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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2019 Feb 27;100(1):41–48. doi: 10.1111/iep.12298

Effects of three‐month streptozotocin‐induced diabetes in mice on blood platelet reactivity, COX‐1 expression and adhesion potential

Tomasz Przygodzki 1,✉, Hassan Kassassir 1, Marcin Talar 1, Karolina Siewiera 1, Cezary Watala 1
PMCID: PMC6463392  PMID: 30811756

Summary

Diabetes is associated with an increased risk of cardiovascular disease. This is partially attributed to an altered activation status of blood platelets in this disease. Previously, alterations have been shown in COX‐1 and protease activated receptor (PAR)‐3 receptor expression in platelets in two animal models of diabetes, there have not been studies which address expression of these proteins in mice with long‐term streptozotocin (STZ)‐induced diabetes. We have also addressed the effect of diabetes on platelet adhesion under flow conditions. With the use of flow cytometry, we have shown that certain markers of platelet basal activation, such as active form of αII bβ3 and of CD40L were increased in STZ‐induced diabetic mice. Platelets from STZ‐induced diabetic mice were also more reactive when stimulated with PAR‐4 activating peptide as revealed by higher expression of active form of αII bβ3, membrane‐bound on vWillebrand Factor and binding of exogenous fluorescein isothyanate‐labelled fibrinogen. Expression of COX‐1 and production of thromboxane A 2 in platelets of STZ‐induced diabetic mice were higher than in control animals. We observed no effect of diabetes on ability of platelets to form stable adhesions with fibrinogen in flow conditions. We conclude that although certain similarities exist between patterns of activation of platelets in animal models of diabetes, the differences should also be taken into account.

Keywords: cell adhesion, diabetes, platelets, prostanoids

1. INTRODUCTION

Diabetes is associated with an increased risk of cardiovascular disease.1 This is partially attributed to an altered activation status of blood platelets in the disease.2 Enhanced platelet activation affects the cardiovascular system in multiple ways. Hyperreactive platelets have been shown to produce larger amounts of thromboxane A2 (TXA2),3 which is not only a platelet activator but also a potent vasoconstrictor. At the same time, diabetes is associated with a decreased production of vasodilating and antiaggregatory prostacyclin by endothelium in certain vascular beds.4 These two phenomena contribute to an imbalance between anti‐ and proaggregatory prostanoids in favour of the latter.5, 6 Another consequence of an increased platelet reactivity is their enhanced potential for adhering to the wall of blood vessels, which in turn sets conditions for development of atherosclerosis.7 Two of the mechanisms which are behind this altered platelet function have been recently studied by our group. Our studies in rats with streptozotocin (STZ)‐induced diabetes that resembles human type 1 diabetes and in db/db mice with a phenotype that resembles human type 2 diabetes revealed increased expression of COX‐1 in diabetic platelets,8, 9 which can explain enhanced TXA2 production. In db/db mice, we have also shown an increased expression of the protease activated receptor (PAR)‐3 receptor, which can explain higher sensitivity of these platelets to low doses of thrombin.10 Another frequently used model of diabetes, which has not been characterized to date in terms of COX‐1 and PAR‐3 expression in platelets, is STZ‐induced diabetes in mice. One of the aims of presented paper was to fill this gap. We were also interested whether platelets from diabetic animals are more prone to interact with proteins which can be found on activated endothelium. Finally, since it is assumed that in some vascular beds production of PGI2 is impaired in diabetes, which accounts for TXA2/PGI2 imbalance, we also measured production of PGI2 in the coronary vascular bed in our experimental model. Our choice of coronary circulation was due to a relevance of this vascular bed to the context of cardiovascular complications in diabetes.

2. MATERIALS AND METHODS

2.1. Ethics statement

All the procedures were approved by the Local Ethical Committee on Animal Experiments, Medical University of Lodz (approval number 65/LB572/2011).

2.2. Mouse husbandry

Male C57BL6/cmdb mice aged 8‐12 weeks were obtained from the Center of Experimental Medicine, Medical University of Bialystok (Bialystok, Poland). During experiments animals were housed in an isolated room with a 12‐hours light‐dark cycle and were given free access to water and standard chow for rodents.

