THROMBOSIS RESEARCH 64; 477-4851991
0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
STUDIES ON FIBRIN NETWORK STRUCTURE IN HUMAN PLASMA. PART II CLINICAL APPLICATION: DIABETES AND ANTIDIABETIC DRUGS.
C.H.Nair, A.Azhar,J.D.Wilson* and D.P.Dhall. and Department of Thrombosis Research Unit Vascular an% Endocrinology , Woden Valley Hospital, P.O. Box 11, Woden, A.C.T. Australia. (Received 28.1.1991; accepted in revised form 14.6.1991 by Editor J. Soria) (Received in final form by Executive Editorial Office 4.9.1991) ABSTRACT. Using measurements of fibrin fibre thickness (VT) derived from turbidity and permeability (r) of clotted plasma, it in vitro added to plasma has been found that glucose decreases permeability of the network despite unaltered fibre thickness (p ) in Fibrin fibrinogen conversion. uncontrolled diabetes is found significantly reduce a . In diabetic plasma the degree of conversion to fibrin is similar to that in age and sex matched plasma from nonthe effect on fibrin network and fibre thickdiabetics: ness probably arises from glycosylation of fibrinogen. Studies with Gliclazide, Metformin, Glibenclamide and insulin have shown that while all other drugs tested have no effect, Gliclazide increases fibrin fibre thickness diminishes tensile strength and (p ) significantly, s permeability. In separate experiments lysability ;;%a I-labelled fibrin networks developed in the presence of all four hypoglycaemic agents by tissue activator was tested. Networks developed in the presence of Metformin were found to lyse more quickly, followed by insulin and Gliclazide. Alterations induced in fibrin networks in diabetes may be nullified by some oral hypoglycaemic agents such as Gliclazide and not by others. Whether nullification of such changes has lonu-term effects in reducing the incidence of cascular disease in diabetics remains to be established.
INTRODUCTION Although
it has been known for a number of years that occlusive
Key words: Diabetes,
fibrin network structure. 477
478
FIBRIN NETWORK STRUCTURE II
Vol. 64, No. 4
vascular disease has a higher incidence in the diabetic patient non-diabetic, the reasons underlying are with compared the several been Over the last decade studies have unclear. undertaken with a view to elucidating underlying mechanism. In the main inconsistent changes in platelet function tests, in coagulation factor concentrations and in tests of fibrinolysis (1) have been identified. Even in the non-diabetic patients there has been little to suggest that any changes detected using tests of platelet function, fibrinolysis or blood several coagulation can be correlated against the incidence of peripheral vascular disease. It has become possible in recent years to study fibrin network characteristics including fibre thickness in clinical situations using permeability and turbidity techniques (2). Clinical tests of compaction of fibrin fibres have also become available in the last decade (2). Little is known about the organisation of fibrin networks formed in the presence of high levels of glucose in both in vitro and in vivo. blood, This investigation was undertaken to examine the effects of glucose in vitro on fibrin structure as well as to examine fibrin network network characteristics in the diabetic patient. Studies were also made on the effect on fibrin network of Insulin, and of hypoglycaemic agents including Metformin, Gliclazide and Glibenclamide. MATERIALS AND METHODS. Blood obtained from Blood Bank donors (9ml) was collected in 3.8% Trisodium citrate (lml) and 35~1 of 10,000 KIU/ml Trasylol It was then centrifuged at 2,400g at room (Bayer, Germany). temperature for ten minutes to obtain platelet-poor plasma, and recentrifuged at 4ooog for minutes to then five obtain essentially platelet-free plasma (PFP platelet count < 3OOO/ul). The concentration of fibrin in each plasma pool was determined by the method of Ratnoff and Menzies (3) as also the Network fibrin content (CN). Bovine thrombin (Parke Davis, USA) was used to clot plasma in all cases. Different concentrations of glucose were added to platelet-free plasma immediately before the addition of thrombin to develop the Network fibre thickness (uT) was determined from network. described and permeability was also turbidity as previously measured (2). Compaction of the network was characterized (6). In a separate study fifty-one diabetic and age and sex-matched fibre fibrin for their subjects were investigated healthy thickness (VT), network permeability (t) and tensile strength of insulin-dependent poorly-controlled Twenty were network. diabetics with a mean blood sugar level of 15 f 0.9mM. investigated as including Insulin were agents Antidiabetic Gliclazide (Diamicron, Servier, Australia), 12ug/ml follows: 5OOuU/ml Insulin (Sigma), 8ug/ml Metformin (Fisons, Australia) and 72ng/ml Glibenclamide (Daonil, Hoechst, Australia) were added in different plasma samples just before the addition of thrombin. Fibre thickness, permeability and compaction were measured.
