THROMBOSIS RESEARCH 65; 469-474,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
PLATELET FUNCTION AND FIBRINOLYTIC ACTIVITY IN CERVICAL SPINAL CORD INJURED PATIENTS.
K. Winther’, G. Gleerup2~K. Snorrason3, F. Biering-Sorensen4. Department of Clinical Chemistry, Glostrup and Hvidovre Hospital’, Department of Clinical Chemistry Frederiksberg Hospital and Department of Medicine Glostrup Hospital*, Department of Plastic Surgery’, and Center of Spinal Cord Injured, Department TH, Rigshospitalet4, University of Copenhagen, Denmark. (Received
26.6.1991;
accepted
in revised form 10.12.1991
by Editor R. Malmgren)
INTRODUCTION The frequent occurrence of deep venous thrombosis (DVT) in acute spinal cord injury has been richly documented, the reported incidence varying according to the detection method used. Two studies employing frequent monitoring of the leg veins with radiolabelled fibrinogen gave the highest incidence, at 78% (1,2). The characteristic feature of patients with cord injury is a paresis, with venous stagnation due to the loss of the muscular pump action. Some of the haemostatic disturbances which express this slowing of the venous stream have been identified as an increase of various factor VIII elements, increased platelet aggregability to collagen (2) and diminished fibrinolysis (1). It has been claimed that the risk of DVT and pulmonary emboli falls off sharply by about 21 days after the injury, and though there are sporadic reports of these complications occurring months later, in the subacute phase (3), there is little information on the final phase of physiologically stable neurological damage. This study therefore concentrated on the changes in haemostasis during this protracted phase of paralysis, to see whether there are any persistent disturbances of haemostasis in the chronic stage of paralysis. Key words: Platelets, Fibrinolysis, Spinal Cord Injury.
469
470
HAEMOSTASIS IN CHRONIC PARALYSIS
Vol. 65, No. 3
SUBJECTS AND METHODS
Included were 10 males and 2 female out-patients aged between 23 and 40 years with cervical spinal cord lesions of traumatic origin of minimum 2 years duration. Patients with any concurrent disease, complication or infection that might influence the laboratory results were excluded. No antibiotic, anticoagulant or blood transfusion was given for a fortnight before the blood sampling. An age-matched group of 12 males were tested in a parallel design and served as a control group. Blood samples invariably were taken from fasting patients and controls between 8 and 11 a.m. after 20 minutes rest in the supine position. When not otherwise stated, blood was always drawn with slight compression, through a 22-gauge needle inserted in an antecubital vein. Beta-thromboglobuiin (B-TG). Blood 2.5 ml was drawn into test tubes containing 100 ,~l EDTA 0.134 M and 100 ~1 theophylline 15 mM and placed in icecold water for 15 min. The samples were then centrifuged at 3000 x g for 20 min at 4°C. A 250 /tl aliquot of the supernatant was stored at -60°C for subsequent determination of B-TG by RIA (Amersham, UK). Platelet aggregation. Citrated blood was centrifuged at 150 x g for 5 min at room temperature to prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was obtained by further centrifugation at 2000 x g for 10 min. Platelet aggregation was studied turbidometrically using a Payton dual channel aggregometer according to Born. In the aggregometer, the PRP was kept at 37°C and was continuously stirred. Aggregation was induced with adrenaline in final concentrations of 0.01, 0.05, 0.10, 0.50 and 1.00 pg/rnl plasma, and by ADP in final concentrations of 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 PM. The threshold value for aggregation was defined as the lowest concentration of adrenaline or ADP which caused irreversible aggregation, with at least an 80% difference in light transmission between PRP and PPP. Euglobulin clot lysis time (ECLT). ECLT was tested before and after standardised venous compression in order to test fibrinolytic activity as well as fibrinolytic reserve capasity. Venous compression was performed for 5 minutes with a cuff of appropriate size, using the mean of systolic and diastolic bloodpressure. PPP was prepared as previously described. To a glass tube containing 1% acetic acid 130 ~1 and 7.5 ml sterile water, 500 ~1 PPP was added. The tubes were placed on ice for 10 min and then centrifuged at 2000 x g for 10 min. The supematant was discharged and the sediment was mixed with 0.9 ml 37°C Michaelis buffer. While keeping the temperature at 37“C, 1.5 IU thrombin reagent (Behring Werke AG, Marburg, Western Germany) was added to produce a fibrin clot. The time of dissolution of the clot was then recorded. Tbromboxane B, and 6-keto-PGF Ia. Glass vials containing 5 ml venous blood collected with minimum stasis, were incubated for 1 hour at 36°C. After centrifugation at 3000 g at 4°C for 20 min, the supematant was transferred to plastic vials and stored at -60°C for subsequent RIA (Amersham U.K.). Catecholamine. Blood 5 ml was collected in pre-cooled glass vials containing 100 ~1 EDTA 0.24 M and 1000 ~1 glutathione 0.195 M and placed on ice for 20 min. After centrifugation at 3000 x g for 20 min, at 4”C, the supematant was placed in plastic vials and stored at -60°C for later assay by an isotope derivative technique [4].
