Platelet Function, Blood Coagulability, and Fibrinolysis in Patients With Low Tension Glaucoma J. Heinz
Joist, MD; Philip Lichtenfeld; Alan
\s=b\ Platelet function in vitro and in vivo and certain measurements of blood coagulability and fibrinolysis were examined in 12 patients with low tension glaucoma and the results were compared with those obtained in 12 normal subjects matched for age, race, and sex. The data obtained seem to indicate that patients with low tension glaucoma cannot be readily distinguished from individuals of similar age and sex not afflicted with this disorder by measuring these variables. Thus, a "hypercoagulable state," ie, a thrombotic tendency, does not appear to be of major importance in the pathogenesis of this disorder.
(Arch Ophthalmol 94:1893-1895, 1976) tension
glaucoma (LTG) is
Lowcomplex disorder whose
Mandell, MD; Allan
E. Kolker MD
based on the finding of either increased platelet adhesiveness and/
was
or an
abnormally prolonged euglobu-
lin lysis time in 16 of 26 patients with LTG. However, in this study', the group of individuals from which the control data for these tests were obtained was not described. In view of the potential therapeutic implications, we examined certain measurements of platelet function in vitro and vivo, blood coagulation, and fibrinolysis in a group of patients with LTG and in a group of appar¬ ently healthy individuals closely matched for age and sex. MATERIALS AND METHODS
a
cause
and pathogenesis are still poorly understood. It has been suggested recently by Drance' that this disor¬ der may be due to an ischemie process of the optic nerve head and that "hypercoagulability," ie a thrombotic ten¬ dency, should be considered among the factors involved in the pathogen¬ esis of this condition. This hypothesis -'
Accepted
I.
for publication April 9, 1976. From the Departments of Medicine, Pathology, and Ophthalmology (Glaucoma Center) Washington University School of Medicine, St Louis. Dr Mandell is now with the Department of Ophthalmology, University of Tennessee, College of Medicine, Memphis. Reprint requests to Hemostasis and Thrombosis Laboratory, Division of Laboratory Medicine, Washington University School of Medicine, Barnes Hospital, St Louis, MO 63110 (Dr Joist).
Twelve and four age
was
patients with LTG (eight women men) were studied. Their mean 66 (range 51 to 81) years. These
individuals had nerve fiber bundle defects on the Goldmann perimeter and character¬ istic glaucomatous cupping observed on repeated examinations. In all cases aver¬ age intraocular pressures over many months were less than 20 mm Hg and no pressure on diurnal testing was over 24 mm Hg. The control group consisted of 12 individuals (eight women and four men) with a mean age of 68 (range 58 to 78) years, who had normal-appearing optic nerves and normal Goldmann visual fields. Patients with LTG and controls were phakic, had no history or signs of uveitis or other ocular disease, and had open angles. None of the patients or controls had elevated fasting blood glucose concentra¬ tions or hyperuricemia. One patient with LTG and two control subjects had mildly
elevated
serum
cholesterol concentrations,
patients with LTG and two control subjects had mildly elevated serum triglyc¬ éride concentrations. Two patients with LTG and one control subject had previous evidence of cardiac insufficiency, which was well controlled by digitilization. None of the LTG patients or control subjects had had signs or symptoms of cerebrovascular or peripheral arterial insufficiency. None
two
of the individuals tested showed clinical or laboratory evidence of hepatic or renal disease. A careful drug history was ob¬ tained from all patients and control subjects studied, and they were instructed not to take aspirin-containing medications for at least seven days prior to testing, and to avoid other medications known to affect platelet function. A list of such agents was given to each individual studied. Informed consent was obtained from all individuals tested. Blood was collected from all individuals after a ten-hour fasting period by clean venipuncture of an antecubital vein, using a double syringe technique as follows: 5 ml into a glass tube containing 1% ethylenediaminetetraacetic acid (EDTA) for deter¬ mination of the whole blood platelet count; 10 ml into a plastic syringe containing no anticoagulant for estimation of platelet adhesiveness; 40 ml into a plastic syringe containing 0.1 volume of 3.8% sodium citrate for studies of platelet aggregation, serotonin release, platelet factor 3 avail¬ ability, plasma fibrinogen, activated par¬ tial thromboplastin time, antithrombin III, and antiheparin activity; 5 ml into a plastic syringe after ten minutes of venous occlu¬ sion of the forearm (blood pressure cuff inflated to 80 mm Hg) for estimation of fibrinolytic activator activity.