Vol. 64, No. 4
FIBRIN NETWORK STRUCTURE II
479
intact networks (labelled with 1251 In separate experiments fibrinogen) developed in the presence of these materials were buffer (pH 7.4, ionic times with Tris-saline washed three tubes separately in test strength 0.153) and then placed containing equal volumes of plasma and Tris-saline buffer. The tubes were then rotated in an elliptical rotator at 37°C and ’ duced with tissue plasminogen activator, and the ~~~~~~~lYos;ISl~~ I fibrin monitored as an index of the rate of lysis. :
Fibrin networks were formed in pre-etched glass tubes 1OOmm in length and of 3mm internal diameter: the individual internal Plasma was clotted with dimensions of each tube were measured. the addition of 1.0 u/ml thrombin (final thrombin concentration) The network was and 25mM Ca2+ (final calcium concentration). perfused with Tris HCl saline buffer, pH 7.35 (5OmM Tris; 103mM NaCl) at a pressure head of 150mm water. The initial 0.5ml of perfusate was collected for determining the fibrin content of the network. The permeability or Darcy constant, calculated from the equation:
‘c,
of the fibrin network was
T = Q
h/Ftp z where Q is the volume flow in time t; t is the viscosity of the liquid; h is the length of the clot; F its cross-section and p the applied pressure.
developed with the The optical density of fibrin networks of lu/ml thrombin (final concentration of thrombin) and additio 25mM Ca4+ (final calcium concentration) in glass cuvettes of lcm path length was measured at a range of wavelengths between 600 and 8001~1, while using unclotted3fibrinogen solution or plasma in the r,ference cell. Then c/TA was plotted as a function of where T is the turbidity (2.303 x optical density),A is l/h the wavelength and c is the concentration of fibrinogen in mg/ml. The intercept A of this plot was used to calculate the average mass-length ratio, uT, according to the equation (5): 10
x 1012 daltons/cm
uT = 1.48A
Compaction was measured according to the method of Dhall et 0.9mls of plasma was clotted with O.lml of thrombin/Ca rtl+ (4). (final concentrations of lu/ml thrombin and 25mM Ca2+). Networks were allowed to form for the same length of time as that taken for optical density readings to reach a plateau in turbidity studies. Networks were then centrifuged in a Zentrifuge
480
FIBRIN NETWORK STRUCTURE II
Vol. 64, No. 4
The volume of (Eppendorf, U.S.A.) at 8000 x g for 45 seconds. expelled serum was then expressed as a percentage of total initial volume of the clotted plasma. RESULTS
As glucose concentration is progressively increased from 5mM to fibre thickness from turbidity (uT) does not alter 25mM, (r) decreases after appreciably. However, network permeability increasing glucose 15mM glucose. Compaction decreases with denoting an increase in the tensile strength of fibrin fibres in tensile strength and reduction in (Figure 1). Increase permeability indicates a greater degree of crosslinkage of within the network. changes in network fibres These characteristics do not arise from altered conversion because (CR) does not alter significantly in the fibrin content concentration range of glucose tested. C 0
‘0
M P
55
A
30
c
T
65
I
60
0 N
‘6
660. 7
600. 660.
T
600’ 36
L----0
6
lOl6P6550
GLUCOSE
(mM)
of glucose on concentration FIGURE 1: Effect of increasing fibrin network developed in plasma. uT is mass-length ratio from turbidity, 'I:is permeability. Results are presented as mean + SEM (n=5).