2.3. Chemicals, antibodies, assay kits

Anaesthetics: Sedazine (20 mg/mL xylazine hydrochloride) and ketamine (100 mg/mL ketamine hydrochloride) were obtained from Biowet (Biowet, Pulawy, Poland). For intraplatelet detection of cyclooxygenase 1, the anti–COX‐1 antibodies and the relevant blocking peptide were provided by Cayman Chemicals (Ann Arbor, MI, USA). Inorganic salts, acetylcholine and STZ were purchased from Sigma‐Aldrich (St. Louis, MO, USA). EIA kits for determination of 6‐keto‐PGF1α (stable metabolite of PGI2) were purchased from Assay Designs (Axxora Platform), while EIA kits for determination of TXB2 were purchased from Cayman Chemicals Europe (Tallinn, Estonia). FITC‐ or PE‐conjugated rat anti‐CD41/61, PE‐conjugated rat anti‐CD62P, PE‐conjugated JON/A antibodies (rat anti‐the active form of αIIbβ3), FITC‐conjugated rat anti‐von Willebrand factor and rat anti‐fibrinogen antibodies were purchased from Emfret Analytics (Eibelstadt, Germany); FITC‐conjugated rat anti‐CD40L antibody was purchased from BioLegend (San Diego, CA, USA); and PE‐conjugated rat anti–PAR‐3 monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). PAR‐4 activating peptide (AYPGKF) was obtained from American Peptide Company (Sunnyvale, CA, USA). Mouse exogenous FITC‐labelled fibrinogen (a full‐length protein) was obtained from Abcam (Cambridge, UK). Low molecular weight heparin (LMWH) was from Sanofi Aventis (Paris, France).

2.4. Induction of experimental diabetes with streptozotocin

Experimental diabetes was induced in C57BL6 eight‐week‐old mice by a single intraperitoneal injection of 200 mg/kg b.w. streptozotocin in 0.1 mol/L citrate buffer (pH 4.5). Control mice were injected with a vehicle. STZ‐induced diabetic animals, in which blood glucose values exceeded 16.6 mmol/L 7 days after STZ injection, were included in the study. Blood glucose was assessed using Accu‐Chek glucometer (Roche Diagnostics Polska Ltd., Warsaw, Poland). The content of glycated haemoglobin (HbA1c) was measured using automatic biochemical analyser Olympus model AV 640 (Olympus, Tokyo, Japan).

2.5. Blood collection and preparation

Three months after induction of diabetes, the mice were anaesthetized with intramuscularly injected ketamine (10 mg/kg body weight) and xylazine (100 mg/kg body weight). Blood was collected on 10 U/ml low molecular weight heparin (LMWH) in TBS buffer (20 mM Tris–HCl, 137 mM NaCl, pH 7.3), from the inferior vena cava. The blood was centrifuged, and cellular blood components were suspended in Tyrode's buffer to obtain blood cells devoid of plasma (washed blood), as described previously.8 Blood used for the determination of the basal concentrations of TXB2 was supplemented with acetylsalicylic acid (500 μmol/L) immediately after collection and centrifuged (1000 g, 15 minutes, 4°C) to obtain plasma. Plasma was deep frozen until further analyses.

2.6. Flow cytometry

The activation of circulating platelets, as well as the platelet reactivity in response to PAR‐4 activating peptide (AYPGKF) at final concentrations of 20 or 100 μmol/L, was evaluated by measuring of the expression of the specific surface membrane antigens, such as CD62P (P‐selectin), active form of αIIbβ3, CD40L, PAR‐3, membrane‐bound fibrinogen and the binding of exogenous fibrinogen. Platelets were gated on the basis of binding anti‐CD41/61 antibodies. The percent fractions of specific FITC‐positive platelets were evaluated after subtracting of non‐specific isotype mouse IgG1 binding (the FL2 gate for isotype control set to 2%). Results were presented as the percent fractions of CD62P‐, the active form of αIIbβ3‐, vWf‐, Fg‐ and PAR‐3‐positive platelets.