Downloaded From: http://archopht.jamanetwork.com/ by a University of California - San Diego User on 06/05/2015
O'Brien et al.1 Native
The stressed template bleeding time from duplicate 9-mm long and 1-mm deep cuts on the forearm was measured with the blood pressure cuff inflated to 80 mm Hg as described by O'Brien et al.' Platelet counting was performed in whole blood or platelet-rich plasma by a semiautomated method.4 Platelet adhesiveness was esti¬ mated by a standard glass bead column re¬ tention method similar to that described by
(nonanticoagulated)
blood issuing from the column between 0 and 10, 10 and 20, and 20 and 30 seconds was collected into separate plastic tubes containing 1% dry EDTA. The pump was then stopped, the glass bead column discon¬ nected from the syringe and replaced by a new glass bead column, and the test was immediately repeated. The duplicate pro¬ cedure was completed in less than two minutes. The platelet counts in the blood before passage and in each of the subsamples after passage of the column were determined, and the percentage platelet retention by the glass-beads column was calculated for each subsample. The results of the duplicate tests were averaged. Platelet aggregation was studied in
passed, immediately after collec¬ tion, into a plastic 10-ml syringe, through a piece of polyvinyl tubing (13 cm long, internal diameter 0.3 mm) containing 1.5 gm of glass beads at a constant rate (transit time 8 seconds), using an infusion pump. At the time the first drop of blood emerged from the end of the glass bead column, a stopwatch was started and the blood
was
Table 1.—Bleeding Time, Platelet Count, and Platelet Adhesiveness in Patients With Low Tension Glaucoma (LTG)* Platelet
Adhesiveness, %
min
Bleeding Time, Controls (12)
SD SD
"All tests were performed in (Student f test) are shown.
Table 2.—Platelet
1,000/cu
10.4 5.3 8.7 4.2
LTG
patients (12)
Platelet Count,
The
means
(i¿)
SD
LTG
X
patients (12) "The are
means
given.
(x)
SD
58.2 13.1
74.1 11.2
238
34.7 16.0
58.5 22.0 > .1
17.2
>
.1
Epinephrine, jimole
28.1 2&5
49.2 2g^
54.0 2&
34.5 28.1 > .1
53.2 25.4 > .1
60.4 23.3 > .1
and standard deviations
(SDs)
47.4
2g£ 49.1 27.9 > .1 of
citrated, platelet-rich plasma (PRP) by
73.7 >
.1
(LTG)*
Collagen, ng/ml
49.9 3Q^
52.3 2ߣ
6.1 28.4 34^3
8.3 39.4 34^g
60.4 17.6 > .1
65.7 15.8 > .1
38.8 28.3 > .1
24.5 > .1
12.5 25 56.2 64.9 2g^ 2a3 60.7 75.1 23.4 14.3 > .1 > .1
values
(Student ( test)
single
observations and
45.3
a
turbidimetric technique,'' using a dualchannel aggregometer and recorder as previously described," except type 0.312 cuvettes, 0.45 ml of PRP, and 0.05 ml of stimulus were used. Platelet-rich plasma was prepared by centrifugation of blood at 180 g for ten minutes at room temperature, stored at room temperature, and prewarmed to 37 C for four minutes before testing. The platelet count was adjusted to 250,000/cu mm with platelet poor plasma (PPP) prepared by centrifugation of the remaining blood (after separation of PRP) at 5,000 g for ten minutes. The maximum height of the aggregation tracing observed within five minutes after the addition of three different stimuli (adenosine diphosphate [ADP], epinephrine, and acid-soluble collagen [prepared according to a method
(x), standard deviations (SDs), and P-values
in Patients With Low Tension Glaucoma
ADP, jjmole
controls
33.0 8.1
37 > .1
duplicate.
