Laboratory of North Carolina &morial Hospital, and
COLWGEN,PWTELETS
Vo1.7,No.3
their
blood coagulation
factor
AND
deficiencies
!+73
CLOTTL\TG
have
been characterized
thoroughly
Test Procedure Dry particles of each different material sufficient to fill a volume of 0.1 ml were placed in a 15 ml silicone-coated conical glass centrifuge tube One half ml of platelet suspension was added to these particles, and the mixture was maintained at 37'C for 10 minutes with gentle mixing every two During this ten minute incubation period, 0.1 ml aliquots were minutes. removed at intervals after thorough mixing and added to 0.2 ml of PPP in The mixture, composed of 10 X 75 mm silicone-coated glass tubes at 23'C. platelets, test material, and plasma, was recalcified by addition of 0.05 ml Clotting times of of 0.1 M CaCl solution and the clotting time recorded. In each case, test mixtures2were determined visually by use of stopwatches. a control was performed simultaneously in which saline was substituted for Plasma clotting the test. material in the platelet-test material mixture. times were recorded in minutes and expressed as percent of the saline control.
Other Stypven time tests (12) were performed on mixtures of platelets, materials, and plasma and on control mixtures with saline substituted material.
test for test
RESULTS
Effects
of Test Materials
on Normal
and Factor
XII-Deficient
PPP
The effects of each of the different test materials investigated on the clotting time of recalcified norm,) and factor XII-deficient PPPs were investigated. In this test system, Ca -free Tyrode's solution alone was substituted for the platelet suspension. Results are shown in Table I. It can be seen that collagen, Silastic, and silicone did not activate the intrinsic blood coagulation system of normal PPP under these test conditions. On the other hand, glass and kaolin produced marked shortening of the clotting time of normal PPP compared to the saline control but failed to activate the intrinsic blood coagulation system of factor XII-deficient PPP. Cuprophane, PE, PVC, and Teflon produced intermediate degrees of activation of the intrinsic blood clotting system of normal PPP but, like glass and kaolin, failed to activate the intrinsic clotting system of factor XIIdeficient PPP. It should be remembered that in this control system, platelets and other sources of thromboplastin are present in only minute quantities.
Effects
of Test Material
on Normal
Platelets
Normal washed platelets were exposed to the various test materials and ef fects of this exposure quantitated by addition of platelet-test material mixtures to normal PPP and to PPPs deficient in factors VII, XI, or XII. Results are shown in Table II. Exposure of normal, factor XI-deficient, and factor XII-deficient
PPPs to mixtures
of test material
and normal
platelets
produced
474
COLLAGEN,PLATEIZ-TS
AND
GLOT'I'mG
nearly identical results. In each case, glass and kaolin produced the greatest shortening of clotting times, while Cuprophane produced a lesser degree of shortening. The amount of shortening of plasma clotting times produced by mixtures of platelets with glass, kaolin, Cuprophane, PVC, or Teflon is proportional to that produced in control studies in which platelets were not present in the test system (Table I). Changes produced by collagen, Silastic,
TABLE I Effects of Test Materials Alone on Clotting Times of Normal and Factor XII-Deficient PPP
Test Material
Clotting time as % of saline control Normal PPP
Collagen
XII-deficient PPP
100
100
Cuprophane
55
100
Glass
27
100
Kaolin
24
100
PE
80
100
PVC
65
100
Silastic
100
100
Silicone
100
100
68
100
Teflon
Each value is the mean of values from three separate experiments. Clotting time of saline control = 92 minutes for normal PPP but 150 minutes for XII-deficient PPP.
silicone, and PE clearly indicate an effect of these materials on platelets, since the degree of shortening of the plasma clotting time exceeded values obtained with test material alone (Table I) in each case. Indeed, collagen, Silastic, and silicone produced no shortening of plasma clotting times in these control tests. Effects of exposure of factor VII-deficient PPP to mixtures of test materials and platelets (Table II) suggested that at least part of the effect of test materials on platelets might be due to an increased availability of platelet tissue factor-like activity. This possible effect of test materials on this tissue factor activity present in the platelet preparation was suggested by the less marked degree of shortening of factor VII-deficient PPP clotting times as compared to values of similar tests with normal PPP. That is, some of the effect seen in tests with normal PPP appears to depend on the
COLLAGEN,
L-01.7,40.3
PLATELFZK
itw
CLO'T"I'L\TG
475
presence of factor VII. Exposure of factor ix-deficient PPP to mixtures of test materials and platelets produced shortening of clotting times only in test mixtures that contained collagen, glass, or kaolin (data not shown). The degree of shortening of plasma clotting times was less than half that observed in tests with normal PPP. Hence, the strong effect of collagen, glass, and kaolin appears to be mediated in part through action of plasma factor XII, and this effect is dependent on the presence of factor IX.