In plasma networks made from poorly controlled diabetics uT and permeability (r) of the network were significantly reduced, when
Vol. 64, No. 4
FIBRIN NETWORK STRUCTURE II
compared with those in the networks in age and sex matched The tensile strength of fibrin fibres which make up controls. the network was not appreciably affected (Figure 2). These findings indicate that the fibrin in the diabetic has inhibited amount of fibrin is polymerization so a greater lateral This network. decreases the minor into the incorporated permeability of the network but the crosslinking in the network is similar to that in the non-diabetic, as evidenced by unchanged compaction. 120 ..P~O.OOI
100
80
60 m
COMWCTION
40
20
0 nkontrol)=31 n(dlabetic)dO
in FIGURE 2: Comparison of fibrin network characteristics diabetics and sex and age matched controls. uT is ratio from turbidity, 'I; is permeability mass-length and CN is the network fibrin content in the group of diabetic patients. Each is expressed as a percentage of the mean value in the control group of non-diabetics.
studies..
.
Fibrin network characteristics were significantly altered by Gliclazide (Figure 3); thickness (uT) was increased fibre significantly and permeability (t) was reduced significantly. None of the other agents altered network characteristics. Changes induced by Gliclazide in mass-length ratio from turbidity are opposite to those seen in the networks from diabetics.
482
FIBRIN NETWORK STRUCTURE It
160
Vol. 64, No. 4
~0.06
??? ?
-6’0.006
-
CONTROL
B
QLIBENCLAYIDE
0
QLICLALIDE
-
METFORYIN
0
INBULIN
60
0
5
COMPACTION
CN
7
FIGURE 3: : Effect of some hypoglycaemic drugs on fibrin network characteristics. Concentrations used are given under Materials and Methods. uT is mass-length ratio from turbidity, t is permeability and C is the network fibrin content (n=5 for each drug teste # ).
tested with the exception of Glibenclamide All the agents (Figure 4). Of the three agents which enhanced fibrinolysis enhanced fibrinolysis, Metformin had the most profound effect. were spectacular effects found with though less Similar Gliclazide and Insulin. 8
k 400 A t 360 : 300 ; 260 p 200 s : 160 j 100 :
P Y
-I 0
30
60
90
120
160
180
210
TIME (MINUTES) I
I
-
CONTROL
+
QLIBENCLAMIDE -+-
-e-
INSULIN
X
METFOAMIN
QLICLAZIDE
FIGURE 4: Lysis by streptokinase of fibrin networks developed drug with some hypoglycaemic agents . tested), quantified by measuring the released
Vol. 64, No. 4
FIBRIN NETWORK STRUCTURE II
DISCUSSION in A causal relationship between the metabolic abnormalities diabetes and vascular disease in diabetics has thus far not been A great deal of work, however, suggests that established. it has not been state. But diabetes is a hypercoagulable primary hypercoagulability is a whether the established abnormality or whether it is secondary to vascular disease. The present study shows that both glucose in vitro and the metabolic abnormality in patients induce alterations in network characteristics. However, effects of glucose on network structure in vitro are different from the abnormalities found in diabetic plasma networks. When glucose is artificially elevated in normal significantly affected, but thickness is not fibre plasma reduced when significantly and compaction are permeability 15mM. These changes are glucose concentration is in excess Of not associated with any alteration in fibrinogen conversion to fibrin or in fibrin content (C,) of the network, indicating that the networks are altered because the fibrin fibres become more highly crosslinked. In the diabetic patient, on the other hand, fibre thickness (uT) and permeability are reduced and compaction remains relatively unaltered. these differences is not clear at underlying The mechanism with artificially added investigation but in the present, before only a few minutes glucose the material is added developing the network - the changes induced are from a direct From our studies it appears that in effect of glucose on fibrin. the presence of glucose added in this fashion the network is more This imparts greater tensile strength and highly crosslinked. In the diabetic, on the diminished permeability to the network. plasma proteins including fibrinogen are the hand, other It has been previously shown that altered plasma glycosylated. proteins like albumin can modify fibrin network characteristics (5). The main abnormality seems to arise from inhibited lateral polymerization or enhanced end-to-end