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The interpretations below are provided by Donna Castellone, MS, MT (ASCP) SH for Aniara Diagnostica, Inc.

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INTRODUCTION:

There are approximately 451 million adults diagnosed with diabetes1. Diabetes is a metabolic disease composed of dyslipidemia, elevated BP, insulin resistance, elevated glucose, a pro-inflammatory state and pro-thrombotic state.² Metabolic disorders such disturb the balance of coagulation and fibrinolysis. This results in a hypercoagulable state resulting in platelet hypersensitivity, coagulation, and hypo fibrinolysis. This is caused by increased glucose levels and insulin resistance resulting in changes in platelet number and activation. Due to the issues of coagulation and fibrinolytic factors the formation of fibrinolytic resistant clots is seen in patients with diabetes. Hypoglycemia, obesity, and dyslipidemia also add to these disorders.¹

The goal of hemostasis is the formation of fibrin clots to stop bleeding as well as the initiation of the fibrinolytic system to resolve the clots to stop widespread thrombus formation and vascular occlusion.

COAGULATION FACTORS:

Coagulation factors in these patients have seen an alteration of both qualitative and quantitative factors. Both hyperglycemia and insulin resistance have demonstrated an effect on the Tissue Factor (TF) pathway resulting in increased FVIIa consumption. Recent studies highlight the role of micro-RNA (miRNAs) in TF expression and diabetes-related coagulation dysfunction. This may inhibit the TF expression in endothelial cells which may possibly result in enhanced vascular TF activity when diabetes is poorly controlled.¹

The intrinsic pathway which involves activation of FXII, FXI and FIX. A study from the Netherlands' Epidemiology of Obesity study showed that increases in FVIII (5.33%, 95%CI: 4.00–6.65), FIX (6.19%, 95%CI: 5.15–7.23) and FXI (2.11%, 95%CI: 1.20–3.02) were observed in fasting plasma glucose. There was also a shorter aPTT seen in patients with impaired insulin sensitivity. Higher levels of fibrinogen were seen in obesity, dyslipidemia and inflammation and may be a contributing factor. Enhanced oxidative stress and sustained inflammatory reactions in patients having DM also alter the structure of fibrinogen and fibrin, thereby leading to fibrinolysis-resistant clots.¹

In a study that looked at 4026 patients with diabetes mellitus (3312 survived after admission to the hospital and 714 died) different results were seen in different groups. A database review found 10 laboratory tests with the strongest association with mortality and included aPTT, platelet count, and the INR. Patients >70 years have higher PT and then a higher risk of mortality, but there was a protective factor when the aPTT level was 31.3-39.6 s. In patients <70, the PT level of 13.2-14.3 s and the aPTT level of 16.9-26.8 s had a higher risk of mortality. Those patients on anticoagulant showed a decreased risk of mortality.²

Clotting factors are elevated in DM patients, such as fibrinogen, factor VII, factor VIII, factor XI, factor XII, von Willebrand factor and kallikrein. While there was a decrease in anticoagulant factor of plasma protein C.3 Some research observed that pro-coagulation biomarker or D-dimer is increased in diabetes patients with microvascular and macrovascular complications. A proinflammatory and procoagulant state can result in endothelial dysfunction and vascular disease.²

Another study (n=84) looked at HbA1c levels and compared coagulation results. Those with HbA1c ≥7 revealed marked variations in coagulation profile when compared to Type 2 Diabetics with HbA1c < 7. Changes were noted in the PTT, fibrinogen and bleeding time. Statistically significant results were seen in those patients with a HbA1c >7 including BT and platelet count. It is believed the platelet count is increased due to the thrombopoietin and nitric oxide produced in diabetes. In this study, no significant changes in PT were noted between the levels of HbA1c (mean 10.23 sec). This is contrary to what has been seen in other studies. Fibrinogen levels were also increased.³ There was no significant variation in DDimer levels of patients (560.01 ng/mL) and (675.34 ng/mL). One study showed that DDimer showed a gradual increase based on the progression from pre-diabetes to diabetes with complications due to the increased formation of fibrin clots and their breakdown.³

