The severity and duration of Hypoglycemia affect platelet-derived protein responses in Caucasians

Abstract

Objective: Severe hypoglycemia is associated with increased cardiovascular death risk, and platelet responses to hypoglycemia (hypo) have been described. However, the impact of deep transient hypo (deep-hypo) versus prolonged milder hypo (mild-hypo) on platelet response is unclear.

Research design and methods: Two hypo studies were compared; firstly, mild-hypo in 18-subjects (10 type-2-diabetes (T2D), 8 controls), blood glucose to 2.8mmoL/L (50 mg/dL) for 1-hour; secondly deep-hypo in 46-subjects (23 T2D, 23 controls), blood glucose to < 2.2mmoL/L (< 40 mg/dL) transiently. Platelet-related protein (PRP) responses from baseline to after 1-hour of hypo (mild-hypo) or at deep-hypo were compared, and at 24-hours post-hypo. Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement was used to determine PRP changes for 13 PRPs.

Results: In controls, from baseline to hypo, differences were seen for four PRPs, three showing increased %change in deep-hypo (Plasminogen activator inhibitor-1(PAI-1), CD40 ligand (CD40LG) and Protein-S), one showing increased %change in mild-hypo (von Willebrand factor (vWF)); at 24-hours in controls, %change for Protein-S remained increased in deep-hypo, whilst % change for vWF and plasminogen were increased in mild-hypo. In T2D, from baseline to hypo, differences were seen for 4 PRPs, three showing increased %change in deep-hypo (PAI-1, platelet glycoprotein VI and Tissue factor), one showing increased %change in mild-hypo (CD40LG); at 24-hours in T2D, %change for CD40LG remained increased, together with vWF, in deep-hypo.

Conclusion: Both mild-hypo and deep-hypo showed marked PRP changes that continued up to 24-hours, showing that both the severity and duration of hypoglycemia are likely important and that any degree of hypoglycemia may be detrimental for increased cardiovascular risk events through PRP changes.

Trial registration: ClinicalTrials.gov NCT02205996 NCT03102801.

Keywords: Hypoglycemia; Inflammation; Platelet-associated proteins; Type 2 diabetes.

Conflict of interest statement

None of the authors have any conflict of interest to declare.

No authors have any conflict of interest or competing interests to declare.

© 2022. The Author(s).

Figures

Fig. 1

 A schematic illustrating the pathways and proteins involved in the natural mechanism that prevents blood from clotting, the activated platelet response and the degradation of fibrin clots. Created with Biorender.com  A.Natural anti-coagulation mechanism. In normal condition, nitric oxide (NO) and prostacyclin (PGI2) secreted from endothelial cells inhibit platelets, keeping them inactive and preventing them from binding to the endothelial lining. Platelet coagulation factors are also inactivated by antithrombin III (ATIII), a protein bound to endothelial cells by a glycose immune glycan (heparin sulfate) and activated protein C (APC) in association with protein S. ATIII cleaves circulating clotting factors (F-II, F-IX, F-X) and inactivates them. Protein C is activated by thrombin (F-II) bound to thrombomodulin (TM) in the endothelial cells. APCs cleaves circulating clotting factors (F-V, F-VIII) and inactivate them B.Platelet activation and aggregation. In response to damage to endothelial cells, circulating platelets migrate to the site of injury and bind to a protein, Von Willebrand factor (vWF), produced by endothelial cells through another platelet-surface protein glycoprotein 1b (GP1b) that activates the platelets. Activated platelets release granules containing adenosine di-phosphate (ADP) and thromboxane A2 (TXA2) which bind to their respective receptors expressed in platelets, allowing more platelets to migrate and form clusters at the site of injury, a process called ‘platelet aggregation’ through which ‘platelet-plaque’ is formed at the injury site. Platelet activation also allows the membrane translocation of CD40 ligand (CD40LG). The translocation of CD40LG seems to coincide with the release α-granule contents, including platelet-derived growth factor (PDGF), transforming growth factor beta (TGFβ) and platelet factor 4 (PF4). The surface-expressed CD40LG is cleaved and shed from the platelet surface in a time-dependent manner as sCD40LG. Platelet-endothelial interactions also promote progressive plaque, and this interaction is facilitated by rolling and adhering of activated platelets to the endothelial cell surface. Rolling of platelets to endothelial cells is mediated by platelet P-selectin glycoprotein ligand − 1 (PSGL-1) binding to endothelial cell P-selectin C. Fibrinolytic system. The final step of the blood homeostasis system involves the natural breakdown of the blood clot (platelet-fibrin plaque). Endothelial cells express a protein tissue plasminogen activator (TPA), which converts plasminogen to plasmin. Plasmin degrades the fibrin mesh in the platelet-fibrin plaque and releases fibrinogen and D-dimer. TPA is inhibited by an endothelial plasminogen activator inhibitor called plasminogen activator inhibitor-1 (PAI-1). D. Cascade pathways of blood coagulation system. The platelet surfaces in the platelet-fibrin clot (shown separately with broken, red-dotted lines) contain the phosphatidyl serine groups which are negatively charged. Coagulation factor XII, secreted from the liver, binds to the negatively charged surface and this contact triggers a conformational change in the XII zymogen, inducing autoactivation (contact activation). The activation leads the cascade pathway activation of XI, IX, VIII (intrinsic pathway). Tissue damage activates coagulation factors III and VII (extrinsic pathway) and, finally, both pathways activate factor X (common pathway) which leads to the conversion of prothrombin to thrombin. Thrombin converts fibrin polymer (insoluble) from fibrinogen monomers (soluble) and, with the help of XIII, fibrin finally forms a fibrin mesh to coagulate blood (with aggregated platelets at the site of injury) E. Comparison of % change of platelet activation-related proteins in type 2 diabetes. The table shows the % change of platelet activation-related proteins in response to mild and deep hypoglycemia in control subjects (C) and in subjects with type 2 diabetes (T2D). The upward red arrows indicate a % increase, and the downward blue arrow indicates a % decrease Hypo, hypoglycemia; 24 h-p-hypo, 24 h post-hypoglycemia; PF3, Platelet factor 3; C, control subjects; T2D, type 2 diabetes subjects

