Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo

Abstract

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

FIGURE 1.

Effect of FPR-ProT on thrombin generation in platelet-rich murine and human plasma. A, thrombin generation was determined by CAT at varying concentrations of FPR-ProT as described under “Experimental Procedures” in a total volume of 60 μl containing 20 μl of platelet-rich murine plasma. Concentrations of reactants were: 5 pm TF, 8 mm added CaCl₂, 0.42 mm fluorogenic thrombin substrate, 40 μg/ml of corn trypsin inhibitor (to inhibit potential contact activation) and final concentrations of FPR-ProT (μm) at 0 (gray), 0.1 (blue), 0.2 (green), 0.3 (orange), 0.4 (red), and 0.5 (violet). B, thrombin generation in platelet-rich human plasma in a total volume of 125 μl containing 80 μl of plasma. Concentrations of reactants were: 1 pm TF, 16 mm added CaCl₂, 0.34 mm fluorogenic substrate, 40 μg/ml of corn trypsin inhibitor, and FPR-ProT (μm) at 0, (gray), 0.2 (blue), 0.4 (green), 0.6 (orange), 0.8 (red), and 1.0 (violet). C, thrombin generation in platelet-rich mouse plasma as in A, but at 10-fold lower TF (0.5 pm) with 0.7 μm of the following ProT derivatives: none (red), F1 (violet), F1.2 (orange), FPR-Pre 1 (blue), or FPR-T (green).

FIGURE 2.

Effect of FPR-ProT on thrombin generation in normal human plasma at varying TF, phospholipid, and plasma ProT levels. Thrombin generation in a total reaction volume of 125 μl containing 80 μl of normal human plasma initiated with varying TF concentrations (A) or with 7 pm TF (B and C) and the effect of increasing FPR-ProT concentration on the relative maximum thrombin concentration (%) determined as described under “Experimental Procedures.” Concentrations of reactants were: A, TF (pm) 1.8 (Δ), 7 (♢), or 40 (♦); 16 mm CaCl₂, 30 μm phospholipid vesicles, 0.34 mm substrate, 40 μg/ml of corn trypsin inhibitor, and the indicated concentrations of FPR-ProT. B, 7 pm TF and 2 (○) or 30 (●) μm phospholipid vesicles with all other reactants as in A. C, 7 pm TF, ProT-deficient plasma reconstituted with purified human ProT representing 25 (Δ), 50 (▴), 100% (□), and 150% (■) of the concentration of ProT present in normal plasma (1.5 μm). All other reactants are as given in A.

FIGURE 3.

Effect of FPR-ProT and ProT^S195A on thrombin generation in platelet-rich human plasma. The effect of varying concentrations of ProT^S195A (A) and FPR-ProT (B) on TF-initiated thrombin generation in a total volume of 125 μl containing 80 μl of platelet-rich human plasma determined by CAT as described under “Experimental Procedures.” Concentrations of reactants were: 0.5 pm TF, 16 mm added CaCl₂, 0.34 mm substrate, 40 μg/ml of corn trypsin inhibitor, and final concentrations: A, ProT^S195A (μm) at 0 (gray), 0.2 (blue), 0.4 (green), 0.6 (orange), 0.8 (red), or 1.0 (violet). B, FPR-ProT (μm) at 0 (gray), 0.1 (not filled), 0.2 (blue), 0.4 (green), 0.6 (orange), 0.8 (red), or 1.0 (violet).

FIGURE 4.

Effect of FPR-ProT, ProT^S195A, and ProT derivatives on the rate of ProT activation by prothrombinase. Initial rates of ProT activation (v_obs) were determined at 37 °C as a function of ProT and FPR-ProT concentrations as described under “Experimental Procedures.” A, changes in initial velocity of 150 nm native ProT activation (Fractional Activity) as a function of total concentrations of FPR-ProT (●), F1 (○), F1.2 (▴), and FPR-Pre 1 (Δ) ([ProT species]). B, initial velocities as a function of total FPR-ProT concentration ([FPR-ProT]) at native ProT concentrations of 50 (●), 150 (○), and 1500 (▴) nm. C, initial velocities as a function of total ProT^S195A ([ProT^S195A]) concentration as in B. Concentrations of reactants were: 25 mm Hepes, pH 7.5, at 37 °C, 175 mm NaCl, 0.5 mg/ml of bovine serum albumin, 2 pm FXa, 5 nm FVa, and 1 μm phospholipid vesicles. Solid lines represent non-linear least squares fits of the data by the competitive inhibition mechanism with values of apparent K_m and K_i for ProT fragments, FPR-ProT, and ProT^S195A given under “Results.”