polymerization. The resulting diminished thickness of fibres increases the total contour length of the fibres thus reducing the permeability, but cross-linking does not seem to be measurably affected in the diabetic. The compaction thus is unchanged. The differences in fibrin network characteristics with added glucose in vitro, on the one hand, and diabetic plasma on the other are not contradictory - they could arise from glycation of plasma proteins such as albumin. This requires detailed investigation. We have previously shown (6) that networks with fibres of reduced thickness and reduced permeability such as those found in the diabetics are resistant to lysis. This resistance to lysis invalidates normal or increased fibrinolytic activity from normal Or even enhanced plasminogen concentration. Thus, such abnormalities in network structure as seen in the diabetic patient provide scope for peripheral vascular disease of both macro- and microangiopathic types. On investigating
the capacity
of pharmacological
materials
used
484
FIBRIN NETWORK STRUCTURE II
Vol. 64, No. 4
in the treatment of diabetes in altering network characteristics it was found that Gliclazide is capable of inducing alterations in fibrin network structure. In its presence fibre thickness was significantly increased but despite a reduction in permeability was found enhanced. Gliclazide modifies network compaction formation in a surprising fashion. Networks show some enhancement lateral polymerization accounting for thickened fibres. of Diminished permeability indicates enhanced crosslinking within the network. However, the crosslinks are not primary crosslinks since they are weaker and readily disrupt on centrifugal stress. It has been previously shown that such changes induced may be induced with Dextran (4) which enhances lateral polymerization thus hiding primary crosslinks within thickened of fibrin fibres. Such fibrin fibres were found more susceptible to lysis. These observations are significant in the light of investigations by Almer (6) on Type II diabetics, who demonstrated that a shift in treatment from Chlopropamide to Gliclazide the induced a significant increase in plasminogen activator activity of the vascular wall in patients with an abnormally low activity, independent of an elevated carbohydrate metabolism. Our study shows relatively independent effects of Gliclazide on the network structure. would seem to promote lysis by Thus, Gliclazide affecting both the fibrinolytic system and the actual structure of the network by influencing the crosslinking arrangement within fibrin. Of the drugs tested all were capable of enhancing fibrinolysis with the exception of Glibenclamide. Metformin, however, had the most spectacular effect. The observations that major changes in fibrin network structure are induced by glucose and by changes in the diabetic decreased metabolic plasma, the lysability of networks in diabetics, and the possibility of reversal of network changes by therapeutic means open a new and management of horizon in research into the pathogenesis vascular disease in diabetics. ACKNOWLEDGEMENTS The authors would like to thank the National Heart Foundation of Administration Practice Hospital Private Australia and the Committee of Woden Valley Hospital for grants supporting this project. REFERENCES 1.
Advanced metabolic disorders. KEEN, H. and JARRET, R.G. In Microangiopathy, its Prevalance in Asymptomatic Diabetes, supp1.13. (1973).
2.
NAIR ,C.H., AZHAR,A. and DHALL, D.P. Studies on fibrin network structure in human plasma Part I: for clinical application. In press, ThromB.Res. (1991).
Methods
Vol. 64, No. 4
3.
FIBRIN NETWORK STRUCTURE II
RATNOFF, O.D. and determination of
J.Lab.Clin.Med.
MENZIES, C. A new method for fibrinogen in small samples of 32, 316 - 320. (1951).
485
the plasma.
4.
DHALL, T.Z., BRYCE, W.A.J. and DHALL, D.P. Effect of dextran on the molecular structure and tensile strength of fibrin. Thromb. and Haemos. 35, 737 - 745. (1976).
5.
GALANAKIS, D.K. and WEIGAND, K. Albumin inhibition of characteristics and lateral fibrin assembly: certain possible physiologic significance. In Fibrinogen and its Derivatives (eds. G.Muller-Berghaus et al), Elsevier Science Publishers B.V. (Biomedical Division), 71 - 79. (1986).
6.
NAIR, C.H., SULLIVAN, J.R., SINGH, D., AZHAR, A, VAN GELDER, Fibrin network structure as a determinant J. and D.P.DHALL. Thromb.Haemos. 62 (l), 86. (1989). of fibrinolysis.
