Multiple protein disulfide isomerases support thrombosis

Abstract

Purpose of review: The present review provides an overview of recent findings on new members of the protein disulfide isomerase (PDI) family required for thrombosis.

Recent findings: Twenty years ago PDI was shown to mediate platelet aggregation, and 10 years ago PDI was shown to support thrombosis in vivo. Subsequently, other members of this endoplasmic reticulum family of enzymes, ERp57 and ERp5, were demonstrated to support thrombosis. A fourth member, ERp72, was recently shown to be required for platelet accumulation and fibrin deposition in vivo. None of these enzymes can individually support these processes. Moreover, aggregation of platelets deficient in a specific PDI is only recovered by the PDI that is missing. This implies that each PDI has a distinct role in activation of the αIIbβ3 fibrinogen receptor and platelet aggregation. Free thiols can be labeled in both subunits of αIIbβ3, suggesting cysteine-based reactions are involved in relaying conformational changes from the cytoplasmic tails to the integrin headpiece of this integrin.

Summary: Multiple members of the PDI family support platelet function, and hemostasis and thrombosis with distinct roles in these processes. The individual cysteine targets of each enzyme and how these enzymes are integrated into a network that supports hemostasis and thrombosis remain to be elucidated.

FIGURE 1.. CGHC motif-containing PDI family in thrombosis.

The a and a′ domains contain the catalytic CGHC active sites. The non-catalytic b and b′ domains are also shown. PDI, ERp57, and ERp72 contain a flexible x-linker domain.

FIGURE 2.. ERp72 generates thiols in αIIb and β3.

Effect of adding ERp72 (with functional a and a′ active sites) to platelets on MPB labeling of thiols in αIIb and β3.

FIGURE 3.

Representative MS/MS spectrum of a doubly-charged ion (m/z 794.87) corresponding to the peptide sequence of 182-TTCLPMFGYK-191 with a biotin-HPDP modification at Cys184 in the β3 subunit. The observed y- and b-ion series confirmed the peptide sequence, and a biotin-HPDP modified Cys (+428.2 amu) found between b² and b³ as well as between y⁷ and y⁸ ions in the spectrum confirmed the modification of Cys184.

FIGURE 4.

Working model of the role of sulfhydryls and PDI in platelet function. The platelet fibrinogen receptor αIIbβ3 (α, β) is shown in three different activation states. The nonactivated state is on the left side. Redox sensitive disulfide bonds in αIIbβ3 are depicted. Agonist induced stimulation leads to cytoplasmic events and talin binding resulting in inside-out signaling and an initial ligand binding interaction of fibrinogen (F) with the receptor (1). A PDI catalyzed event then converts αIIbβ3 to the high affinity conformation (2) represented by secondary platelet aggregation. During platelet activation, sulfhydryls are generated in αIIbβ3 as well as in the active site of PDI from cytoplasmic reducing equivalents supplied by NAD(P)H. GSH or other low molecular weight thiols in the external redox environment also generate sulfhydryls in both αIIbβ3 and PDI facilitating the reactions shown.