This is a study done many years ago using a unique colony of rhesus monkeys that had been made diabetic using streptozotocin, a drug that is highly toxic to pancreatic islet cells. This colony had been maintained on daily insulin injections for many years thereafter and had been studied as a model of human diabetes to great advantage by the Jonasson group. For the present study, we examined the generation of chemotaxis factors derived from normal and diabetic monkey platelets. The significance of the study related to the long term vascular and related tissue lesions often seen in human diabetes.
Interestingly, this colony had to be discontinued eventually when the considerable funding required for its ongoing maintenance was lost. It seemed like a very valuable resource at the time primarily because animal models for long-term effects of diabetes were rare and a primate model was exceedingly rare. I don't know if such a model even exists at present in view of the great expense of maintaining it and the political incorrectness of using primates for research. However, diabetes marches on despite political correctness and is now a far greater public health problem in the US than it was when this study was performed.
Below is the full paper and a link to the PDF.
Release of Chemotactic Activity by Platelets from Diabetic Monkeys
Agonist-Induced Release of Chemotactic Activity by
Platelets Isolated from Diabetic Rhesus Monkey
Glenn S. Takimoto,1Robert
J. Walter, James D. Jeffery,
Anne F. Bauman and Olga
Jonasson
Department of Surgery
University of Illinois College of Medicine
and
1 Department of Surgery, Cook County
Hospital
Chicago, IL
60612
Send
all correspondence to:
Robert J. Walter, Ph.D.
Department
of Surgery
Hektoen
Institute for Medical Research
627 South Wood St.
Chicago, IL
60612
ABSTRACT
Agonist-induced liberation of chemotactic and
chemokinetic activity from normal rhesus monkey platelets was characterized and
compared to activity liberated from platelets obtained from streptozotocin-treated,
insulin-deficient diabetic (STZ-ID) monkeys. The STZ-ID monkeys were severely
hyperglycemic with minimal residual c-peptide secretion, hyperglucagonemic, and
had elevated glycosylated hemoglobin levels. Fifteen control and 7 STZ-ID
monkeys were studied. Platelet-free supernatants isolated from washed, intact
platelet preparations incubated with agonist were applied to a microchemotaxis
system employing mouse 3T3 fibroblasts as target cells. Using platelets from
control monkeys, dose-dependent increases in aggregation response and
liberation of chemotactic activity were obtained when thrombin and arachidonic
acid were employed as agonists. These responses were attenuated by known
platelet activation inhibitors that increase cAMP levels or mimic its cellular
actions. When control and STZ-ID monkey groups were compared, thrombin-induced
liberation of chemotactic activity was greater with platelets isolated from the
STZ-ID group. Arachidonic acid-induced liberation of chemotactic activity was
similar for both groups. There were also no differences between control and
STZ-ID groups in platelet aggregation responses to either thrombin or
arachidonic acid. Supernatants from platelet suspensions exposed to either thrombin
or arachidonic acid showed markedly stimulated 3T3 cell chemokinesis, but no
differences in agonist-induced liberation of chemokinetic activity were
observed when comparing results from control and STZ-ID groups. In summary, we
have demonstrated using agonist stimulation of intact, washed platelets that
the liberation of chemotactic activity but not aggregation is enhanced with
chronic hyperglycemia.