Aggregation
mm
247 70
Table 3.—Serotonin Release, Platelet Factor 3 Availability, and Antiheparin Activity in Plasma in Patients With Low Tension Glaucoma (LTG)* PF 3
Release of "C-Serotonin (% of Total) 3
Controls
SD
LTG SD
patients (12)
Availability (% of Total)
ADP,
Epinephrine, 3 fimole
Collagen, 25 ;g/ml
21.7 19.8 27.1 20.7
21.0 21.7 37.3 23.4 > .05
24.0 16.5
mole
>
.1
Intact PRP
0.05 0.04 0.05 0.05
34.0 19.9
After Collagen, 25 jig/ml 1.11 1.29 0.94 0.83
Antiheparin Activity In Plasma, Units/ml 2.84 0.2 2.69 0.4
*The means (x) and standard deviations (SDs) of single (MC'-serotonin release, PF3 availability) or duplicate (antiheparin activity) observations and the Pvalues (Student t test) are shown.
Table 4.—Measurements of Blood Coagulability and Fibrinolytic Activator Activity in Patients With Low Tension Glaucoma (LTG) aPTT,
Fibrinogen, mg/100 ml .„„,.,„, Controls (12)
LTG
x
Antithrombin Ill-Activity, % of Normal
25~7
Ï06
Fibrinolytic Activator Activity (Fibrin Plate Lysis sq mm)
Í42
sp_33_^_^_^_ 23.9 231 x
patients (12)
271
Sec
107
250
SD_68_2J3_17_124_ > .2 > .2 .1 > .05 >
'The
means
(x)
and standard deviations
(SDs)
of
duplicate
observations and the P-values
(Student t test)
are
Downloaded From: http://archopht.jamanetwork.com/ by a University of California - San Diego User on 06/05/2015
shown.
described elsewhere']) was measured in chart paper units and the results were
expressed as percentage change in optical density with the difference between PRP and platelet-free plasma (PFP) taken as 100%. Serotonin release induced by colla¬ gen
was
measured in connection with the
aggregation experiments after labeling of the platelets by incubation of PRP with 5hydroxy [side chain-2l4C] tryptamine cre¬ atinine sulfate (50 mCi/m M, 0.03 pCi/ml PRP) for 15 minutes
at
room
temperature
described in detail elsewhere." Platelet factor 3 availability in intact PRP and in PRP, stirred in the aggregometer for five minutes after addition of collagen, was examined with a Stypven clotting time assay as previously described." The results for both serotonin release and platelet factor 3 availability were expressed as a percentage of that measured in a sample of sonicated PRP. Measurements of blood coagulation included determination of fibrinogen," the activated partial thrombo¬ plastin time (aPTT),'1 plasma antithrombin III,'" and plasma antiheparin (platelet factor 4) activity. The latter test was a modification of the method previously described" and consisted of the following: 0.2 ml of PRP containing heparin sodium was mixed with 0.1 ml of various dilutions (TRIS-saline) of protamine sulfate (PS) (100 ug/ml). Thrombin (40 units/ml, 0.05M TRIS-saline) was added to this mixture and the clotting time was recorded. The concentration of heparin (0.2 to 0.4 units/ ml) in the substrate PFP was adjusted so that the clotting times with the various concentrations of PS ranged between 10 and 60 seconds. The test was then repeated with platelet-free test plasma (defibrinated by incubation at 56 C for 15 minutes and centrifugation at 15,000 g for ten minutes), replacing PS, and the clotting time obtained was expressed in units per 1 Mg PS) read from the milliliter (1 unit PS dilution curve. Fibrinolytic activator activity after venous occlusion (80 mm Hg, for ten minutes) was determined by measuring the lysis area in square milli¬ meters produced by 0.03-ml aliquots of plasma euglobulin precipitate redissolved in saline on untreated bovine fibrin plates1during 18 hours of incubation at 37 C. All of the measurements of blood coagulability and fibrinolysis were performed in dupli¬ cate (using PPP, immediately frozen and stored at -70 C until testing unless indi¬ cated otherwise), and the results were as
=
averaged.
