Vitamin K antagonist use induces calcification and atherosclerotic plaque progression resulting in increased hypercoagulability

Figures

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  Figure 1. Warfarin treatment increases plaque calcification. (A) 18 weeks of treatment demonstrated that warfarin increased calcification compared to control treatment detected by Alizarin Red S staining. Scale bar = 250 µm. Control (n = 11) and warfarin (n = 11) (longitudinal arch sections; A, Adventitia; L, Lumen). (B) Plaque size was significant increased after 18 weeks of warfarin treatment compared to control treatment. Scale bar = 500 µm. Control (n = 10) and warfarin (n = 12) (black arrows indicate atherosclerotic plaque areas). Number of analysis varied from 10 to 12 due to technical errors and missing of data. Data were analysed using Mann–Whitney U test.

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  Figure 2. Vascular calcification induces a pre-thrombotic state. (A) Circulating FIXa:AT levels remained similar between warfarin and control treated mice after 18 weeks. Control (n = 20) and warfarin (n = 22). (B) Warfarin treated mice had significantly increased circulating TAT levels as compared to control mice. Control (n = 19) and warfarin (n = 22). (C) Stratification of all mice by calcification revealed a significant increase in circulating FIXa:AT ratio levels in mice with higher than the median calcification score. Median < (n = 18) and median > (n = 21). (D) Stratification results in a borderline significant increase (P = 0.068) of circulating TAT levels in mice with higher than median calcification score. Median < (n = 17) and median > (n = 21). Data were analysed using Mann–Whitney U test.

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  Figure 3. Thrombogenicity of vascular smooth muscle cell phenotype in normal plasma. (A) Using human normal pooled plasma, both synthetic (orange) and contractile (red) VSMC were inducers of thrombin generation. Synthetic VSMC showed a shorter lag time (n = 8) (B), slightly increased endogen thrombin potential (ETP) (n = 8) (C) and higher peak height (n = 8) (D) and velocity index (n = 8) (E) compared to contractile VSMC. (F) Calcified VSMC (black) demonstrated a shorter lag time (n = 12) (G), similar ETP (n = 12) (H), higher peak height (n = 12) (I) and velocity index (n = 12) (J) as compared to synthetic VSMC (green). All experiments were done with at least three biological replicates, with quadriplo technical replicates. Data were analysed using Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001).

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  Figure 4. Thrombogenicity of vascular smooth muscle cell phenotype in Factor XII deficient plasma. The contribution of the phenotype of vascular smooth muscle cells to intrinsic and extrinisic activation of coagulation was assessed in factor XII deficient plasme. Thrombin generations of contractile, synthetic and calcified VSMC in factor XII deficient plasma were comparable to those obtained in normal pooled plasma (A and F, and Figure 3). Inhibition of the extrinsic pathway through addition of activated site inhibitor seven (ASIS) in factor XII deficient plasma prolonged the lag time (n = 8; B and n = 12; G) and reduced endogen thrombin potential (ETP) (n = 8; C and n = 12; H), peak height (n = 8; D and n = 12; I) and velocity index (n = 8; E and n = 12; J) of all VSMC phenotypes synthetic. (K) Contractile VSMC showed significant lower pro-coagulant TF activity levels as compared to synthetic and calcified VSMC. Calcified VSMC show a significant increase in pro-coagulant TF activity as compared to synthetic VSMC. Control (n = 8), synthetic (n = 8), calcified (n = 8). All experiments were done with at least three biological replicates, with quadriplo technical replicates. Data were…

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  Figure 5. Thrombogenicity of vascular smooth muscle cells under flow conditions. (A) Representative images of platelet thrombus formation (brightfield), VSMC nuclei (Hoechst33342, blue) and AF647-fibrin formation (red) on calcified synthetic VSMC under blood flow. (B) Quantification of fluorescent fibrin formation indicates an increase in the presence of calcification, and a reduction in the presence of either CTI or ASIS. (C) Platelet deposition on calcified VSMC was reduced in the presence of ASIS. Scale bar = 20 µm. (D) Quantification of fluorescent fibrin indicates an increase of fibrin generation in VSMCs treated with warfarin and calcium (P = 0.0337). (B) Quantification of platelet deposition of synthetic VSMCs under flow. Cells treated with warfarin and calcium show an increase in platelet deposition (P = 0.0059). All experiments were done with at least three biological replicates, with duplicate technical replicates. Data were analysed using Mann–Whitney U test and Dunnett’s multiple comparison test.

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  Figure 6. Vascular calcification promotes a pre-thrombotic state in patients on vitamin K-antagonist (VKA). Patient data include 54 patients on VKA (acenocoumerol) treatment with a mean duration of 44 ± 26 month. VKA treated patients with coronary calcification (Agatston score > 0; n = 35) compared to no coronary calcification (Agatston score = 0; n = 19) showed no difference on circulating TAT levels (B), but significantly increased circulating FIXa:AT levels (A). Dividing the Agatston scores >0 in low (Agatston score 1–50) and high (150–647) did not show any significance for TAT levels (D) or FIXa:AT (C). Data were analysed using Mann–Whitney U test.

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  Figure 7. Short-term treatment of VKA induces systemic and local vitamin K deficiency. Long-term VKA treatment aggravates atherogenesis and results in increased vascular calcification. Vascular calcification induces a pre-thrombotic state priming for an increased risk on atherothrombosis. (A) (1) VKA induce local vitamin K deficiency. (2) Vitamin K deficiency initiates micro-calcification in the vessel wall. (3) Vascular calcification promotes vessel stiffening causing local wall damage and stress. (4) Plaque development is initiated by local inflammation. (B) (1) Calcification causes vessel wall stiffening. (2) VSMCs proliferate and are activated thereby expressing tissue factor. (3) VKA treatment causes further plaque progression and calcification. (4) Local mechanical changes occur which promotes flow perturbations. (C) (1) Calcified plaques favour a pre-thrombotic coagulation state due to the formation of FIXa:AT and TAT complexes. (2) This pre-thrombotic state feeds back into the vessel thereby promoting further plaque progression resulting in (3) VSMC proliferation and (4) calcification of the vasculature. (D) (1) Upon plaque erosion, calcified material and activated VSMCs are…

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