TABLE II Effects of Mixtures of Test Materials and Platelets from Normal Subjects on the Clotting Time of Normal and Factor VII-, XI-, or XII-Deficient PPP
Clotting time as X of saline control
Test material
Normal platelets and test materials added to PPP from: Normal VII-deficient XI-deficient XII-deficient Collagen
7124
89 +_ 9
76 +_ 6
68 + 11
Cuprophane
58 + 4
86 2 1
64 +_ 3
63 +_ 5
Glass
20 + 4
33 + 6
35 + 1
30 + 6
Kaolin
18 +_ 2
24 + 3
2124
21 +_ 7
PE
70 + 9
87 +_ 5
75 + 2
71 + 16
PVC
68 + 4
80 +, 10
66 +_ 15
67 +_ 16
Silastic
77 + 3
89 +_4
70 +_ 19
80 +_ 9
Silicone
76 + 3
80 t 6
77 +, 10
83 +_ 13
Teflon
69 + 8
87 + 11
76 +_4
75 +, 12
Number of different Saline control clotting time (min.)
4
4
35
38
4
16
60
70
Effect of Test Materials on Platelets from Patients Congenitally Deficient in Factors XI or XII Exposure of platelets from a patient congenitally deficient in factor XI to test materials and subsequent exposure of this mixture to normal PPP pre duced marked shortening of clotting times in tests with glass and kaolin and intermediate degrees of shortening in tests with Cuprophane, PE, PVC and Teflon (Table III). Under these test conditions, collagen, Silastic, and sili-
474
COLLAGEN,PLATELETS
Vo1.7,No.3
ANIl CLOTTING
cone produced little if any shortening of plasma clotting times. These data suggest that collagen, Silastic, and silicone mediate their effect to shorten plasma clotting times mainly through factor XI present on the surface of platelets. The remaining test materials appear to mediate their effects primarily by other mechanisms. Exposure of normal PPP to mixtures of test materials and platelets from a patient congenitally deficient in factor XII produced results (Table III) similar to those obtained in tests with normal PPP and normal platelets (Table II). Exposure of factor XII-deficient PPP to mixtures of test materials and factor XII-deficient platelets produced varying degrees of shortening of plasma clotting times (Table III). The greatest decrease in plasma clotting
TABLE III Effects of Mixtures lets on the Clotting
of Test Materials and Factor XI- or XII-Deficient Time of Normal or Factor XII-Deficient PPP
Plate-
Test material
Clottine times as a of saline XI-deficient XII-deficient platelets and platelets and normal PPP normal PPP
control XII-deficient platelets and XII-deficient PPP
Collagen
93 + 14
73 +_ 12
62 +_ 13
Cuprophane
62 +_ 16
66 + 4
78 +, 6
Glass
29 +_ 1
26 & 1
35 + 4
Kaolin
19 +_ 6
20 f. 5
24 5 3
PE
77 + 19
77 +, 6
74 2 6
PVC
71 +, 12
70 +_ 17
76 +, 9
Silastic
92 +_ 1.3
78 + 11
80 +, 7
Silicone
94 +_ 14
78 2 13
79 t
Teflon
73 f
68 +, 4
55 +, 5
Number of different exper imen t s Saline control, clotting time (min.)
12
4
4
4
30
32
102
11
but these changes were not times was obtained in tests with glass and kaolin, degrees of shortening of as marked as in tests with normal PPP. Intermediate The plasma clotting times were obtained in tests with collagen and Teflon. least amount of change occurred in tests with Cuprophane, PE, PVC, Silastic, of plasma clotting times and silicone. In each case, the degree of shortening is presumed to be produced primarily by mechanisms other than activation of The mechanisms of shortening of factor XII-deficient Plasplasma factor XII.
vo1,7,ro.3
COLIAGEN,
PLATELETS
AXi
CLOTTP;G
$77
ma clotting times by the various test materials mixed with factor XII-deficient platelets could be by activation of tissue factor activity present in the platelet preparation, or by effects on platelet factor 3. Effects of Mixtures of Test Material and Normal or Factor XI-Deficient Platelets on Stvnven Time Values of Normal and Factor IX- or XII-Deficient PPP Effects of mixtures of test materials with normal or factor XI-deficient platelets on Stypven time values are shown in Table IV. These data indicate that all surfaces activate platelet factor 3 to some extent with glass and kaolin producing the most marked change. Experiments performed with normal platelets and factor IX- or XII-deficient PPP and with factor XI-deficient platelets and normal PPP indicate that the test materials activate platelet factor 3 by mechanisms independent of the presence of blood coagulation factors IX, XI, or XII.
TABLE IV Effects of Mixtures of Test Materials and Normal or Factor XI-Deficient Platelets on Stypven Time Values of Tests Performed with Normal or Factor IX- or XII-Deficient PPP
ST as % of saline control Test material Normal PPP
Normal platelets and: IX-deficient XII-deficient PPP PPP
XI-deficient platelets and Normal PPP
Collagen
68
64
72 2 5
66 + 7
Cuprophane
61
61
65 t 3
61 +_ 1
Glass
53
56
55 2 3
54 + 3
Kaolin
32
33
37 &4
30 + 0
PE
74
67
69 5 6
69 +_ 4
PVC
69
68
70 + 5
68 +_ 6
Silastic
68
67
70 t 8
69 +_4
Silicone
78
76
74 + 7
71 + 4
Teflon
75
76
74 +_ 5
78 +_ 6
2
2
6
4
53
54
48
Number of different experiments Saline control Stypven times (sec.)