Vol. 65, No. 3
471
HAEMOSTASIS IN CHRONIC PARALYSIS
Plasma fibrinogen, activated partial thromboplastin time, thrombin time, pro-thrombin time, anti-thrombin III, and platelet counts were estimated using normal laboratory routine. The Mann-Whitney matched-pairs test was used for statistical evaluation. p < 0.05 was regarded as statistically significant. RESULTS There was a considerable, statistically significant, increase in the ECLT in the patients (mean 869 min.) as compared with the controls (mean 192 min.), and this persisted after venous compression, though both values were reduced by just over 40% (figure). These values indicate substantially reduced fibrinolytic activity in the tetraplegic patients. The aggregability of platelets by adrenaline or ADP, and the platelet release of B-TG was not different in tetraplegic and their controls (Table), nor was there difference in any of the coagulation parameters studied, namely activated partial thromboplastin time, thrombin and prothrombin times, plasma fibrinogen, or anti-thrombin III (detailed results not given).
BEFORE A-
COMPRESS.
AFTER
COMPRESS.
Patients-Controls
-Patients-Controls
-
1000
-
a 2 2 ._
750
E
500
-
5
M 25C
-
I
-
Median Range *
=
869 (120
- 2850)
113
192 (115 - 320)
(50
- 1800)
min
(45 - 190)
P < 0.01
Legend Fig. Euglobulin clot lysis time (ECLT) is shown before venous compression (left panel) and after venous compression (right panel) in tetraplegics and controls.
472
HAEMOSTASIS IN CHRONIC PARALYSIS
Vol. 65, No. 3
Table Tetraple&
Contr&
Heart rate (beats/mm)
(?OE)
70.00 * (68.90)
Blood pressure (systolic mm Hg)
108 (90-115)
120 * (100-125)
Blood pressure (diastolic mm Hg)
(6Z5)
Platelet aggregation @TIP-thresholds PM)
(24108)
(657-38s) (24:)
0.75 (0.05-l)
0.55 (0.05-l)
Platelet count ( 109/1)
229 (151-312)
279* (227-385)
Plasma B-TG (ng/m0
(3&O)
Platelet aggregation (ADR-thresholds pg/ml)
(3422-64)
Plasma adrenaline (nmol/l)
0.15 (0.15-0.40)
0.25 * (0.15-0.42)
Plasma noradrenaline (nmol/l)
0.51 (0.30-1.52)
2.50 * (1.10-2.91)
Plasma CAMP (ng/mU
(6.4Y8.8)
14.2 * (12.2-W.1)
Serum Thromboxane B, (ng/mU
114 (38-322)
227 * (152-361)
’ P-CO.05
Lwnd
table
Heart rate, blood pressures, platelet aggregation, platelet count, platelet release of B-TG, plasma adrenaline, plasma noradrenaline, plasma CAMP, serum thromboxane B, and serum 6-keto PGF,, are shown for tetraplegics and controls. Data given are median and range.