Key
Words: chemotaxis, chemokinesis, platelet, rhesus monkey, diabetes, platelet
aggregation, 3T3 fibroblasts
INTRODUCTION
Platelets from humans with Type I diabetes
exhibit increased sensitivity to proaggregatory agents [1-4]. This sensitivity is thought to contribute to
the development of macro- and microvascular lesions seen in these patients
[5,6]. It was initially postulated that
the role of platelets in lesion development was mediated principally through
enhanced platelet aggregate formation. However, subsequent studies have
suggested that an alteration in the formation and release of platelet-derived
bioactive agents may also be involved. Many platelet products are liberated during
agonist-induced activation but their precise functions are only partially
understood. Prostaglandin endoperoxides
or thromboxanes and dense granule constituents are known to promote platelet
aggregation [7], but little is known about the function of other granule-stored
constituents or about lipoxygenase enzyme products of arachidonic acid. Recent studies by several investigators
demonstrate that the alpha granule constituents (platelet-derived growth factor
(PDGF), β-thromboglobulin, platelet factor 4) and
12-L-hydroxy-5,8,10,14-eicosatetraenoic acid promote migration of a variety of
cell types including vascular smooth muscle, endothelial cells [8,9],
fibroblasts [10], and neutrophils [11]. Since these substances may be derived
from activated platelets they are relevant to vascular lesion development and
might also affect host immune response to infections and wound healing. However, the actual involvement of such
platelet-derived substances in the pathophysiology of diabetes remains to be
clarified.
A large colony of streptozotocin-treated insulin
dependent (STZ-ID) diabetic rhesus monkeys is available for our study [12]. The progression of disease in this diabetic
monkey colony has been systematically followed for several years. During this time, glycemic and hormonal
changes as well as functional and morphological alterations of kidney and
retina have been observed. The character
and extent of these alterations closely resemble those seen in the early,
non-proliferative stages of human Type I diabetes [12-14].
Our principal aim here was to determine whether
platelets from normal and diabetic monkeys differed in their ability to
liberate products affecting cell migration. Washed platelet suspensions were incubated
with a variety of agonists, the platelets were removed, and platelet-free
supernatants were applied to mouse 3T3 fibroblasts in a microchemotaxis system.
All platelet supernatants stimulated
fibroblast chemokinesis (random cellular movement) and moreover, chemotaxis
(directed movement). Furthermore,
certain agonists induced the appearance of significantly greater chemotactic
activity in platelet suspension supernatants (PSS) from STZ-ID than control
monkeys.
Abbreviations used: PSS, platelet
suspension supernatants; STZ-ID, streptozotocin-treated insulin dependent;
HEPES, N-2-hydroxyethylpiperazine-N-2`-ethanesulfonic acid; DMEM, Dulbecco`s
modified Eagle`s medium; EDTA, ethylenediamine tetraacetic acid; platelet-derived
growth factor, PDGF.
METHODS AND MATERIALS
Reagents and biochemicals were obtained as
follows: ADP (sodium salt), arachidonic acid, thrombin (human plasma), ± verapamil
(sodium salt), indomethacin, acetylsalicylic acid (aspirin), prostaglandin E1,
and HEPES (Sigma Chemical Co., St. Louis, MO); 3-isobutyl-1-methylxanthine
(Aldrich Chemical Co., Milwaukee, WI); streptozotocin was a generous gift from
The Upjohn Co. (Kalamazoo, MI); human c-peptide standard, 125I-c-peptide
and M1230 guinea pig anti-human c-peptide antiserum (Novo Research Institute,
Bagsvaerd, Denmark); beef-pork glucagon standard was a gift from Eli Lilly and
Co. (Indianapolis, IN); 30K rabbit anti-beef-pork glucagon antiserum was
obtained from Dr. R. Unger, University of Texas Medical School (Dallas, Texas);
125I-glucagon was obtained from Cambridge Medical Diagnostics Inc.
(Billerica, MA); hemoglobin A1 electrophoresis kit (American Scientific
Products, McGaw Park, IL); trypsin, EDTA, Dulbecco`s modified Eagle`s medium
(DMEM) (Gibco Supply, Grand Island, NY).
Animals
Age-matched control and STZ-ID rhesus monkeys
were studied. Monkeys were made diabetic by intravenous injection of 45-55
mg/kg streptozotocin. STZ-ID monkeys
were severely hyperglycemic and required exogenous insulin (2-15 U/day) to
maintain body weight and prevent ketoacidosis. All monkeys were fed standard
Purina monkey chow with the STZ-ID group receiving a fruit supplement. (Refer
to Table 1 for additional information).
