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Interaction of Collagen with the factor VIII antigen-activity - von Willebrand factor complex
THROMBOSIS
Printed
RESEARCH in Great
BRIEF
Vol.
Britain
11, pp. 433-438, Pergamon Press,
1977 Ltd.
COMMUNICATION
INTERACTIONOF COLLAGEN WITH THE FACTOR VIII ANTIGEN-ACTIVITY - VON WILLEBRAND FACTOR COMPLEX
Dag Nyman Department of Blood Coagulation Disorders Karolinska Institutet, Stockholm, Sweden
(Received
7.5.1977; in revised form 12.7.1977. Accepted by Editor H.C. Godal)
INTRODUCTION In von Willebrand's disease the bleeding time is greatly prolonged. This abnormality is caused by a deficient platelet function which has been connected to a plasma factor deficiency (1). This plasma factor, von Willebrand factor, is closely related to the factor VIII antigen-activity complex (2). In vitro studies have demonstrated that the functional defect is caused by absent adhesion of the platelets to the collagen fibers subendothelium (3). In spite of this, the platelet interaction with collagen fibers has been found normal when studied in stirred platelet-rich plasma from patients with von Willebrand's disease. Interaction of collagen with coagulation factors in vitro have mainly shown an adsorption and activation of the contact system (4). This study was undertaken in order to investigate the possibility of interaction between collagen and the factor VIII antigen-activity complex.
MATERIAL AND METHODS Citrated platelet poor plasma (PPP) from4healthy
individuals and from 4
patients with von Willebrand's disease was used for the experiments. Fibrillar collagen, Hormon-Chemie, Munich, GFR, in a suspension containing 1 mg/ml. Further dilutions in order to give the final concentrations of colla. gen as indicated below were done with Tris-0.05 M-NaCl 0.1 M buffer of
433
pH 7.4
COLLAGEN
434
- F. VIII - INTERACTION
Vol.ll,No.3
Soluble collagen, Laboratoire Stago, Asnilres s/Seine, France, dissolved in distilled water and diluted with buffer to 200 pg/ml and subsequentlyincubated at 33' C for polymerizationduring 30 minutes. Factor VIII activity (VIIIC)was assayed according to Nilsson et al (5) against a standard of pooled plasma from 20 healthy individualsstored at -7O'C. Factor VIII related antigen (VIIIgAG)was assayed inmunochemically(6). The effect of collagen on these parameterswas studied by adding dilutions of the collagen preparationsor buffer (control)to equal volumes of PPP. After 10 min at 37'C and with gentle agitation the tubes were centrifugedat 2700 g for 20 minand the supernatantwas collected and stored at -7O'C until the assays were done. Statisticaltesting of significancewas made by the t-test and by the coefficient of regression. REXJLTS Incubationof normal platelet poor plasma with collagen suspensionsfor 10 min caused marked changes in both VIIIC and VIIIgAG in the supernatantwhen collagen 'Stage'was used, whereas collagen 'Hone' did not produce any significant changes over the range of concentrationstested (Table I). Collagen 'Stage' adsorbed VIIISAG in significantamounts, directly proportionalto the amount of collagen (p
100
50
'Stago' VIIIC VIIIkAG
'Horm' VIIIC VIIIRAG
96 + 14
22 + 5
56 + 9
80217
36 + 6
60 + 14 59 + 7
60 + 8
25
75 + 19
49 _+ 10
58 + 10
61 + 11 .
12.5
69 + 16
56 _ + 9
54+9 -
59 -+ 9
46 + 4
55 + 7
46+4
55 -. + 7
0
The adsorption experimentswere also carried out on the plasma of 4 patients with von Willebrand'sdisease of varying severity.The results are given in Tables IIa and IIb. Collagen 'Stage'gives the same pattern for VIIQ and
- F.