There is a lack of reliable coagulation markers to evaluate the vascular complication of diabetes.⁴ Normal vascular endothelium has anticoagulant and antiplatelet functions such as heparin sulfate proteoglycan, thrombomodulin (TM) and antithrombin. Having an imbalance of phosphorylation and dephosphorylation in DM, hyperhomocysteinemia, hypercholesterolemia, or hyperuricemia patients could cause dysfunction of endothelium by modulation of vascular endothelial L-arginine/nitric oxide synthetase.⁵

Routine coagulation testing only measures the time to clot formation which is less than 5% of thrombin generation. Global assays which look at the final components of the coagulation cascade may provide a more complete assessment of the hemostatic profile.⁴

A study looked at 147 adult patients with diabetes. Thromboelastography (TEG), thrombin generation using calibrated automated thrombogram using the CAT assay, and fibrin generation and fibrinolysis using the overall hemostatic potential (OHP) assay. The results were compared with healthy controls. Patients with diabetes when compared to 153 healthy controls showed higher maximum amplitude (68.6 vs 60.2 mm, p < 0.001) on TEG, and higher OHP (9.3 vs 6.4, p < 0.001) with comparable CAT parameters. Patients with T2DM were more hypercoagulable than those with T1DM on most biomarkers. Higher maximum amplitude, velocity index, and OHP were associated with increased risk of complications (C-stat 0.82). Patients with history of microvascular complications appear to have more hypercoagulable thrombin and fibrin generation than without.

This study highlighted the increased hypercoagulable profile in patients with diabetes and those with microvascular complications, which appear to be even more hypercoagulable.⁴

PLATELETS:

Platelets circulate for five to seven days and a normal count is 150.000–450.000 per microliter. After an injury, platelets activation to aggregate, form occlusive thrombosis and stop bleeding. Hypersensitivity results in an increased number and enhanced aggregation which contributes to a thrombotic state. In patients with diabetes platelet counts are increased by about 10% as well has an increased MPV are seen in patients with a glycated hemoglobin, HbA1c > 8%1. A possible mechanism may be an upregulated expression of pro-aggregatory factors like P-selectin, thromboxane A2 and von Willebrand factor (vWF) antigen, amplifying the aggregation and adhesion of platelets.⁶

The signalling and factors that are involved with platelet hyperresponsiveness and coagulation factors are essential to understand and to be able to target the prothromotic state while preserving hemostasis. Insulin resistance may play a role in coagulation dysfunction due to the binding of insulin receptors on platelet surfaces resulting in an activation of downstream pathways which result in higher adenosine monophosphate (cAMP) generation and lower intracellular calcium inside platelets, thereby inhibiting their aggregation. Also, drugs used to treat diabetes may also change gut microbiome and contribute to platelet responsiveness.¹

There is also the deterioration of the physiologic reaction of platelets due to the anti-aggregatory effects of nitric oxide (NO), prostaglandin I2 (PGI2) and insulin by interfering with downstream signalling pathways. They also upregulate the adhesion molecules on the surface of the platelet such as CD31, CD49b and CD63, which is reversable when glucose is under control1. Protease-activated receptor 4 also promotes the release of activated platelet-derived microparticles (PMPs) via the Ca2+-calpain pathway. Released PMPs then trigger the secretion of interleukin-6, a pro-thrombotic and pro-inflammatory mediator in diabetes.⁷

FIBRINOLYTIC SYSTEM:

Alterations in the fibrinolytic system have also been attributed to diabetes. The precursor to plasmin is plasminogen. Tissue plasminogen activator (tPA), the key catalysator of plasmin generation, which then initiates fibrinolysis and restrains excessive thrombus formation. A negative correlation of tPA was associated with HbA1c. Higher glucose levels were found to inhibit tPA leading to reduced plasmin levels. Also, due to plasminogen being a pro-inflammatory factor, and elevated in diabetes results in a prothrombotic state.⁸ Hyperglycemia also leads to the elevation of plasminogen activator inhibitor-1 (PAI-1) and thrombin-activator fibrinolysis inhibitor (TAFI). These are inhibitors the prevent plasminogen to plasmin. The increase in these factors lead to a reduction in the availability of these fibrinolytic factors.¹

OTHER CONTRIBUTING FACTORS:

Hypoglycemia is associated with an increased thrombosis formation based on platelet activation, and alteration of coagulation factors and impaired fibrinolysis.¹ Obesity is related to an increased hazard ratio for thrombosis of 3.4. This mechanism may be caused by the increased number and size of adipocytes in obese individuals, accompanied by enhanced secretion of TF and PAI-1, finally leading to hyper-coagulation and hypo-fibrinolysis.⁹

Patients with dyslipidemia have demonstrated higher fibrinogen levels and shortened aPTTs. This may be due to increased blood lipids which can impair the function of hepatic cells which is where coagulation factors are produced.