Plasma glucose,
immunoreactive c-peptide and glucagon, and glycosylated hemoglobin
Fasting plasma glucose, immunoreactive c-peptide
and immunoreactive glucagon, as well as hemoglobin A1 measurements, were
obtained from control and STZ-ID monkeys. The STZ-ID monkey group was severely
hyperglycemic (337 ± 22 mg/dL) as compared to the control group (66 ± 2 mg/dL).
Sufficient exogenous insulin was provided to the STZ-ID monkeys to prevent
ketoacidosis and to allow growth in juvenile monkeys and maintain weight in
adult monkeys. Indicative of extensive STZ-induced beta cell destruction with
minimal residual insulin secretion were measurements of depressed plasma
immunoreactive c-peptide levels in the STZ-ID (74 ± 30) as compared to the
control (235 ± 42) group under severe fasting hyperglycemia. Plasma
immunoreactive glucagon in the STZ-ID group was also elevated, presumably as a
result of reduced beta cell secretion of insulin. Moreover, consistently
elevated hemoglobin A1 levels reflect the stability of the hyperglycemia in the
diabetic monkey group. Similar findings have been reported previously for this
monkey colony [12].
Blood collection and
isolation of washed platelets
Blood samples were taken from monkeys that were
lightly anesthetized with ketamine after an overnight fast. Blood from a femoral
vein was drawn into a plastic syringe through a 19 gauge needle, immediately
transferred in 3 ml aliquots to siliconized tubes containing 0.5 ml of 0.129 M
buffered citrate and 1.25 ml of saline. A washed platelet fraction was prepared
by a modification of the method described by Baenziger and Majerus [15]. Briefly,
citrated whole blood was centrifuged at 200xg for 10 min at 20ºC to obtain
platelet-rich plasma. Prostaglandin E1 was added to the platelet-rich plasma
isolate at a final concentration of 1 µg/ml to prevent platelet activation
during further preparative procedures. The isolated platelet-rich plasma was
centrifuged again at 200xg to remove contaminating erythrocytes, and the
resulting supernatant centrifuged at 3000xg for 15 min at 20ºC. The platelet
pellet was resuspended in phosphate buffer, pH 6.5, containing 1 µg/ml
prostaglandin E1 and centrifuged again. The final pellet was suspended in serum
free Dulbecco`s modified Eagle`s medium (DMEM) containing 25 mM HEPES buffer at
a final count of 500x106 platelets/ml.
Platelet aggregation and
isolation of activation products
Platelet aggregation was measured by a
conventional transmission spectrophotometric method. Aliquots (500 µl) of
washed platelet suspensions were added to siliconized glass aggregation tubes,
warmed to 37ºC for 2 min and placed in an aggregation chamber with stirring for
an additional 1 min. Agonist (5 µl) was then added and the resulting
aggregation response monitored for 3 min against a blank containing
platelet-free, serum free DMEM/HEPES. After 3 min, aggregation and release were
terminated by rapidly transferring the experimental sample to a 1.5 ml
microfuge tube and pelleting the platelets by microfuge centrifugation for 2
min. Aliquots of the resulting supernatant (PSS) were placed on ice prior to
chemotaxis or chemokinesis measurements. In studies examining the effects of
platelet activation inhibitors upon agonist-induced aggregation and liberation
of chemotactic activity, washed platelet suspensions were preincubated with
inhibitor in aggregation tubes for 14 min at 37ºC without stirring. Tubes were
then transferred to the aggregation chamber where samples were treated as
described above. Control samples not containing inhibitor were also subjected
to a 14 min preincubation period.
Cell culture conditions
NIH 3T3 cells were grown in continuous culture
in DMEM supplemented with amino acids (2X), glutamine (2X), 100 U/ml
penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum. Cells were
maintained at pH 7.2 in a water-jacketed C02 incubator at 37ºC and
harvested from flasks using 0.1% trypsin with 0.2 mM EDTA. They were
centrifuged at 500xg for 5 min, resuspended in fresh suspension medium, counted
using a hemocytometer, and kept at 4ºC in suspension until used for chemotaxis
studies.