RESULTS There was no apparent difference in the bleeding time, whole blood platelet count, or platelet adhesive¬ ness between the patient and control group (Table 1). Measurements of platelet aggregation induced by serial concentrations of ADP, epinephrine, and collagen tended to be higher in patients with LTG but none of the apparent differences were statisti¬ cally significant (Table 2). Serotonin release from T4C-labeled platelets in¬ duced by ADP, collagen, or epineph¬ rine was not significantly different in the two groups (Table 3). Platelet factor 3 availability, both in intact PRP and after exposure of PRP to collagen, was also similar and no difference was observed between pa¬ tients and control subjects with
respect
to
measurements.
COMMENT
The data obtained in this study to indicate that patients with LTG cannot be readily distinguished from individuals not affected with this disorder by a variety of specific measurements of platelet function in vivo and in vitro, blood coagulability, seem
fibrinolysis. In particular, we were unable to find evidence that would support the concept that patients with LTG have increased platelet adhesive¬ ness or diminished fibrinolytic activi¬ ty as suggested by Drance.4 In fact, patients with LTG tended to have greater fibrinolytic activity in re¬ sponse to venous occlusion than the control subjects, although the differ¬ or
statistically significant. patients studied was relatively small, the patients examined were comparative-
Although
This investigation was supported in part by grant EY 00336 from the National Eye Insti¬ tute.
S.
Ogden provided technical
plasma antiheparin (pla¬
telet factor 4) activity (Table 3). The results of measurements of plasma fibrinogen, aPTT, antithrombin III activity, and fibrinolytic activator activity after venous occlusion are shown in Table 4. No differences between patient and control groups could be shown for any of these
ence was
free of any other major clinical conditions or factors known or sus¬ pected to affect platelet function, blood coagulability, or fibrinolysis, and they were compared with a group of control subjects well matched for age, sex, race, and associated clinical disease. Thus, it seems reasonable to conclude from the data presented here that LTG is not commonly associated with a "hypercoagulable state" and that factors such as increased platelet function, blood hypercoagulability, or defective fibrinolysis are unlikely to be of major importance in the patho¬ genesis of this disorder.
ly
not
the number of
assistance.
References 1. Drance SM: Studies of factors involved in the production of low tension glaucoma. Arch Ophthalmol 89:457-465, 1973. 2. Drance SM: Some factors in the production of low tension glaucoma. Br J Ophthalmol 56:229\x=req-\ 242, 1972. 3. O'Brien JR, Jamieson S, Etherington M, et al: Stressed template bleeding-time and other platelet function tests in myocardial infarction. Lancet 1:694-696, 1973. 4. Brittin GF, Dew S, Fewell EK: Automated optical counting of blood platelets. Blood 38:442\x=req-\ 430, 1971. 5. Mustard JF, Hegardt B, Rowsell HC, et al: Effect of adenosine nucleotides on platelet aggregation and clotting time. J Lab Clin Med 64:548-559, 1964. 6. Joist JH, Dolezel G, Lloyd JV, et al: Platelet factor 3 availability and the platelet release reaction. J Lab Clin Med 84:474-482, 1974. 7. Cazenave J-P, Packham MA, Mustard JF: Adherence of platelets to a collagen coated surface: Development of a quantitative method. J Lab Clin Med 82:978-990, 1973. 8. Claus A: Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 17:237-246, 1957. 9. Proctor RR, Rapaport SI: The patial thromboplastin time with kaolin: A simple screening test for first stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212-219, 1961. 10. Howie PW, Prentice CRM, McNicol GP: A method of antithrombin estimation using plasma defibrinated with Ancrod. Br J Haematol 25:101\x=req-\ 110, 1973. 11. Nath N, Niewiarowski S, Joist JH: Platelet factor 4-antiheparin protein releasable from platelets: Purification and properties. J Lab Clin Med 82:754-768, 1973. 12. Astrup T, M\l=u"\llertzS: The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346-352, 1952.