7.
and gliclazide on ALMER, L.O. Effect of chlopropamide plasminogen activator activity in vascular walls in patients with maturity onset diabetes. Thromb.Res., 35, 19-25. (1984)
0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
STUDIES ON FIBRIN NETWORK STRUCTURE IN HUMAN PLASMA. PART II CLINICAL APPLICATION: DIABETES AND ANTIDIABETIC DRUGS.
C.H.Nair, A.Azhar,J.D.Wilson* and D.P.Dhall. and Department of Thrombosis Research Unit Vascular an% Endocrinology , Woden Valley Hospital, P.O. Box 11, Woden, A.C.T. Australia. (Received 28.1.1991; accepted in revised form 14.6.1991 by Editor J. Soria) (Received in final form by Executive Editorial Office 4.9.1991) ABSTRACT. Using measurements of fibrin fibre thickness (VT) derived from turbidity and permeability (r) of clotted plasma, it in vitro added to plasma has been found that glucose decreases permeability of the network despite unaltered fibre thickness (p ) in Fibrin fibrinogen conversion. uncontrolled diabetes is found significantly reduce a . In diabetic plasma the degree of conversion to fibrin is similar to that in age and sex matched plasma from nonthe effect on fibrin network and fibre thickdiabetics: ness probably arises from glycosylation of fibrinogen. Studies with Gliclazide, Metformin, Glibenclamide and insulin have shown that while all other drugs tested have no effect, Gliclazide increases fibrin fibre thickness diminishes tensile strength and (p ) significantly, s permeability. In separate experiments lysability ;;%a I-labelled fibrin networks developed in the presence of all four hypoglycaemic agents by tissue activator was tested. Networks developed in the presence of Metformin were found to lyse more quickly, followed by insulin and Gliclazide. Alterations induced in fibrin networks in diabetes may be nullified by some oral hypoglycaemic agents such as Gliclazide and not by others. Whether nullification of such changes has lonu-term effects in reducing the incidence of cascular disease in diabetics remains to be established.
INTRODUCTION Although
it has been known for a number of years that occlusive
Key words: Diabetes,
fibrin network structure. 477
478
FIBRIN NETWORK STRUCTURE II
Vol. 64, No. 4
vascular disease has a higher incidence in the diabetic patient non-diabetic, the reasons underlying are with compared the several been Over the last decade studies have unclear. undertaken with a view to elucidating underlying mechanism. In the main inconsistent changes in platelet function tests, in coagulation factor concentrations and in tests of fibrinolysis (1) have been identified. Even in the non-diabetic patients there has been little to suggest that any changes detected using tests of platelet function, fibrinolysis or blood several coagulation can be correlated against the incidence of peripheral vascular disease. It has become possible in recent years to study fibrin network characteristics including fibre thickness in clinical situations using permeability and turbidity techniques (2). Clinical tests of compaction of fibrin fibres have also become available in the last decade (2). Little is known about the organisation of fibrin networks formed in the presence of high levels of glucose in both in vitro and in vivo. blood, This investigation was undertaken to examine the effects of glucose in vitro on fibrin structure as well as to examine fibrin network network characteristics in the diabetic patient. Studies were also made on the effect on fibrin network of Insulin, and of hypoglycaemic agents including Metformin, Gliclazide and Glibenclamide. MATERIALS AND METHODS. Blood obtained from Blood Bank donors (9ml) was collected in 3.8% Trisodium citrate (lml) and 35~1 of 10,000 KIU/ml Trasylol It was then centrifuged at 2,400g at room (Bayer, Germany). temperature for ten minutes to obtain platelet-poor plasma, and recentrifuged at 4ooog for minutes to then five obtain essentially platelet-free plasma (PFP platelet count < 3OOO/ul). The concentration of fibrin in each plasma pool was determined by the method of Ratnoff and Menzies (3) as also the Network fibrin content (CN). Bovine thrombin (Parke Davis, USA) was used to clot plasma in all cases. Different concentrations of glucose were added to platelet-free plasma immediately before the addition of thrombin to develop the Network fibre thickness (uT) was determined from network. described and permeability was also turbidity as previously measured (2). Compaction of the network was characterized (6). In a separate study fifty-one diabetic and age and sex-matched fibre fibrin for their subjects were investigated healthy thickness (VT), network permeability (t) and tensile strength of insulin-dependent poorly-controlled Twenty were network. diabetics with a mean blood sugar level of 15 f 0.9mM. investigated as including Insulin were agents Antidiabetic Gliclazide (Diamicron, Servier, Australia), 12ug/ml follows: 5OOuU/ml Insulin (Sigma), 8ug/ml Metformin (Fisons, Australia) and 72ng/ml Glibenclamide (Daonil, Hoechst, Australia) were added in different plasma samples just before the addition of thrombin. Fibre thickness, permeability and compaction were measured.
