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Several high-density lipoprotein (HDL)-associated proteins are present in a clot prepared from plasma, suggesting that HDL particles are bound to fibrin [1]. Proteomic studies with purified HDL reveal up to 75 HDL-associated proteins that are involved in processes such as lipid metabolism, hemostasis, protease inhibition, the immune system and the complement system [2, 3]. These proteins are differentially distributed over HDL subspecies that vary in size and composition [4, 5]. The diverse biological functions described for HDL [6] may be mediated by the distinct HDL subspecies with their specific associated proteins. Low plasma levels of HDL are a risk factor for arterial and venous thrombosis [7-9] and thrombosis patients could have a low quantity of HDL-associated proteins in their thrombi [10]. As it is not yet clear how HDL affects hemostasis and thrombosis, the aim of this study was to further investigate this function of HDL. The role of HDL in reverse cholesterol transport is regarded as the most important function that contributes to the negative association between HDL level and arterial thrombosis. However, HDL has additional properties, including anti-oxidant properties, anti-inflammatory properties, anticoagulant properties and favorable effects on endothelial function [6]. HDL is thought to be anticoagulant by acting as a cofactor for the activated protein C pathway together with protein S [11]. Another mechanism by which HDL could be anticoagulant is that phosphatidyl serine loses its procoagulant properties when incorporated into HDL particles, because the surface area of HDL is too small to accommodate the prothrombinase complex [12].

The possible function of HDL in coagulation and fibrinolysis was investigated by thromboelastography using ROTEM® analysis with tissue factor. Additional methodological details and results are provided as online supplementary information. We added different amounts (1–8 mg mL−1) of commercially available HDL (comHDL) to citrated blood of healthy volunteers. Figure 1(A) shows a typical ROTEM® curve in the absence and presence of 4 mg mL−1 comHDL. There was no effect of comHDL on the coagulation parameters (Fig. 1B). The maximum clot firmness (MCF, Fig. 1C) and the amplitude at 30 min, 45 min and 60 min (Fig. 1D) were decreased in the presence of 4 mg mL−1 comHDL (n = 8), indicating decreased blood clot firmness and apparent lysis. The decrease in clot firmness and the apparent lysis were concentration dependent (data not shown).

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Figure 1. Thromboelastography analysis of whole blood from eight healthy subjects in the absence and presence of 4 mg mL −11 comHDL. (A) A typical thromboelastogram from whole blood with added comHDL (4 mg mL −11) or 150 mM NaCl/0.2 mM EDTA, pH 7.4 as control. (B) The mean (± SD) clotting time (CT) and clot formation time (CFT). (C) The mean (± SD) maximum clot firmness (MCF). (D) The mean (± SD) amplitude at 30 min, 45 min and 60 min. The P-value indicates the significance of the difference with the control at the same time-point.

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The data presented in Fig. 1 were obtained with one batch of comHDL. When we tested more batches, not every batch of comHDL had a similar effect on the clot firmness. SDS-PAGE analysis of two comHDL batches that showed an effect on clot firmness and two batches that did not show any effect on clot firmness using ROTEM® analysis revealed that the effect was correlated with a shift of the apolipoprotein AI protein band around 25 kDa and an apparent smear in the gels above apolipoprotein AI (Fig. S1A). This smear on SDS-PAGE of comHDL was similar to the smear of oxidized HDL (oxHDL) that was prepared from native HDL (nHDL) isolated in-house (Fig. S1B).

We also investigated the effect of nHDL and oxHDL on clot firmness using ROTEM® analysis. The clot firmness reduced during the thromboelastography by the addition of oxHDL, but not by nHDL. The mean amplitude (± SD) at 30 min, at 45 min, and at 60 min was decreased from 100 (± 0)%, 96 (± 2)% and 91 (± 3)% to 63 (± 25)%, 51 (± 24)% and 45 (± 24)%, respectively (P < 0.01), by the addition of 4 mg mL−1 oxHDL (Fig. S2A). This decrease in clot firmness was specific for oxHDL because oxLDL did not show any effect (Fig. S2B). The effect on clot firmness was not inhibited by ε-aminocaproic acid (EACA) (Fig. S3A) and we did not measure increased fibrin degradation products after ROTEM® analysis in the presence of oxHDL (Fig. S3C). In addition, in the presence of cytochalasin D, which inhibits platelet function and decreases the MCF, the effect of oxHDL was not seen (Fig. S3B). Taken together this suggested that the decrease in clot firmness induced by oxHDL was not caused by fibrin breakdown but predominantly involved platelets.

