Relationship Between Vitamin D Supplementation and Platelet Parameters, Platelet Aggregation and Thrombosis in Healthy Adult Male Dogs: A Pilot Study

Abstract

Vitamin D has well‐documented antithrombotic effects on coagulation system components, and platelet‐associated vitamin D receptors (VDR) play an important role. Adenosine 5′‐diphosphate (ADP) induced platelet activation, and thrombin induced a change in the VDR shape, allowing it to bind to fibrinogen and stimulate the aggregation cascade. The effect of vitamin D supplementation on thrombosis in dogs has not been investigated. Eight mixed‐breed adult male dogs were selected, and oral vitamin D3 at 50 IU/kg was administered for 42 days. Serum levels of 25‐hydroxyvitamin D concentration, platelet number, mean platelet volume (MPV), fibrinogen, prothrombin time (PT) and partial thrombin time were measured before treatment (Day 0) and on Days 14, 28 and 42. The results identified a significant reduction in platelet count following the administration of oral vitamin D (p < 0.0001). There was no significant change in ADP‐induced platelet aggregation, MPV, fibrinogen, PT and partial thromboplastin time (PTT). The results suggest that oral administration of vitamin D daily at a dose of 50 IU/kg BW for 42 days leads to a reduction in platelet count; larger studies are necessary to confirm the results and to elucidate potential clinical implications.

GRAPHICAL ABSTRACT

1. Vitamin D reduced in platelet count.

2. Vitamin D increased in MPV levels.

Abbreviations

25(OH)D

25 hydroxy vitamin D

MPV

mean platelet volume

PLt

platelet

PT

prothrombin time

PTT

partial thromboplastin time

RM ANOVA

repeated measure analysis of variance

1. Introduction

The plasma concentration of vitamin D metabolites and associated components influence the regulation of thrombosis. Reports indicate that vitamin D has a direct or indirect influence on the expression of around 200 genes, including epidermal growth factor receptor, phospholipase C, gamma‐1, insulin‐like growth factor‐binding protein 3 and others (Lee et al. 2007). These genes play a crucial role in thrombosis regulation. Vitamin D has well‐documented antithrombotic effects on several coagulation system components and assists in clot homeostasis, a dynamic process that balances clot formation and inhibition. Platelets, prothrombin, fibrinogen, collagen and platelet‐activating factor are thrombogenic (Mohammad et al. 2019). Vitamin D receptors (VDR) have been identified in the mitochondria of thrombocytes, which play a crucial role in regulating protein synthesis and thrombocyte functions. Megakaryocytes, the precursor cells to thrombocytes, also express VDR. Activation of these receptors is instrumental in modulating cell maturation and promoting megakaryocyte proliferation. Vitamin D deficiency has been shown to promote megakaryocyte maturation and increase thrombocyte counts (Song 1996). The precise role of VDR in the differentiation of progenitors and mature platelets remains to be fully elucidated. Control of calcium homeostasis is likely the most prominent non‐genomic function of platelet VDR, considering the critical role of calcium fluxes in platelet formation, aggregation and granule content release. As a modulator of calcium fluxes, VDR may play a crucial role in megakaryocytopoiesis, platelet activation and apoptosis, all of which are calcium‐dependent processes (Silvagno et al. 2010; Gawaz and Borst 2019). In dogs, platelet activation is mediated by adenosine 5′‐diphosphate (ADP)‐activated platelet receptors such as P2Y12. ADP from platelet‐dense granules amplifies the aggregation signal elicited by other platelet agonists, ensuring irreversible platelet aggregation. Activation of platelets with ADP and thrombin changes the receptor's shape, allowing it to bind to fibrinogen and stimulate the aggregation cascade (Oliver 2016; Cortese et al. 2020). Platelet size and function can be determined by mean platelet volume (MPV) (Harrison and Goodall 2016). MPV correlates with platelet function and activation, whether as aggregation, thromboxane synthesis, β‐thromboglobulin release, procoagulant function or adhesion molecule expression. Korniluk et al. (2019) highlighted a nonlinear inverse relationship between PLT and MPV, with additional contributions from variables such as age, gender, race, lifestyle and genetic determinants. Moreover, lifestyle interventions, such as weight reduction and increased physical activity, were reported to effectively lower MPV (Korniluk et al. 2019). The platelet aggregation approach is suggested to be an acceptable diagnostic indication for platelet function compared with MPV levels. In addition, previous studies have shown that oral vitamin D supplementation may have clinically significant effects on coagulation in both patients with and without vitamin D deficiency (Sultan et al. 2019; Vuthaluru and Goyal 2022).