Chemotaxis and
chemokinesis measurements
Cells harvested by trypsinization were suspended
in serum free DMEM/HEPES (106 cells/ml). Supernatants derived from
PSS that were undiluted or diluted 1:3 with this medium were placed into the
lower wells of a 48 well chemotaxis chamber (Neuroprobe, Cabin John, MD). A
polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA)
with 5 µm pores was pretreated with 0.001% poly-L-lysine for 10 min, rinsed,
dried, and placed over these lower wells. Suspended cells (25 µl; 25,000 cells)
were placed into the upper wells and either additional media, platelet buffer,
or PSS was also added (25 µl). Chambers were then incubated at 37ºC in a
humidified incubator for 4 hours. This incubation was terminated by removing
the filter from the chamber, fixing it in methanol, scraping the adherent,
non-migrated cells off the top of the filter, and staining the migrated cells
on the underside of the filter with Lillie`s hematoxylin. Filters were then
dried onto glass slides, mounted, and the cells counted at 400x magnification. Samples
were run in triplicate and 5 microscope fields in each well were counted. Heat inactivated
fetal calf serum (1%) was used as a positive control (see Table 2).
In experiments designed to measure chemokinesis,
PSS were added to upper (25 µl) and lower (29 µl) chambers while suspended 3T3
cells (25 µl; 25,000 cells) were added to the upper chamber. The number of
cells penetrating the polycarbonate filter was then measured as described
above.
Statistical analysis
Student`s two-tailed t-test was employed for all
statistical analyses.
.
RESULTS
Animals
Characteristics of the animals employed in the
present study are shown in Table 1. Fifteen control (6 male and 9 female)
monkeys ranging in age from 7.4 to 21.9 years with a mean (± SEM) age of 11.5 ±
1.2 years, and 7 STZ-ID (2 male and 5 female) monkeys ranging in age from 6.2
to 16.9 years with a mean (± SEM) age of 10.0 ± 1.3 years were studied. The duration
of insulin dependence for the STZ-ID group ranged from 1.2 to 10.3 years with a
mean ± SEM) duration of 4.4 ± 1.2 years.
Platelet aggregation and
release of chemotactic activity in control monkeys
Thrombin- and arachidonic acid-induced aggregation
and release of chemotactic activity from washed platelets isolated from control
monkeys was assessed using cultured mouse 3T3 fibroblasts as target cells in a
microchemotaxis assay system. Dose-dependent increases in chemotactic activity
and aggregation response were obtained for both agonists (Figure 1). Appearance
of chemotactic activity was platelet-dependent since no additional activity was
obtained when agonist was added directly to PSS incubated with buffer. However,
at high thrombin and arachidonic acid concentrations (above 0.5 U/ml and 30 µM,
respectively) residual agonist present in the PSS contributed significantly to
the chemotactic activity measured. Serial dilution of PSS from either thrombin-
or arachidonic acid-stimulated platelets with serum free DMEM/HEPES led to
progressive decreases in chemotactic activity. At concentrations of arachidonic
acid above 60 µM the aggregation responses became inversely proportional to
agonist concentration, giving the aggregation response curve a bell-shaped
appearance.
Chemotactic activity in control PSS incubated
with 0.1 U/ml thrombin was inhibited by preincubation with
3-isobutyl-1-methylxanthine, 8-bromo-cAMP and prostaglandin E1, although
aspirin had little effect. On the other hand, both 3-isobutyl-1-methylxanthine
and aspirin inhibited the liberation of chemotactic substances from arachidonic
acid-treated platelets. Inhibition of the thrombin- and arachidonic
acid-induced appearance of chemotactic activity in PSS was also accompanied by
an inhibition of the corresponding aggregation response.
