A sensitive and adaptable method to measure platelet‐fibrin clot contraction kinetics

Figures

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  FIGURE 1. Assay configuration and examples of data. (A) Image of the imaging setup used. (B) Diagram of the samples and the under‐sample lighting system. (C) Representative images at 0 (upper panel) and 70 min (lower panel) of clot formation in resting (R) and thrombin (50–200 mU/ml) stimulated mouse platelet‐rich plasma (PRP). (D) Representative contraction curves from a thrombin titration of clot contraction of mouse PRP in (C). (E) Extent of clot contraction percent calculated as (100 − % area at tend). Error bars indicate the SEM for n = 3. GraphPad Prism and Excel were used for all calculations

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  FIGURE 2. Analysis of clot contraction: platelet dependence. (A) A representative contraction curve with green line showing clot area (extent of clot contraction percent calculated as 100 − % area at tend and red line showing the calculated first derivative smoothed with 10 neighbors on each side. The three phases of the curve were identified by the maxima (t 1) and minima (t 2) of the first derivative curve. Lag time was calculated as the time point at which point‐to‐point area measurements decreased by more than 1%. Extent was the contraction at 100 min (t end). Area under the curve (AUC) was calculated using GraphPad Prism. (B) A representative dataset recovered from a mouse platelet titration stimulated with thrombin (20 mU/ml, 0.5 mM CaCl2, and 250 μg/ml fibrinogen). The lag time (min; C) average velocity (%/min; D) extent of contraction (100 − % area at t end; %; E) and AUC (F) were calculated and plotted versus platelet concentration. Error bars show SEM for n = 2–4; not significant: ns; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 when compared with 5 × 106 platelets/ml. Statistical significance was calculated as described in the Methods section using GraphPad Prism

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  FIGURE 3. Analysis of clot contraction: calcium dependence. Shown are representative contraction curves (A), lag time (min; B), average velocity (%/min; C), the extent of clot contraction (100 − % area at tend; %; D), and AUC (E) as determined with constant numbers of washed platelets (50 × 106/ml platelets) and the indicated concentrations of CaCl2 or 1 mM EGTA. The concentrations of thrombin (20 mU/ml) and fibrinogen (250 μg/ml) were held constant. Error bars show SEM for n = 3; not significant: ns; ***p ≤ 0.001; ****p ≤ 0.0001 when compared with no calcium. Statistical significance was calculated as described in the Methods section using GraphPad Prism

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  FIGURE 4. Analysis of clot contraction: thrombin dependence. Shown are representative contraction curves (A), lag time (min; B), average velocity (%/min; C), the extent of clot contraction (100 − % area at tend; %; D), and AUC (E) as determined with constant numbers of washed platelets (50 × 106/ml mouse platelets) and the indicated concentrations of thrombin. The concentrations of CaCl2 (0.5 mM), and fibrinogen (250 μg/ml) were held constant. Error bars show SEM for n = 3; not significant: ns; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 when compared with no thrombin. Statistical significance was calculated as described in the Methods section using GraphPad Prism

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  FIGURE 5. Analysis of clot contraction: fibrinogen dependence. Shown are representative contraction curves (A), lag time (min; B), average velocity (%/min; C), the extent of clot contraction (100 − % area at tend; %; D), and AUC (E) as determined with constant numbers of washed platelets (50 × 106/ml mouse platelets) and the indicated concentrations of fibrinogen (μg/ml). The concentrations of CaCl2 (0.5 mM), and thrombin (20 mU/ml) were held constant. Error bars show SEM for n = 3–5; not significant: ns; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 when compared with no fibrinogen. Statistical significance was calculated as described in the Methods section using GraphPad Prism

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  FIGURE 6. Role of myosin IIA in clot contraction. (A) Contraction in the presence of the indicated concentrations of blebbistatin (μM), under standard conditions (20 mU/ml thrombin, 250 μg/ml fibrinogen, 0.5 mM CaCl2, and 50 × 106/ml washed mouse platelets). The lag time (min; B), average velocity of contraction (%/min; C), extent of contraction (100 − % area at tend; %; D), and AUC (E) were calculated and plotted. Error bars show SEM for n = 3; not significant: ns; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 when compared with no blebbistatin. Statistical significance was calculated as described in the Methods section using GraphPad Prism

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  FIGURE 7. Role of αIIbβ3 integrin in clot contraction. (A) Contraction in the presence of the indicated concentrations of eptifibatide (μM), under standard conditions (20 mU/ml thrombin, 250 μg/ml fibrinogen, 0.5 mM CaCl2, and 50 × 106/ml washed mouse platelets). The lag time (min; B), average velocity of contraction (%/min; C), extent of contraction (100 − % area at tend; %; D); and AUC (E) were calculated and plotted. Error bars show SEM for n = 3; not significant: ns; *p ≤ 0.05; **p ≤ 0.01 when compared with no eptifibatide. Statistical significance was calculated as described in the Methods section using GraphPad Prism

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  FIGURE 8. Role of glucose metabolism in clot contraction. (A) Contraction in the presence of the indicated concentrations of 2‐deoxyglucose (2DG; mM) under standard conditions (20 mU/ml thrombin, 250 μg/ml fibrinogen, 0.5 mM CaCl2, and 50 × 106/ml platelets). The lag time (min; B), average velocity of contraction (%/min; C); extent of contraction (100 − % area at tend; %; D) and AUC (E) were calculated and plotted. Error bars show SEM for n = 3; not significant: ns; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 when compared with no 2DG. Statistical significance was calculated as described in the Methods section using GraphPad Prism