Generation of PDI fragments and PDI mutants

Fragments of PDI were generated (Fig. 2A) with the boundaries of the domains as described [16]. Different PDI mutants were also generated from full length human PDI construct by site-directed mutagenesis using Quickchange Stratagen kit and the mutated sequences were verified by DNA sequencing. Truncated or mutated constructs were synthesized with an N-terminal His tag (MHHHHHH) to facilitate the purification and subcloned into pET30a vector. The plasmids were transformed into E. coli BL21 (DE3) competent cells. A single colony was inoculated into LB medium containing kanamycin and cultured in 37°C at 200 rpm. The cells were harvested by centrifugation and the pellets were lysed by sonication. The target proteins were purified by one-step purification using a Ni column. Fractions were pooled together and dialyzed against phosphate buffered saline (PBS). The purified proteins were run on 4%−20% SDS-PAGE gels and the purity of the proteins was analyzed by Coomassie staining. The purity was > 85–90%. Protein concentrations were determined by Bradford protein assay with bovine serum albumin as a standard (Supplemental Fig. 1).

PDI fragments containing the b′ domain inhibit Alexa-PDI binding to platelets and the abb′x fragment inhibits platelet aggregation. (A) Schematic of PDI fragments. (B) The fragments of PDI that inhibit Alexa-PDI binding to platelets contain the b′ domain of PDI. Thrombin-activated human platelets were incubated with PBS, unlabeled wild-type PDI, abb′x, b′xa′, ab or a′c fragment for 10 min followed by the incubation of Alexa 488-labeled PDI for 10 min. Surface binding of Alexa-PDI was detected by flow cytometry; mean ± SEM, n ≥ 3, ***p < 0.001, NS = non significant, ANOVA. (C and D) Platelets were incubated with the indicated fragments of PDI and collagen (1 μg/ml) (C) or thrombin (0.5 U/ml) (D) was added to induce aggregation and secretion. Representative aggregation and secretion tracings (left panel) and cumulative results (right panels), with platelet aggregation normalized to results obtained in control samples; mean ± SEM, n = 3, **p < 0.01, ***p < 0.001, ANOVA.

The point mutations in PDI we used (Figs. 3A and ​and4A)4A) have been previously used to study PDI binding to other proteins [4, 17, 18]. Using a variety of biophysical analysis mutant PDI proteins were shown not to cause significant alterations in PDI structure with the exception of W111I, which had a marginally different form than the wild-type protein [4]. The range of biophysical analysis used to screen for significant structural changes in the recombinant proteins included binding to somatostatin (a function of the b′ domain), protease stability (proteinase K and V8 protease digestion), and CD spectra in the far UV region [4]. We employed the W111I, F258W, I272W/A and L386W mutations in our studies (amino acids are numbered without the 17 amino acid signal peptide).

The a and a′ domains contribute to PDI binding to platelets and platelet aggregation. (A) Schematic of PDI mutants. (B) Thrombin-activated human platelets were incubated with unlabeled wild-type (WT) PDI or different mutants for 10 min followed by the addition of Alexa 488-labeled PDI for 10 min. Surface binding of Alexa-PDI was detected by flow cytometry; mean ± SEM, n = 6, ***p < 0.001, ANOVA. (C) PDI-null mouse platelets were incubated with WT PDI or the triple mutant PDI (W111I/I272W/L386W) for 10 min and platelet aggregation was induced by thrombin. Representative aggregation and secretion tracings (left panel) and cumulative results (right panel); mean ± SEM, n = 3, *p < 0.05; **p < 0.01, ***p < 0.001, ANOVA. (D) The triple mutant (W111I/I272W/L386W) has little effect on aggregation of human platelets. Platelets were incubated with WT PDI or W111I/I272W/L386W PDI for 10 min and aggregation was induced by adding thrombin. Representative aggregation and secretion tracings (left panel) and cumulative results (right panel); mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ANOVA.

The a and a′ domains functionally cooperate with the b′ domain in PDI binding to platelets. (A) Schematic of PDI mutants. (B) Thrombin-activated human platelets were incubated with unlabeled wild-type (WT) PDI or different mutants for 10 min followed by addition of Alexa 488-labeled PDI for 10 min. Surface binding of Alexa PDI was detected by flow cytometry; mean ± SEM, n ≥ 3, **p < 0.01, ***p < 0.001, ANOVA. Wild-type PDI but not the W111I/F258W/I272A/L386W quadruple mutant supports aggregation of mouse platelets (C) and potentiates aggregation of human platelets (D); mean ± SEM, n ≥ 3, *p < 0.05, **p < 0.01, ANOVA.