Platelets are vital determinants of fibrin clot architecture which is modifiable by standard anti-platelet therapies

BACKGROUND: The formation of blood clots at sites of atherosclerotic plaque rupture remains a huge clinical burden despite advances in anti-thrombotic therapies. There exists a close temporal and spatial coupling between the coagulation cascade and platelet aggregation. Clot microstructure is a major determinant of the balance between bleeding and thrombotic risk. Dense clots composed of thinner fibres and increased branch points are more related to thrombosis and associated with coronary artery disease. High on treatment platelet reactivity despite anti-platelet therapies represents another risk factor for adverse clinical outcome. Despite the interwoven nature of the coagulation system and platelet activation in thrombosis, few studies relate analyses of the protein and cellular parts of coagulation in the same population. Here we establish interactions between platelets and anti-platelet therapies on fibrin clot microstructure.

METHODS: Two groups of 8 healthy male volunteers received either aspirin (75mg) or prasugrel (10mg) for 7 days. Fibrin clot structure, determined by the viscoelastic properties, fractal analysis (Df) and relative mass (RM) 1 of whole blood pre- and post-therapy was associated to platelet responses assessed by light transmission aggregometry (LTA), lumi-aggregometry and flow cytometry. Platelet and fibrin interactions during DAPT (aspirin+prasugrel, or aspirin+ticagrelor (90mg)) were then further characterised in two additional study groups of 8 volunteers.

RESULTS: Aspirin caused inhibition of platelet responses to arachidonic acid (AA), but had no effect on ATP release (6.3±0.6 to 4.7±0.5 nmol, p=0.07), P-selectin (geometric mean fluorescence index; MFI, 32±6 to 28±8 units, p=0.71) or PAC-1 binding (19±3 to 15±4 units, p=0.35). Aspirin also caused no significant changes in Df(1.71±0.01 to 1.69±0.01, p=0.41) or RM (-8±19%, p=0.47). Prasugrel significantly reduced (p<0.05) ATP release (8.6±0.5 to 5.2±0.4 nmol), PAC-1 (19±4 to 2±1 units) and P-selectin expression (25±6 to 9±3 units), as well as Df (1.72±0.02 to 1.67±0.01, p =0.03) and RM (-40±11%, p=0.03). The two DAPT regimes significantly inhibited (p<0.05) platelet responses to AA, ADP, collagen, U46619 and TRAP-6. Aspirin+prasugrel and aspirin+ticagrelor also inhibited P-selectin expression (29±8 to 2±1 units) and (99±7 to 55±12 units), respectively. Df and RM values were also significantly reduced by DAPT: 1.73±0.02 to 1.68±0.02 (p=0.03) and -35±16% (p=0.04) by aspirin+prasugrel; 1.72±0.03 to 1.62±0.02 (p=0.04) and -45±14% (p=0.04) by aspirin+ticagrelor.

CONCLUSION: Here we have identified the key importance of platelets as regulators of clot microstructure and the effectiveness of standard anti-platelet therapies as modifiers of clot architecture. There exists a significant relationship between platelet function and the complex, highly disordered microstructure of incipient clots with increasing platelet inhibition being associated with more permeable fibrin networks. We suggest that the therapeutic benefits of anti-platelet agents are likely to depend on their ability to reduce thrombus density and furthermore that it is activation of P2Y12 receptors rather than TX (A2) receptors that is critical in clot formation. This provides further evidence to suggest that in the presence of strong P2Y12 receptor blockade aspirin produces little or no additional therapeutic benefit 2. These mechanistic insights are important to our understanding of thrombus formation, to the characterisation of thrombotic risk, and to the optimisation of anti-thrombotic therapies for individual patients.

REFERENCES:

1. Evans, P.A., et al., Blood 116:17, 2010,

2. Warner, T.D., et al., Heart 96: 21, 2010