One study validates the inverse association between poor glycaemic control and elevated platelet aggregation, as well as between low vitamin D25 levels and high aggregation. Calcitriol demonstrates a novel and direct effect in reducing platelet aggregation, potentially benefiting vascular complications related to diabetes. They suggested the therapeutic role of vitamin D in mitigating platelet‐mediated inflammatory responses in diabetes (Sultan et al. 2019). Vuthaluru et al. (2022) highlighted the relationship between serum vitamin D levels and platelet aggregation in hypertensive patients and assessed its potential role in reducing morbidity and mortality in this population. This study demonstrated that platelet aggregation is a significant physiological process in hypertension, which may contribute to complications such as cerebrovascular and ischaemic heart diseases. Lower serum vitamin D levels were associated with increased platelet aggregation (Vuthaluru and Goyal 2022).

The significance of vitamin D in preventing thrombosis suggests a potential correlation between vitamin D and the factors associated with thrombosis. This study examines the antithrombotic impact of daily vitamin D supplementation (50 IU [1.25 µg] per kg bodyweight for 42 days) on platelet count, MPV, aggregation, prothrombin time (PT), partial thromboplastin time (PTT) and fibrinogen in healthy dogs.

2. Materials and Methods

2.1. Animals

Eight intact male adult mixed‐breed dogs (mean weight 20 kg, body condition scores 3–4 on a 9‐point scale) were recruited. All dogs were screened to confirm good health (complete blood count and serum biochemistry) and were then adapted for 2 weeks before entering the study. The 2‐week acclimation period was implemented to evaluate the dogs’ health, minimize stress and facilitate their adjustment to the new environment. This process involved comprehensive clinical assessments alongside provisions for adequate nutrition and daily physical activity. Additionally, interactions between the caregivers and the dogs promoted socialization and fostered the development of positive relationships.

During the adaptation period, oral administration of antiparasitic treatments was conducted using mebendazole (22 mg/kg; Parazol, Zagros Pharmed, Iran) and praziquantel (10 mg/kg; Lorensit, Zagros Pharmed, Iran) on Days 1 and 14.

The dogs were fed 300 g/20 kg BW dry dog food (Adult Nutripet dry dog food for moderate physical activity, Behintash Co., Tehran, Iran) and tap water ad libitum in individual pens. The diet composition included 21% crude protein, 9% fat, 3% fibre and 10% vitamins (A, E, K, B, D) and minerals. After the adaptation period, all eight dogs received a vitamin D supplement in a commercial form (D‐Vigel 1000, Daana Pharma Co., Iran) orally at a dose of 50 IU/kg BW per day. The supplement was fed with food and was placed in a small treat. After the trial, all dogs were neutered and placed in a non‐profit shelter for adoption.

2.2. Study Design

During the 42‐day study period, all eight dogs received 50 IU/kg BW vitamin D (D‐Vigel 1000, Daana Pharma Co., Iran) once daily by mouth. Venous blood samples were collected from the jugular vein on Days 0, 14, 28 and 42. A maximum blood volume of 5 mL was collected and was transferred immediately to sodium citrate, EDTA anticoagulant and plain tubes. Whole blood for platelet indices, aggregation and coagulation testing was stored at room temperature, and all assays were performed within 3 h of blood collection. The remaining whole blood was centrifuged within 30 min of collection, and the plasma was harvested for vitamin D and fibrinogen testing.

2.3. Assay for 25‐Hydroxy Vitamin D

Whole blood was centrifuged at a speed of 750 g for 10 min, following which the serum was decanted and stored at −20°C. Assays for 25‐hydroxy vitamin D were performed within 3 days using a commercial kit (Biorex Fars, Fars, Iran) according to the manufacturer's instructions.

