Correlation of Platelet Aggregation, Plasma Factor Activity, and Megathrombocytes in Diabetic Subjects With and Without Vascular Disease John A. Colwell,
Julius Sagel,
Lynn Crook, Arthur
Second-phase platelet aggregation induced by adenosine diphosphate (ADP) and epinephrine was measured in fasting platelet-rich plasma in normals, “prediabetics,” and diabetics with or without vascular disease. “Plasma factor” potentiation of ADP-induced second-phase platelet aggregation was also estimated, as were megathrombocyte numbers in the same patient groups. There was an increased sensitivity of second-phase platelet aggregation noted with both aggregating agents in all diabetic groups except for the prediabetics. This activity was
Chambers,
and Marta
Laimins
paralleled by an increase in plasma factor activity. In vivo evidence of an increased turnover of platelets in fmnk diabetics was suggested by increased numbers of megathrombocytes. These studies demonstrate that platelets from diabetics are sensitive to aggregating agents and that this sensitivity may be related to plasma factor(s) present in diabetics. In vivo platelet aggregation may be present in diabetics. Longitudinal studies will be necessary to establish the relationship of these findings to the genesis of diabetic vascular disease.
P
LATELETS FROM DIABETIC SUBJECTS display an increased sensitivity to platelet aggregating agents, according to mounting evidence. Heath et al. showed that patients with deteriorating retinopathy had increased sensitivity to adenosine diphosphate (ADP)-induced platelet aggregation.’ Bensoussan et al. found similar results in a group of diabetics with retinopathy.2 O’Malley et al. showed that there was an increased sensitivity of platelets from diabetics with peripheral neuropathy to ADP- and epinephrine-induced aggregation.3 Leone et al. showed that diabetics with retinopathy, peripheral vascular thrombosis, or abnormal ECG patterns had increased platelet aggregation to ADP.4 A similar finding in some diabetics without apparent vascular disease was also reported by this group, where 10 of 39 diabetics of this type showed irreversible second-phase aggregation to 0.5 PM ADP, as compared to only 2 of 35 control subjects. Finally, Sage1 et al. have recently reported an abnormal sensitivity to aggregating agents in latent diabetics and in diabetics without vascular disease.5 Controversy exists regarding the role of “plasma factors” in this abnormal response of platelets from diabetic subjects. Kwaan et al. have previously reported that plasma factor activity is present in diabetic subjects.6 This material potentiates ADP-induced platelet aggregation and results in irreversible secondphase aggregation. This finding has been confirmed by Leone et al.,4 while others could find no evidence for such a plasma factor.2 In all cases, when From the Charleston VA Hospital and the Endocrinology. Metabolism and Nutrition Division, Department of Medicine, Medical Universiiy of South Carolina, Charleston. S.C. Received for publication January 6. 1976. Supported by VA Research Funds. Reprint requests should be addressed to Dr. John A. Colwell. Professor of Medicine, VA Hospiial, 109 Bee St.. Charleston, S.C. 29403. 0 1977 bv Grune & Srrarton. Inc. Metabolism, Vol. 26, No. 3 (March), 1977
279
280
COLWELL ET Al.
present, plasma factor activity has been found to be highest in patients with diabetic angiopathy. 4,6To date, there has been no evidence of increased plasma factor activity in latent diabetics or in those without clinically apparent vascular disease. Furthermore, no papers have correlated plasma factor activity with platelet aggregation in the same groups of diabetic patients. Finally, the in vivo significance of these in vitro findings has remained in question. Although increased platelet turnover’ and a large number of megathrombocytes8s9 have been found in diabetic subjects, there are no studies which relate platelet aggregation, plasma factor activity, and indices of platelet turnover in diabetic subjects. In view of these facts, we felt that it would be important to assess platelet aggregation, plasma factor activity, and platelet turnover in one group of diabetic subjects. In order to acquire some information which might bear on the importance of these abnormalities in the genesis of diabetic vascular disease, we have performed such studies in “prediabetics,” latent diabetics, and diabetic subjects with and without vascular disease. MATERIALS
AND METHODS
Hospitalized male patients with a diagnosis of diabetes mellitus, made by repetitive fasting hyperglycemia (above 130 mg/dl) or by glucose tolerance testing according to the method of Fajans and Conn,‘u were studied. A diagnosis of latent diabetes was made if plasma glucose values exceeded 185 (1 hr), 160 (I$ hr), and 140 mg/dl (2 hr) under age 50. For each decade over age 50, 10 mg/dl was added to each figure. A diagnosis of “prediabetes” was made if the subject had a normal glucose tolerance and both parents were unequivocal diabetics. All medications. except for insulin or oral hypoglycemic agents, were discontinued for at least I wk. Studies were performed in the morning after a l4-hr fast and a 30-min rest period. Informed Subjects.