Vol. 64, No. 4
FIBRIN NETWORK STRUCTURE II
479
intact networks (labelled with 1251 In separate experiments fibrinogen) developed in the presence of these materials were buffer (pH 7.4, ionic times with Tris-saline washed three tubes separately in test strength 0.153) and then placed containing equal volumes of plasma and Tris-saline buffer. The tubes were then rotated in an elliptical rotator at 37°C and ’ duced with tissue plasminogen activator, and the ~~~~~~~lYos;ISl~~ I fibrin monitored as an index of the rate of lysis. :
Fibrin networks were formed in pre-etched glass tubes 1OOmm in length and of 3mm internal diameter: the individual internal Plasma was clotted with dimensions of each tube were measured. the addition of 1.0 u/ml thrombin (final thrombin concentration) The network was and 25mM Ca2+ (final calcium concentration). perfused with Tris HCl saline buffer, pH 7.35 (5OmM Tris; 103mM NaCl) at a pressure head of 150mm water. The initial 0.5ml of perfusate was collected for determining the fibrin content of the network. The permeability or Darcy constant, calculated from the equation:
‘c,
of the fibrin network was
T = Q
h/Ftp z where Q is the volume flow in time t; t is the viscosity of the liquid; h is the length of the clot; F its cross-section and p the applied pressure.
developed with the The optical density of fibrin networks of lu/ml thrombin (final concentration of thrombin) and additio 25mM Ca4+ (final calcium concentration) in glass cuvettes of lcm path length was measured at a range of wavelengths between 600 and 8001~1, while using unclotted3fibrinogen solution or plasma in the r,ference cell. Then c/TA was plotted as a function of where T is the turbidity (2.303 x optical density),A is l/h the wavelength and c is the concentration of fibrinogen in mg/ml. The intercept A of this plot was used to calculate the average mass-length ratio, uT, according to the equation (5): 10
x 1012 daltons/cm
uT = 1.48A
Compaction was measured according to the method of Dhall et 0.9mls of plasma was clotted with O.lml of thrombin/Ca rtl+ (4). (final concentrations of lu/ml thrombin and 25mM Ca2+). Networks were allowed to form for the same length of time as that taken for optical density readings to reach a plateau in turbidity studies. Networks were then centrifuged in a Zentrifuge
480
FIBRIN NETWORK STRUCTURE II
Vol. 64, No. 4
The volume of (Eppendorf, U.S.A.) at 8000 x g for 45 seconds. expelled serum was then expressed as a percentage of total initial volume of the clotted plasma. RESULTS
As glucose concentration is progressively increased from 5mM to fibre thickness from turbidity (uT) does not alter 25mM, (r) decreases after appreciably. However, network permeability increasing glucose 15mM glucose. Compaction decreases with denoting an increase in the tensile strength of fibrin fibres in tensile strength and reduction in (Figure 1). Increase permeability indicates a greater degree of crosslinkage of within the network. changes in network fibres These characteristics do not arise from altered conversion because (CR) does not alter significantly in the fibrin content concentration range of glucose tested. C 0
‘0
M P
55
A
30
c
T
65
I
60
0 N
‘6
660. 7
600. 660.
T
600’ 36
L----0
6
lOl6P6550
GLUCOSE
(mM)
of glucose on concentration FIGURE 1: Effect of increasing fibrin network developed in plasma. uT is mass-length ratio from turbidity, 'I:is permeability. Results are presented as mean + SEM (n=5).
