Activated Monocytes Enhance Platelet-Driven Contraction of Blood Clots via Tissue Factor Expression

Figures

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  Figure 1. Effects of activated monocytes on clot contraction. Isolated non-activated monocytes or PMA-activated monocytes were supplemented to whole citrated blood. Clotting and clot contraction were initiated with 2 mM CaCl2 and 1 U/ml thrombin followed by optical tracking of clot size. (A) Representative raw images showing an initially uncontracted clot and clots after 20-minute contraction obtained from the same blood sample under standard experimental conditions after addition of non-activated and PMA-activated monocytes. (B) Average extent of clot contraction and (C) average velocity of contraction at 20 minutes, determined after addition of non-activated and PMA-activated monocytes (n = 8). (D) The averaged kinetic contraction curves (n = 8) with and without monocyte activation were used to determine the rates of the three phases of clot contraction separated by the vertical lines. Insert – the comparative rates of Phase 2. The results are presented as mean ± SEM. **p < 0.01.

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  Figure 3. Flow cytometry of isolated monocytes. Representative raw data of a flow cytometry experiment with a freshly isolated monocyte preparation incubated with mouse anti-human CD14 antibodies (monocytes are CD14-positive) and mouse anti-human CD41 antibodies (platelets are CD41-positive) for 30 min at 4 °C in the dark. The preparation contains ~98% CD14+ monocytes ((A) dark grey) of all mononuclear cells ((A) light grey). The ratio of CD14+ monocytes ((B) dark grey) to CD41+ platelets ((B) light grey) is roughly 1:10.

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  Figure 2. Effects of cell-expressed or purified TF on clot contraction. (A) Average extent of clot contraction and (B) average velocity of clot contraction at 20 minutes after addition to whole blood of non-activated monocytes (normal contraction), PMA-activated monocytes, and PMA-activated monocytes treated with a PPARα agonist, an inhibitor of TF expression, prior to PMA activation (n = 24). (C,D) The fraction of TF-expressing (CD142-positive) monocytes determined by flow cytometry in the CD14-positive monocyte populations under various experimental conditions indicated. (C) Raw flow cytometry peaks from a representative experiment are shown. The monocyte population is shown in light gray peaks, while the fractions of TF-expressing monocytes are shown in dark gray peaks and numbers in italics. (D) The average percent of TF-expressing monocytes under various experimental conditions indicated (n = 3). (E) Average extent of clot contraction and (F) average velocity of clot contraction at 20 minutes after addition to whole blood of non-activated monocytes (normal contraction), PMA-activated monocytes and PMA-activated monocytes treated with inhibitory anti-TF antibodies (n = 3). (G) Average…

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  Figure 4. Relationship between the degree of vessel obstruction and blood flow. Thrombotic vessel obstruction increases the vascular resistance as described by Poiseuille’s law, which says that resistance is inversely related to the radius to the fourth power. Therefore, if the radius (or diameter) of a vascular segment is reduced by one-half, the resistance or the volumetric blood flow within that narrowed segment increases by 16-fold, which is a dramatic effect relative to the change in degree of vessel obstruction. Based on the Poiseuille’s equation: Volume flow rate = π*(pressure difference)*(radius)4/8*(viscosity)*(length) the change in the cross-sectional area corresponding to a decrease of the degree of vessel obstruction from 56% to 48% will result in an increase in the volumetric blood flow by about 30%.