consent was routinely obtained. Peripheral vascular disease was diagnosed when there had been either gangrene or amputation of a lower extremity for gangrene. All patients in the retinopathy-nephropathy group had retinal findings compatible with diabetic retinopathy and unequivocal proteinuria. Absence of these findings, plus no historical, physical, or electrocardiographic evidence for any form of diabetic vascular disease, was necessary for classification in the group with no apparent vascular disease (NAVD).
Normal
subjects
were drawn
equally
from hospitalized
patients
without
glucose
intoler-
ance and from ambulatory volunteers. Plateler sfudies. Blood was drawn in the laboratory, without stasis, using 3.8”,, sodium citrate (1:9), using plastic syringes and a two-syringe technique. All glassware coming in contact with the blood was siliconized. Platelet-rich plasma (PRP) was obtained by centrifugation at 100 g for 10 min at 26°C. and platelet-poor plasma (PPP) was obtained by centrifugation at 1000 g at 4’C for 20 min. Platelet aggregation was performed as previously described’ in a Chronolog aggregometer at 37°C. stirring at 1000 rpm. Aggregation was induced with ADP or epinephrine. In view of the possibility that platelet aggregation response could change with time, all samples were tested between I and 2 hr after obtaining PRP. The sequence of aggregating agents was kept constant: ADP. 0. 0.5. 1.0, and 2.0 rM: epinephrine, 0. 0.5. 1.0, and 2.0 pM. Concentrations are shown as the final concentrations in PRP. The time lag for determining aggregation response from the time of drawing the blood was the same at each ADP and epinephrine concentration in the normal and diabetic groups. Plasma factor was determined according to the method of Kwaan et al.6 using titrated PPP. This method estimated the additive affect of diabetic or normal plasma on second-phase platelet aggregation of platelets from a second nondiabetic donor after adding an amount of ADP that was just below the threshold of the amount for second-phase platelet aggregation. The final ratio of diabetic or normal plasma to nondiabetic plasma was 1:8. In order to exclude the possibility that time from drawing the blood affected the results, plasma factor activity in plasma from the diabetic subject was assayed first, followed within 5 min by assay of plasma factor activity from
PLATELETS, PLASMA FACTOR, AND MEGATHROMBOCYTES
281
the normal donor. All of these studies were done between 1 and 2 hr after drawing the second nondiabetic donor’s blood. In all cases, the percentage change in optical density was measured comparing PRP and PPP 4 min after adding the aggregating agent. Platelet counts in PRP were 300,00&400,000/cu mm. Platelet sizing was done according to the method of Karpatkin,s*’ using the EDTA smear method. Biochemical studies. Plasma concentrations of glucose, triglycerides, and cholesterol were determined using AutoAnalyzer techniques6 RESULTS