54
478
COLIAGEN,PLATELETS
AND
CLOT'ITNG
Vo1.7,No.3
DISCUSSION
Under the conditions of the test system used here in which a large surface area of test material was exposed to washed platelets and the materialplatelet mixture was added subsequently to PPP, collagen and each of the artificial surfaces tested were shown to influence blood clotting. Collagen, Silastic, and silicone did not activate the intrinsic blood clotting system under these experimental conditions when platelets were omitted from the test system. These same three materials did appear to accelerate clotting times by a mechanism dependent on the presence of factor XI or a similar factor on the surface of washed platelets. The other artificial surfacsstested also appeared to react with various components of platelets and plasma to produce shortening of test plasma clotting times. One known platelet component in addition to factor XI that may have been influenced by the artificial surfaces tested was platelet factor 3. In addition, the platelet preparation contained a tissue factor-like activity after exposure to test materials. Reference to activation of various factors on platelets is made with the full understanding that evidence for activation of any specific factor is indirect; final proof awaits purification and characterization of these platelet-bound or platelet-associated factors. The most clear cut examples of the mechanisms of action of artificial surfaces on platelet constituents were the effects of collagen, Silastic, and silicone. These three surfaces could be shown to shorten the plasma clotting time by activation of a system dependent on the presence of factor XI or a similar factor on the platelet regardless of whether tests were performed with normal plasma or with plasmas deficient in factors XI or XII. That the major effect of collagen, Silastic, or silicone was mediated through factor XI present on platelets was demonstrated by the use of platelets deficient in factor XI. However, tests with normal platelets and plasma deficient in factor VII indicated that collagen, Silastic, and silicone each may have a minor effect of activating or making available a tissue factor activity present in the platelet preparation. Similarly, Stypven time tests indicate that each of these three materials activates or makes available detectable quantities of platelet factor 3 in a manner independent of factor XI present on platelets. The mechanisms by which Cuprophane, glass, kaolin, PE, PVC, and Tef!on influence blood coagulation were less clearly delineated than was the case with collagen, Silastic, and silicone. Cuprophane, glass, kaolin, PE, PVC, and Teflon were shown to activate factor XII in plasma confirming the work of several other workers (5,6). In addition, the presence of each of these materials in tests with both platelets and PPP deficient in factor XII were shown to produce a decrease in plasma clotting times. Such a decrease could have been brought about by activation of factor XI or a similar factor present on the factor XII-deficient platelets, by activation of platelet factor 3, or by activation of the tissue factor activity. The relative contributions of activated platelet factor 3 or the tissue factor activity to the overall effects of Cuprophane, glass, kaolin, PE, PVC, and Teflon in the test system are difficult to gauge with methods currently available. The present studies can be criticized with regard to the total surface area of the various test materials to which platelets were exposed. The total surface areas of glass, PE, PVC, Silastic, silicone, and Teflon are approximately equal. The total surface areas of Cuprophane and kaolin are much great. er than those of all of the other materials tested except collagen, which probably had a total surface area exceeding that of any of the other test materials. It is of interest that glass and kaolin produced similar degrees
Vo1.7,To.3
COLUGEN,
PLATELETS
AND CLOTTING
479
of shortening of plasma clotting times, even though the total surface area of each must have differed considerably. Contributions of the silicone-coated glassware in which tests were conducted should have been negligible on a surface area basis and would have been neutralized by the controls, if present. The present studies can be criticized also in that experimental design may have permitted transfer of protein from the platelet suspension medium to the surfaces of the dry test particles. These studies indicate that collagen and a number of different artificial surfaces can influence both the intrinsic and extrinsic blood coagulation systems in multiple ways. This multiplicity of activating or enhancing effects may well explain why the search for an artificial surface highly compatible with blood has been notably unsuccessful. A major contribution to our understanding of the effects of artificial surfaces on blood coagulation has been the recent work of Walsh (l), who showed that both kaolin and collagen could activate factor XI or a similar factor present on platelets. Effects of certain artificial surfaces on platelet factor 3 have been known for a number of years (3,4). The fact that platelets may possess tissue factor activity has been appreciated only recently (12). The tissue factor-like activity measured in these experiments may be mediated by a platelet-leukocyte interaction reported recently (13). b the other hand, platelets may possess tissue factor-like activity demonstrable only upon adherence of platelets to a surface as occurs with fibroblasts (14). The present findings and the work of others (1,2) serve mainly to indicate the complexity of the interactions of artificial surfaces with blood, and point out that a truly blood-compatible artificial surface may be nearly impossible to fabricate.
ACKNOWLEDGEMENTS Supported in part by Contract PH-43-68-977 with the Artificial KidneyChronic Uremia Program of the National Institute for Arthritis, Metabolism, and Digestive Diseases, and by grants HL46351, HL13296, and HL14228 from the National Heart and Lung Institute, National Institutes of Health.
REFERENCES 1.
