Both platelet- and endothelial cell–derived ERp5 support thrombus formation in a laser-induced mouse model of thrombosis

Abstract

• ERp5, like its family members PDI and ERp57, accumulates at sites of vessel wall injury.• Both platelets and endothelium secrete ERp5 on activation and contribute ERp5 necessary for thrombus formation in vivo.Protein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5. Anti-ERp5 antibody inhibited ERp5-dependent platelet and endothelial cell disulfide reductase activity in vitro. ERp5 release at the thrombus site was detected after infusion of Alexa Fluor 488-labeled anti-ERp5 antibody at 0.05 mg/g body weight, a dose that does not inhibit thrombus formation. Anti-ERp5 at 3 mg/g body weight inhibited laser-induced thrombus formation in vivo by causing a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (P < .01). ERp5 binds to b3 integrin with an equilibrium dissociation constant (K D ) of 21 mM, measured by surface plasmon resonance. The cysteine residues in the ERp5 active sites are not required for binding to b3 integrin. These results provide evidence for a novel role of ERp5 in thrombus formation, a function that may be mediated through its association with aIIbb3.

Vascular thiol isomerases

“…41 Several other groups subsequently demonstrated that thiol isomerases traffic to secretory granules/membrane systems and are released from platelets in an activation-dependent manner. 36,[42][43][44][45] Kim et al showed that platelets lacking PDI have reduced activation-dependent aggregation. 46 Platelet surface PDI has been postulated to modulate a IIb b 3 function, 47 Thiol isomerases have also been found on the surface of the endothelium.…”

“…69,70 Since the discovery of the importance of PDI in thrombus formation, similar observations have been extended to other thiol isomerases, including ERp57 and ERp5 (Table 2). 45,56,57 ERp57, with a domain organization parallel to PDI, is also secreted by platelets and endothelial cells on cell activation. Three groups have related ERp57 to thrombus formation using (1) blocking antibodies to ERp57 56 ; (2) genetic alterations of megakaryocyte/platelet PDIA3, 71 the gene that encodes ERp57; and (3) in vivo morpholinos in mice to decrease expression of ERp57.…”

Thiol Isomerases – A Possible Target for Thrombosis Control

“…Среди большого семейства из двадцати тиоловых изомераз к формированию тромба in vivo причастны протеин дисульфид изомераза (ПДИ) [4,7,8] и ERp57 [9][10][11][12][13]). Интегрины αIIbb3 тромбоцитов и αVb3 эндо-телия играют ключевую роль в этом процессе и напря-мую взаимодействуют с ПДИ, ERp5 и ERp57 [13].…”

“…Интегрины αIIbb3 тромбоцитов и αVb3 эндо-телия играют ключевую роль в этом процессе и напря-мую взаимодействуют с ПДИ, ERp5 и ERp57 [13].…”

“…Только что высвобожденная ПДИ связывает αIIbb3 на по-верхности тромбоцитов и αVb3 на поверхности эндо-телиальных клеток [13]. В модели на живой мыши показано, что появление ПДИ на поверхности эндоте-лия, и впоследствии -на поверхности связанных тромбоцитов следует сразу после лазерной травмы, после чего следует стремительная экспрессия ткане-вого фактора [4,5].…”