Downloaded From: http://archopht.jamanetwork.com/ by a University of California - San Diego User on 06/05/2015
Joist, MD; Philip Lichtenfeld; Alan
\s=b\ Platelet function in vitro and in vivo and certain measurements of blood coagulability and fibrinolysis were examined in 12 patients with low tension glaucoma and the results were compared with those obtained in 12 normal subjects matched for age, race, and sex. The data obtained seem to indicate that patients with low tension glaucoma cannot be readily distinguished from individuals of similar age and sex not afflicted with this disorder by measuring these variables. Thus, a "hypercoagulable state," ie, a thrombotic tendency, does not appear to be of major importance in the pathogenesis of this disorder.
(Arch Ophthalmol 94:1893-1895, 1976) tension
glaucoma (LTG) is
Lowcomplex disorder whose
Mandell, MD; Allan
E. Kolker MD
based on the finding of either increased platelet adhesiveness and/
was
or an
abnormally prolonged euglobu-
lin lysis time in 16 of 26 patients with LTG. However, in this study', the group of individuals from which the control data for these tests were obtained was not described. In view of the potential therapeutic implications, we examined certain measurements of platelet function in vitro and vivo, blood coagulation, and fibrinolysis in a group of patients with LTG and in a group of appar¬ ently healthy individuals closely matched for age and sex. MATERIALS AND METHODS
a
cause
and pathogenesis are still poorly understood. It has been suggested recently by Drance' that this disor¬ der may be due to an ischemie process of the optic nerve head and that "hypercoagulability," ie a thrombotic ten¬ dency, should be considered among the factors involved in the pathogen¬ esis of this condition. This hypothesis -'
Accepted
I.
for publication April 9, 1976. From the Departments of Medicine, Pathology, and Ophthalmology (Glaucoma Center) Washington University School of Medicine, St Louis. Dr Mandell is now with the Department of Ophthalmology, University of Tennessee, College of Medicine, Memphis. Reprint requests to Hemostasis and Thrombosis Laboratory, Division of Laboratory Medicine, Washington University School of Medicine, Barnes Hospital, St Louis, MO 63110 (Dr Joist).
Twelve and four age
was
patients with LTG (eight women men) were studied. Their mean 66 (range 51 to 81) years. These
individuals had nerve fiber bundle defects on the Goldmann perimeter and character¬ istic glaucomatous cupping observed on repeated examinations. In all cases aver¬ age intraocular pressures over many months were less than 20 mm Hg and no pressure on diurnal testing was over 24 mm Hg. The control group consisted of 12 individuals (eight women and four men) with a mean age of 68 (range 58 to 78) years, who had normal-appearing optic nerves and normal Goldmann visual fields. Patients with LTG and controls were phakic, had no history or signs of uveitis or other ocular disease, and had open angles. None of the patients or controls had elevated fasting blood glucose concentra¬ tions or hyperuricemia. One patient with LTG and two control subjects had mildly
elevated
serum
cholesterol concentrations,
patients with LTG and two control subjects had mildly elevated serum triglyc¬ éride concentrations. Two patients with LTG and one control subject had previous evidence of cardiac insufficiency, which was well controlled by digitilization. None of the LTG patients or control subjects had had signs or symptoms of cerebrovascular or peripheral arterial insufficiency. None
two
of the individuals tested showed clinical or laboratory evidence of hepatic or renal disease. A careful drug history was ob¬ tained from all patients and control subjects studied, and they were instructed not to take aspirin-containing medications for at least seven days prior to testing, and to avoid other medications known to affect platelet function. A list of such agents was given to each individual studied. Informed consent was obtained from all individuals tested. Blood was collected from all individuals after a ten-hour fasting period by clean venipuncture of an antecubital vein, using a double syringe technique as follows: 5 ml into a glass tube containing 1% ethylenediaminetetraacetic acid (EDTA) for deter¬ mination of the whole blood platelet count; 10 ml into a plastic syringe containing no anticoagulant for estimation of platelet adhesiveness; 40 ml into a plastic syringe containing 0.1 volume of 3.8% sodium citrate for studies of platelet aggregation, serotonin release, platelet factor 3 avail¬ ability, plasma fibrinogen, activated par¬ tial thromboplastin time, antithrombin III, and antiheparin activity; 5 ml into a plastic syringe after ten minutes of venous occlu¬ sion of the forearm (blood pressure cuff inflated to 80 mm Hg) for estimation of fibrinolytic activator activity.