Vol. 65, No. 3
HAEMOSTASIS IN CHRONIC PARALYSIS
473
Other positive findings were: sizeable and significant reductions in the plasma levels of adrenaline, noradrenaline and CAMP, and in the production of thromboxane B, a stable metabolite of thromboxane A*, and 6-keto-PGF,, a stable metabolite of prostacyclin, in the patients, as compared with controls (Table). The heart rate and systolic blood pressure were significantly lower in the patients than in controls (Table).
DISCUSSION The salient positive finding of this study is that fibrinolytic activity is markedly less in patients with chronic traumatic tetraplegia than in their controls. This applies both under normal resting conditions and when fibrinolysis acclerate by the manoeuvre of venous compression. The control of fibrinolytic activity is only dimly understood, and it is not possible to give a comprehensive explanation of why it is disturbed in chronic paraplegia. Sluggishness of the venous return due to the enfeebled pumping action of the paralysed muscles is probably important, but other factors, in particular the reduced level of circulating catecholamines (present in the acute stage of acute injury too) may also contibute. The level of circulating noradrenaline in the patients was only 34% of the control value, and adrenaline was 60%, a change in line with previous reports (6,7). Raising catecholamine levels by infusion has been reported to enhance fibrinolytic activity, and it seems coherent that the lowered catecholamine levels observed in our paraplegic patients would give reduced fibrinolytic activity. Reduced catecholamine stimulus of the beta-adrenoceptor system could be responsible for the much lower plasma level of cyclic AMP in the patients. This “second messenger” of the beta-adrenoceptor system has been reported to stimulate the release or production of tissue plasmin activator (t-PA) and it’s lowering could, conversely, be expected to diminish fibrinolysis (8). Another effect which might contribute to the fall of fibrinolysis is the observed sharp reduction of prostacyclin in the tetraplegic patients (table 1). Experimental infusion of prostacyclin has been reported to increase fibrinolytic activity (9). Whether depression of prostacyclin would lead to a decrease in fibrinolytic activity has not been experimentally verified: it is, however, a reasonable speculation, which fits with the present result. In conclusion: Fibrinolytic activity is substantially decreased in patients with chronic spinal cord injury, even after venous compression. The observed sharp reduction of circulating catecholamines and prostacyclin could be significant factors in the disturbed haemostasis.
ACKNOWLEDGEMENTS We would like to thank Gitte Hagel and Anne Brock-Nielsen for excellent secretarial assistance.
474
HAEMOSTASIS IN CHRONIC PARALYSIS
Vol. 65, No. 3
REFERENCES
1.
Petaia J, Myllynen P, Rokkanen P, Nokelainen M. Fibrinolysis and spinal injury. Relationship to post-traumatic deep vein thrombosis. Acta Chir Sa; U: 251-256, 1989.
2.
Green D, Rossi E C, Yao J S T, Flinn W R, Spies S M. Deep vein thrombosis in spinal cord injury: Effect of prophylaxis with calf compression, aspirin, and dipyridamole. Paraplegia; 24: 227-234, 1982.
3.
Perkash A, Prakash V, Perkash I. Experience with the management of thromboembolism in patients with spinal cord injury: Part I. incidence, diagnosis and role of some risk factors. Paraolegia: 16: 322-331, 1978-79.
4.
Peuler J D, Johnson G A. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci; 2: 625-636, 1977.
5.
Kral J G, Ablad B, Johnsson G, Korsan-Bengtsen K. Effects of adrenaline and alprenolol on blood coagulation and fibrinolysis in man. Eur J Clin Pham; 2: 144-147, 1971.
6.
Debarge 0, Christensen N J, Corbett J L, Eidelman B H, Frankel H L, Mathias C J. Plasma catecholamines in tetraplegics. Para&&; 12: 44-49, 1974.
7.
Frewin D B, Levitt M, Myers S J, Downey J A. Catecholamine responses in paraplegia. ParaDlegia;a: 238-244, 1973.
8.
Winther K. Fibrinolyse und Kardioprotektive Therapie, Hlmos@seolo&; 8: 100-101, 1988.
9.
Winther K. Snorrason K. Knudsen J B, Medgyesi S. The effect of prostacyclin infusion on tissue plasminogen activator. Thrombosis Research; a: 741-745, 1987.