Expression of COX‐1 in platelets was assayed as previously described.8 Briefly, blood platelets in fixed washed blood samples were permeabilized and incubated with anti–COX‐1 antibodies. To control for non‐specific fluorescence, anti–COX‐1 antibodies were incubated with saturating concentrations of their blocking peptides. Results were presented as MFI values for COX‐1–positive platelets.

Flow cytometric measurements were performed using the FACSCanto II instrument (BD Biosciences, San Diego, CA, USA). At least 10,000 cells were analysed per sample. All data were processed using FACSDiva ver. 6.0 software (BD Biosciences, San Diego, CA, USA).

2.7. Blood platelet adhesion in flow conditions

Blood platelet adhesion was assessed with the use of VenaFlux platform (Cellix, Dublin, Ireland). Channels of Vena8 Fluo+ biochip were coated with mouse fibrinogen (200 μg/mL) overnight at 4°C and blocked with BSA (1 mg/mL) for 1 hour at 4°C. Biochip was mounted on a thermo‐controlled stage of inverted AxioVert microscope (Carl Zeiss, Oberkochen, Germany), assuring the constant temperature of 37°C throughout the experiment. Prior to measurements, the channels were washed with PBS with heparin (1 U/mL) for 2 minutes at 2 dynes/cm2. Washed blood was perfused through the channel at 4 dynes/cm2, which corresponds to 400 s−1 for 1 minute. The channel was then perfused with PBS with heparin for 2 minutes at 2 dynes/cm2 to remove non‐adherent platelets. Anti‐CD41/61/FITC‐conjugated antibodies were aspired to the channel, and incubation was held for 5 minutes. The channel was then washed for 2 minutes at 2 dynes/cm2 to remove unbound antibodies. Pictures of labelled platelets were taken with a 40× objective at 5 different sites using a filter set dedicated for FITC. Adherent platelets were counted with the use of ZEN software (Carl Zeiss, Oberkochen, Germany).

2.8. Prostacyclin synthesis in the coronary vascular bed

The mice were anaesthetized as described above. A thoracotomy was performed, and the heart was rapidly excised and transferred to cold Krebs‐Henseleit (K‐H) buffer (NaCl 118 mmol/L, NaHCO3 25.0 mmol/L, KCl 4.7 mmol/L, KH2PO4 1.2 mmol/L, CaCl2 1.2 mmol/L, MgSO4 1.2 mmol/L, glucose 11.0 mmol/L; pH 7.4). The aorta was cannulated, and heart was connected to a Langendorff apparatus (ADInstruments, Dunedin, New Zealand). Both atria were excised, and the heart was then retrograde‐perfused with K‐H buffer (saturated with carbogen: 95% O2, 5% CO2). Perfusion pressure (mm Hg) was constantly monitored and recorded with the use of PowerLab ML870 transducer and PowerLab Recorder software (ADInstruments). The system adjusted the flow rate of perfusate to maintain a constant pressure of 80 mm Hg. The flow was generated by means of peristaltic pump, which was under the control of the system. The actual flow rate was calculated by the system based on the extrapolation of the conditions of flow rate calibration performed prior to the experiment. To maintain a constant temperature of 37°C during experiments, the hearts were placed in a water‐jacketed chamber.

The hearts were perfused with K‐H buffer for 15 minutes to stabilize flow parameters. Perfusate samples were collected for assaying of the products of basal prostaglandin synthesis. Afterwards, acetylcholine was administered by infusion pump connected via a Y luer stopcock mounted above the cannula. The rate of acetylcholine administration was always adjusted to the actual perfusion rate to maintain a constant concentration of acetylcholine at 300 nmol/L. After 5 minutes, the aliquot of perfusate was collected for the subsequent assaying of the products of acetylcholine‐induced prostaglandin synthesis. All the collected perfusate samples were deep frozen at −80°C immediately after collection for further analysis. After termination of the protocol, the hearts were dried at 200°C for 8 hours to determine their dry weights.