Downloaded From: http://archopht.jamanetwork.com/ by a University of California - San Diego User on 06/05/2015
O'Brien et al.1 Native
The stressed template bleeding time from duplicate 9-mm long and 1-mm deep cuts on the forearm was measured with the blood pressure cuff inflated to 80 mm Hg as described by O'Brien et al.' Platelet counting was performed in whole blood or platelet-rich plasma by a semiautomated method.4 Platelet adhesiveness was esti¬ mated by a standard glass bead column re¬ tention method similar to that described by
(nonanticoagulated)
blood issuing from the column between 0 and 10, 10 and 20, and 20 and 30 seconds was collected into separate plastic tubes containing 1% dry EDTA. The pump was then stopped, the glass bead column discon¬ nected from the syringe and replaced by a new glass bead column, and the test was immediately repeated. The duplicate pro¬ cedure was completed in less than two minutes. The platelet counts in the blood before passage and in each of the subsamples after passage of the column were determined, and the percentage platelet retention by the glass-beads column was calculated for each subsample. The results of the duplicate tests were averaged. Platelet aggregation was studied in
passed, immediately after collec¬ tion, into a plastic 10-ml syringe, through a piece of polyvinyl tubing (13 cm long, internal diameter 0.3 mm) containing 1.5 gm of glass beads at a constant rate (transit time 8 seconds), using an infusion pump. At the time the first drop of blood emerged from the end of the glass bead column, a stopwatch was started and the blood
was
Table 1.—Bleeding Time, Platelet Count, and Platelet Adhesiveness in Patients With Low Tension Glaucoma (LTG)* Platelet
Adhesiveness, %
min
Bleeding Time, Controls (12)
SD SD
"All tests were performed in (Student f test) are shown.
Table 2.—Platelet
1,000/cu
10.4 5.3 8.7 4.2
LTG
patients (12)
Platelet Count,
The
means
(i¿)
SD
LTG
X
patients (12) "The are
means
given.
(x)
SD
58.2 13.1
74.1 11.2
238
34.7 16.0
58.5 22.0 > .1
17.2
>
.1
Epinephrine, jimole
28.1 2&5
49.2 2g^
54.0 2&
34.5 28.1 > .1
53.2 25.4 > .1
60.4 23.3 > .1
and standard deviations
(SDs)
47.4
2g£ 49.1 27.9 > .1 of
citrated, platelet-rich plasma (PRP) by
73.7 >
.1
(LTG)*
Collagen, ng/ml
49.9 3Q^
52.3 2ߣ
6.1 28.4 34^3
8.3 39.4 34^g
60.4 17.6 > .1
65.7 15.8 > .1
38.8 28.3 > .1
24.5 > .1
12.5 25 56.2 64.9 2g^ 2a3 60.7 75.1 23.4 14.3 > .1 > .1
values
(Student ( test)
single
observations and
45.3
a
turbidimetric technique,'' using a dualchannel aggregometer and recorder as previously described," except type 0.312 cuvettes, 0.45 ml of PRP, and 0.05 ml of stimulus were used. Platelet-rich plasma was prepared by centrifugation of blood at 180 g for ten minutes at room temperature, stored at room temperature, and prewarmed to 37 C for four minutes before testing. The platelet count was adjusted to 250,000/cu mm with platelet poor plasma (PPP) prepared by centrifugation of the remaining blood (after separation of PRP) at 5,000 g for ten minutes. The maximum height of the aggregation tracing observed within five minutes after the addition of three different stimuli (adenosine diphosphate [ADP], epinephrine, and acid-soluble collagen [prepared according to a method
(x), standard deviations (SDs), and P-values
in Patients With Low Tension Glaucoma
ADP, jjmole
controls
33.0 8.1
37 > .1
duplicate.