Comparison of
aggregation and release of chemotactic activity in control and STZ-ID monkey
groups
Thrombin- and arachidonic acid-induced release
of chemotactic activity was then compared using washed platelets from control
and STZ-ID monkeys (Table 2). Thrombin-induced appearance of chemotactic
activity in PSS was greater with platelets isolated from STZ-ID than control
monkeys. Since chemoattractant stimulation of 3T3 cell migration is known to
exhibit bell-shaped dose-response curves, we also examined effects of diluting
PSS upon measured chemotactic activity. A significant increase in chemotactic
activity was obtained with the STZ-ID group upon 1:2 through 1:8 dilutions of
PSS. Data obtained for the 1:3 dilution are shown in Table 2. However, the corresponding aggregation
responses recorded for thrombin-stimulated platelets from control and STZ-ID
monkeys were not significantly different. No significant differences were observed in
the arachidonic acid-induced appearance of chemotactic activity in undiluted or
diluted PSS, or in the corresponding aggregation responses with platelets from
control and STZ-ID monkeys (Table 2). Chemotactic activity obtained with PSS
from platelets incubated in the presence of buffer alone was similar for
platelets isolated from control and STZ-ID monkeys. It constituted 13-24% of
the activity derived from platelets incubated in the presence of agonist.
Platelet-derived
chemokinetic activity in control and STZ-ID monkeys
In addition to measuring chemotactic (directed)
movement of cultured mouse 3T3 cells, we monitored the effects of PSS upon
chemokinetic (stimulated but not directed) movement (Table 3). PSS exposed to
thrombin (0.1 U/ml) or arachidonic acid (0.67 µM) stimulated 3T3 chemokinesis
markedly (2-8X) over baseline levels obtained with buffer alone. No significant
differences in chemokinetic activity were obtained with PSS from STZ-ID and
control monkey groups, irrespective of which agonist was employed. Using similar methods to those used in the
evaluation of under-agarose chemotaxis assays, values obtained for chemotaxis were
reduced by a factor equal to the chemokinesis to yield a “corrected” chemotaxis
value (Table 3).
DISCUSSION
In the present study, we have found that: 1) Washed
platelet suspensions stimulated with thrombin or arachidonic acid liberated
factors that are both chemokinetic and chemotactic for 3T3 fibroblasts. 2) PSS
from diabetic monkeys released more chemotactic activity than did control monkey
platelets in response to thrombin. Similarly,
previous studies with extracts of whole platelets, platelet-enriched plasma
[16], or purified platelet products [9-11] have presented compelling evidence
that platelet-derived compounds are capable of inducing chemotaxis or
chemokinesis. Several platelet-derived
products liberated consequent to agonist-induced activation are, in fact, known
to have chemotactic activity when added in purified form to a chemotaxis assay
system. For example, the alpha granule constituents,
platelet factor 4, PDGF and β-thromboglobulin,
are all strongly chemotactic for human and bovine fibroblasts [10]. Similarly, the lipoxygenase metabolite of
arachidonic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, which may be
produced by platelets in vivo has
been shown to be chemotactic for human neutrophils [11] and rat smooth muscle
cells [9]. Whether these substances
mediate chemotactic activity following platelet activation in vivo has not been determined.
As a step toward understanding the importance of
in vivo platelet activation products,
we measured the release of chemotactic activity resulting from exposure of
isolated, intact platelets to physiologically relevant agonists. Using this
approach, agonist-induced release of chemotactic activity could be studied
relative to the concomitant activation of other pathways, such as aggregation
and granule release. We first examined
the effects of platelet products formed consequent to agonist stimulation of
washed platelet suspensions obtained from control monkeys upon fibroblast cell
migration. Dose-dependent increases in
chemotactic activity paralleled corresponding increases in aggregation response
when either thrombin or arachidonic acid was employed as agonist. Known inhibitors of platelet activation, such
as 3-isobutyl-1-methylxanthine, 8-bromo-cAMP and prostaglandin E1 that increase
or mimic cellular cAMP, inhibited both thrombin and arachidonic acid-induced
liberation of chemotactic activity and also corresponding aggregation
responses. The cyclooxygenase enzyme
inhibitor, aspirin, inhibited arachidonic acid-induced increases in chemotactic
activity and aggregation but had no effect on either response to thrombin. These results suggest that aggregation and
also release of chemotactic activity from agonist-activated platelets are both
sensitive to antagonists of established platelet activation pathways.