2.4. Determination of Food Vitamin D Content

A sample of food was used for analysis of vitamin D3 content as described previously by Official Methods of Analysis (2005).

2.5. Assay for Platelet Count and MPV

Platelet count and MPV were measured using the veterinary haematology analyser (Nihon Kohden, MEK‐6450 Celltac Alpha, Tokyo, Japan) on whole blood samples containing EDTA.

2.6. Assay for Fibrinogen

The Clauss method was used to measure fibrinogen in sodium citrate plasma. The Clauss method detects the conversion of fibrinogen to fibrin in the presence of high amounts of thrombin, resulting in a quick, sensitive and precise measurement (Guven et al. 2022).

2.7. Assay for PT and PTT

PT and PTT were measured using an automatic coagulometer CC‐4000 on sodium‐citrated blood.

2.8. Generation of Platelet‐Rich Plasma (PRP)

To obtain PRP for measurement of platelet aggregation, sodium citrate anticoagulated whole blood was centrifugated with a force of 150 g for 15 min at room temperature (25°C). The PRP supernatant was then extracted, and the platelets quantified using a veterinary haematology analyser (Nihon Kohden, MEK‐6450 Celltac Alpha, Tokyo, Japan). To ensure consistent measurement of platelet aggregation, it was essential to adjust the PRP to reach a standardized platelet count of 300 × 10^9/L. In cases where the PRP platelet count was ≤300 × 10^9/L, the platelets were concentrated through a second or third centrifugation at 100 g for 15 min. If the PRP platelet count exceeded this threshold, it was diluted using platelet‐poor plasma (PPP). The PPP was obtained by centrifugation of the PRP for an extra 15 min at a force of 10,000 g.

2.9. Platelet Aggregation

Platelet aggregation was assessed using the turbidimetric technique described by Born (1962). Changes in the optical density of PRP were assessed via measurement of light transmission before and after adding adenosine 5′‐diphosphate (ADP‐Reagent, Hyphen‐Biomed) as an aggregation agonist. Measurements were carried out within 4 h of blood collection using an aggregometer (Chrono Log) with computer‐based curve analysis. The speed of the paper feed was set at 7.5 mm/min. Analysis of the aggregation curve concerning the parameter maximum aggregation (%) was calculated automatically. To measure platelet aggregation with the agonist, 200 µL aliquots of ADP were reconstituted with 200 µL of distilled water and then allowed to stabilize at room temperature for 30 min. Two measurement channel cuvettes were each filled with 500 µL PRP, and the recording of aggregation was started. After incubation for 2 min at 37°C, aggregation was induced with a final concentration of 10 µM ADP solution.

2.10. Statistical Analysis

The formula presented by Arifin and Zahiruddin (2017) was used for the calculation of sample size in an animal study using the same subjects and repeated measures—one within factor, repeated‐measures analysis of variance (RM ANOVA): [minimum sample size N = 10/(r − 1) + 1, where r is the number of measurements]. The study design required four measurements; thus, a minimum of five dogs were required (N = 4.3).

The data were entered into SPSS version 26. The normal distribution of the data was assessed and confirmed using the Shapiro–Wilk test. Mean and standard deviation (SD) of the quantitative variables were reported. Unpaired t‐test and one‐way repeated measures ANOVA tests were used to compare data among different sampling days, and Tukey's multiple comparison tests were employed to compare the data between sampling days. A two‐tailed p value <0.05 was considered significant.

3. Results

3.1. 25‐Hydroxy Vitamin D Serum Level

The results of 25‐hydroxyvitamin D concentration on Days 0, 14, 28 and 42 are shown in Table 1.

TABLE 1.

25‐hydroxy vitamin D serum concentration, platelet count, mean platelet volume, prothrombin time, partial thromboplastin time and fibrinogen comparisons of dogs (n = 8) at baseline (Day 0) and after vitamin D supplementation (Days 14, 28 and 42).