Fig. 2

Comparison of percent (%) changes of protein levels in response to hypoglycemia in two different prospective studies in CONTROL subjects. Line graphs showing changes as percentage of six platelet activation related proteins, Plasminogen activator inhibitor 1 (PAI-1) (A) Soluble CD-40 ligand (B), Vitamin K-dependent protein S (C), Von Willebrand factor (D), Plasminogen (E), D-Dimer (F) from baseline (BL) to hypoglycemia and to 24 h post-hypoglycemia in study 1 (open white square) and study 2 (open white circle). Baseline protein levels were normalized to 1 to show the % change from baseline to subsequent timepoints. PAI-1 and soluble CD-40 ligand showed a significant differential percentage change from BL to hypoglycemia. Vitamin K-dependent protein S and Von Willebrand factor showed a significant differential percentage change from BL to hypoglycemia and from BL to 24 h post-hypoglycemia as well. Plasminogen and D-Dimer showed significant differential percentage change from BL to 24 h post-hypoglycemia only. Two-way arrows in the graphs indicate the duration of hypoglycemia for study 1. Data are presented here as mean % Change of proteins ± SEM. *p < 0.01, ***p < 0.001, % change of BL to hypo between study 2 vs. study 1 in control; #p < 0.05, ###p < 0.001, % change of BL to 24 h post-hypoglycemia between study 2 vs. study 1 in control. BL, baseline; Hypo, hypoglycemia

Fig. 3

Comparison of percent (%) changes of protein levels in response to hypoglycemia in two different prospective studies in T2D subjects. Line graphs showing changes as percentage of six platelet activation related proteins, Platelet glycoprotein VI (A) Plasminogen activator inhibitor 1 (PAI-1) (B), Tissue factor (C), Soluble CD-40 ligand (D), Von Willebrand factor (E), D-Dimer (F) from baseline (BL) to hypoglycemia and to 24 h post-hypoglycemia in study 1 (open blue square) and study 2 (open blue circle). Baseline protein levels were normalized to 1 to show the % change from baseline to subsequent timepoints. Platelet glycoprotein VI and Plasminogen activator inhibitor 1 (PAI-1) showed a significant differential percentage change from BL to hypoglycemia. Tissue factor and Soluble CD-40 ligand showed a significant differential percentage change from BL to hypoglycemia and from BL to 24 h post-hypoglycemia as well. Von Willebrand factor and D-Dimer showed significant differential percentage change from BL to 24 h post-hypoglycemia only. Two-way arrows in the graphs indicate the duration of hypoglycemia for study 1. Data are presented here as mean % Change of proteins ± SEM. $p < 0.01, % change of BL to hypo between study 2 vs. study 1 in T2D; &p < 0.05, % change of BL to 24 h post-hypoglycemia between study 2 vs. study 1 in T2D. BL, baseline; Hypo, hypoglycemia