However, when we compared agonist-induced
platelet activation in control and STZ-ID monkey groups, it seemed that the pathways
mediating aggregation and liberation of chemotactic activity were affected
differently in STZ-ID monkeys. Following
exposure of washed platelet suspensions from control and STZ-ID groups to
thrombin, greater chemotactic activity was liberated using platelets obtained
from the STZ-ID group, whereas aggregation responses were similar for platelets
from both groups. Using identical
incubation conditions, we also found that the release of granule-stored 14C-5-hydroxytryptamine
is similar for control and STZ-ID groups [Takimoto et al., unpublished
observations]. Since 5-hydroxytryptamine
release from platelet granules usually parallels the aggregation response,
these data further emphasize that the liberation of chemotactic activity by
STZ-ID monkey platelets seems to be independent of aggregation and total
granule release.
The enhanced chemotactic activity observed here
was attributable primarily to an increase in directed rather than random
(chemokinetic) cellular movement (see Table 2). It was also agonist-specific, since no
difference in chemotactic activity or aggregation response was observed between
control and STZ-ID groups when arachidonic acid was employed as agonist. These findings indicate that: 1) a complete
assessment of the pathophysiological changes in platelet function cannot be
achieved through aggregation and granule release measurements alone, and 2)
that the in vitro liberation of
platelet-derived proaggregatory and chemotactic activity may be independently
regulated. Further investigation of
these platelet properties will require identification of platelet products
mediating the chemotactic response and characterization of pathways regulating
the liberation of chemotactic activity.
Platelets from humans with Type I diabetes display
increased thrombin-induced aggregation and granule release [8,17], although
agonist-induced release of chemotactic activity has not been measured. The reason for the difference in platelet
aggregation and granule release characteristics between diabetic humans and our
STZ-ID monkey model is unclear. Hypertension
and hyperlipidemia are frequently associated with diabetes in humans [18,19],
and have been shown to increase platelet aggregation, granule release and
formation of prostaglandin/thromboxane even in non-diabetic subjects [20,21]. However, our STZ-ID monkeys have thus far
failed to exhibit hypertension and hyperlipidemia as well as the advanced,
proliferative macro- and microvascular changes which frequently arise in
long-term diabetic humans [12].
In summary, our findings demonstrate that
agonist-induced aggregation and liberation of chemotactic and chemokinetic
activity can be measured using intact, washed platelets from rhesus monkey. We have also shown that thrombin-induced
liberation of chemotactic activity is selectively enhanced with chronic
hyperglycemia without alteration of the corresponding aggregation response. We speculate that the enhanced liberation of
chemotactic activity from STZ-ID monkey platelets may be related to hyperglycemia,
since secondary complications normally associated with diabetes in humans are
absent in the diabetic monkeys. We have
recently induced mineralocorticoid/salt hypertension and diet-induced
hyperlipidemia in subsets of our control and diabetic monkeys allowing further
study of this problem. The differential
effects of STZ-induced diabetes upon aggregation and liberation of chemotactic
activity suggest that these platelet actions are independently regulated. The significance of the increased in vitro release of platelet-derived
chemotactic activity to the development of diabetes-related vascular, immune
and tissue repair complications requires further investigation.
ACKNOWLEDGMENTS
This work was supported by USPHS grant AM 18284
and an American Diabetes Association, Northern Illinois Affiliate Young
Investigator Award. The authors wish to thank John Krewer for excellent
technical assistance.
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Thorgeirsson, G., Robertson, A.L., Cowan, D.H. (1979) Lab. Invest. 41, 51-62.
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W.S. (1975) Nature 257, 680-681.
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Ann. Surg. 201, 27-39.
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Chmielewski, J., Farbiszewski, R. (1978) Cells 14, 203-205.
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Mogensen, C.E. (1980) Acta Endocrinol. 94 (Suppl. 238), 103-108.