Aggregation
mm
247 70
Table 3.—Serotonin Release, Platelet Factor 3 Availability, and Antiheparin Activity in Plasma in Patients With Low Tension Glaucoma (LTG)* PF 3
Release of "C-Serotonin (% of Total) 3
Controls
SD
LTG SD
patients (12)
Availability (% of Total)
ADP,
Epinephrine, 3 fimole
Collagen, 25 ;g/ml
21.7 19.8 27.1 20.7
21.0 21.7 37.3 23.4 > .05
24.0 16.5
mole
>
.1
Intact PRP
0.05 0.04 0.05 0.05
34.0 19.9
After Collagen, 25 jig/ml 1.11 1.29 0.94 0.83
Antiheparin Activity In Plasma, Units/ml 2.84 0.2 2.69 0.4
*The means (x) and standard deviations (SDs) of single (MC'-serotonin release, PF3 availability) or duplicate (antiheparin activity) observations and the Pvalues (Student t test) are shown.
Table 4.—Measurements of Blood Coagulability and Fibrinolytic Activator Activity in Patients With Low Tension Glaucoma (LTG) aPTT,
Fibrinogen, mg/100 ml .„„,.,„, Controls (12)
LTG
x
Antithrombin Ill-Activity, % of Normal
25~7
Ï06
Fibrinolytic Activator Activity (Fibrin Plate Lysis sq mm)
Í42
sp_33_^_^_^_ 23.9 231 x
patients (12)
271
Sec
107
250
SD_68_2J3_17_124_ > .2 > .2 .1 > .05 >
'The
means
(x)
and standard deviations
(SDs)
of
duplicate
observations and the P-values
(Student t test)
are
Downloaded From: http://archopht.jamanetwork.com/ by a University of California - San Diego User on 06/05/2015
shown.
described elsewhere']) was measured in chart paper units and the results were
expressed as percentage change in optical density with the difference between PRP and platelet-free plasma (PFP) taken as 100%. Serotonin release induced by colla¬ gen
was
measured in connection with the
aggregation experiments after labeling of the platelets by incubation of PRP with 5hydroxy [side chain-2l4C] tryptamine cre¬ atinine sulfate (50 mCi/m M, 0.03 pCi/ml PRP) for 15 minutes
at
room
temperature
described in detail elsewhere." Platelet factor 3 availability in intact PRP and in PRP, stirred in the aggregometer for five minutes after addition of collagen, was examined with a Stypven clotting time assay as previously described." The results for both serotonin release and platelet factor 3 availability were expressed as a percentage of that measured in a sample of sonicated PRP. Measurements of blood coagulation included determination of fibrinogen," the activated partial thrombo¬ plastin time (aPTT),'1 plasma antithrombin III,'" and plasma antiheparin (platelet factor 4) activity. The latter test was a modification of the method previously described" and consisted of the following: 0.2 ml of PRP containing heparin sodium was mixed with 0.1 ml of various dilutions (TRIS-saline) of protamine sulfate (PS) (100 ug/ml). Thrombin (40 units/ml, 0.05M TRIS-saline) was added to this mixture and the clotting time was recorded. The concentration of heparin (0.2 to 0.4 units/ ml) in the substrate PFP was adjusted so that the clotting times with the various concentrations of PS ranged between 10 and 60 seconds. The test was then repeated with platelet-free test plasma (defibrinated by incubation at 56 C for 15 minutes and centrifugation at 15,000 g for ten minutes), replacing PS, and the clotting time obtained was expressed in units per 1 Mg PS) read from the milliliter (1 unit PS dilution curve. Fibrinolytic activator activity after venous occlusion (80 mm Hg, for ten minutes) was determined by measuring the lysis area in square milli¬ meters produced by 0.03-ml aliquots of plasma euglobulin precipitate redissolved in saline on untreated bovine fibrin plates1during 18 hours of incubation at 37 C. All of the measurements of blood coagulability and fibrinolysis were performed in dupli¬ cate (using PPP, immediately frozen and stored at -70 C until testing unless indi¬ cated otherwise), and the results were as
=
averaged.
