Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis

Abstract

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4(-/-) mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4(-/-) mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4(-/-) mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4(-/-) thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4(-/-) and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.

Fig. 1.

Thrombosis in cremaster arterioles after laser-induced injury in wild-type and Par4^−/− mice. Shown are digital fluorescence and brightfield images of representative thrombi in a wild-type (Upper) and a Par4^−/− (Lower) mouse at 5, 16, 74, 180, and 300 s after laser-induced injury of the blood vessel wall. Platelets were labeled with anti-mouse CD41 Fab fragments conjugated to Alexa-488. Red pseudo color, platelets.

Fig. 2.

Platelet accumulation after laser injury in wild-type and Par4^−/− mice. Platelets were labeled as in Fig. 1. (A) Median integrated platelet fluorescence for 33 thrombi in four wild-type mice and for 30 thrombi in three Par4^−/− mice plotted versus time after laser-induced injury of the cremaster arteriole vessel wall. (B) Distribution of the integrated platelet fluorescence for each of the 33 thrombi in wild-type mice and 30 thrombi in Par4^−/− mice at 16 s after injury. No significant difference is seen between the thrombi in the two genotypes by the Wilcoxon rank sum test (P < 0.33). (C) Distribution of the integrated platelet fluorescence for each of 33 wild-type and 30 Par4^−/− thrombi at maximal size. Wild-type thrombi are significantly larger than Par4^−/− thrombi by the Wilcoxon rank sum test (P < 0.0001). (D) Distribution of the maximal integrated platelet fluorescence for each thrombus in both genotypes placed in order of ascending thrombus size. Fifty-nine wild-type thrombi and 56 Par4^−/− thrombi were ranked for determination of the quartile distribution. The percentage of thrombi of each genotype was determined independently in each quartile of the rank order. Black bars, wild-type; gray bars, Par4^−/−.

Fig. 3.

P-selectin expression in thrombi generated in wild-type and Par4^−/− mice. Platelets were labeled with Alexa-647-conjugated Fab fragments of anti-mouse CD41 antibodies. P-selectin expression was detected with Alexa-488-conjugated anti-mouse P-selectin antibodies. Confocal fluorescent images (2-μm spacing) were captured every 30 s for 12 min after laser-injury of the arteriolar vessel wall. (A) Representative digital fluorescent confocal image of a thrombus in a wild-type mouse and in a Par4^−/− mouse 6 min after laser injury. Red pseudo color, platelets; blue pseudo color, P-selectin; magenta pseudo color, overlap. For orientation, brightfield images of the corresponding vessels after injury are superimposed on the digital fluorescent images. (B) Median platelet specific P-selectin exposure, measured as the integrated anti-P-selectin fluorescence colocalized with anti-CD41 fluorescence divided by the integrated colocalized anti-CD41 fluorescence, plotted as a function of time for each genotype. Twenty-eight thrombi in four mice of each genotype were analyzed for wild-type and Par4^−/−, and seven thrombi in one P-selectin null mouse were analyzed as a control for the specificity of P-selectin antibody. Wild-type and Par4^−/− thrombi were different at 6 min after injury by the Wilcoxon rank sum test (P = 0.001).

Fig. 4.

Fibrin deposition in thrombi in Par4^−/− and wild-type mice after laser injury. Platelets were labeled as in Fig. 1. Fibrin was detected by using Alexa-647-conjugated anti-human fibrin antibodies. (A) For quantification of fibrin deposition, widefield fluorescent images were captured every 500 ms for 5 min after laser injury of the arteriolar vessel wall. The median anti-fibrin antibody fluorescence accumulation of 26 thrombi in four wild-type mice and of 23 thrombi in three Par4^−/− mice is plotted versus time. The median fibrin accumulation of six thrombi in a wild-type mouse and of five thrombi in a Par4^−/− mouse determined in the presence of a direct thrombin inhibitor, lepirudin, is also shown. (B) Distribution of maximum fibrin deposition of the 26 thrombi in wild-type mice and the 23 thrombi in Par4^−/− mice. No significant difference is seen between the thrombi in wild-type and Par4^−/− mice by the Wilcoxon rank sum test (P = 0.43). (C) Representative digital fluorescent confocal image of a thrombus in a wild-type mouse and a thrombus in a Par4^−/− mouse 90 s after laser injury. Platelets, fibrin, and overlap are in red, green, and yellow pseudo color, respectively. For orientation, brightfield images of the corresponding vessels after injury are superimposed on the digital fluorescent images.