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K. Jimenez et al.
theorized that there could be a defect in von Willebrand factor, and immunohistochemical staining of arterial thrombi did appear to be patchier in Def rats than in Con rats (Online Supplementary Figure S8A), which was not the case for venous thrombi (Online Supplementary Figure S8B).
In summary, baseline platelet activity was higher in ID, and platelet response to thrombin and clot dynamics were also augmented. Iron replacement therapy normalized these changes. Platelet aggregation to collagen and ADP and static platelet adhesion appeared to be similar across the groups. Platelet adhesion under shear flow appeared to be impaired, which could explain why occlusion times in the arterial model were similar across the groups. However, the net effect resulted in a greater thrombus size in both venous and arterial models.
Discussion
In this study we used animal models to investigate thrombotic tendency in ID. We found that IDA increases platelet numbers, blood coagulability, as well as baseline platelet activity. While these effects were protective, by augmenting hemostasis in response to tail injury, they also led to larger thrombi in both venous and arterial models of thrombosis. Alterations increasing the thrombotic tenden- cy reversed upon iron replacement therapy, supporting the notion that IDA is a prothrombotic state.
We showed that platelet counts are consistently elevat-
ed in iron-deficient rats, confirming our previous find-
ings,16 and further showed that these changes are
reversible. Similar ID-induced thrombocytosis is common
in humans and iron replacement therapy has been shown
to reduce platelet counts in various groups of patients,
including those with inflammatory bowel disease or
chronic kidney disease, and even in iron-depleted blood donors.18,19,26-28
We have also shown that IDA in the absence of any other confounding illness can increase thrombus size in both arte- rial and venous animal models of thrombosis. While we are limited by current models in which thrombosis is induced rather than spontaneous, numerous clinical studies have found that ID may indeed be a risk factor for thrombotic events.7-13 In clinical studies, a large thrombus burden con- tributes to poor clinical outcome by increasing recurrence or mortality.29-31 As anemia and ID itself are known to be detri- mental in cardiovascular disease,3 the alterations in platelet numbers and function could be other aspects of ID that contribute to adverse sequelae.
ID led to an increase in some parameters of the in vitro assays. Clot dynamics were augmented by ID, with an increase in clot formation as well as clot strength. Similar results were found in children, in whom IDA increased clot firmness on rotational thromboelastometry.32 Another study using a thrombin generation assay also found a decrease in coagulability in response to iron replacement therapy.33 This was likely due to an increase in the contri- bution of platelets to clot formation, which is also reflect- ed by the increased platelet area seen on thrombus immunohistochemistry.
Baseline expression of P-selectin by platelets was also higher in ID, which suggests that circulating platelets are already partially activated. Stimulation with thrombin, but not ADP, increased P-selectin expression further. Baseline expression in control and iron-treated animals
was similar, and the response to thrombin was diminished in comparison to that of animals with ID but still higher than that of controls. Our group previously showed that baseline and ADP-stimulated platelet P-selectin expression was higher in patients with inflammatory bowel disease who had ID than in those after iron replacement therapy.19 In another study on children with IDA, P-selectin was likewise elevated on agonist stimulation.34 Apart from platelet P-selectin, we also found in the current study that plasma levels of soluble P-selectin were increased in ID and diminished upon iron replacement therapy. Increasing soluble P-selectin by genetic engineering in mice leaves the animals in a procoagulant-prothrombotic state.35,36 This is relevant, as thrombocytosis and soluble P-selectin are predictive of VTE in cancer patients,37 who may also suffer from functional ID and anemia of chronic disease.
We did not find a significant difference in platelet aggre- gability to ADP or collagen in ID, although our previous study showed increased aggregability to ADP in platelets from rats after 2 weeks on an iron-deficient diet; this aggregability then diminished by 3 weeks.16 Several stud- ies of patients have actually found impaired aggregability to ADP or collagen in IDA,34 which improved with iron replacement therapy.38-40 This occurred despite an increased platelet count in ID in some studies.38,39 We found only one study that recorded an increase in aggre- gability.41 This could be due to methodological differences, as platelets resuspended in plasma were used in the previ- ous animal study,16 whereas we used whole blood in order to mimic the intravascular environment better. However, it could also be due to differences in timing: perhaps aggregability increases in early ID, and diminishes over time.
Remarkably, we found impaired adhesion of platelets from iron-deficient animals under flow (as determed using the PFA-100), with no impairment in static adhesion tests. While hematocrit influences PFA-100 results, none of the samples tested exceeded the 25% cutoff,42 suggesting that the results may be due to a functional defect of the platelets. Similar results have been found in children with IDA, whose PFA-100 closure times were significantly longer than those of controls.34,43 This defect in adhesion under flow could explain why there were no differences in occlusion times, as the model of arterial thrombosis is sub- ject to high shear flow. Nevertheless, both hemostasis and arterial thrombus size were augmented in ID. Perhaps the defect is compensated for by the increased platelet count.
We hypothesize that apart from increased platelet counts, there is also onging platelet activation in ID, result- ing in an overall increased thrombotic tendency. Results of standard in vitro tests may be incongruous, but those of the thrombosis models are quite distinct. In one study, the risk of VTE in cancer patients was associated with a reduction in platelet responsiveness to PAR-1 and GPVI agonists.44 Further studies are needed to better define how ID influ- ences platelet function, and what tests may be appropriate in this context. It is also unclear how ID augments platelet function, and whether this is a general feature of reactive thrombocytosis, or due to something specific to ID.
In conclusion, our study is the first to show that ID alone is sufficient to increase thrombotic tendency. ID consistently increases platelet counts, modulates platelet function, and increases thrombus size in both arterial and venous models of thrombosis. Iron replacement therapy reverses these changes, making it a viable strategy for the
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haematologica | 2021; 106(3)