In plasma networks made from poorly controlled diabetics uT and permeability (r) of the network were significantly reduced, when
Vol. 64, No. 4
FIBRIN NETWORK STRUCTURE II
compared with those in the networks in age and sex matched The tensile strength of fibrin fibres which make up controls. the network was not appreciably affected (Figure 2). These findings indicate that the fibrin in the diabetic has inhibited amount of fibrin is polymerization so a greater lateral This network. decreases the minor into the incorporated permeability of the network but the crosslinking in the network is similar to that in the non-diabetic, as evidenced by unchanged compaction. 120 ..P~O.OOI
100
80
60 m
COMWCTION
40
20
0 nkontrol)=31 n(dlabetic)dO
in FIGURE 2: Comparison of fibrin network characteristics diabetics and sex and age matched controls. uT is ratio from turbidity, 'I; is permeability mass-length and CN is the network fibrin content in the group of diabetic patients. Each is expressed as a percentage of the mean value in the control group of non-diabetics.
studies..
.
Fibrin network characteristics were significantly altered by Gliclazide (Figure 3); thickness (uT) was increased fibre significantly and permeability (t) was reduced significantly. None of the other agents altered network characteristics. Changes induced by Gliclazide in mass-length ratio from turbidity are opposite to those seen in the networks from diabetics.
482
FIBRIN NETWORK STRUCTURE It
160
Vol. 64, No. 4
~0.06
??? ?
-6’0.006
-
CONTROL
B
QLIBENCLAYIDE
0
QLICLALIDE
-
METFORYIN
0
INBULIN
60
0
5
COMPACTION
CN
7
FIGURE 3: : Effect of some hypoglycaemic drugs on fibrin network characteristics. Concentrations used are given under Materials and Methods. uT is mass-length ratio from turbidity, t is permeability and C is the network fibrin content (n=5 for each drug teste # ).
tested with the exception of Glibenclamide All the agents (Figure 4). Of the three agents which enhanced fibrinolysis enhanced fibrinolysis, Metformin had the most profound effect. were spectacular effects found with though less Similar Gliclazide and Insulin. 8
k 400 A t 360 : 300 ; 260 p 200 s : 160 j 100 :
P Y
-I 0
30
60
90
120
160
180
210
TIME (MINUTES) I
I
-
CONTROL
+
QLIBENCLAMIDE -+-
-e-
INSULIN
X
METFOAMIN
QLICLAZIDE
FIGURE 4: Lysis by streptokinase of fibrin networks developed drug with some hypoglycaemic agents . tested), quantified by measuring the released
Vol. 64, No. 4
FIBRIN NETWORK STRUCTURE II
DISCUSSION in A causal relationship between the metabolic abnormalities diabetes and vascular disease in diabetics has thus far not been A great deal of work, however, suggests that established. it has not been state. But diabetes is a hypercoagulable primary hypercoagulability is a whether the established abnormality or whether it is secondary to vascular disease. The present study shows that both glucose in vitro and the metabolic abnormality in patients induce alterations in network characteristics. However, effects of glucose on network structure in vitro are different from the abnormalities found in diabetic plasma networks. When glucose is artificially elevated in normal significantly affected, but thickness is not fibre plasma reduced when significantly and compaction are permeability 15mM. These changes are glucose concentration is in excess Of not associated with any alteration in fibrinogen conversion to fibrin or in fibrin content (C,) of the network, indicating that the networks are altered because the fibrin fibres become more highly crosslinked. In the diabetic patient, on the other hand, fibre thickness (uT) and permeability are reduced and compaction remains relatively unaltered. these differences is not clear at underlying The mechanism with artificially added investigation but in the present, before only a few minutes glucose the material is added developing the network - the changes induced are from a direct From our studies it appears that in effect of glucose on fibrin. the presence of glucose added in this fashion the network is more This imparts greater tensile strength and highly crosslinked. In the diabetic, on the diminished permeability to the network. plasma proteins including fibrinogen are the hand, other It has been previously shown that altered plasma glycosylated. proteins like albumin can modify fibrin network characteristics (5). The main abnormality seems to arise from inhibited lateral polymerization or enhanced end-to-end polymerization. The resulting diminished thickness of fibres increases the total