THROMBOSIS RESEARCH 61; 469-475,199l 0049-3848191 $3.00 + .OO Pn’nted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
COMMUNICATION
BRIEF
THBHYPERSENSITIVITY To TBRONBIN OF PIATELEISFRC@lDIABlFMCRATS INCREASED TBRU’IBIN BINDING ISNDTDUETD P.D. Winocour, D.W. Perry, M.W.C. Hatton, and R.L. Kinlough-Rathbone, Department of Pathology, McMasterUniversity, Hamilton, Ontario, CANADA. (Received
24.9.1990;
accepted
in revised form 13.11 .1990 by Editor V. Gurewich)
Platelets from diabetic humans and animals are hypersensitive to Washed platelets from diabetic aggregating agents, including thrombin (l-5). rats are hypersensitive to thrombin through a mechanism(s) that is independent of effects of released ADP or products of arachidonate metabolism (1,2); similar observations have been made with platelets from diabetic patients with An enhanced response to thrombin of platelets from diabetic retinopathy (6). humans and animals could be related to an alteration in the binding of thrombin to its receptor on platelets or to an alteration in the sensitivity of a pathway(s) involved in platelet activation in response to thrombin. In the present study we have examined whether the enhanced aggregation in response to thrombin of platelets from diabetic rats results from an increase in the amount of thrombin that becomes associated with the platelet membrane. Animals Male BB Wistar rats with spontaneous diabetes weighed 450-500 g; these animals were a gift from Animal Resources Division, Health and Welfare, Canada. The diabetic animls had had glycosuria and had been treated with protamine zinc insulin daily for at least 3 months before the study. The dose of insulin was determined according to the degree of glycosuria and loss of weight, but animals were maintained in a hyperglycaemic state. Weightmatched, normoglycaemic rats from the same diabetic colony (non-diabetic littermates) were used as controls as well as a group of control Wistar rats bred from the mother colony from which the diabetic colony was derived. Preparation of suspensions of washed platelets Blood Was collected from the exposed hearts of rats anaesthetized with ether. The anticoagulant used was the ACD solution of Aster and Jandl (7). Suspensions of washed platelets were prepared from blood pooled from two rats from the same group. Platelets were washed as described previously with KEY VKEIDS:
PLATELETS, THROMBIN, DIAB.BTES, RATS. 469
470
PLATELETS, THROMBIN AND DIABETES
Vol. 61, No. 4
calcium-free Tyrode’s solution (pH 6.5) containing 0.35% bovine ~~$umalbumin For studies of aggregation in response to thrombin and I-thrombin (8). binding, platelets were incubated with acetylsalicylic acid (aspirin; 0.55 mmol/L final concentration) in the first washing solution and resuspended in Tyrode’s solution (pH 7.35) containing 0.35% bovine serum albumin, and creatine phosphate (CP) (4.25 mmol/L)/creatine phosphokinase (CPK) (8.5 U/mL). Platelet counts were determined with a Coulter Counter (Coulter Electronics of Canada Ltd. , Oakville, Ontario, Canada) with a 70 ,umdiameter orifice. The platelet count was adjusted to 5 x 10’ platelets/ml. For determination of platelet protein, cholesterol and phospholipid, the platelets from 1.5 mL of suspension (first washing fluid) were washed twice in phosphate-free, calcium-free Tyrode’s solution (pH 6.5) (5 II&), resuspended in this buffer (1 mL) and the platelet count determined; to prevent the platelets from undergoing secretion in the absence of protein in the medium, the initial washing solution also contained PGE1 (10 ,~oMol/Lfinal concentration). Purification and radio-iodination of thrombin for studies of amount of thrombin bound to platelets Thrombin was purified from crude bovine thrombin (Thrombostat, Parke-Davis Co., Detroit, MI) by an anion-exchange procedure according to a modification (9) of the method of Lundblad et al. (10). The purified thrombin had a specific activity of approximately 2,000 IU/mg and appeared largely (>90%) as a-thrombin (Mr=36 Kd) with a small quantity of (Whrombin (5-lo%, M =28Kd), when analyzed by polyacrylamide gel electrophoresis in the presence ok 0 .l% sodium dodecyl sulphate (11). Reaction of thrombin with an excess of rabbit antithrombin III caused formation of thrombin-antithrombin III complexes by bo,tzhso- and 8-thrombin in the mixture. The thrombin was iodinated with I using Enzymobeads, (Biorad Ltd., Misissauga, Ontario) according to the directions of the supplier. A solution containing approximately 100-200 pugof thrombin was labelled for 30 min at 23”C, centrifuged, and then dialyzed against four changes of 250 mL of Eagles’ Minimal Essential ~#i.um for 16 to 20 hr at 4’C as previously d:scribed (12). Dialyzed I-labelled thrombin was stored in aliquots at1740 C and used within 10 days iodination. The specific radioactivity of I-thrombin was 400 cpm/ng. Pf1 I-albumin ( 20P2$pm/ng; New