Parameter	Time point	Mean+/−SD	One‐way repeated measures ANOVA			Tukey post hoc comparisons			CI
			F	df between/within groups	p	Time point comparisons	Standard error	p
25 hydroxy vitamin D (ng/mL)	Day 0	62.12 ± 10.07d	365.9	7/4	<0.001	0–14	3.20	0.011	−27.04 to −4.13
	Day 14	78.94 ± 4.84bc				0–28	3.66	0.017	−29.48 to −3.26
	Day 28	77.70 ± 5.05c				0–42	3.23	0.001	−33.93 to −10.78
	Day 42	84.47 ± 3.22a				14–28	1.66	0.987	−6.74 to 5.17
						14–42	1.88	0.049	−13.52 to −0.013
						28–42	1.64	0.046	−11.87 to −0.097
Day 0	323.10 ± 154.70a	27.84	7/4	<0.001	0–14	38.45	0.76	−157.4 to 66.66
PLT (×1000/µL)	Day 14				368.50 ± 119a	0–28	38.45	0.98	−91.16 to 132.9
	Day 28				302.30 ± 99.21a	0–42	38.45	0.60	−56.16 to 167.9
						14–28	38.45	0.43	−45.79 to 178.3
						14–42	38.45	0.09	−10.79 to 213.3
	Day 42				244.6 ± 136a	28–42	38.45	0.89	−77.04 to 147.0
	Day 0	6.52 ± 0.73a	3.00	7/4	0.12	0–14	0.065	0.31	−0.371 to 0.096
Day 14	6.66 ± 0.61a	0–28				0.086	0.08	−0.583 to 0.033
									MPV (fL)
Day 28	6.80 ± 0.68a	0–42				0.131	0.07	−0.897 to 0.047
Day 42	6.88 ± 0.55a	14–28				0.080	0.48	−0.423 to 0.148
		14–42				0.117	0.20	−0.706 to 0.131
		28–42				0.096	0.56	−0.494 to 0.194
	Day 0	7.30 ± 1.26a	2.46	7/4	0.13	0–14	0.572	0.92	−1.596 to 2.496
PT (s)	Day 14	6.85 ± 1.04a				0–28	0.873	0.99	−3.248 to 2.99
	Day 28	7.44 ± 1.53a				0–42	0.552	0.99	−1.741 to 2.216
	Day 42	7.06 ± 1.31a				14–28	0.620	0.87	−2.795 to 1.645
						14–42	0.484	0.99	−1.948 to 1.523
			28–42			0.866	0.99	−2.739 to 3.464
	Day 0	16.93 ± 1.90a	0.22	7/3	0.80	0–14	0.884	0.99	−2.804 to 3.054
PTT (s)
	Day 14	16.24 ± 1.42a				0–28	0.787	0.93	−2.156 to 3.056
	Day 28	16.56 ± 1.20a				0–42	1.15	0.99	−4.052 to 3.577
	Day 42	16.69 ± 1.69a				14–28	0.763	0.97	−2.203 to 2.853
						14–42	0.749	0.96	−2.844 to 2.119
						28–42	0.662	0.73	−2.882 to 1.507
	Day 0	301.60 ± 172.20a	0.27	7/3	0.79	0–14	60.22	0.84	−150.6 to 248.1
Fibrinogen (mg/dL)
	Day 14	348.80 ± 178.80a				0–28	76.23	0.99	−238.6 to 266.1
	Day 28	313.80 ± 147.60a				0–42	73.50	0.83	−182.4 to 304.2
	Day 42	362.50 ± 159.80a				14–28	68.22	0.95	−260.8 to 190.8
						14–42	78.73	0.99	−248.5 to 272.7
						28–42	103.9	0.96	−296.8 to 391.1

Note: Values in columns with no common superscript letter are significantly different at p < 0.05. F = ANOVA test statistic. p = Statistical significance set at p.

Abbreviations: ANOVA, analysis of variance; df, degrees of freedom; SD, standard deviation.

Significantly, 25‐hydroxyvitamin D levels increased from 62.12 ± 10.07 ng/mL before supplementation to 84.47 ± 3.228 ng/mL on the 42nd day (p < 0.0001). Notable increases in 25‐hydroxy vitamin D serum levels were detected between Days 0 and 14 (p = 0.01), 28 (p = 0.01) and 42 (p = 0.001), and additionally between Days 28 and 42 (p = 0.04).