WALSH, P.N. The effects of collagen and kaolin on the intrinsic coagulant activity of platelets. u. 2. Haematol. 22, 393, 1972.
2.
SCHIFFMAN, S., RAPAPORT, S.I., and CHOUG, M.M.Y. Platelets and initiation of intrinsic clotting. m. 2. Haematol. 24, 633, 1973.
3.
SPAET, T.H. and CINTRON, J. m.
Studies on platelet factor -3 availability. 2. Haematol. 11, 269, 1965.
4.
HARDISTY, R.M. and HUTTON, R.A. Platelet aggregation and the availability of platelet factor 3. w. J. Haematol. 12, 764, 1966.
5.
NIEWIARaWSKI, S., BAN’KCMSKI, E., and ROG(;WICKA,I. Studies on the adsorption and activation of the Hageman factor (Factor XII) by collagen and elastin. Thromb. -. Diath Haemorrh. 14, 387, 1965.
6.
WILNER, G.D., NOSSEL, H.L., and LEROY, E.C. Activation of Hageman factor by collagen. 2. -Clin. Invest. 47, 2608, 1968.
480
COLLAGEN,PLATEld?TS
AND CLCYMXNG
Vo1.7,No*3
7.
SALZMAN, E.W. Surface effects in hemostasis and thrombosis. In, The Chemistry of Biosurfaces, vbl. 2, M.E. Hair editor, Marcel Dekker,xc., New York, 1972, p. 489.
8.
MASON, R.G. The interaction of blood hemostatic elements with artificial surfaces. &. Hemost. Thromb. 1, 141, 1972. --
9.
WALSH, P.N. Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. m. J. Haematol. 22, 205, 1972.
10.
HOVIG, T. Release of a platelet-aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline "extract" of tendons Thromb. Diath. Haemorrh. 9, 264, 1963.
11.
FAREY, M.G., and BERNARD, G.T. Determination of surface areas by an improved continuous flow method. Analvt. Chem. 43, 1307, 1971.
12.
BIGGS, R., DENSON, K.W.E., RIESENBERG, I).,and MCINTYRE, C. The coagulant activity of platelets. _Brit. J. Haematol. 15, 283, 1968.
13.
NIEMETZ, J. and MARCUS, A.J. The stimulatory effect of platelets and platelet membranes on the procoagulant activity of leukocytes. J. m. Invest. 54, 1437, 1974.
14.
ZACHARSKI, L.R. and MCINTYRE, O.R. Membrane-mediated synthesis of tissue factor (thromboplastin) in cultured fibroblasts. Blood. 41, 679, 1973.
vol. States
7, pp. 471-480, Pergamon Press,
1975 Inc.
EFFECTS OF COLLAGEN AND ARTIFICIAL SURFACES ON PLATELETS THAT INFLUENCE BLOOD COAGULATION
R.G. Mason and KS. Read Department of Pathology University of North Carolina Chapel Hill, N. C. 27514
in revised (Received 8.6.1975; Accepted by Editor A.L.
form 4.8.1975. Copley)
ABSTRACT Human platelets were isolated by albumin density gradient technique. Isolated platelets were exposed to collagen or particles of polymeric and other materials and then assayed for the effects that this exposure might have upon the clotting time when the various mixtures were added to platelet-poor plasmas. Normal plasma and plasmas from patients deficient in factors VII, IX, or XII were tested. Normal platelets and platelets from patients deficient in factors XI or XII were used in these studies. Collagen, Silastic, and silicone shortened clotting times of platelet-poor plasmas primarily through their effect on factor XI present on the surface of test platelets. Cuprophane, glass, kaolin, polyethylene, polyvinyl chloride, and Teflon produced shortening of clotting times of platelet-poor plasma by diverse means. These means included probable activation of factor XI on the platelet surface, probable activation of plasma factor XII, activation of platelet factor 3 and activation or unmasking of a tissue factor-like activity in the platelet preparation. These findings indicate the complexity of the inter action of artificial surfaces with blood and point out that a truly blood compatible artificial surface may be nearly impossible to fabricate.
INTRODUCTION The ability of collagen preparations and of kaolin to activate factor XI or a similar factor present on the outer surfaces of the platelet plasma membrane has been reported recently (1,2). Collagen and kaolin are known also to increase the amount of platelet factor 3 "available" on platelets (3,4). In addition, both collagen and kaolin activate factor XII present in plasma (5,6). Hence, collagen and kaolin, two "foreign" surfaces not normally in contact with blood, can influence the blood coagulation mechanisms at at least three different points. The increased use of numerous artificial materials with diverse surface
471
472
CGLUGET,PLATELETS
AND
CLOTTING
Vo1.7,No.3
properties in cardiovascular prostheses and in various extracorporeal devices that contact flowing blood is well documented (7,8). Most, if not all, artificial surfaces in contact with native blood activate the intrinsic blood clotting system. The mechanisms by which such activation takes place are only partially understood at present. Certain effects of a number of different artificial or "foreign" surfaces on the blood clotting system were investigated in order to gain a better understanding of blood-artificial surface interactions. Surfaces studied included collagen and kaolin as well as a number of materials commonly used in cardiovascular prostheses and in other devices that contact blood ex vivo. Effects of these foreign surfaces on activation of factor XII in plasma, activation of factor XI on platelets, activation of platelet factor 3, and possible activation of tissue factor on platelets was quantitated.