The concentration of 6‐keto‐PGF1α, the stable metabolite of prostacyclin, was assayed in the collected perfusate samples with the use of competitive ELISA tests. The tests were performed according to the manufacturer's instructions. The numerical values of the concentrations of all the analysed samples, expressed in pg/mL, were multiplied by the actual flow rate recorded during the collection of the samples in mL/min to determine the quantities of prostaglandin secreted by myocardium over a normalized time interval (pg/min). The above values were divided by the dry weights (mg) of particular heart muscles to normalize prostaglandin secretion to the weight of the heart. The final results of 6‐keto‐PGF1α secretion were expressed in pg/min/mg dry weight.

2.9. Thromboxane B2 concentration assays in plasma

The TXB2 concentration was measured using a thromboxane B2 ELISA kit from Cayman Chemicals, according to the manufacturer's recommendation.

2.10. Statistical analysis

The normal distributions of data were verified with the Shapiro‐Wilk test, and variance homogeneity was tested with Levene's test. Data are expressed as mean ± SD for normally distributed variables, or as median and interquartile range (IQR: lower [25%] to upper [75%] quartile) otherwise. Unpaired Student's t test was employed to reason on the significance of differences when comparing two independent groups with distributions not departing from normality. When normality assumption and/or variance homogeneity assumption was violated, the significance of differences was tested with Mann‐Whitney's U test. Statistical analysis was performed with the use of Statistica v. 12.5.

3. RESULTS

3.1. Streptozotocin‐induced diabetes

Mice injected with STZ were characterized by hyperglycaemia. Postprandial glucose levels on the day of the experiment were significantly higher in mice treated with STZ than those treated with citrate: 28.9 (22.6; 33.3) mmol/L vs. 12.3 (11.0; 13.9) mmol/L respectively (median [IQR]) (one‐sided, Mann‐Whitney's U test; P < 0.0001, n = 15). Also, levels of glycated haemoglobin were higher in STZ‐induced diabetic mice (9.8 [8.2; 10.0]%) than in control mice (4.2 [4.1; 4.3]%) (median [IQR]) (one‐sided, Mann‐Whitney's U test; P < 0.0001, n = 14).

3.2. Platelet activation and reactivity—flow cytometry and plasma markers

Among parameters of platelet activation assessed by flow cytometry in non‐stimulated platelets, only expression of active form of αIIbβ3 (Figure 1A) and that of CD40L (Figure 1F) were significantly increased in STZ‐induced diabetic mice when compared to the non‐diabetic group. In turn, parameters such as membrane‐bound vWF, membrane‐bound Fbg, binding of exogenous Fbg and PAR‐3 expression were not significantly different (Figure 1B, C, D and E).

Figure 1.

Figure 1

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Flow cytometry analysis of the changes in selected markers of platelet activation in resting platelets and in response to protease‐activated receptor‐4 (PAR‐4) activating peptide (AYPGKF). Concentration “0” refers to the basal activation of platelets. Percentage of platelets positive for an active form of αII bβ3 (A), von Willebrand factor (B), bound fibrinogen (C), binding of exogenous fibrinogen (D), PAR‐3 (E) and CD40L in resting platelets (F). Results are shown as median (horizontal line) with interquartile range (box) for non‐diabetic (white) and STZ‐induced diabetic mice (light‐grey). Differences between means were tested with the use of Mann‐Whitney's U test. Statistical significances were the following: AYPGKF peptide 0 μmol/L: αII bβ3, P 1,α < 0.05, STZ > control; AYPGKF peptide 20 μmol/L: αII bβ3, P 1,α < 0.05, STZ > control; Fbg exog, P 1,α < 0.0001, STZ > control; AYPGKF peptide 100 μmol/L: αII bβ3, P 1,α < 0.05, STZ > control; vWF, P 1,α < 0.05, STZ > control; Fbg exog, P 1,α < 0.05, STZ > control