3.2. Vitamin D3 Content of the Food

The vitamin D3 content of the food was determined to be 72.5 µg/kg or 2900 IU/kg dry matter.

3.3. Platelet Parameters

The changes in platelet count, MPV and platelet aggregation over the 42‐day trial are shown in Table 1. A significant reduction in platelet count, from 323.1 ± 136 × 1000/µL before supplementation to 244.6 ± 154.70 × 1000/µL on Day 42 (p < 0.0001), was documented. No significant difference in platelet count was noted between the other time points. MPV increased from 6.525 ± 0.7305 to 6.886 ± 0.5551 fL on the 42nd day of the study. No significant change in MPV was noted at any time point.

3.4. Platelet Aggregation

Mean ADP‐induced platelet aggregation results for each dog before and 42 days after oral vitamin D supplementation are shown in Table 2. There was no difference in mean platelet aggregation pre‐ (34.8 ± 35.39) and post‐ (18.25 ± 26.41) ADP‐induced aggregation (p = 0.003). In seven of eight dogs, the percentage of platelet aggregation decreased with supplement administration. In the remaining dog, the percentage of aggregation on Day 42 was higher than that on Day 0.

TABLE 2.

Platelet aggregation percentage of eight dogs before and after oral supplementation with vitamin D for 42 days at 50 IU (1.25 µg) per kg body weight.

Dog number	Day 0		Day 42		Unpaired t‐test (Day 0 to Day 42 comparison)
	Platelet aggregation (%)	Mean platelet aggregation (%) +/−SD	Platelet aggregation (%)	Mean platelet aggregation (%) +/−SD	t	df	p
Dog 1	78	34.88 ± 35.39a	71	18.25 ± 26.41a	1.06	14	0.30
Dog 2	48		1
Dog 3	96		46
Dog 4	22		0
Dog 5	7		1
Dog 6	17		8
Dog 7	8		18
Dog 8	3		1

Note: Values in columns with no common superscript letter are significantly different at p < 0.05. t = Test statistic. p = statistical significance set at p < 0.05.

Abbreviations: df, degrees of freedom; SD, standard deviation.

3.5. Thrombosis Factors

A trend for initial decline followed by subsequent increase was observed for PT, PTT and fibrinogen over the 42‐day study period; however, no statistically significant differences were observed. Similarly, no significant differences were found across individual time points (Table 1).

4. Discussion

In this study, vitamin D3 supplementation at a dose of 50 IU/kg body weight daily for 42 days resulted in a significant reduction in platelet count. There was no significant change in ADP‐induced platelet aggregation, MPV, fibrinogen, PT and PTT.

Consistent with these results, previous research has suggested that vitamin D supplementation can reduce platelet counts. Alanli et al. (2020) showed a significant negative correlation between 25‐hydroxyvitamin D3 levels and platelet counts in patients with severe 25‐hydroxyvitamin D3 deficiency, and lower vitamin D concentrations were associated with higher platelet counts. A study from Korea demonstrated a significant negative correlation between serum vitamin D concentration and platelet counts. This relationship was attributed to the anti‐thrombogenic, anti‐inflammatory, antioxidant and anticoagulant properties of vitamin D (Park et al. 2017). This supports the theory that vitamin D inhibits the maturation of megakaryocytes and the generation of thrombocytes via activating VDR (Silvagno et al. 2010; Song 1996).

MPV, a parameter associated with platelet size and activation, correlates with platelet function markers such as aggregation, thromboxane synthesis, β‐thromboglobulin release and adhesion molecule expression. The association between MPV and vitamin D levels has been variable across previous studies, and further research is needed to determine if prolonged supplementation affects platelet reactivity (Alanli et al. 2020). Some studies have found an inverse relationship between MPV and vitamin D levels, whereas others have found no association between the two variables. For example, Talebzadeh et al. (2024) found that there was a negative correlation between MPV and vitamin D levels in COVID‐19‐infected patients, whereas Cure et al. (2014) and Alanli et al. (2020) found no association between vitamin D insufficiency and MPV in healthy adults. They suggested that these discordant results could be attributed to variations in measuring procedures, sample storage times and/or the choice of anticoagulant (Alanli et al. 2020; Cure et al. 2014); however, innate differences in the effects of vitamin D in healthy versus diseased populations should also be considered. In the current study comprising healthy dogs, there was no significant change in MPV following vitamin D supplementation, but a debatable trend for increasing MPV was noted. The effect of vitamin D supplementation on MPV in diseased dog population thus warrants additional investigation.