MATERIALS AND METHODS Platelets in Plasma Platelets were prepared by the albumin density gradient procedure of Walsh (9) from blood of normal subjects and patients with congenital bloo$+ coagulation defects or hemostatic disorders and finally resuspended in Ca free Tyrode's solution. Platelet-poor plasma (PPP) was prepared from whole blood by centrifugation at 39,000 g for 30 minutes at 4'C; only the middle two quarters of the PPP column were collected. All blood was anticoagulated with acid-citrate-dextrose (ACD) (7.5 ml ACD to 42.5 ml of blood). Washed or isolated platelets and plasmas were kept in silicone-coated glass tubes at 28'C for not longer than one hour before use. Test Materials Collagen was prepared from human Achilles tendon by the method of Hovig (10). Cuprophane PT 150 was obtained from Gulf South Research Institute, New Orleans, La. Glass beads measuring 0.25 to 0.30 mm in diameter were obtained from B. Braun Co., &lsungen, Germany. Kaolin was obtained from Fisher Scientific Co., Raleigh, N.C. and used in the form obtained. Polyethylene (PE) low density, polyvinyl chloride (PVC), and Teflon TFE were obtained from Commercial Plastics, Raleigh, N.C. Silastic (Dow Corning Co., Midland, Mich.) was prepared by mixing 10 ml of MDX4-4092 and 6 drops of catalyst. Siliconecoated glass beads and laboratory glassware were prepared by expsing glass to a 10% solution of Dri-film SC-87 (Pierce Chemical Co., Rockford, Ill.) in toluene for 15 min. followed by rinsing in 0.001 N NaOH solution, then in tap water, and finally in distilled water. All test materials were washed in a dilute solution of detergent (FL-70, Fisher Scientific Co., Raleigh, N.C.) and rinsed 5 times in tap water and twice in demineralized water. Washed materials were dried in air. All polymeric materials were ground in a Wiley mill with a 0.3 mm mesh screen. Only polymer particles which would pass through a 297 micron sieve but not through a 210 micron sieve were used. Surface area values for certain test materials as determined by gas adsorption techniques (11) (Nu lear Materials and Rqui ment Corp., Appollo, Penn.) were: 4 glass beads, 0.77 m5 /gm; and Teflon, 0.08 m /gm. Blood was obtained from donors congenitally deficient in factors IX, q or XII. PPP from a patient congenitally deficient in factor VII was obtained commercially (Medical Sciences International, Stoneham, Mass.). All patients except the one deficient in factor VII have been examined repeatedly in the Clinical Coagulation Laboratory of North Carolina &morial Hospital, and
COLWGEN,PWTELETS
Vo1.7,No.3
their
blood coagulation
factor
AND
deficiencies
!+73
CLOTTL\TG
have
been characterized
thoroughly
Test Procedure Dry particles of each different material sufficient to fill a volume of 0.1 ml were placed in a 15 ml silicone-coated conical glass centrifuge tube One half ml of platelet suspension was added to these particles, and the mixture was maintained at 37'C for 10 minutes with gentle mixing every two During this ten minute incubation period, 0.1 ml aliquots were minutes. removed at intervals after thorough mixing and added to 0.2 ml of PPP in The mixture, composed of 10 X 75 mm silicone-coated glass tubes at 23'C. platelets, test material, and plasma, was recalcified by addition of 0.05 ml Clotting times of of 0.1 M CaCl solution and the clotting time recorded. In each case, test mixtures2were determined visually by use of stopwatches. a control was performed simultaneously in which saline was substituted for Plasma clotting the test. material in the platelet-test material mixture. times were recorded in minutes and expressed as percent of the saline control.
Other Stypven time tests (12) were performed on mixtures of platelets, materials, and plasma and on control mixtures with saline substituted material.
test for test
RESULTS
Effects
of Test Materials
on Normal
and Factor
XII-Deficient
PPP
The effects of each of the different test materials investigated on the clotting time of recalcified norm,) and factor XII-deficient PPPs were investigated. In this test system, Ca -free Tyrode's solution alone was substituted for the platelet suspension. Results are shown in Table I. It can be seen that collagen, Silastic, and silicone did not activate the intrinsic blood coagulation system of normal PPP under these test conditions. On the other hand, glass and kaolin produced marked shortening of the clotting time of normal PPP compared to the saline control but failed to activate the intrinsic blood coagulation system of factor XII-deficient PPP. Cuprophane, PE, PVC, and Teflon produced intermediate degrees of activation of the intrinsic blood clotting system of normal PPP but, like glass and kaolin, failed to activate the intrinsic clotting system of factor XIIdeficient PPP. It should be remembered that in this control system, platelets and other sources of thromboplastin are present in only minute quantities.