BRIEF
COMMUNICATION
PLATELET FUNCTION AND FIBRINOLYTIC ACTIVITY IN CERVICAL SPINAL CORD INJURED PATIENTS.
K. Winther’, G. Gleerup2~K. Snorrason3, F. Biering-Sorensen4. Department of Clinical Chemistry, Glostrup and Hvidovre Hospital’, Department of Clinical Chemistry Frederiksberg Hospital and Department of Medicine Glostrup Hospital*, Department of Plastic Surgery’, and Center of Spinal Cord Injured, Department TH, Rigshospitalet4, University of Copenhagen, Denmark. (Received
26.6.1991;
accepted
in revised form 10.12.1991
by Editor R. Malmgren)
INTRODUCTION The frequent occurrence of deep venous thrombosis (DVT) in acute spinal cord injury has been richly documented, the reported incidence varying according to the detection method used. Two studies employing frequent monitoring of the leg veins with radiolabelled fibrinogen gave the highest incidence, at 78% (1,2). The characteristic feature of patients with cord injury is a paresis, with venous stagnation due to the loss of the muscular pump action. Some of the haemostatic disturbances which express this slowing of the venous stream have been identified as an increase of various factor VIII elements, increased platelet aggregability to collagen (2) and diminished fibrinolysis (1). It has been claimed that the risk of DVT and pulmonary emboli falls off sharply by about 21 days after the injury, and though there are sporadic reports of these complications occurring months later, in the subacute phase (3), there is little information on the final phase of physiologically stable neurological damage. This study therefore concentrated on the changes in haemostasis during this protracted phase of paralysis, to see whether there are any persistent disturbances of haemostasis in the chronic stage of paralysis. Key words: Platelets, Fibrinolysis, Spinal Cord Injury.
469
470
HAEMOSTASIS IN CHRONIC PARALYSIS
Vol. 65, No. 3
SUBJECTS AND METHODS
Included were 10 males and 2 female out-patients aged between 23 and 40 years with cervical spinal cord lesions of traumatic origin of minimum 2 years duration. Patients with any concurrent disease, complication or infection that might influence the laboratory results were excluded. No antibiotic, anticoagulant or blood transfusion was given for a fortnight before the blood sampling. An age-matched group of 12 males were tested in a parallel design and served as a control group. Blood samples invariably were taken from fasting patients and controls between 8 and 11 a.m. after 20 minutes rest in the supine position. When not otherwise stated, blood was always drawn with slight compression, through a 22-gauge needle inserted in an antecubital vein. Beta-thromboglobuiin (B-TG). Blood 2.5 ml was drawn into test tubes containing 100 ,~l EDTA 0.134 M and 100 ~1 theophylline 15 mM and placed in icecold water for 15 min. The samples were then centrifuged at 3000 x g for 20 min at 4°C. A 250 /tl aliquot of the supernatant was stored at -60°C for subsequent determination of B-TG by RIA (Amersham, UK). Platelet aggregation. Citrated blood was centrifuged at 150 x g for 5 min at room temperature to prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was obtained by further centrifugation at 2000 x g for 10 min. Platelet aggregation was studied turbidometrically using a Payton dual channel aggregometer according to Born. In the aggregometer, the PRP was kept at 37°C and was continuously stirred. Aggregation was induced with adrenaline in final concentrations of 0.01, 0.05, 0.10, 0.50 and 1.00 pg/rnl plasma, and by ADP in final concentrations of 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 PM. The threshold value for aggregation was defined as the lowest concentration of adrenaline or ADP which caused irreversible aggregation, with at least an 80% difference in light transmission between PRP and PPP. Euglobulin clot lysis time (ECLT). ECLT was tested before and after standardised venous compression in order to test fibrinolytic activity as well as fibrinolytic reserve capasity. Venous compression was performed for 5 minutes with a cuff of appropriate size, using the mean of systolic and diastolic bloodpressure. PPP was prepared as previously described. To a glass tube containing 1% acetic acid 130 ~1 and 7.5 ml sterile water, 500 ~1 PPP was added. The tubes were placed on ice for 10 min and then centrifuged at 2000 x g for 10 min. The supematant was discharged and the sediment was mixed with 0.9 ml 37°C Michaelis buffer. While keeping the temperature at 37“C, 1.5 IU thrombin reagent (Behring Werke AG, Marburg, Western Germany) was added to produce a fibrin clot. The time of dissolution of the clot was then recorded. Tbromboxane B, and 6-keto-PGF Ia. Glass vials containing 5 ml venous blood collected with minimum stasis, were incubated for 1 hour at 36°C. After centrifugation at 3000 g at 4°C for 20 min, the supematant was transferred to plastic vials and stored at -60°C for subsequent RIA (Amersham U.K.). Catecholamine. Blood 5 ml was collected in pre-cooled glass vials containing 100 ~1 EDTA 0.24 M and 1000 ~1 glutathione 0.195 M and placed on ice for 20 min. After centrifugation at 3000 x g for 20 min, at 4”C, the supematant was placed in plastic vials and stored at -60°C for later assay by an isotope derivative technique [4].