No significant changes were detected in ADP‐induced platelet aggregation, fibrinogen levels, PT or PTT. Previous studies have demonstrated an inverse relationship between serum vitamin D concentration and platelet aggregation suggesting that vitamin D may play a role in maintaining optimal platelet function and reducing the risk of excessive clot formation (Vuthaluru and Goyal 2022; Park et al. 2023; Johny et al. 2022). Vuthaluru et al. (2022) measured platelet aggregation using a chronolog aggregometer and PRP, induced by ADP and epinephrine. They found that lower vitamin D levels were associated with increased platelet aggregation suggesting that vitamin D supplementation might reduce thrombotic complications in hypertensive patients, thereby improving morbidity and mortality outcomes (Vuthaluru and Goyal 2022). However, our study yielded no evidence to support this hypothesis. Consistent with our findings, one study examined the impact of vitamin D supplementation on serum fibrinogen concentrations in adults aged 64 and older. They suggested that vitamin D supplementation does not influence fibrinogen concentrations (Kattula et al. 2017) or on coagulation profiles in women with polycystic ovary syndrome. They found no significant differences in coagulation or anticoagulation protein levels between patients with vitamin D deficiency and those with sufficient vitamin D, indicating that vitamin D deficiency does not increase the risk of coagulopathy in individuals with polycystic ovary syndrome (Moin et al. 2021). Moinuddin et al. (2017) demonstrated that vitamin D supplementation does not significantly influence the coagulation profile (PT, PTT, fibrinogen and platelet count) in healthy adults. Their results indicate that routine vitamin D administration does not affect conventional coagulation parameters in this population (Moinuddin et al. 2017). A study was conducted to investigate the association between serum 25‐hydroxyvitamin D levels and platelet activity among patients with a history of acute coronary syndrome. The results demonstrated that there was no significant correlation between serum 25‐hydroxyvitamin D concentration and platelet activity indices in this patient (Dziedzic et al. 2022).

However, various human studies have provided evidence that vitamin D supplementation can have an effect on coagulation factors in patients with vitamin D deficiency. In one study, it was observed that high‐dose vitamin D supplementation (one‐time 300,000 IU followed by 800 IU daily) was associated with reduced thrombin production in individuals with severe vitamin D deficiency (25‐hydroxyvitamin D3 < 25 nmol/L) suggesting that high‐dose cholecalciferol supplementation could reverse a prothrombotic phenotypes, associated with severe vitamin D deficiency (Barnes et al. 2010). Further studies are therefore required in vitamin D‐deficient canine populations.

Limitation of this study includes the lack of a control population or cross over design. Given the paucity of previous canine studies, the optimal vitamin D supplementation dose is also unclear, and studies using incremental doses would help to ascertain whether more aggressive supplementation could lead to larger effects. Indeed, mean serum 25‐hydroxyvitamin D concentrations were within normal limits both at baseline and on Day 42 of the study (Hazewinkel and Tryfonidou 2002; Selting et al. 2016; Alizadeh et al. 2022). Similarly, studies employing longer durations of supplementation are warranted.

A further limitation is that platelet aggregation was investigated using a single agonist, and it is advisable to employ agonists of varying concentrations in subsequent investigations.

5. Conclusions

This pilot study provides early evidence that vitamin D administration can reduce platelet numbers in healthy dogs. Additional studies are required to confirm this finding and to assess its clinical relevance and possible therapeutic applications.

Author Contributions

All authors have seen and approved the final version of the manuscript being submitted. All authors contributed to all aspects of the study from designing the study to writing and preparing the manuscript and contributed to study design, performing the study, sampling, laboratory metabolites analysis, data collection and analysis and preparing the manuscript.

Ethics Statement

The ethical approval of this study was sought from supervision of the Iranian Society for the Prevention of Animal Cruelty and the Research Council of Shiraz University (IACUC No. 6387/63).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.70530.

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.