Effects
of Test Material
on Normal
Platelets
Normal washed platelets were exposed to the various test materials and ef fects of this exposure quantitated by addition of platelet-test material mixtures to normal PPP and to PPPs deficient in factors VII, XI, or XII. Results are shown in Table II. Exposure of normal, factor XI-deficient, and factor XII-deficient
PPPs to mixtures
of test material
and normal
platelets
produced
474
COLLAGEN,PLATEIZ-TS
AND
GLOT'I'mG
nearly identical results. In each case, glass and kaolin produced the greatest shortening of clotting times, while Cuprophane produced a lesser degree of shortening. The amount of shortening of plasma clotting times produced by mixtures of platelets with glass, kaolin, Cuprophane, PVC, or Teflon is proportional to that produced in control studies in which platelets were not present in the test system (Table I). Changes produced by collagen, Silastic,
TABLE I Effects of Test Materials Alone on Clotting Times of Normal and Factor XII-Deficient PPP
Test Material
Clotting time as % of saline control Normal PPP
Collagen
XII-deficient PPP
100
100
Cuprophane
55
100
Glass
27
100
Kaolin
24
100
PE
80
100
PVC
65
100
Silastic
100
100
Silicone
100
100
68
100
Teflon
Each value is the mean of values from three separate experiments. Clotting time of saline control = 92 minutes for normal PPP but 150 minutes for XII-deficient PPP.
silicone, and PE clearly indicate an effect of these materials on platelets, since the degree of shortening of the plasma clotting time exceeded values obtained with test material alone (Table I) in each case. Indeed, collagen, Silastic, and silicone produced no shortening of plasma clotting times in these control tests. Effects of exposure of factor VII-deficient PPP to mixtures of test materials and platelets (Table II) suggested that at least part of the effect of test materials on platelets might be due to an increased availability of platelet tissue factor-like activity. This possible effect of test materials on this tissue factor activity present in the platelet preparation was suggested by the less marked degree of shortening of factor VII-deficient PPP clotting times as compared to values of similar tests with normal PPP. That is, some of the effect seen in tests with normal PPP appears to depend on the
COLLAGEN,
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475
presence of factor VII. Exposure of factor ix-deficient PPP to mixtures of test materials and platelets produced shortening of clotting times only in test mixtures that contained collagen, glass, or kaolin (data not shown). The degree of shortening of plasma clotting times was less than half that observed in tests with normal PPP. Hence, the strong effect of collagen, glass, and kaolin appears to be mediated in part through action of plasma factor XII, and this effect is dependent on the presence of factor IX.
TABLE II Effects of Mixtures of Test Materials and Platelets from Normal Subjects on the Clotting Time of Normal and Factor VII-, XI-, or XII-Deficient PPP
Clotting time as X of saline control
Test material
Normal platelets and test materials added to PPP from: Normal VII-deficient XI-deficient XII-deficient Collagen
7124
89 +_ 9
76 +_ 6
68 + 11
Cuprophane
58 + 4
86 2 1
64 +_ 3
63 +_ 5
Glass
20 + 4
33 + 6
35 + 1
30 + 6
Kaolin
18 +_ 2
24 + 3
2124
21 +_ 7
PE
70 + 9
87 +_ 5
75 + 2
71 + 16
PVC
68 + 4
80 +, 10
66 +_ 15
67 +_ 16
Silastic
77 + 3
89 +_4
70 +_ 19
80 +_ 9
Silicone
76 + 3
80 t 6
77 +, 10
83 +_ 13
Teflon
69 + 8
87 + 11
76 +_4
75 +, 12
Number of different Saline control clotting time (min.)
4
4
35
38
4
16
60
70
Effect of Test Materials on Platelets from Patients Congenitally Deficient in Factors XI or XII Exposure of platelets from a patient congenitally deficient in factor XI to test materials and subsequent exposure of this mixture to normal PPP pre duced marked shortening of clotting times in tests with glass and kaolin and intermediate degrees of shortening in tests with Cuprophane, PE, PVC and Teflon (Table III). Under these test conditions, collagen, Silastic, and sili-
474
COLLAGEN,PLATELETS
Vo1.7,No.3
ANIl CLOTTING
cone produced little if any shortening of plasma clotting times. These data suggest that collagen, Silastic, and silicone mediate their effect to shorten plasma clotting times mainly through factor XI present on the surface of platelets. The remaining test materials appear to mediate their effects primarily by other mechanisms. Exposure of normal PPP to mixtures of test materials and platelets from a patient congenitally deficient in factor XII produced results (Table III) similar to those obtained in tests with normal PPP and normal platelets (Table II). Exposure of factor XII-deficient PPP to mixtures of test materials and factor XII-deficient platelets produced varying degrees of shortening of plasma clotting times (Table III). The greatest decrease in plasma clotting
TABLE III Effects of Mixtures lets on the Clotting
of Test Materials and Factor XI- or XII-Deficient Time of Normal or Factor XII-Deficient PPP
Plate-
Test material
Clottine times as a of saline XI-deficient XII-deficient platelets and platelets and normal PPP normal PPP
control XII-deficient platelets and XII-deficient PPP
Collagen
93 + 14
73 +_ 12
62 +_ 13
Cuprophane
62 +_ 16
66 + 4
78 +, 6
Glass
29 +_ 1
26 & 1
35 + 4
Kaolin
19 +_ 6
20 f. 5
24 5 3
PE
77 + 19
77 +, 6
74 2 6
PVC
71 +, 12
70 +_ 17
76 +, 9
Silastic
92 +_ 1.3
78 + 11
80 +, 7
Silicone
94 +_ 14
78 2 13
79 t
Teflon
73 f
68 +, 4
55 +, 5
Number of different exper imen t s Saline control, clotting time (min.)
