Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds⁻¹) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α_IIbβ₃, GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds⁻¹ (peak shear, 19 224 seconds⁻¹). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Recent in vitro studies have shown that shear stress can cause platelet activation by agonist-independent pathways. However, no studies have assessed the extent of shear-induced platelet activation within human coronary arteries. We sampled blood from the coronary arteries proximal and distal to coronary lesions and from the coronary sinus in humans with stable coronary disease who were taking both aspirin and clopidogrel. A novel, computationally based technique for estimating shear stress from 3-dimensional coronary angiographic images of these arteries was developed, and the effect of stenosis severity and calculated shear stress on in vivo platelet and related leukocyte activation pathways were determined. We provide evidence of intracoronary up-regulation of platelet P-selectin, platelet-monocyte aggregation, and monocyte CD11b without platelet glycoprotein IIb-IIIa activation or soluble P-selectin up-regulation. This correlates with intracoronary stenosis severity and calculated shear stress and occurs despite the concurrent use of aspirin and clopidogrel. Our results show for the first time shear-related platelet and monocyte activation in human coronary arteries and suggest this as a potential therapeutic target that is resistant to conventional antiplatelet agents.

Essentials Procoagulant platelets can be detected using GSAO in human whole blood. Stable coronary artery disease is associated with a heightened procoagulant platelet response. Agonist‐induced procoagulant platelet response is not inhibited by aspirin alone. Collagen plus thrombin induced procoagulant platelet response is partially resistant to clopidogrel. Summary Background Procoagulant platelets are a subset of highly activated platelets with a critical role in thrombin generation. Evaluation of their clinical utility in thrombotic disorders, such as coronary artery disease (CAD), has been thwarted by the lack of a sensitive and specific whole blood assay. Objectives We developed a novel assay, utilizing the cell death marker, GSAO [(4‐(N‐(S‐glutathionylacetyl)amino)phenylarsonous acid], and the platelet activation marker, P‐selectin, to identify procoagulant platelets in whole blood by flow cytometry. Patients/Methods Using this assay, we characterized the procoagulant platelet population in healthy controls and a cohort of patients undergoing elective coronary angiography. Results In patients with CAD, compared with patients without CAD, there was a heightened procoagulant platelet response to thrombin (25.2% vs. 12.2%), adenosine diphosphate (ADP) (7.8% vs. 2.7%) and thrombin plus collagen (27.2% vs. 18.3%). The heightened procoagulant platelet potential in CAD patients was not associated with other markers of platelet function, including aggregation, dense granule release and activation of α_2bβ₃ integrin. Although dual antiplatelet therapy (DAPT) was associated with partial suppression of procoagulant platelets, this inhibitory effect on a patient level could not be predicted by aggregation response to ADP and was not fully suppressed by clopidogrel. Conclusions We report for the first time that procoagulant platelets can be efficiently detected in a few microliters of whole blood using the cell death marker, GSAO, and the platelet activation marker, P‐selectin. A heightened procoagulant platelet response may provide insight into the thrombotic risk of CAD and help identify a novel target for antiplatelet therapies in CAD.