Vol. 65, No. 3
471
HAEMOSTASIS IN CHRONIC PARALYSIS
Plasma fibrinogen, activated partial thromboplastin time, thrombin time, pro-thrombin time, anti-thrombin III, and platelet counts were estimated using normal laboratory routine. The Mann-Whitney matched-pairs test was used for statistical evaluation. p < 0.05 was regarded as statistically significant. RESULTS There was a considerable, statistically significant, increase in the ECLT in the patients (mean 869 min.) as compared with the controls (mean 192 min.), and this persisted after venous compression, though both values were reduced by just over 40% (figure). These values indicate substantially reduced fibrinolytic activity in the tetraplegic patients. The aggregability of platelets by adrenaline or ADP, and the platelet release of B-TG was not different in tetraplegic and their controls (Table), nor was there difference in any of the coagulation parameters studied, namely activated partial thromboplastin time, thrombin and prothrombin times, plasma fibrinogen, or anti-thrombin III (detailed results not given).
BEFORE A-
COMPRESS.
AFTER
COMPRESS.
Patients-Controls
-Patients-Controls
-
1000
-
a 2 2 ._
750
E
500
-
5
M 25C
-
I
-
Median Range *
=
869 (120
- 2850)
113
192 (115 - 320)
(50
- 1800)
min
(45 - 190)
P < 0.01
Legend Fig. Euglobulin clot lysis time (ECLT) is shown before venous compression (left panel) and after venous compression (right panel) in tetraplegics and controls.
472
HAEMOSTASIS IN CHRONIC PARALYSIS
Vol. 65, No. 3
Table Tetraple&
Contr&
Heart rate (beats/mm)
(?OE)
70.00 * (68.90)
Blood pressure (systolic mm Hg)
108 (90-115)
120 * (100-125)
Blood pressure (diastolic mm Hg)
(6Z5)
Platelet aggregation @TIP-thresholds PM)
(24108)
(657-38s) (24:)
0.75 (0.05-l)
0.55 (0.05-l)
Platelet count ( 109/1)
229 (151-312)
279* (227-385)
Plasma B-TG (ng/m0
(3&O)
Platelet aggregation (ADR-thresholds pg/ml)
(3422-64)
Plasma adrenaline (nmol/l)
0.15 (0.15-0.40)
0.25 * (0.15-0.42)
Plasma noradrenaline (nmol/l)
0.51 (0.30-1.52)
2.50 * (1.10-2.91)
Plasma CAMP (ng/mU
(6.4Y8.8)
14.2 * (12.2-W.1)
Serum Thromboxane B, (ng/mU
114 (38-322)
227 * (152-361)
’ P-CO.05
Lwnd
table
Heart rate, blood pressures, platelet aggregation, platelet count, platelet release of B-TG, plasma adrenaline, plasma noradrenaline, plasma CAMP, serum thromboxane B, and serum 6-keto PGF,, are shown for tetraplegics and controls. Data given are median and range.