12
4
4
4
30
32
102
11
but these changes were not times was obtained in tests with glass and kaolin, degrees of shortening of as marked as in tests with normal PPP. Intermediate The plasma clotting times were obtained in tests with collagen and Teflon. least amount of change occurred in tests with Cuprophane, PE, PVC, Silastic, of plasma clotting times and silicone. In each case, the degree of shortening is presumed to be produced primarily by mechanisms other than activation of The mechanisms of shortening of factor XII-deficient Plasplasma factor XII.
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COLIAGEN,
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$77
ma clotting times by the various test materials mixed with factor XII-deficient platelets could be by activation of tissue factor activity present in the platelet preparation, or by effects on platelet factor 3. Effects of Mixtures of Test Material and Normal or Factor XI-Deficient Platelets on Stvnven Time Values of Normal and Factor IX- or XII-Deficient PPP Effects of mixtures of test materials with normal or factor XI-deficient platelets on Stypven time values are shown in Table IV. These data indicate that all surfaces activate platelet factor 3 to some extent with glass and kaolin producing the most marked change. Experiments performed with normal platelets and factor IX- or XII-deficient PPP and with factor XI-deficient platelets and normal PPP indicate that the test materials activate platelet factor 3 by mechanisms independent of the presence of blood coagulation factors IX, XI, or XII.
TABLE IV Effects of Mixtures of Test Materials and Normal or Factor XI-Deficient Platelets on Stypven Time Values of Tests Performed with Normal or Factor IX- or XII-Deficient PPP
ST as % of saline control Test material Normal PPP
Normal platelets and: IX-deficient XII-deficient PPP PPP
XI-deficient platelets and Normal PPP
Collagen
68
64
72 2 5
66 + 7
Cuprophane
61
61
65 t 3
61 +_ 1
Glass
53
56
55 2 3
54 + 3
Kaolin
32
33
37 &4
30 + 0
PE
74
67
69 5 6
69 +_ 4
PVC
69
68
70 + 5
68 +_ 6
Silastic
68
67
70 t 8
69 +_4
Silicone
78
76
74 + 7
71 + 4
Teflon
75
76
74 +_ 5
78 +_ 6
2
2
6
4
53
54
48
Number of different experiments Saline control Stypven times (sec.)
54
478
COLIAGEN,PLATELETS
AND
CLOT'ITNG
Vo1.7,No.3
DISCUSSION
Under the conditions of the test system used here in which a large surface area of test material was exposed to washed platelets and the materialplatelet mixture was added subsequently to PPP, collagen and each of the artificial surfaces tested were shown to influence blood clotting. Collagen, Silastic, and silicone did not activate the intrinsic blood clotting system under these experimental conditions when platelets were omitted from the test system. These same three materials did appear to accelerate clotting times by a mechanism dependent on the presence of factor XI or a similar factor on the surface of washed platelets. The other artificial surfacsstested also appeared to react with various components of platelets and plasma to produce shortening of test plasma clotting times. One known platelet component in addition to factor XI that may have been influenced by the artificial surfaces tested was platelet factor 3. In addition, the platelet preparation contained a tissue factor-like activity after exposure to test materials. Reference to activation of various factors on platelets is made with the full understanding that evidence for activation of any specific factor is indirect; final proof awaits purification and characterization of these platelet-bound or platelet-associated factors. The most clear cut examples of the mechanisms of action of artificial surfaces on platelet constituents were the effects of collagen, Silastic, and silicone. These three surfaces could be shown to shorten the plasma clotting time by activation of a system dependent on the presence of factor XI or a similar factor on the platelet regardless of whether tests were performed with normal plasma or with plasmas deficient in factors XI or XII. That the major effect of collagen, Silastic, or silicone was mediated through factor XI present on platelets was demonstrated by the use of platelets deficient in factor XI. However, tests with normal platelets and plasma deficient in factor VII indicated that collagen, Silastic, and silicone each may have a minor effect of activating or making available a tissue factor activity present in the platelet preparation. Similarly, Stypven time tests indicate that each of these three materials activates or makes available detectable quantities of platelet factor 3 in a manner independent of factor XI present on platelets. The mechanisms by which Cuprophane, glass, kaolin, PE, PVC, and Tef!on influence blood coagulation were less clearly delineated than was the case with collagen, Silastic, and silicone. Cuprophane, glass, kaolin, PE, PVC, and Teflon were shown to activate factor XII in plasma confirming the work of several other workers (5,6). In addition, the presence of each of these materials in tests with both platelets and PPP deficient in factor XII were shown to produce a decrease in plasma clotting times. Such a decrease could have been brought about by activation of