COLLAGEN
Vol.ll,No.3
VIII - INTEZUCTION
435
VIII~AC as in normal plasma but in this experiment collagen 'Horm' promotes a rise in VIIIC of about 100 per cent compared to control values. This rise is, however, independantof the collagen concentration.
collagen factor VIII~AC after adsorption
12.5
25
50
100
COLLAGEN
%a90
rg/ml
TABLE II a VIIIC in per cent of Standard after Incubationwith Collagen in von Willebrand's Disease Pat
Control
Collagen 'Stago' 100 )Ig/ml 25 50
Collagen 'Harm' 25 50 100 pg/ml
1
19
36
59
60
42
39
40
2
1
2.0
2.0
2.5
3.8
3.9
4.1
3
10
21
31
57
20
23
30
4
14
43
50
66
27
28
31
TABLE II b ~:~k Pat
1
in per cent of Standard after Incubationwith Collagen in von WilleDisease Control
20
Collagen 'Stago' 100 ug/ml 25 50 9
9
0
Collagen 'Harm' 25 50 100 ug/ml 11
12
9
2
0
0
0
0
0
0
0
3
12
8
5
0
13
14
13
4
15
9
8
0
17
17
16
COLLAGEN
436
- F. VIII - INTEmCTION
Vol.ll,No.3
In order to exclude a possible thromboplasticeffect of the collagen preparations the factor IX activity after incubationwith the collagen preparations was tested on two occasions.No effect was found, Table III. TABLE III Factor IX Activity in per cent of Standard after Incubationwith Collagen. Mean Values of Double Determinationsin Two Different Plasma Samples. Collagen ug/ml final cone
'Stago'
‘HOITI’
54,64
86,60
50
70,65
82,70
25
63,53
79,53
0
64,62
64,62
100
DISCUSSION The simultaneousrise in VIIIC by approx 100 per cent in normal plasma connected with an adsorption of VIIIkAG indicatesa change in the VIII~AG - VIIIC complex caused by incubationwith collagen 'Stago'.The rise in VIIIC can be considered specific, as incubationof plasma from a patient with severe von Willebrand'sdisease did not show any appreciableamount of activation.Also, the results obtained in the factor IX assay did not show significantactivation due to thromboplasticactivity of the collagen preparation. In contrast to this, collagen 'Harm' did not adsorb measurable amounts of VIIIgAG nor did any significant rise in VIII occur. C The results obtained in the same experimentsusing von Willebrand plasma inshows the same stead of normal plasma are similar, The behaviour of VIII RAG pattern as in the case of normal plasma. A complete dissociationof VIIIFAG can be achieved when a high amount of collagen 'Stago' is added. Despite insignificantadsorption of VIIIFAG collagen 'Horm' also produced a rise in VIIIc, however, moderate compared with collagen 'Stago'. Since these collagen preparationsare not of known compositionand most certainly are mixtures of various species of collagens,however, in different proportions,the rise in VIIIc induced by collagen 'Worm' clearly observed in von Willebrand plasma is possibly due to contaminationwith the main type of collagen represented in collagen 'Stage'.Assuming a greater load of VIIIc on VIIIgAG in von Willebrand plasma, even a small interactioncould be ex-
COLLAGEN
Vol.11,No.Y
- F. VIII
- INTERACTION
437
petted to give such an effect. It is of interest that these collagen preparations have appeared to have different properties as inductors of platelet aggregation (7). The tentative explanation of the findings reported is that certain types of collagen dissociates VIIIC from VII&G
which serves as carrier. The load of
VIIIC on VIIIRAG may differ and may be greater when the amount of the carrier molecule is restricted as in von Willebrand's disease. It has been shown that VIIIC is in a state of dynamic equilibrium with VII&G (8). This equilibrium is disturbed by the coagulation process. It has also been shown that the distribution of VIIIC can be influenced by chylomicrons (9). The present results show that a certain type of collagen strongly adsorbs the VII&G
and at the same time liberates VIIIC. In view of the apparent rise
VII&G
from the carrier c is a prerequisite for coagulant activity. The shown interaction between
VIIIUG
and certain collagens could be of importance for normal platelet ad-
in VIIIC it might be possible that dissociation of VIII
hesion. The fact that such a dependency is not observed in platelet aggregation studies can be explained by the existence of at least two pathways for the induction of platelet aggregation of collagen.
ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council (No 19X-520) and the Karolinska Institutet Funds. REFERENCES 1. NILSSON, I.M., BLOmCK, M., JORPES, E., BLOMBACK, B., and JORANSSON, S.A.: Von Willebrand's disease and its connection with human fraction I-O. Acta Med. Stand. 159, 179, 1957. 2. MEYER, D., JENKINS, C., DREYFUS, M., and LARRIEU, M.J.: An experimental model for von Willebrand's disease. Nature. 243, 293, 1973. 3. BAUMGARTNER, H.R., TSCOPP, T.B., and WEISS, H.J.: Platelet interaction with collagen fibrils in flowing blood II. Impaired adhesion-aggregation in bleeding disorders, A comparison with subendothelium. Thromb. Raemostas. 37, 17, 1977. 4. NIEWIAROWSKI, S., BANKOWSKI, E., and ROGOWICKA, I.: Studies on the adsorption and activation of the Hageman factor (factor XII) by collagen and elastin. Thromb. Diathes. Raemorrh. 14, 387, 1965. 5. NILSSON, I.M., BLOMB&K, M., and VON FRANCKEN, I.: On an inherited autosomal hemorrhagic diathesis with antihemophilic globulin (ARG) deficiency and prolonged bleeding time. Acta Med. Stand. 159, 35, 1957.
438
COLLAGEN
- F. VIII - INTERACTION
Vol.ll,No.3
6. HOLMBERG, L. and NILSSON, I.M.: ARP-relatedprotein in clinical praxis. Stand. J. Raematol. 12, 221, 1974. 7. NYMAN, D.: Collagen-inducedplatelet aggregation:Evidence of several mechanisms for the inductionof platelet release by collagen. Thromb.Res. in press, 1977. 8. KODTTS, J., GUDE, N., and FIRKIN, B.: The dynamic interrelationshipbetween factor VIII and von Willebrand factor. Thromb. Res. 8, 533, 1976. H.L.M.A., SPAAN, PIB., DE JONG, 9. PADLSSEN, M.&P., VANDENBUSSCRK-SCREPPERS, T ., and PLANJE, M.C.: Studies on the characterizationof factor VIII and a co-factor VIII. Thromb. Diathes. Raemorrh. 31, 328, 1974.
Reprint requests should be made to the Departmentof Blood Coagulation Disorders, Karolinska sjukhuset, S-104 01 Stockholm,Sweden.
Printed
RESEARCH in Great
BRIEF
Vol.
Britain
11, pp. 433-438, Pergamon Press,
1977 Ltd.
COMMUNICATION
INTERACTIONOF COLLAGEN WITH THE FACTOR VIII ANTIGEN-ACTIVITY - VON WILLEBRAND FACTOR COMPLEX
Dag Nyman Department of Blood Coagulation Disorders Karolinska Institutet, Stockholm, Sweden
(Received
7.5.1977; in revised form 12.7.1977. Accepted by Editor H.C. Godal)
INTRODUCTION In von Willebrand's disease the bleeding time is greatly prolonged. This abnormality is caused by a deficient platelet function which has been connected to a plasma factor deficiency (1). This plasma factor, von Willebrand factor, is closely related to the factor VIII antigen-activity complex (2). In vitro studies have demonstrated that the functional defect is caused by absent adhesion of the platelets to the collagen fibers subendothelium (3). In spite of this, the platelet interaction with collagen fibers has been found normal when studied in stirred platelet-rich plasma from patients with von Willebrand's disease. Interaction of collagen with coagulation factors in vitro have mainly shown an adsorption and activation of the contact system (4). This study was undertaken in order to investigate the possibility of interaction between collagen and the factor VIII antigen-activity complex.