RESULTS There was no apparent difference in the bleeding time, whole blood platelet count, or platelet adhesive¬ ness between the patient and control group (Table 1). Measurements of platelet aggregation induced by serial concentrations of ADP, epinephrine, and collagen tended to be higher in patients with LTG but none of the apparent differences were statisti¬ cally significant (Table 2). Serotonin release from T4C-labeled platelets in¬ duced by ADP, collagen, or epineph¬ rine was not significantly different in the two groups (Table 3). Platelet factor 3 availability, both in intact PRP and after exposure of PRP to collagen, was also similar and no difference was observed between pa¬ tients and control subjects with
respect
to
measurements.
COMMENT
The data obtained in this study to indicate that patients with LTG cannot be readily distinguished from individuals not affected with this disorder by a variety of specific measurements of platelet function in vivo and in vitro, blood coagulability, seem
fibrinolysis. In particular, we were unable to find evidence that would support the concept that patients with LTG have increased platelet adhesive¬ ness or diminished fibrinolytic activi¬ ty as suggested by Drance.4 In fact, patients with LTG tended to have greater fibrinolytic activity in re¬ sponse to venous occlusion than the control subjects, although the differ¬ or
statistically significant. patients studied was relatively small, the patients examined were comparative-
Although
This investigation was supported in part by grant EY 00336 from the National Eye Insti¬ tute.
S.
Ogden provided technical
plasma antiheparin (pla¬
telet factor 4) activity (Table 3). The results of measurements of plasma fibrinogen, aPTT, antithrombin III activity, and fibrinolytic activator activity after venous occlusion are shown in Table 4. No differences between patient and control groups could be shown for any of these
ence was
free of any other major clinical conditions or factors known or sus¬ pected to affect platelet function, blood coagulability, or fibrinolysis, and they were compared with a group of control subjects well matched for age, sex, race, and associated clinical disease. Thus, it seems reasonable to conclude from the data presented here that LTG is not commonly associated with a "hypercoagulable state" and that factors such as increased platelet function, blood hypercoagulability, or defective fibrinolysis are unlikely to be of major importance in the patho¬ genesis of this disorder.
ly
not
the number of
assistance.
References 1. Drance SM: Studies of factors involved in the production of low tension glaucoma. Arch Ophthalmol 89:457-465, 1973. 2. Drance SM: Some factors in the production of low tension glaucoma. Br J Ophthalmol 56:229\x=req-\ 242, 1972. 3. O'Brien JR, Jamieson S, Etherington M, et al: Stressed template bleeding-time and other platelet function tests in myocardial infarction. Lancet 1:694-696, 1973. 4. Brittin GF, Dew S, Fewell EK: Automated optical counting of blood platelets. Blood 38:442\x=req-\ 430, 1971. 5. Mustard JF, Hegardt B, Rowsell HC, et al: Effect of adenosine nucleotides on platelet aggregation and clotting time. J Lab Clin Med 64:548-559, 1964. 6. Joist JH, Dolezel G, Lloyd JV, et al: Platelet factor 3 availability and the platelet release reaction. J Lab Clin Med 84:474-482, 1974. 7. Cazenave J-P, Packham MA, Mustard JF: Adherence of platelets to a collagen coated surface: Development of a quantitative method. J Lab Clin Med 82:978-990, 1973. 8. Claus A: Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 17:237-246, 1957. 9. Proctor RR, Rapaport SI: The patial thromboplastin time with kaolin: A simple screening test for first stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212-219, 1961. 10. Howie PW, Prentice CRM, McNicol GP: A method of antithrombin estimation using plasma defibrinated with Ancrod. Br J Haematol 25:101\x=req-\ 110, 1973. 11. Nath N, Niewiarowski S, Joist JH: Platelet factor 4-antiheparin protein releasable from platelets: Purification and properties. J Lab Clin Med 82:754-768, 1973. 12. Astrup T, M\l=u"\llertzS: The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346-352, 1952.
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