Reactivity of blood platelets stimulated with AYPGKF peptide was significantly higher in STZ‐induced diabetic mice. Expression of active form of αIIbβ3 was higher in response to both concentrations of AYPGKF peptide (Figure 1A). The level of membrane‐bound vWF was higher in platelets of mice with STZ‐induced diabetes when stimulated with 100 μmol/L AYPGKF peptide (Figure 1B), while the binding of exogenous FITC‐labelled fibrinogen to platelets of mice with STZ‐induced diabetes was significantly increased for both concentrations of AYPGKF peptide (Figure 1D). In contrast, neither bound fibrinogen (Figure 1C) nor expression of PAR‐3 receptors differed between the two groups when stimulated with AYPGKF peptide (Figure 1E).

COX‐1 expression was elevated in the platelets of mice with STZ‐induced diabetes (Figure 2). TXB2 levels in plasma of STZ‐induced diabetic mice were increased (Figure 3).

Figure 2.

Figure 2

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Expression of COX‐1 in platelets of control and STZ‐induced diabetic mice. Left panel: Results are shown as median (horizontal line) with interquartile range (box) and raw data (n = 13). Statistical significant differences, estimated with Mann‐Whitney U test were P < 0.05, STZ > control. Right panel: Representative flow cytometric histograms showing detection of COX‐1 in platelets from non‐diabetic (control) and diabetic (STZ) mice. The parent gate was set on the platelet population, according to the presence of CD41/61, a unique antigen for platelets (gate P1) for control (A) and STZ (B) mice. Within such a gated platelet cohort, the expression of COX‐1 (gate P2) in blood samples taken from non‐diabetic (E) and diabetic mice (F) was identified. Non‐specific fluorescence was estimated according to samples incubated with peptide blocking the binding of anti–COX‐1 antibodies to platelets surface (C for control, D for STZ‐induced diabetic mice) [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3.

Figure 3

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Concentrations of TXB 2 in plasma of control and STZ‐induced diabetic mice. Results are shown as median (horizontal line) with interquartile range (box) and raw data (n = 8). Statistical significant differences, estimated with Mann‐Whitney U test, were P < 0.05, STZ > control

3.3. Platelet adhesion to fibrinogen in flow conditions

The number of platelets forming firm adhesions with fibrinogen was not significantly different between STZ‐induced diabetic (169.0 [111.0; 259.5] plt/mm2) and non‐diabetic mice (48.0 [26.5; 217.0] plt/mm2) (median [IQR]) (one‐sided, Mann‐Whitney's U test; P = 0.0794, n = 9).

3.4. Prostacyclin synthesis in coronary vascular bed

Secretion of PGI2 assayed as 6‐keto‐PGF1α in effluent collected from coronary perfusion during flow stabilization period was 11.21 (10.48; 16.34) (median [IQR]) (pg/min/mg of dry weight) in control mice and 13.85 (10.86; 15.37) in STZ‐induced diabetic mice. These values did not differ significantly. During stimulation with ACh secretion of PGI2 was significantly lower in STZ‐induced diabetic mice when compared to non‐diabetic mice (Figure 4A). Coronary flow was lower in STZ‐induced diabetic animals (Figure 4B).

Figure 4.

Figure 4

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PGI 2 release in coronary vascular bed in control and in STZ‐induced diabetic mice. A, Production of PGI 2 measured as 6‐keto‐PGF 1α in coronary vascular bed in response to acetylcholine in concentration of 300 nmol/L. Significance of differences tested with Mann–Whitney U test was P < 0.05, n = 8; B, coronary flow. Significance of differences tested with Mann–Whitney U test was P < 0.05, n = 7; Results are shown as median (horizontal line) with interquartile range (box) and raw data

4. DISCUSSION

Results of studies on alterations in basal activation and reactivity of blood platelets in animal models of diabetes are not consistent. Some researchers report that these parameters are increased in platelets in diabetes,8, 10, 11, 12, 13 but others do not support this concept.14 Significant differences have been also shown between models of diabetes in the same species.15 In our model of 3‐month STZ‐induced diabetes, some of the hallmarks of platelet status showed their higher activation and reactivity, but the extent of these alterations was lower than that described previously in 1‐month STZ‐induced diabetic mice and db/db mice.10, 11 Compared to previously described models, we have observed minor differences in the expressions of the active form of αIIbβ3 and CD40L and no differences in terms of the bound fibrinogen and binding of exogenous fibrinogen. In terms of platelet reactivity in response to AYPGKF peptide, although the differences were significant in case of selected markers, they were not as large in their extent as observed in db/db mice.10

