Plasma Tissue Factor Plus Activated Peripheral Mononuclear Cells Activate Factors VII and X in Cardiac Surgical Wounds
Takashi Hattori, MD*,
Mohammad M.H. Khan, MD, PhD ,
Robert W. Colman, MD and
L. Henry Edmunds, Jr, Md*,*
* Harrison Department of Surgical Research, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Sol Sherry Thrombosis Research Center, Hematology Division of the Departments of Medicine and Physiology, Temple University, Philadelphia, Pennsylvania
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Figure 1 (A) Concentrations of soluble plasma tissue factor (pTF) (filled bars) and MCP-1 (open bars) in plasma harvested before cardiopulmonary bypass (CPB) (Pre) and simultaneously from the perfusion circuit (Perf) and pericardial wound (Wnd) during CPB. Values are means ± SEM, n = 7. p = 0.0002 and 0.0009 for Wnd pTF versus Pre pTF and Perf pTF, respectively. p = 0.000017 and 0.0000093 for Wnd MCP-1 versus Pre MCP-1 and Perf MCP-1, respectively. p = 0.18 for Pre pTF versus Perf pTF and for Pre MCP-1 versus Perf MCP-1. (B) Wound pTF with C5a-stimulated mononuclear cells (2 x 106/ml) from all locations (open bars) and unstimulated wound mononuclear cells (filled bars) convert all available factor VII (fVII) to fVIIa. Unstimulated monocytes from pre-bypass and perfusate samples partially activate fVII. Values are means ± SEM, n = 6.
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Figure 2 (A) Tissue factor antigen in microparticle pTF (mpTF) and supernatant pTF (spTF) derived from the same wound pTF processed as described by Nieuwland et al. (16) (filled bars) or Bogdanov et al. (17) (open bars). p = 0.021, mpTF versus spTF, filled bars; p = 0.00041, mpTF versus spTF, open bars, n = 4). (B) fVIIa generated from unstimulated and C5a-stimulated donor mononuclear cells incubated with 1.46 pmoles/l of wound pTF, wound microparticle TF (mpTF) or wound supernatant TF (spTF) (n = 3). p values for fVIIa production between stimulated and unstimulated monocytes are 0.002, wound mpTF; 0.043, wound spTF; and 0.069, wound pTF. p values for wound mpTF versus wound spTF or wound pTF for stimulated and unstimulated monocytes ranged from 0.000 to 0.005; p values for wound spTF versus wound pTF were 0.270 and 0.407. Solid bars = unstimulated; open bars = stimulated. Abbreviations as in Figure 1.
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Figure 3 Western blots probed using anti-tissue factor (TF) (left panel) and anti-factor VII(fVII) (right panel) from fractionated samples of plasma tissue factor (pTF) taken from three locations in a representative patient (one of three). Molecular standards appear at the left in each panel. Plasma TF from each location was separated as described (16) into microparticle (mpTF) and supernatant (spTF) fractions. Note that all TF proteins migrate at 47.5 kDa except for recombinant soluble TF (rTF). The highest amount of TF is in the supernatant fraction of wound pTF. In contrast, the microparticle fraction of wound pTF contains the largest amount of fVII/fVIIa complex (right panel).
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Figure 4 Activation of fX by fractionated wound pTF and C5a-stimulated donor mononuclear cells (open bars) and unstimulated monocytes (filled bars) taken from each location. Paired t test p values for fXa generation by each sample mpTF, spTF pair (n = 6 pairs) ranged from 0.0004 to 0.060, with only the unstimulated pre-bypass pair >p = 0.05. Abbreviations as in Figure 3.
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Figure 5 Correlation between wound pTF and prothrombin fragment F1.2 (A) and thrombin antithrombin complex (B) in wound samples during CPB. Abbreviations as in Figure 1.
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