Clinical characteristics of the normals and the six groups of diabetics studied are given in Table 1. It can be seen that the normal and prediabetic groups were younger than the diabetic subjects. There was no significant height difference in any group when compared to normals. In both the prediabetics and the diabetics without clinical evidence of vascular disease, mean weights exceeded those of the normal group. Known duration of diabetes was longest in the diabetics with peripheral vascular disease and retinopathy-nephropathy. Results of fasting plasma concentrations of glucose, triglycerides, and cholesterol are given in Table 1. Mean fasting plasma glucose levels were higher than normal in the diabetics without clinical evidence of vascular disease and in those with peripheral vascular disease or retinopathy-nephropathy. Increased fasting triglyceride levels were seen only in diabetics without clinical evidence of vascular disease, and in those with retinopathy-nephropathy. No increased levels of plasma cholesterol were seen in any diabetic group when compared to normals. Although fewer patients without vascular disease were on insulin, no effect of therapy on platelet aggregation with diet, oral hypoglycemic agents, or insulin could be found within any diabetic group or for the disease group as a whole. Platelet aggregation results are given in Fig. 1. Increased sensitivity to epinephrine and ADP is apparent in all the diabetic groups, except for the prediabetics, when compared to normals. As is also shown in Fig. 1, all diabetic groups, except for prediabetics, had increased plasma factor activity. There was Table 1. Clinical Chorocteristics
ond loberotory Values of Subjects Diabetes Mellitur
Normals
Variable
Prediobetic
Latent
NAVD
Peripheral VosculorDisease
N
25
10
13
26
15
13
Age (Yr)
40 (3)
34 (3)
58 (3)*
61 (7)*
49 (3)t
wt (kg) Duration of
75 (2)
89 (6)*
73 (4)
47 (2)t 86 (4)*
77 (3)
80 (4)
4 (1)
‘5 (2) 84
diabetes mellitus (yr) Insulin Rx (%)
38
‘3 (2) 80
Oral Rx (%)
24
13
Diet Rx (%)
38
13
Triglycerides (mg/dl)
98 (2) 109 (17)
Cholesterol (mg/dl)
203 (10)
Gluco~ (mg/dl)
V&es
lp
shown ore meon (SEM).
< 0.01 versus normals.
tp < 0.05 versus normals.
83 (3)
112 (9)
138 (lo)*
132 (18) 198 (16)
98 (7) 207 (30)
8 8
170 (15)*
251 (36)’
191 (32)’
127 (13)
243 (41)*
216 (8)
202 (22)
222 (22)
COLWELL ET Al.
282
(-Jo.5
* P
Julius Sagel,
Lynn Crook, Arthur
Second-phase platelet aggregation induced by adenosine diphosphate (ADP) and epinephrine was measured in fasting platelet-rich plasma in normals, “prediabetics,” and diabetics with or without vascular disease. “Plasma factor” potentiation of ADP-induced second-phase platelet aggregation was also estimated, as were megathrombocyte numbers in the same patient groups. There was an increased sensitivity of second-phase platelet aggregation noted with both aggregating agents in all diabetic groups except for the prediabetics. This activity was
Chambers,
and Marta
Laimins
paralleled by an increase in plasma factor activity. In vivo evidence of an increased turnover of platelets in fmnk diabetics was suggested by increased numbers of megathrombocytes. These studies demonstrate that platelets from diabetics are sensitive to aggregating agents and that this sensitivity may be related to plasma factor(s) present in diabetics. In vivo platelet aggregation may be present in diabetics. Longitudinal studies will be necessary to establish the relationship of these findings to the genesis of diabetic vascular disease.