contour length of the fibres thus reducing the permeability, but cross-linking does not seem to be measurably affected in the diabetic. The compaction thus is unchanged. The differences in fibrin network characteristics with added glucose in vitro, on the one hand, and diabetic plasma on the other are not contradictory - they could arise from glycation of plasma proteins such as albumin. This requires detailed investigation. We have previously shown (6) that networks with fibres of reduced thickness and reduced permeability such as those found in the diabetics are resistant to lysis. This resistance to lysis invalidates normal or increased fibrinolytic activity from normal Or even enhanced plasminogen concentration. Thus, such abnormalities in network structure as seen in the diabetic patient provide scope for peripheral vascular disease of both macro- and microangiopathic types. On investigating
the capacity
of pharmacological
materials
used
484
FIBRIN NETWORK STRUCTURE II
Vol. 64, No. 4
in the treatment of diabetes in altering network characteristics it was found that Gliclazide is capable of inducing alterations in fibrin network structure. In its presence fibre thickness was significantly increased but despite a reduction in permeability was found enhanced. Gliclazide modifies network compaction formation in a surprising fashion. Networks show some enhancement lateral polymerization accounting for thickened fibres. of Diminished permeability indicates enhanced crosslinking within the network. However, the crosslinks are not primary crosslinks since they are weaker and readily disrupt on centrifugal stress. It has been previously shown that such changes induced may be induced with Dextran (4) which enhances lateral polymerization thus hiding primary crosslinks within thickened of fibrin fibres. Such fibrin fibres were found more susceptible to lysis. These observations are significant in the light of investigations by Almer (6) on Type II diabetics, who demonstrated that a shift in treatment from Chlopropamide to Gliclazide the induced a significant increase in plasminogen activator activity of the vascular wall in patients with an abnormally low activity, independent of an elevated carbohydrate metabolism. Our study shows relatively independent effects of Gliclazide on the network structure. would seem to promote lysis by Thus, Gliclazide affecting both the fibrinolytic system and the actual structure of the network by influencing the crosslinking arrangement within fibrin. Of the drugs tested all were capable of enhancing fibrinolysis with the exception of Glibenclamide. Metformin, however, had the most spectacular effect. The observations that major changes in fibrin network structure are induced by glucose and by changes in the diabetic decreased metabolic plasma, the lysability of networks in diabetics, and the possibility of reversal of network changes by therapeutic means open a new and management of horizon in research into the pathogenesis vascular disease in diabetics. ACKNOWLEDGEMENTS The authors would like to thank the National Heart Foundation of Administration Practice Hospital Private Australia and the Committee of Woden Valley Hospital for grants supporting this project. REFERENCES 1.
Advanced metabolic disorders. KEEN, H. and JARRET, R.G. In Microangiopathy, its Prevalance in Asymptomatic Diabetes, supp1.13. (1973).
2.
NAIR ,C.H., AZHAR,A. and DHALL, D.P. Studies on fibrin network structure in human plasma Part I: for clinical application. In press, ThromB.Res. (1991).
Methods
Vol. 64, No. 4
3.
FIBRIN NETWORK STRUCTURE II
RATNOFF, O.D. and determination of
J.Lab.Clin.Med.
MENZIES, C. A new method for fibrinogen in small samples of 32, 316 - 320. (1951).
485
the plasma.
4.
DHALL, T.Z., BRYCE, W.A.J. and DHALL, D.P. Effect of dextran on the molecular structure and tensile strength of fibrin. Thromb. and Haemos. 35, 737 - 745. (1976).
5.
GALANAKIS, D.K. and WEIGAND, K. Albumin inhibition of characteristics and lateral fibrin assembly: certain possible physiologic significance. In Fibrinogen and its Derivatives (eds. G.Muller-Berghaus et al), Elsevier Science Publishers B.V. (Biomedical Division), 71 - 79. (1986).
6.
NAIR, C.H., SULLIVAN, J.R., SINGH, D., AZHAR, A, VAN GELDER, Fibrin network structure as a determinant J. and D.P.DHALL. Thromb.Haemos. 62 (l), 86. (1989). of fibrinolysis.
7.
and gliclazide on ALMER, L.O. Effect of chlopropamide plasminogen activator activity in vascular walls in patients with maturity onset diabetes. Thromb.Res., 35, 19-25. (1984)