Vol. 65, No. 3
HAEMOSTASIS IN CHRONIC PARALYSIS
473
Other positive findings were: sizeable and significant reductions in the plasma levels of adrenaline, noradrenaline and CAMP, and in the production of thromboxane B, a stable metabolite of thromboxane A*, and 6-keto-PGF,, a stable metabolite of prostacyclin, in the patients, as compared with controls (Table). The heart rate and systolic blood pressure were significantly lower in the patients than in controls (Table).
DISCUSSION The salient positive finding of this study is that fibrinolytic activity is markedly less in patients with chronic traumatic tetraplegia than in their controls. This applies both under normal resting conditions and when fibrinolysis acclerate by the manoeuvre of venous compression. The control of fibrinolytic activity is only dimly understood, and it is not possible to give a comprehensive explanation of why it is disturbed in chronic paraplegia. Sluggishness of the venous return due to the enfeebled pumping action of the paralysed muscles is probably important, but other factors, in particular the reduced level of circulating catecholamines (present in the acute stage of acute injury too) may also contibute. The level of circulating noradrenaline in the patients was only 34% of the control value, and adrenaline was 60%, a change in line with previous reports (6,7). Raising catecholamine levels by infusion has been reported to enhance fibrinolytic activity, and it seems coherent that the lowered catecholamine levels observed in our paraplegic patients would give reduced fibrinolytic activity. Reduced catecholamine stimulus of the beta-adrenoceptor system could be responsible for the much lower plasma level of cyclic AMP in the patients. This “second messenger” of the beta-adrenoceptor system has been reported to stimulate the release or production of tissue plasmin activator (t-PA) and it’s lowering could, conversely, be expected to diminish fibrinolysis (8). Another effect which might contribute to the fall of fibrinolysis is the observed sharp reduction of prostacyclin in the tetraplegic patients (table 1). Experimental infusion of prostacyclin has been reported to increase fibrinolytic activity (9). Whether depression of prostacyclin would lead to a decrease in fibrinolytic activity has not been experimentally verified: it is, however, a reasonable speculation, which fits with the present result. In conclusion: Fibrinolytic activity is substantially decreased in patients with chronic spinal cord injury, even after venous compression. The observed sharp reduction of circulating catecholamines and prostacyclin could be significant factors in the disturbed haemostasis.
ACKNOWLEDGEMENTS We would like to thank Gitte Hagel and Anne Brock-Nielsen for excellent secretarial assistance.
474
HAEMOSTASIS IN CHRONIC PARALYSIS
Vol. 65, No. 3
REFERENCES
1.
Petaia J, Myllynen P, Rokkanen P, Nokelainen M. Fibrinolysis and spinal injury. Relationship to post-traumatic deep vein thrombosis. Acta Chir Sa; U: 251-256, 1989.
2.
Green D, Rossi E C, Yao J S T, Flinn W R, Spies S M. Deep vein thrombosis in spinal cord injury: Effect of prophylaxis with calf compression, aspirin, and dipyridamole. Paraplegia; 24: 227-234, 1982.
3.
Perkash A, Prakash V, Perkash I. Experience with the management of thromboembolism in patients with spinal cord injury: Part I. incidence, diagnosis and role of some risk factors. Paraolegia: 16: 322-331, 1978-79.
4.
Peuler J D, Johnson G A. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci; 2: 625-636, 1977.
5.
Kral J G, Ablad B, Johnsson G, Korsan-Bengtsen K. Effects of adrenaline and alprenolol on blood coagulation and fibrinolysis in man. Eur J Clin Pham; 2: 144-147, 1971.
6.
Debarge 0, Christensen N J, Corbett J L, Eidelman B H, Frankel H L, Mathias C J. Plasma catecholamines in tetraplegics. Para&&; 12: 44-49, 1974.
7.
Frewin D B, Levitt M, Myers S J, Downey J A. Catecholamine responses in paraplegia. ParaDlegia;a: 238-244, 1973.
8.
Winther K. Fibrinolyse und Kardioprotektive Therapie, Hlmos@seolo&; 8: 100-101, 1988.
9.
Winther K. Snorrason K. Knudsen J B, Medgyesi S. The effect of prostacyclin infusion on tissue plasminogen activator. Thrombosis Research; a: 741-745, 1987.