England Nuclear, Markham,Ontario) was used as a space marker in i2;-thrombin binding experiments. Determination of I-thrombin associ.a$_edwith platelets 125Mixtures of a fixed amount of *JAI-albumin (2 ,ug) and a variable amount I-thrombin (20-200 ng) were made up to 200 ,YLwith Tyrode;35buffer and of I-thrombin added to 1 mL samples of platelet suspension. The amount of associated with the piqfelets was determined using methods described previously for,meaf@ng I-fibrinogen.binding to platelets (13). Platelets were stirred rlth I-thrombin (20-200 ng) at 37°C. Thirty seconds following addition of r ’I-thrombin, the platelet suspension was rapidly centrifuged for 1 min at 12,000 x g in an Eppend;r;;z;r$uL$ld 53%IraErdi;ynv’dt;; platelet pellet was measured. the platelet pellet was calculateidz5using 13’I-albumin as a space marker and I-thrombin associated with the pellet. was subtracted ffyS\ the amount of l$Tthrombin binding was determined frpy5 the difference Specific I-thrombin bound and the non-specific I-thrombin bound between the total measured after the addition of 10,000 ng of unlabelled thrombin simultaneously with labelled thrombin. The non-specific binding of thrombin to rat platelets was not saturable in the concentration range used; with humanplatelets under the same conditions saturation did occur (not shown). Platelet analysis Platelet protein was determined (14) using 40 NL of the platelet suspension (without added albumin). Lipids were extracted from an aliquot of Platelet this platelet suspension (0.5 mL) as previously described (15). cholesterol and phosphorous contents were measured on samples of lipid extract
Vol. 61, No. 4
PLATELETS, THROMBIN AND DIABETES
471
using methods previously described (15,16). (160 and 20 ,uL, respectively) Plasma glucose concentrations Plasma glucose concentrations were determined in blood samples taken under ether anaesthesia from the freshly cut ends of the tails of the rats. Plasma glucose concentrations were measured by the glucose oxidase method (17) with a Kodak Ektachem (Eastman Kodak Co., Rochester, NY). Analysis of data comparisons were Results are expressed as the Mean f S!ZM. Statistical when no statistical done by paired or unpaired t-tests where indicated. significance is shown, results were not significantly different (p>O.O5). RESULTS The mean plasma glucose concentration in rats with spontaneous diabetes higher than in their non-diabetic (418 + 32 mg/dL, n-22) was significantly littermates (169 + 14 mg/dL, n=22, p
COMMUNICATION
BRIEF
THBHYPERSENSITIVITY To TBRONBIN OF PIATELEISFRC@lDIABlFMCRATS INCREASED TBRU’IBIN BINDING ISNDTDUETD P.D. Winocour, D.W. Perry, M.W.C. Hatton, and R.L. Kinlough-Rathbone, Department of Pathology, McMasterUniversity, Hamilton, Ontario, CANADA. (Received
24.9.1990;
accepted
in revised form 13.11 .1990 by Editor V. Gurewich)
Platelets from diabetic humans and animals are hypersensitive to Washed platelets from diabetic aggregating agents, including thrombin (l-5). rats are hypersensitive to thrombin through a mechanism(s) that is independent of effects of released ADP or products of arachidonate metabolism (1,2); similar observations have been made with platelets from diabetic patients with An enhanced response to thrombin of platelets from diabetic retinopathy (6). humans and animals could be related to an alteration in the binding of thrombin to its receptor on platelets or to an alteration in the sensitivity of a pathway(s) involved in platelet activation in response to thrombin. In the present study we have examined whether the enhanced aggregation in response to thrombin of platelets from diabetic rats results from an increase in the amount of thrombin that becomes associated with the platelet membrane. Animals Male BB Wistar rats with spontaneous diabetes weighed 450-500 g; these animals were a gift from Animal Resources Division, Health and Welfare, Canada. The diabetic animls had had glycosuria and had been treated with protamine zinc insulin daily for at least 3 months before the study. The dose of insulin was determined according to the degree of glycosuria and loss of weight, but animals were maintained in a hyperglycaemic state. Weightmatched, normoglycaemic rats from the same diabetic colony (non-diabetic littermates) were used as controls as well as a group of control Wistar rats bred from the mother colony from which the diabetic colony was derived. Preparation of suspensions of washed platelets Blood Was collected from the exposed hearts of rats anaesthetized with ether. The anticoagulant used was the ACD solution of Aster and Jandl (7). Suspensions of washed platelets were prepared from blood pooled from two rats from the same group. Platelets were washed as described previously with KEY VKEIDS:
PLATELETS, THROMBIN, DIAB.BTES, RATS. 469
470
PLATELETS, THROMBIN AND DIABETES
Vol. 61, No. 4