factor XI or a similar factor present on the factor XII-deficient platelets, by activation of platelet factor 3, or by activation of the tissue factor activity. The relative contributions of activated platelet factor 3 or the tissue factor activity to the overall effects of Cuprophane, glass, kaolin, PE, PVC, and Teflon in the test system are difficult to gauge with methods currently available. The present studies can be criticized with regard to the total surface area of the various test materials to which platelets were exposed. The total surface areas of glass, PE, PVC, Silastic, silicone, and Teflon are approximately equal. The total surface areas of Cuprophane and kaolin are much great. er than those of all of the other materials tested except collagen, which probably had a total surface area exceeding that of any of the other test materials. It is of interest that glass and kaolin produced similar degrees
Vo1.7,To.3
COLUGEN,
PLATELETS
AND CLOTTING
479
of shortening of plasma clotting times, even though the total surface area of each must have differed considerably. Contributions of the silicone-coated glassware in which tests were conducted should have been negligible on a surface area basis and would have been neutralized by the controls, if present. The present studies can be criticized also in that experimental design may have permitted transfer of protein from the platelet suspension medium to the surfaces of the dry test particles. These studies indicate that collagen and a number of different artificial surfaces can influence both the intrinsic and extrinsic blood coagulation systems in multiple ways. This multiplicity of activating or enhancing effects may well explain why the search for an artificial surface highly compatible with blood has been notably unsuccessful. A major contribution to our understanding of the effects of artificial surfaces on blood coagulation has been the recent work of Walsh (l), who showed that both kaolin and collagen could activate factor XI or a similar factor present on platelets. Effects of certain artificial surfaces on platelet factor 3 have been known for a number of years (3,4). The fact that platelets may possess tissue factor activity has been appreciated only recently (12). The tissue factor-like activity measured in these experiments may be mediated by a platelet-leukocyte interaction reported recently (13). b the other hand, platelets may possess tissue factor-like activity demonstrable only upon adherence of platelets to a surface as occurs with fibroblasts (14). The present findings and the work of others (1,2) serve mainly to indicate the complexity of the interactions of artificial surfaces with blood, and point out that a truly blood-compatible artificial surface may be nearly impossible to fabricate.
ACKNOWLEDGEMENTS Supported in part by Contract PH-43-68-977 with the Artificial KidneyChronic Uremia Program of the National Institute for Arthritis, Metabolism, and Digestive Diseases, and by grants HL46351, HL13296, and HL14228 from the National Heart and Lung Institute, National Institutes of Health.
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WALSH, P.N. The effects of collagen and kaolin on the intrinsic coagulant activity of platelets. u. 2. Haematol. 22, 393, 1972.
2.
SCHIFFMAN, S., RAPAPORT, S.I., and CHOUG, M.M.Y. Platelets and initiation of intrinsic clotting. m. 2. Haematol. 24, 633, 1973.
3.
SPAET, T.H. and CINTRON, J. m.
Studies on platelet factor -3 availability. 2. Haematol. 11, 269, 1965.
4.
HARDISTY, R.M. and HUTTON, R.A. Platelet aggregation and the availability of platelet factor 3. w. J. Haematol. 12, 764, 1966.
5.
NIEWIARaWSKI, S., BAN’KCMSKI, E., and ROG(;WICKA,I. Studies on the adsorption and activation of the Hageman factor (Factor XII) by collagen and elastin. Thromb. -. Diath Haemorrh. 14, 387, 1965.
6.
WILNER, G.D., NOSSEL, H.L., and LEROY, E.C. Activation of Hageman factor by collagen. 2. -Clin. Invest. 47, 2608, 1968.
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COLLAGEN,PLATEld?TS
AND CLCYMXNG
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7.
SALZMAN, E.W. Surface effects in hemostasis and thrombosis. In, The Chemistry of Biosurfaces, vbl. 2, M.E. Hair editor, Marcel Dekker,xc., New York, 1972, p. 489.
8.
MASON, R.G. The interaction of blood hemostatic elements with artificial surfaces. &. Hemost. Thromb. 1, 141, 1972. --
9.
WALSH, P.N. Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. m. J. Haematol. 22, 205, 1972.
10.
HOVIG, T. Release of a platelet-aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline "extract" of tendons Thromb. Diath. Haemorrh. 9, 264, 1963.
11.
FAREY, M.G., and BERNARD, G.T. Determination of surface areas by an improved continuous flow method. Analvt. Chem. 43, 1307, 1971.
12.
BIGGS, R., DENSON, K.W.E., RIESENBERG, I).,and MCINTYRE, C. The coagulant activity of platelets. _Brit. J. Haematol. 15, 283, 1968.
13.
NIEMETZ, J. and MARCUS, A.J. The stimulatory effect of platelets and platelet membranes on the procoagulant activity of leukocytes. J. m. Invest. 54, 1437, 1974.
14.
ZACHARSKI, L.R. and MCINTYRE, O.R. Membrane-mediated synthesis of tissue factor (thromboplastin) in cultured fibroblasts. Blood. 41, 679, 1973.
