Abstract
Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.
Highlights
Human clinical studies consistently link greater blood leukocyte count to increased risk of atherothrombotic disease events, such as myocardial infarction (MI) and stroke [1,2,3]
The pro-aggregatory effect of neutrophils appeared to be mediated in part by the action of cathepsin G on platelet protease activated receptor-4 (PAR4) because the cathepsin G inhibitor failed to reduce aggregation when samples were simultaneously activated with TRAP and PAR4 agonist peptide (Fig. 1B)
The ability of neutrophil cathepsin G to promote platelet aggregation and vascular occlusion was demonstrated in human platelet rich plasma (PRP) in vitro, after tissue trauma in the mouse in vivo, under arterial flow conditions in mouse microvessels, and in a vascular occlusive stroke model
Summary
Human clinical studies consistently link greater blood leukocyte count to increased risk of atherothrombotic disease events, such as myocardial infarction (MI) and stroke [1,2,3]. Neutrophils demonstrate the most robust association with thrombotic disease. Increased numbers of leukocyte-platelet conjugates in the peripheral blood of patients with acute coronary syndromes are associated with risk of myocardial injury [7,8]. Similar associations between leukocyte count and arterial thrombotic risk, MI, have been described for patients with myeloproliferative disorders [9]. Despite an abundance of observational data, a causal relation between blood leukocytes and thrombotic vascular morbidity has not been established because a clear mechanism to explain this association has not been defined
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