Divergent Effect of Insulin On Whole-Blood Tissue Factor Procoagulant Activity in the Presence and Absence of Lipopolysaccharide

Individuals with diabetes mellitus (DM) have accelerated atherosclerosis and are prone to acute vascular events. Both hyperglycemia (HG) and hyperinsulinemia (HI) are independent risk factors for mortality in Type 2 DM (T2DM). Tissue factor (TF) is the principal initiator of blood coagulation. There is a circulating pool of TF in blood, which is thrombogenic. We have shown that membrane-bound TF levels are elevated in T2DM (J Clin Endocrinol Metab 2007, 92; 4352–8), and T1DM (Diabetes Care, 2012;35:1322–7). Infusion studies in normal healthy subjects showed that HG (∼200 mg/dl), HI and combined HG+HI elevated whole blood TF-procoagulant activity (TF-PCA) by 2, 6 and 9-fold, respectively (Diabetes 2006, 55; 202–8). In T2DM patients, the elevation by HI was 30%, and by HG+HI was 80% (J Clin Endocrinol Metab 2007, 92; 4352–8). However, in contrast to the findings in T2DM, our studies in T1DM patients (Diabetes Care, 2012;35:1322–7) showed that neither acutely raising blood glucose or insulin for 24 h, nor raising blood glucose together with insulin for up to 24 h increased TF-PCA. In fact, TF-PCA levels decreased during combined HG and HI and selective HG. To better understand the roles of HG and HI on TF-PCA, we studied their effects in vitro in whole blood from healthy individuals under both unstimulated and lipopolysaccharide (LPS)-stimulated conditions; the latter was done because patients with T2DM have evidence of platelet/monocyte activation as well as elevated plasma LPS levels.

Glucose (200 mg/dL, HG) and insulin (1, 10 or 100 nM, HI) were added either alone or in combination (HG+HI) to blood from 6 healthy subjects and incubated for 4 hr at 37°C. These studies were performed in the presence or absence of 1 mg/ml LPS. Membrane bound TF-PCA was measured in whole blood lysates by a two-stage clotting assay (Key et al, Blood 1998, 91; 4216–23).

Basal glucose and insulin levels were 75±4 mg/dL and 99±24 pmol/L (n=6, mean±SE), respectively. With added glucose (HG) TF-PCA increased from baseline (22±5 U/ml mean ± SE) to 41±7 U/ml (p=0.006) at 2 hr and to 57±10 U/ml (p=0.0005) at 4 hr. Similarly, at 2 hr there was an increase with insulin 10 nM (33±8, p=0.033) and 100 nM (44±12, p=0.04). A combination of HG with increasing insulin concentrations (1, 10 or 100 nM) increased TF-PCA at 2 hr to 42±6 (p=0.04), 50±9 (p=0.02), and 63±12 (p=0.002), respectively. At 4 hr the corresponding levels were 71±16 (p=0.031), 69±15 (p=0.003), and 93±20 (p=0.004), respectively. Thus, HG, HI and HG+HI increased TF-PCA levels with highest levels being with the combination. This is in line with the findings from our infusion studies in healthy subjects.

LPS alone induced a marked increase in TF-PCA from 22±5 U/ml to 826±93 (p<0.001) by 2 hr and to 591±113 (p<0.01) by 4 hr. The small increase with HG in presence of LPS was not statistically significant. With added insulin TF-PCA at 2 hr was lower than with LPS alone (826±93 U/ml) with 10 nM insulin (587±119, p=0.037) and 100 nM insulin (552±207, p=0.025). This was noted at 4 hr with 10 nM insulin (591±113 vs 472±83, p=0.014). This is in contrast to the findings in the absence of LPS-stimulation where HI stimulated TF-PCA. Notably, in the presence of LPS, the combination of HG with increasing HI caused a marked increase in TF-PCA at 2 hr over that with LPS alone (826±93 U/ml) at 1 nM insulin (2369±234, p=0.003) and 100 nM insulin (1992±288, p=0.009). This persisted at 4 hr (LPS: 591±113 vs 2595±49, p=0.006; and vs 1936±347, p= 0.005). TF-PCA with HG + HI at 2 and 4 hr was higher than with LPS plus glucose or insulin alone.

In non-diabetic subjects HG and HI individually and in combination, increased whole blood TF-PCA. However, insulin inhibited the marked increase in TF-PCA induced by LPS. These findings suggest that the insulin effect is influenced by the cellular activation state. This may be relevant because platelets/monocytes are activated and endogenous LPS levels are elevated in DM. In LPS-stimulated samples, combined HG + HI induced the highest TF-PCA levels suggesting that HG may override the inhibitory effect of insulin in the presence of LPS. Further studies, including in patients with T1DM and T2DM, are needed to understand the effects of HG and HI and the cellular activation state on TF expression.

No relevant conflicts of interest to declare.