P
LATELETS FROM DIABETIC SUBJECTS display an increased sensitivity to platelet aggregating agents, according to mounting evidence. Heath et al. showed that patients with deteriorating retinopathy had increased sensitivity to adenosine diphosphate (ADP)-induced platelet aggregation.’ Bensoussan et al. found similar results in a group of diabetics with retinopathy.2 O’Malley et al. showed that there was an increased sensitivity of platelets from diabetics with peripheral neuropathy to ADP- and epinephrine-induced aggregation.3 Leone et al. showed that diabetics with retinopathy, peripheral vascular thrombosis, or abnormal ECG patterns had increased platelet aggregation to ADP.4 A similar finding in some diabetics without apparent vascular disease was also reported by this group, where 10 of 39 diabetics of this type showed irreversible second-phase aggregation to 0.5 PM ADP, as compared to only 2 of 35 control subjects. Finally, Sage1 et al. have recently reported an abnormal sensitivity to aggregating agents in latent diabetics and in diabetics without vascular disease.5 Controversy exists regarding the role of “plasma factors” in this abnormal response of platelets from diabetic subjects. Kwaan et al. have previously reported that plasma factor activity is present in diabetic subjects.6 This material potentiates ADP-induced platelet aggregation and results in irreversible secondphase aggregation. This finding has been confirmed by Leone et al.,4 while others could find no evidence for such a plasma factor.2 In all cases, when From the Charleston VA Hospital and the Endocrinology. Metabolism and Nutrition Division, Department of Medicine, Medical Universiiy of South Carolina, Charleston. S.C. Received for publication January 6. 1976. Supported by VA Research Funds. Reprint requests should be addressed to Dr. John A. Colwell. Professor of Medicine, VA Hospiial, 109 Bee St.. Charleston, S.C. 29403. 0 1977 bv Grune & Srrarton. Inc. Metabolism, Vol. 26, No. 3 (March), 1977
279
280
COLWELL ET Al.
present, plasma factor activity has been found to be highest in patients with diabetic angiopathy. 4,6To date, there has been no evidence of increased plasma factor activity in latent diabetics or in those without clinically apparent vascular disease. Furthermore, no papers have correlated plasma factor activity with platelet aggregation in the same groups of diabetic patients. Finally, the in vivo significance of these in vitro findings has remained in question. Although increased platelet turnover’ and a large number of megathrombocytes8s9 have been found in diabetic subjects, there are no studies which relate platelet aggregation, plasma factor activity, and indices of platelet turnover in diabetic subjects. In view of these facts, we felt that it would be important to assess platelet aggregation, plasma factor activity, and platelet turnover in one group of diabetic subjects. In order to acquire some information which might bear on the importance of these abnormalities in the genesis of diabetic vascular disease, we have performed such studies in “prediabetics,” latent diabetics, and diabetic subjects with and without vascular disease. MATERIALS
AND METHODS
Hospitalized male patients with a diagnosis of diabetes mellitus, made by repetitive fasting hyperglycemia (above 130 mg/dl) or by glucose tolerance testing according to the method of Fajans and Conn,‘u were studied. A diagnosis of latent diabetes was made if plasma glucose values exceeded 185 (1 hr), 160 (I$ hr), and 140 mg/dl (2 hr) under age 50. For each decade over age 50, 10 mg/dl was added to each figure. A diagnosis of “prediabetes” was made if the subject had a normal glucose tolerance and both parents were unequivocal diabetics. All medications. except for insulin or oral hypoglycemic agents, were discontinued for at least I wk. Studies were performed in the morning after a l4-hr fast and a 30-min rest period. Informed Subjects.
consent was routinely obtained. Peripheral vascular disease was diagnosed when there had been either gangrene or amputation of a lower extremity for gangrene. All patients in the retinopathy-nephropathy group had retinal findings compatible with diabetic retinopathy and unequivocal proteinuria. Absence of these findings, plus no historical, physical, or electrocardiographic evidence for any form of diabetic vascular disease, was necessary for classification in the group with no apparent vascular disease (NAVD).