In spite of this heterogeneity between models, studies have confirmed a role for PAR‐4, the receptor that mediates a response to AYPGKF peptide in hyperreactivity of platelets. This appears to be a common feature in different animal models of diabetes. This conclusion is of a great importance and especially when taking into account that inhibitors of PARs consist a new family of antiplatelet drugs.16 It also has the implication that mice with 3‐month STZ‐induced diabetes constitute a relevant model to study the efficacy of PAR‐4 inhibitors in ameliorating platelet hyperreactivity in animal models of metabolic disorders. Interestingly, we did not detect a significant difference between diabetic and non‐diabetic mice in PAR‐3 expression in response to AYPGKF peptide. In mice this receptor has been found to play an auxiliary role during activation of PAR‐4 by thrombin.17 We have recently described the increased expression of PAR‐3 in db/db mice in response to AYPGKF peptide when compared to heterozygous mice,10 which to our understanding explained higher reactivity of these platelets to low thrombin concentration. Lack of such an effect in mice with 3‐month STZ‐induced diabetes points at possible differences behind the mechanisms of platelet hyperreactivity between these two models. Such a difference in a pattern of platelet reactivity between models of diabetes has been previously observed. It has been shown that two different models of diabetes in rats show different phenotypes of platelet response.15 Also, recently published report on alterations in profile of protein expression in megakaryocytes and platelet generation in STZ‐induced diabetic and in ob/ob diabetic mice revealed that these two models differ in this aspect.18 It implicates that platelet hyperreactivity in diabetes is a complex phenomenon, which is influenced by a plethora of factors which vary with the type of diabetes and its associated alterations.

Another expression of altered platelet function in diabetes is an increased production of TXA2. As we have previously shown in rats with long‐term untreated STZ‐induced diabetes and in db/db mice, this effect may be attributed to higher expression and activity of COX‐1 in platelets.8, 9 In line with these studies, mice with 3‐month STZ‐induced diabetes had increased plasma concentration of TXA2 and level of COX‐1 in platelets was elevated. Although concentration of TXA2 in plasma is not a direct measure of platelet production of the prostanoid, it has been shown that platelets are the main source of TXA2 in plasma.19, 20 It implicates that enhanced expression of the enzyme in platelets is a common feature among different animal models of the disease. The evidence of increased COX‐1 expression in human diabetic patients is rather scarce. In one of the published studies, no differences were reported,21 while the other showed significantly higher expression in diabetic platelets.22 Animal models of disease are usually more homogenous in terms of confounding factors which influence intra‐population variation than patients, which is why the differences are easier to be detected. How diabetes influences expression of COX‐1 in platelets remains to be elucidated. It has been recently published that bovine serum albumin modified by advanced glycation end products significantly altered expression of certain proteins including platelet factor 4 in cultured megakaryocytes.18 It suggests that long‐term hyperglycaemia can indirectly affect protein profile in platelets produced by megakaryocytes.

It is known that diabetes is associated with imbalance between prostacyclin and thromboxane.5 Two phenomena which apparently contribute to this effect are increased synthesis of TXA2 by platelets on the one hand and lowered production of PGI2 in some vascular beds in diabetes on the other.4 In line with this concept, we have observed a decreased production of PGI2 from the cardiovascular bed in our model of diabetes in response to ACh. This could contribute to lower coronary flow in STZ‐induced diabetic mice as it has been previously shown that prostacyclin is a major mediator of vasodilatory response to ACh in coronary endothelium in the mouse.23 Since basal production of prostacyclin did not significantly differ between the two groups, one can assume that the lower production in response to ACh was not due to an altered function of any COX enzyme isoforms and that other mechanism should be taken into account. Acetylcholine stimulates PGI2 synthesis by increasing intracellular pool of calcium.24 It has been shown that diabetes affects calcium signalling in cardiac endothelial cells in rats.25 Thus, it may be suggested that worsened PGI2 release in response to ACh in diabetic animals is related to dysfunctional calcium signalling.

