Platelet‐targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow

Abstract

To cite this article: Zhu S, Welsh JD, Brass LF, Diamond SL. Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow. J Thromb Haemost 2016; 14: 1070-81.See also Flaumenhaft R. Probing for thiol isomerase activity in thrombi. This issue, pp 1067-9. Essentials• Protein disulfide isomerases may have an essential role in thrombus formation.• A platelet-binding sensor (PDI-sAb) was developed to detect thiol reductase activity under flow.• Primary human platelet adhesion to collagen at 200 s À1was correlated with the PDI-sAb signal.• Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice.Summary. Background: Protein disulfide isomerases (PDIs) may regulate thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results: Using click chemistry to link anti-CD61 and a C-terminal azido disulfide-linked peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for thiol reductase activity detection in whole blood under flow conditions. PDI-sAb was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI) and detected thiol reductase activity on P-selectin/phosphatidylserine-positive platelets activated with convulxin/PAR1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 lg mL À1 corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s À1 ), the PDI-sAb signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI-sAb signal. Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb signal only when thrombin production was inhibited with PPACK, suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti-mouse CD41 PDI-sAb used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. Conclusion: PDI-sAb is a sensitive and real-time reporter of plateletand vascular-derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients.

Dynamics of Thrombin Generation and Flux from Clots during Whole Human Blood Flow over Collagen/Tissue Factor Surfaces

“…However, clotting under hemodynamic flow is an open system involving platelet adhesion and activation on a surface as well as rapid build-up of a dense platelet core surrounded by less activated platelets in surrounding shell of the clot. The core of the clot is highly contracted with P-selectin-positive platelets (21,22), localized thrombin and fibrin (23,24), and localized disulfide reductase activity (25). The kinetics of thrombin generation are less well understood in this hierarchical structure where intrathrombus transport and binding effects may control reactions (26 -30).…”

Vascular thiol isomerases

“…In addition to these in vivo murine models, a role for thiol isomerases in platelet activation and thrombus formation has also been shown in vitro using human samples. 72 Several surprises can be noted. First, these thiol isomerases reside in the endoplasmic reticulum and, in the case of PDI, also the T-granule 43 and yet have extracellular function; second, they have important function with regard to thrombus formation.…”

“…In summary, a series of thiol isomerase has been identified in the vessel wall and in platelets that, on cell activation, stimulates activation of extracellular thiol isomerase activity 72 and accompanies thrombus formation in vivo. 45,67 Each of these thiol isomerases is either absolutely required or partially required for thrombus formation.…”

Probing for thiol isomerase activity in thrombi

“…Although PDI has been shown to accumulate at sites of vascular injury following laser ablation, the enzymatic activity of PDI during thrombus formation had not previously been evaluated. Following infusion of PDI‐sAb and laser injury of arterioles, a fluorescent signal corresponding to PDI‐sAb cleavage was observed in the thrombus core near the endothelium . P‐selectin demonstrated co‐localization with the PDI‐sAb signal, indicating that thiol isomerase activity is concentrated in the core region of the thrombus.…”

“…Antagonists of PDI blocked activation‐dependent increases in the PDI‐Abs signal. PDI‐sAb also emitted a signal in thrombi formed when whole blood was flowed over collagen .…”