Normal
subjects
were drawn
equally
from hospitalized
patients
without
glucose
intoler-
ance and from ambulatory volunteers. Plateler sfudies. Blood was drawn in the laboratory, without stasis, using 3.8”,, sodium citrate (1:9), using plastic syringes and a two-syringe technique. All glassware coming in contact with the blood was siliconized. Platelet-rich plasma (PRP) was obtained by centrifugation at 100 g for 10 min at 26°C. and platelet-poor plasma (PPP) was obtained by centrifugation at 1000 g at 4’C for 20 min. Platelet aggregation was performed as previously described’ in a Chronolog aggregometer at 37°C. stirring at 1000 rpm. Aggregation was induced with ADP or epinephrine. In view of the possibility that platelet aggregation response could change with time, all samples were tested between I and 2 hr after obtaining PRP. The sequence of aggregating agents was kept constant: ADP. 0. 0.5. 1.0, and 2.0 rM: epinephrine, 0. 0.5. 1.0, and 2.0 pM. Concentrations are shown as the final concentrations in PRP. The time lag for determining aggregation response from the time of drawing the blood was the same at each ADP and epinephrine concentration in the normal and diabetic groups. Plasma factor was determined according to the method of Kwaan et al.6 using titrated PPP. This method estimated the additive affect of diabetic or normal plasma on second-phase platelet aggregation of platelets from a second nondiabetic donor after adding an amount of ADP that was just below the threshold of the amount for second-phase platelet aggregation. The final ratio of diabetic or normal plasma to nondiabetic plasma was 1:8. In order to exclude the possibility that time from drawing the blood affected the results, plasma factor activity in plasma from the diabetic subject was assayed first, followed within 5 min by assay of plasma factor activity from
PLATELETS, PLASMA FACTOR, AND MEGATHROMBOCYTES
281
the normal donor. All of these studies were done between 1 and 2 hr after drawing the second nondiabetic donor’s blood. In all cases, the percentage change in optical density was measured comparing PRP and PPP 4 min after adding the aggregating agent. Platelet counts in PRP were 300,00&400,000/cu mm. Platelet sizing was done according to the method of Karpatkin,s*’ using the EDTA smear method. Biochemical studies. Plasma concentrations of glucose, triglycerides, and cholesterol were determined using AutoAnalyzer techniques6 RESULTS
Clinical characteristics of the normals and the six groups of diabetics studied are given in Table 1. It can be seen that the normal and prediabetic groups were younger than the diabetic subjects. There was no significant height difference in any group when compared to normals. In both the prediabetics and the diabetics without clinical evidence of vascular disease, mean weights exceeded those of the normal group. Known duration of diabetes was longest in the diabetics with peripheral vascular disease and retinopathy-nephropathy. Results of fasting plasma concentrations of glucose, triglycerides, and cholesterol are given in Table 1. Mean fasting plasma glucose levels were higher than normal in the diabetics without clinical evidence of vascular disease and in those with peripheral vascular disease or retinopathy-nephropathy. Increased fasting triglyceride levels were seen only in diabetics without clinical evidence of vascular disease, and in those with retinopathy-nephropathy. No increased levels of plasma cholesterol were seen in any diabetic group when compared to normals. Although fewer patients without vascular disease were on insulin, no effect of therapy on platelet aggregation with diet, oral hypoglycemic agents, or insulin could be found within any diabetic group or for the disease group as a whole. Platelet aggregation results are given in Fig. 1. Increased sensitivity to epinephrine and ADP is apparent in all the diabetic groups, except for the prediabetics, when compared to normals. As is also shown in Fig. 1, all diabetic groups, except for prediabetics, had increased plasma factor activity. There was Table 1. Clinical Chorocteristics
ond loberotory Values of Subjects Diabetes Mellitur
Normals
Variable
Prediobetic
Latent
NAVD
Peripheral VosculorDisease
N
25
10
13
26
15
13
Age (Yr)
40 (3)
34 (3)
58 (3)*
61 (7)*
49 (3)t
wt (kg) Duration of
75 (2)
89 (6)*
73 (4)
47 (2)t 86 (4)*
77 (3)
80 (4)
4 (1)
‘5 (2) 84
diabetes mellitus (yr) Insulin Rx (%)
38
‘3 (2) 80
Oral Rx (%)
24
13
Diet Rx (%)
38
13
Triglycerides (mg/dl)
98 (2) 109 (17)
Cholesterol (mg/dl)
203 (10)
Gluco~ (mg/dl)
V&es
lp
shown ore meon (SEM).
< 0.01 versus normals.
tp < 0.05 versus normals.
83 (3)
112 (9)
138 (lo)*
132 (18) 198 (16)
98 (7) 207 (30)
8 8
170 (15)*
251 (36)’
191 (32)’
127 (13)
243 (41)*
216 (8)
202 (22)
222 (22)
COLWELL ET Al.
282
(-Jo.5
* P