TREATMENT:

Antiplatelet medication such as aspirin is used to prevent the generation of prostaglandin H2 from arachidonic acid, thus inhibiting thromboxane A2 formation and platelet activation since it binds irreversibly to cyclooxygenase-1 (COX-1). This is the most widely used drug in patients with diabetes and a history of cardiovascular disease.

CONCLUSION:

It is important to understand the impact that diabetes has on coagulation factors, platelets and the fibrinolytic system. Testing should be part of a workup in diabetes patients. Knowing the risks to the hemostatic system in diabetic patients and being aware of the possible abnormalities in coagulation testing can aid in treatment as well as the possibility of alternative diagnosis.

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REFERENCES

1. Xiaoling Li, Nina C Weber, Danny M Cohn, Markus W Hollmann, J Hans DeVries, Jeroen Hermanides, Benedikt Preckel, Effects of Hyperglycemia and Diabetes Mellitus on Coagulation and HemostasisJ Clin Med, 2021 May 29;10(11):2419, https://pmc.ncbi.nlm.nih.gov/articles/PMC8199251/
2. Yingxin Huang, MM https://orcid.org/0000-0002-8854-4007, Zhihua Zhong, MSc, and Fanna Liu, PhD, The Association of Coagulation Indicators and Coagulant Agents With 30-Day Mortality of Critical Diabetics, Clinical and Applied hemostasis, July 2021 https://journals.sagepub.com/doi/full/10.1177/10760296211026385
3. BiIniIa SheriIn, Bhuvanamha DeviI Rramamurthy, Mmuthu SuDalaImuthu, ShIvaShekar Shivashekar GanapPathy, Comparison of Coagulation Profile in Type 2 Diabetic Patients with Good Glycaemic Control and Poor Glycaemic Control, Journal of Clinical and Diagnostic Research. 2020 Jul, Vol-14(7).
4. Hui Yin Lim, Brandon Lui, Mark Tacey, Anna Kwok, Suresh Varadarajan, Geoffrey Donnan, Harshal Nandurkar, Prahlad Ho Global Coagulation assays in patients with diabetes mellitus
Res Pract Thromb Haemost. 2021 Nov 8;5(7)https://pmc.ncbi.nlm.nih.gov/articles/PMC8576266/
5. Jamwal S., Sharma S. Vascular endothelium dysfunction: a conservative target in metabolic disorders. Inflamm Res. 2018;67(5):391–405
6. Zaccardi F., Rocca B., Rizzi A., Ciminello A., Teofili L., Ghirlanda G., De Stefano V., Pitocco D. Platelet indices and glucose control in type 1 and type 2 diabetes mellitus: A case-control study. Nutr. Metab. Cardiovasc. Dis. 2017;27:902–909
7. Giannella A., Ceolotto G., Radu C.M., Cattelan A., Iori E., Benetti A., Fabris F., Simioni P., Avogaro A., Vigili de Kreutzenberg S. PAR-4/Ca(2+)-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes. Cardiovasc. Diabetol. 2021;20:77.
8. Canecki-Varzic S., Prpic-Krizevac I., Bilic-Curcic I. Plasminogen activator inhibitor-1 concentrations and bone mineral density in postmenopausal women with type 2 diabetes mellitus. BMC Endocr. Disord. 2016;16:14.
9. Christiansen S.C., Lijfering W.M., Naess I.A., Hammerstrom J., van Hylckama Vlieg A., Rosendaal F.R., Cannegieter S.C. The relationship between body mass index, activated protein C resistance and risk of venous thrombosis. J. Thromb. Haemost. 2012;10:1761

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