ROTEM® analysis suggested a time-dependent inhibitory effect of oxHDL on blood clot firmness (Fig. 1A). During thromboelastography platelets are activated and aggregate, as shown by the increase in clot firmness at the beginning. In the presence of oxHDL the clot firmness started to decline after about 10 min, suggesting that platelet aggregates fall apart. This decline stopped at an amplitude of around 9 mm, which was similar to the amplitude reached when the effect of platelets was eliminated by cytochalasin D. These data suggested that the platelet contribution to the clot firmness was lost completely when oxHDL was added to the blood. Studies in the past observed similar thromboelastographic patterns and suggested either the relaxation of the retracted clot [13] or the release of the clot from the wall of the cup due to increased clot retraction [14].

High-density lipoprotein (HDL) can inhibit platelet aggregation via binding to the scavenger receptor B type I (SR-BI) on platelets. However, whether this is true for nHDL, specific HDL subfractions or oxidatively-modified HDL is still controversial [15-18]. The possible involvement of platelets was studied by investigating the effect of oxHDL on both plasma clot retraction and platelet aggregation. OxHDL (2–4 mg mL−1) did not have any effect on plasma clot retraction using plasma with 150 * 109 platelets L1 (Fig. S4A). Only at a low platelet concentration (75 × 109 platelets L−1) and a high oxHDL concentration (4 mg mL−1) was a minimal decrease in plasma clot retraction observed (data not shown). OxHDL as well as nHDL (2 mg mL−1) slightly decreased platelet aggregation induced by thrombin; however, there was no difference between nHDL and oxHDL (Fig. S4B). These data are in line with Valiyaveettil et al. [15], though other studies concluded that nHDL had no effect at all and that oxHDL can even activate platelets [16, 19]. The reasons for these discrepancies are not clear. Differences in lipoprotein isolation methods, platelet agonists and oxidation conditions may play a part in the inconsistent results.

In conclusion, we did not observe any effect of nHDL on coagulation and fibrinolysis using thromboelastography. However, oxHDL diminished the blood clot firmness by an unknown mechanism involving platelets. The small decreases in clot retraction at low platelet concentrations and in platelet aggregation could probably not explain the diminished clot firmness. We rejected the hypothesis that in the presence of oxHDL the clot retraction becomes stronger and results in the release of the clot from the cup wall and in that way apparently decreases the clot firmness [14]. Unfortunately, using standard plasma clot retraction assays we could not determine the effect of oxHDL on clot relaxation. More studies are needed to elucidate the mechanism by which oxHDL diminishes the blood clot firmness. The concentrations of oxHDL used here are most likely higher than in the circulation. However, it cannot be excluded that locally the concentration of oxHDL can be much higher than in the circulation.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information

We wish to thank A. van Tol and T. van Gent for their help with the isolation and handling of lipoproteins.

Disclosure of Conflict of Interests

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information

The authors state that they have no conflict of interests.

References

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information
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Supporting Information

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  5. Supporting Information
FilenameFormatSizeDescription
jth12125-sup-0001-DataS1-FigS1-S4.pdfapplication/PDF602K

Data S1. Materials and methods.

Fig. S1. SDS-PAGE analysis of different HDL preparations after staining with colloidal blue.

Fig. S2. Effect of different HDL and LDL preparations on blood clot firmness, investigated using thromboelastography analysis.

Fig. S3. Effect of oxHDL on blood clot firmness and fibrinolysis.

Fig. S4. The effect of oxHDL on clot retraction and platelet aggregation.

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