FIGURE 5.

Time courses of ProT, ProT^S195A, and FPR-ProT cleavage by prothrombinase. Aliquots of reaction mixtures containing ProT derivatives (5.2 μm) incubated with 0.7 nm prothrombinase (0.7 nm FXa, 50 nm FVa, and 50 μm PS/PC/PE vesicles) were quenched at times (min) 0, 0.33, 0.67, 1, 1.33, 1.67, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 16, 20, 26, and 32. Relative concentrations of native ProT activation (A) and ProT^S195A (B) or FPR-ProT (C) cleavage products. ProT, ProT^S195A, or FPR-ProT (●), meizothrombin F1.2-A (○), F1.2 (▴), thrombin B-chain (Δ), and Pre 2 (■). D–F, SDS gels (4–12%), corresponding to the reactions for A (native ProT), B (ProT^S195A), and C (FPR-ProT). Lanes 1–18 are samples taken at the above reaction times except for native ProT, where the 32-min sample was deleted because of a gel anomaly. Bands corresponding to ProT, ProT^S195A, or FPR-ProT (ProT), F1.2-A, F1.2, Pre 2, thrombin B-chain (B chain), F1, F2, and thrombin A-chain (A chain) are indicated on the right. Positions of molecular weight markers are indicated on the left with the molecular weights in thousands. Reactions were performed and analyzed as described under “Experimental Procedures.”

FIGURE 6.

Bright-field and wide-field fluorescence microscopy of platelet and [AF660]FPR-ProT accumulation during thrombosis. A, wide-field fluorescence intensity of platelets (green) in the absence of [AF660]FPR-human ProT with corresponding bright-field images and composite images at 75 and 225 s after injury. B, wide-field, bright-field, and composite images of platelets (green) in the presence of 0.36 μm [AF660]FPR-human ProT ([AF660]-ProT) (red). Postinjury recording of A and B began at ∼66 and ∼25 s, respectively. Note the smaller size of the thrombus in the presence of labeled ProT. Experiments were performed and analyzed as described under “Experimental Procedures.”

FIGURE 7.

Effect of [AF660]FPR-ProT on the rate and size of thrombus formation. A, time to maximum platelet accumulation following laser injury obtained for controls (2 mice, 13 thrombi) (♢), 0.36 μm [AF660]FPR-human ProT ([AF660]-ProT) (4 mice, 44 thrombi) (Δ), 1 μm [AF660]FPR-human Pre 1 ([AF660]-Pre 1) (3 mice, 26 thrombi) (○), and 0.36 μm [AF660]FPR-mouse ProT ([AF660]-mProT) (2 mice, 10 thrombi) (●). B, maximum thrombus size (same thrombi analyzed in A). Controls (♢), 0.36 μm [AF660]FPR-human ProT (Δ), 1 μm [AF660]FPR-human Pre 1 (○), and 0.36 μm [AF660]FPR-mouse ProT (●) are indicated. The bars denote the medians. Experiments were performed and analyzed as described under “Experimental Procedures.”

FIGURE 8.

Colocalization of [AF660]FPR-ProT and platelets. Z-plane images were digitally recorded every 3 μm for 23 slices where the total Z-step distance was 70 μm. A full set of images of the 23 slices was collected every 8 s. A, selected maximum intensity projection images from confocal microscopy of [AF660]FPR-mouse ProT (red) and platelet (green) localization at the indicated times after vessel wall injury. B, platelets (green points) and [AF660]FPR-mouse ProT (red points) total fluorescence intensity for the volume through 70 μm. C, colocalization of platelet fluorescence (green points) and [AF660]FPR-mouse ProT (red points) expressed as percent of the sum of the intensities as a function of time after injury. Inset, selected confocal images of the colocalization (yellow) of platelets (green) and [AF660]FPR-mouse ProT (red). Experiments were performed and analyzed as described under “Experimental Procedures.”