calcium-free Tyrode’s solution (pH 6.5) containing 0.35% bovine ~~$umalbumin For studies of aggregation in response to thrombin and I-thrombin (8). binding, platelets were incubated with acetylsalicylic acid (aspirin; 0.55 mmol/L final concentration) in the first washing solution and resuspended in Tyrode’s solution (pH 7.35) containing 0.35% bovine serum albumin, and creatine phosphate (CP) (4.25 mmol/L)/creatine phosphokinase (CPK) (8.5 U/mL). Platelet counts were determined with a Coulter Counter (Coulter Electronics of Canada Ltd. , Oakville, Ontario, Canada) with a 70 ,umdiameter orifice. The platelet count was adjusted to 5 x 10’ platelets/ml. For determination of platelet protein, cholesterol and phospholipid, the platelets from 1.5 mL of suspension (first washing fluid) were washed twice in phosphate-free, calcium-free Tyrode’s solution (pH 6.5) (5 II&), resuspended in this buffer (1 mL) and the platelet count determined; to prevent the platelets from undergoing secretion in the absence of protein in the medium, the initial washing solution also contained PGE1 (10 ,~oMol/Lfinal concentration). Purification and radio-iodination of thrombin for studies of amount of thrombin bound to platelets Thrombin was purified from crude bovine thrombin (Thrombostat, Parke-Davis Co., Detroit, MI) by an anion-exchange procedure according to a modification (9) of the method of Lundblad et al. (10). The purified thrombin had a specific activity of approximately 2,000 IU/mg and appeared largely (>90%) as a-thrombin (Mr=36 Kd) with a small quantity of (Whrombin (5-lo%, M =28Kd), when analyzed by polyacrylamide gel electrophoresis in the presence ok 0 .l% sodium dodecyl sulphate (11). Reaction of thrombin with an excess of rabbit antithrombin III caused formation of thrombin-antithrombin III complexes by bo,tzhso- and 8-thrombin in the mixture. The thrombin was iodinated with I using Enzymobeads, (Biorad Ltd., Misissauga, Ontario) according to the directions of the supplier. A solution containing approximately 100-200 pugof thrombin was labelled for 30 min at 23”C, centrifuged, and then dialyzed against four changes of 250 mL of Eagles’ Minimal Essential ~#i.um for 16 to 20 hr at 4’C as previously d:scribed (12). Dialyzed I-labelled thrombin was stored in aliquots at1740 C and used within 10 days iodination. The specific radioactivity of I-thrombin was 400 cpm/ng. Pf1 I-albumin ( 20P2$pm/ng; New England Nuclear, Markham,Ontario) was used as a space marker in i2;-thrombin binding experiments. Determination of I-thrombin associ.a$_edwith platelets 125Mixtures of a fixed amount of *JAI-albumin (2 ,ug) and a variable amount I-thrombin (20-200 ng) were made up to 200 ,YLwith Tyrode;35buffer and of I-thrombin added to 1 mL samples of platelet suspension. The amount of associated with the piqfelets was determined using methods described previously for,meaf@ng I-fibrinogen.binding to platelets (13). Platelets were stirred rlth I-thrombin (20-200 ng) at 37°C. Thirty seconds following addition of r ’I-thrombin, the platelet suspension was rapidly centrifuged for 1 min at 12,000 x g in an Eppend;r;;z;r$uL$ld 53%IraErdi;ynv’dt;; platelet pellet was measured. the platelet pellet was calculateidz5using 13’I-albumin as a space marker and I-thrombin associated with the pellet. was subtracted ffyS\ the amount of l$Tthrombin binding was determined frpy5 the difference Specific I-thrombin bound and the non-specific I-thrombin bound between the total measured after the addition of 10,000 ng of unlabelled thrombin simultaneously with labelled thrombin. The non-specific binding of thrombin to rat platelets was not saturable in the concentration range used; with humanplatelets under the same conditions saturation did occur (not shown). Platelet analysis Platelet protein was determined (14) using 40 NL of the platelet suspension (without added albumin). Lipids were extracted from an aliquot of Platelet this platelet suspension (0.5 mL) as previously described (15). cholesterol and phosphorous contents were measured on samples of lipid extract
Vol. 61, No. 4
PLATELETS, THROMBIN AND DIABETES
471
using methods previously described (15,16). (160 and 20 ,uL, respectively) Plasma glucose concentrations Plasma glucose concentrations were determined in blood samples taken under ether anaesthesia from the freshly cut ends of the tails of the rats. Plasma glucose concentrations were measured by the glucose oxidase method (17) with a Kodak Ektachem (Eastman Kodak Co., Rochester, NY). Analysis of data comparisons were Results are expressed as the Mean f S!ZM. Statistical when no statistical done by paired or unpaired t-tests where indicated. significance is shown, results were not significantly different (p>O.O5). RESULTS The mean plasma glucose concentration in rats with spontaneous diabetes higher than in their non-diabetic (418 + 32 mg/dL, n-22) was significantly littermates (169 + 14 mg/dL, n=22, p