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Shattil, S.J., Anaya-Galindo, R., Bennett, J., Colman, R.W., Cooper, R.A. (1975)
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Table 1
Demographics of monkey subpopulations studied
Animal # Studied
Age Gender a Duration of ID
Status (range) (range)
_______________________________________________________________
Control 15
11.5 ± 1.2 9F,
6M
(7.4 ± 21.9)
STZ-ID 7
10.0 ± 1.3 5F,
2M 4.4 ± 1.2
(6.2-16.9) (1.2-10.3)
_______________________________________________________________
STZ-ID
= streptozotocin-treated, insulin-dependent. Values for Age (years) and
Duration of ID (insulin dependence; years) represent the mean ± SEM.
a Duration of
insulin-dependence
Table 2
Agonist-induced aggregation and release of chemotactic and
chemokinetic activity
using washed platelets isolated from control and diabetic
monkeys
Animal Agonist Aggregation Chemotaxis (# cells/400x field)
Status a
undiluted 1:3 dilution N
____________________________________________________________________
Control Buffer 0 7.2
± 1.6 <1
15
STZ-ID 0 8.0
± 2.7 <1
6
Control Thrombin
26.1 ± 3.5 38.4 ± 4.8 14.4 ± 3.3 15
STZ-ID (0.1 U/ml) 31.0 ± 7.2 *63.3 ± 9.1 **38.0
± 8.5 8
Control Arach.
Acid 10.2 ± 2.8 30.0
± 4.8 15.8 ± 2.5 11
STZ-ID (0.67 µM) 13.0 ± 3.9 47.2
± 7.7 22.1 ± 5.5 5
b CX FCS 23.2 ± 1.7 ---
--- 15
Control
(1.0%)
_____________________________________________________________________
Buffer
or agonist (5 µl) were added to washed platelet suspensions (500 µl), and the
aggregation
response was recorded over the ensuing 3 min. The aggregation period was
terminated
by microfuge centrifugation and the resulting supernatant applied to the
microchemotaxis
chamber. All values represent the mean ± SEM. N = number of different
monkeys
from which measurements were obtained.
a Platelet supernatants
were used either undiluted or diluted 1:3 with serum-free DMEM/ HEPES media.
b In chemotaxis control
wells, no platelet supernatants were present. Instead, 1% fetal calf
serum was added to the lower wells and 3T3 cells in serum-free DMEM/HEPES added
to the
upper wells. Such control wells were normally present in the 48-well assay
described here.
N = number of different experiments in which measurements for 1% fetal calf
serum controls
were obtained.
*
significantly different from control at
p< 0.05
**
significantly different from control at p< 0.02
Table 3
Agonist-induced release of chemokinetic activity using
platelets isolated from control and diabetic monkeys
Animal Agonist Concentration Chemokinesis Corrected
Status # cells/400x field (n) Chemotaxis
___________________________________________________________________
Control Buffer --- 4.2 ± 1.3
(8) 3.0
STZ-ID --- 12.6 ± 4.1
(5) 0
Control Thrombin 0.1 U/ml
34.0 ± 7.7 (13) 4.4
STZ-ID 27.3 ± 8.7 (6) *36.0
Control Arachidonic
0.67
µM 19.4 ± 2.5 (9) 10.6
STZ-ID Acid 29.9 ± 5.3
(6) *17.3
____________________________________________________________________
Incubation
conditions and preparation of platelet-free supernatants were as
described in
Table 2.
All
values represent the mean ± SEM.
(n)
= number of different monkeys from which measurements were obtained.
FIGURES
Figure 1. Platelet aggregation and released chemotactic
activity in response to thrombin (A) and arachidonic acid (B). Buffer or
agonist (5 µl) was added to washed platelet suspensions (500 µl), and the
aggregation response was recorded over the ensuing 3 min. The aggregation
period was terminated by microfuge centrifugation and the resulting supernatant
applied to the microchemotaxis chamber. All values represent the mean ± SEM of
data obtained from 3-15 control monkeys.
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