MATERIAL AND METHODS Citrated platelet poor plasma (PPP) from4healthy
individuals and from 4
patients with von Willebrand's disease was used for the experiments. Fibrillar collagen, Hormon-Chemie, Munich, GFR, in a suspension containing 1 mg/ml. Further dilutions in order to give the final concentrations of colla. gen as indicated below were done with Tris-0.05 M-NaCl 0.1 M buffer of
433
pH 7.4
COLLAGEN
434
- F. VIII - INTERACTION
Vol.ll,No.3
Soluble collagen, Laboratoire Stago, Asnilres s/Seine, France, dissolved in distilled water and diluted with buffer to 200 pg/ml and subsequentlyincubated at 33' C for polymerizationduring 30 minutes. Factor VIII activity (VIIIC)was assayed according to Nilsson et al (5) against a standard of pooled plasma from 20 healthy individualsstored at -7O'C. Factor VIII related antigen (VIIIgAG)was assayed inmunochemically(6). The effect of collagen on these parameterswas studied by adding dilutions of the collagen preparationsor buffer (control)to equal volumes of PPP. After 10 min at 37'C and with gentle agitation the tubes were centrifugedat 2700 g for 20 minand the supernatantwas collected and stored at -7O'C until the assays were done. Statisticaltesting of significancewas made by the t-test and by the coefficient of regression. REXJLTS Incubationof normal platelet poor plasma with collagen suspensionsfor 10 min caused marked changes in both VIIIC and VIIIgAG in the supernatantwhen collagen 'Stage'was used, whereas collagen 'Hone' did not produce any significant changes over the range of concentrationstested (Table I). Collagen 'Stage' adsorbed VIIISAG in significantamounts, directly proportionalto the amount of collagen (p
100
50
'Stago' VIIIC VIIIkAG
'Horm' VIIIC VIIIRAG
96 + 14
22 + 5
56 + 9
80217
36 + 6
60 + 14 59 + 7
60 + 8
25
75 + 19
49 _+ 10
58 + 10
61 + 11 .
12.5
69 + 16
56 _ + 9
54+9 -
59 -+ 9
46 + 4
55 + 7
46+4
55 -. + 7
0
The adsorption experimentswere also carried out on the plasma of 4 patients with von Willebrand'sdisease of varying severity.The results are given in Tables IIa and IIb. Collagen 'Stage'gives the same pattern for VIIQ and
- F.
COLLAGEN
Vol.ll,No.3
VIII - INTEZUCTION
435
VIII~AC as in normal plasma but in this experiment collagen 'Horm' promotes a rise in VIIIC of about 100 per cent compared to control values. This rise is, however, independantof the collagen concentration.
collagen factor VIII~AC after adsorption
12.5
25
50
100
COLLAGEN
%a90
rg/ml
TABLE II a VIIIC in per cent of Standard after Incubationwith Collagen in von Willebrand's Disease Pat
Control
Collagen 'Stago' 100 )Ig/ml 25 50
Collagen 'Harm' 25 50 100 pg/ml
1
19
36
59
60
42
39
40
2
1
2.0
2.0
2.5
3.8
3.9
4.1
3
10
21
31
57
20
23
30
4
14
43
50
66
27
28
31
TABLE II b ~:~k Pat
1
in per cent of Standard after Incubationwith Collagen in von WilleDisease Control
20
Collagen 'Stago' 100 ug/ml 25 50 9
9
0
Collagen 'Harm' 25 50 100 ug/ml 11
12
9
2
0
0
0
0
0
0
0
3
12
8
5
0
13
14
13
4
15
9
8
0
17
17
16
COLLAGEN
436
- F. VIII - INTEmCTION
Vol.ll,No.3
In order to exclude a possible thromboplasticeffect of the collagen preparations the factor IX activity after incubationwith the collagen preparations was tested on two occasions.No effect was found, Table III. TABLE III Factor IX Activity in per cent of Standard after Incubationwith Collagen. Mean Values of Double Determinationsin Two Different Plasma Samples. Collagen ug/ml final cone
'Stago'
‘HOITI’
54,64
86,60
50
70,65
82,70
25
63,53
79,53
0
64,62
64,62
100
DISCUSSION The simultaneousrise in VIIIC by approx 100 per cent in normal plasma connected with an adsorption of VIIIkAG indicatesa change in the VIII~AG - VIIIC complex caused by incubationwith collagen 'Stago'.The rise in VIIIC can be considered specific, as incubationof plasma from a patient with severe von Willebrand'sdisease did not show any appreciableamount of activation.Also, the results obtained in the factor IX assay did not show significantactivation due to thromboplasticactivity of the collagen preparation. In contrast to this, collagen 'Harm' did not adsorb measurable amounts of VIIIgAG nor did any