There is a concept that platelets facilitate interaction of leucocytes with dysfunctional endothelium and thus perpetuate the process of inflammation of the vascular wall and atherosclerosis development.26, 27 The process of platelet adhesion to activated endothelium has been shown to be dependent inter alia on fibrinogen bound to ICAM on surface of endothelial cells.28 We have previously shown that platelets in mice with 1‐month STZ‐induced diabetes adhere transiently more often to intact vascular wall in diabetic than in non‐diabetic animals.11 Thus, we hypothesized that platelets from STZ‐induced diabetic mice which express higher levels of active form of αIIbβ3 would adhere to fibrinogen‐coated surface to higher extent. However, we were not able to detect such an effect. Our suggested explanation for this discrepancy is that an increase in fraction of platelets positive for active form of αIIbβ3, which was observed in STZ‐induced diabetic mice, was relatively low (from 1.7% in the control mice to 5.1% in STZ‐induced diabetic mice), and these subtle change had possibly a minor effect on the number of adhering platelets.

Interestingly, it has been recently reported that in the same model of diabetes, contrary to our findings, blood platelets were adhering in vitro to fibrinogen in flow conditions to a higher extent.14 A possible explanation for this discrepancy may lie in the difference in criteria applied to define platelets as adherent between the two studies. In the referenced report, platelets were considered adherent if they did not displace for more than two‐seconds. In our studies, only those platelets were counted which remained adherent after washing, that is those which formed more stabilized interactions. It suggests that although platelets from diabetic animals are more prone to form transient interaction with immobilized fibrinogen via αIIbβ3, they do not have an increased potential to form stable adhesion in this conditions. Ju et al have also shown that recruitment of discoid, non‐activated platelets to growing thrombus in vivo in STZ‐induced diabetic mice was more effective than in non‐diabetic counterparts and it was fully dependent on αIIbβ3. Thus, diabetes may affect platelets readiness to transient interactions via αIIbβ3 but it still needs elucidation how it actually contributes to platelet‐dependent cardiovascular complication in diabetes. At the same time, it implicates that enhanced adhesion of platelets in diabetes observed in vivo may be to some extent an effect of impaired endothelial function rather than of altered platelet reactivity. In our previously published in vivo studies, we have shown that enhanced adhesion of platelets to the vascular wall in mice with 1‐month STZ‐induced diabetes was driven to the largest extent by interaction of GPIb with vWF on endothelium.11 Although, platelets from these animals were characterized by higher basal activation and reactivity, they did not adhere to a larger extent to vWF in vitro in flow conditions.

5. CONCLUSIONS

To conclude, we show that platelets in mice with 3‐month STZ‐induced diabetes show similarities to other animal models of diabetes, such as increase in COX‐1 expression and thromboxane A1 production. At the same time, they differ in terms of the extent of basal activation and reactivity when compared to db/db model of diabetes in mice. These differences should be taken into account when considering choice of particular model for certain studies. Blood platelets in this model were not more prone to form stable adhesion to fibrinogen in flow conditions despite expressing more active form of αIIbβ3.

FUNDING SOURCE

This work was supported by the National Science Centre grant MAESTRO (No. UMO‐2012/06/A/N25/00069). Karolina Siewiera and Hassan Kassassir were supported by Etiuda scholarship funded by the National Science Center (2016/20/T/NZ3/00505 and 2016/20/T/NZ3/00508).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Przygodzki T, Kassassir H, Talar M, Siewiera K, Watala C. Effects of three‐month streptozotocin‐induced diabetes in mice on blood platelet reactivity, COX‐1 expression and adhesion potential. Int. J. Exp. Path. 2019;100:41–48. 10.1111/iep.12298

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