**5.2 Blood glucose and platelet functions in T1DM**

Although platelet hyperactivity in T1DM has partly been ascribed to hyperglycemia, however, empirical evidence has been conflicting. Study results have ranged from no effects, decreased to increased platelet functions. The ambiguity may be attributed to confounders, depending on study designs, dose, and duration of exposure. Specifically, non-diabetic or healthy controls do not have the coexisting inflammatory biomarker seen in diabetics (T1DM from T2DM) who additionally have hyperlipidemia. Thus, in diabetics, duration of disease determines the level of nonenzymatic glycation. The route of glucose administration also matters. Oral intake

is accompanied by post-prandial secretion of gut and counter regulatory hormones while direct or parenteral administration bypasses the gut, with *in vitro* mixing with platelet-rich plasma excluding the leukocytes contribution.

Bridges and coworkers [19] first demonstrated the link between blood glucose and platelet function. They showed that either a postprandial oral glucose load or intravenous infusion increased platelet stickiness in both diabetics and nob-diabetics. The observation has since been confirmed by others mostly in T2DM [68–71]. Surprisingly, in one study of hyperglycemia platelet p-selectin expression was decreased during hyperglycemia compared with levels before oral intake [72]. These results are confounded other factors concurrently activating platelets such as postprandial hyperglycemia on counter regulatory hormones [73], procoagulant platelet microparticles [74], combined effect with insulin [75, 76], neutrophil-megakaryocyte thrombopoiesis [16], and hyperlipidemia [25, 77, 78].

In order to avoid the many confounders inherent in *in vivo* hyperglycaemia, *in vitro* studies designed to define the particular role of glucose have yielded mixed results. It has been shown that *in vitro* incubation of healthy PRP platelets with glucose upto 50 mmol has no effect on agonist-induced aggregation to ADP, AA or collagen, secretion of MDA or TXB2 [79] indicating no direct effects on platelets. Many *in vitro* studies involving short incubation periods with varying concentrations of glucose have shown no effects [80, 81]. However, studies have shown that glucose paradoxically increases platelet aggregation or surface P-selectin expression depending on the type of agonist [82–85].

The discordance in effects of glucose on platelets can be explained by the duration of exposure. While acute or short duration of exposure is associated with no or minimal effects, longer duration or chronic exposure stimulates thrombopoiesis in which reticulocytes increase adhesive receptors and are hyperactive to agonists [16]. The duration of glucose exposure correlates with HbA1c levels [86]. Contrary to popular belief, hyperactive platelets are evident even before observable metabolic features in individuals predisposed to T1DM with normal blood [41]. Early evidence of the influence of glucose on platelet functions via metabolic reactions was provided by Murer et al. [87] and Chaudhry et al. [88]. The two groups reported the presence of aggregation in media containing glucose or substitute substrates, the extent of which was related to glucose consumption and carbon dioxide production due to metabolism. In these experiments, addition of metabolic inhibitors inhibited aggregation. Moreover, alterations of membrane properties and energy requiring processes have been noted upon incubation of platelets with varying glucose concentrations [18] suggesting other mechanisms are at play.

Several preclinical animal studies have confirmed the dependency of platelet functions on glucose uptake and subsequent metabolism [33, 89]. Under resting conditions, the plasma membrane localized GLUT-1 glucose transporter facilitates glucose entry into platelets, activation glycolysis, and ATP production [90]. Upon stimulation with agonists such as thrombin, alpha granules containing both P-selectin and GLUT-3 translocate and fuse with plasma membrane to increase glucose uptake [91]. The resulting alpha granules degranulation, glucose influx, and increased ATP synthesis lead to P-selectin expression and aggregation [33, 92].

It appears that hyperglycemia in itself has little bearing on platelet activation unless accompanied by other changes. In a study of T1DM and T2DM, despite postprandial elevation of blood glucose in both types, however, markers of platelet activation were only increased in T2DM. Interestingly, insulin levels did not change in T1DM unlike in T2DM. Administration of insulin pre-meal was accompanied

by increase of postprandial platelet activation markers [74, 76, 93]. However, platelet-activating effects of insulin in T1DM are evident only when there is concurrent hypoglycemia [94]. At low extracellular glucose, insulin potentiates action of thrombin to increase glucose entry via Glut-3 with concomitant increase in surface expression of P-selectin and aggregation. Conversely, at high glucose concentrations, insulin becomes inhibitory through its action on protein kinase B (PKB) and hampers thrombin activation mechanisms leading to decreased platelet activation and aggregation [92].