significant rise in VIII occur. C The results obtained in the same experimentsusing von Willebrand plasma inshows the same stead of normal plasma are similar, The behaviour of VIII RAG pattern as in the case of normal plasma. A complete dissociationof VIIIFAG can be achieved when a high amount of collagen 'Stago' is added. Despite insignificantadsorption of VIIIFAG collagen 'Horm' also produced a rise in VIIIc, however, moderate compared with collagen 'Stago'. Since these collagen preparationsare not of known compositionand most certainly are mixtures of various species of collagens,however, in different proportions,the rise in VIIIc induced by collagen 'Worm' clearly observed in von Willebrand plasma is possibly due to contaminationwith the main type of collagen represented in collagen 'Stage'.Assuming a greater load of VIIIc on VIIIgAG in von Willebrand plasma, even a small interactioncould be ex-
COLLAGEN
Vol.11,No.Y
- F. VIII
- INTERACTION
437
petted to give such an effect. It is of interest that these collagen preparations have appeared to have different properties as inductors of platelet aggregation (7). The tentative explanation of the findings reported is that certain types of collagen dissociates VIIIC from VII&G
which serves as carrier. The load of
VIIIC on VIIIRAG may differ and may be greater when the amount of the carrier molecule is restricted as in von Willebrand's disease. It has been shown that VIIIC is in a state of dynamic equilibrium with VII&G (8). This equilibrium is disturbed by the coagulation process. It has also been shown that the distribution of VIIIC can be influenced by chylomicrons (9). The present results show that a certain type of collagen strongly adsorbs the VII&G
and at the same time liberates VIIIC. In view of the apparent rise
VII&G
from the carrier c is a prerequisite for coagulant activity. The shown interaction between
VIIIUG
and certain collagens could be of importance for normal platelet ad-
in VIIIC it might be possible that dissociation of VIII
hesion. The fact that such a dependency is not observed in platelet aggregation studies can be explained by the existence of at least two pathways for the induction of platelet aggregation of collagen.
ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council (No 19X-520) and the Karolinska Institutet Funds. REFERENCES 1. NILSSON, I.M., BLOmCK, M., JORPES, E., BLOMBACK, B., and JORANSSON, S.A.: Von Willebrand's disease and its connection with human fraction I-O. Acta Med. Stand. 159, 179, 1957. 2. MEYER, D., JENKINS, C., DREYFUS, M., and LARRIEU, M.J.: An experimental model for von Willebrand's disease. Nature. 243, 293, 1973. 3. BAUMGARTNER, H.R., TSCOPP, T.B., and WEISS, H.J.: Platelet interaction with collagen fibrils in flowing blood II. Impaired adhesion-aggregation in bleeding disorders, A comparison with subendothelium. Thromb. Raemostas. 37, 17, 1977. 4. NIEWIAROWSKI, S., BANKOWSKI, E., and ROGOWICKA, I.: Studies on the adsorption and activation of the Hageman factor (factor XII) by collagen and elastin. Thromb. Diathes. Raemorrh. 14, 387, 1965. 5. NILSSON, I.M., BLOMB&K, M., and VON FRANCKEN, I.: On an inherited autosomal hemorrhagic diathesis with antihemophilic globulin (ARG) deficiency and prolonged bleeding time. Acta Med. Stand. 159, 35, 1957.
438
COLLAGEN
- F. VIII - INTERACTION
Vol.ll,No.3
6. HOLMBERG, L. and NILSSON, I.M.: ARP-relatedprotein in clinical praxis. Stand. J. Raematol. 12, 221, 1974. 7. NYMAN, D.: Collagen-inducedplatelet aggregation:Evidence of several mechanisms for the inductionof platelet release by collagen. Thromb.Res. in press, 1977. 8. KODTTS, J., GUDE, N., and FIRKIN, B.: The dynamic interrelationshipbetween factor VIII and von Willebrand factor. Thromb. Res. 8, 533, 1976. H.L.M.A., SPAAN, PIB., DE JONG, 9. PADLSSEN, M.&P., VANDENBUSSCRK-SCREPPERS, T ., and PLANJE, M.C.: Studies on the characterizationof factor VIII and a co-factor VIII. Thromb. Diathes. Raemorrh. 31, 328, 1974.
Reprint requests should be made to the Departmentof Blood Coagulation Disorders, Karolinska sjukhuset, S-104 01 Stockholm,Sweden.