Hemophilia A is an X-linked, recessive bleeding disorder caused by congenital factor VIII (FVIII) deficiency. Although the bleeding tendency largely depends on residual FVIII activity (FVIII:C), there is tremendous heterogeneity in bleeding frequency and severity among individuals with similar FVIII:C plasma levels. It is therefore likely that additional factors modulate thrombin generation and fibrin deposition in patients with hemophilia A. PDI is an abundant oxidoreductase with chaperone activity that is also present in human platelets and released upon activation. Preclinical studies indicate that extracellular PDI is critical to hemostasis, thrombosis and vascular inflammation. In particular, PDI has been implicated in monocyte/macrophage tissue factor activation, integrin regulation and platelet-associated thrombin generation. Furthermore, impaired PDI release has most recently been shown to contribute to the bleeding tendency of Hermansky-Pudlak syndrome, an inherited platelet function defect. To explore the role of platelet PDI in hemophilia A, we studied 24 patients (15 severely, 5 moderately and 4 mildly affected) in comparison to 12 age- and sex-matched controls. Expression of PDI antigen on resting platelets and platelets stimulated with either 20 µM ADP or 50 µM thrombin receptor activator peptide 6 (TRAP-6) was assessed by flow cytometry using a fluorescently labeled monoclonal antibody. Analysis of CD41 and CD62P (P-selectin) served as positive controls for constitutive platelet antigen expression and α-granule secretion, respectively. In addition, release of soluble PDI antigen into platelet supernatants was measured by ELISA. There was no significant difference in baseline CD41, CD62P and PDI antigen expression between patients and controls. Furthermore, ADP- and TRAP-6-induced CD62P expression was similar between the two groups (percent positive platelets in patients vs. controls: 28±14 vs. 32±15% and 80±12 vs. 83±9% for ADP- and TRAP-6-treated platelets, respectively). However, expression of PDI antigen on platelets stimulated with either ADP (3.3±2.1 vs. 1.5±1.2%, P<0.01) or TRAP-6 (3.4±1.7 vs. 2.1±1.3%, P<0.05) was significantly increased in patients compared to controls. While ADP-induced release of PDI antigen into platelet supernatants was similar between the two groups and not significantly different from baseline, stimulation with TRAP-6 resulted in significantly increased PDI antigen levels in platelet releasates from patients vs. controls (median, range): 1.5, 0.2-23.2 ng/mL vs. 0.4, 0.2-1.9 ng/mL (P<0.01). Importantly, in two patients with exceedingly high TRAP-6-induced PDI release over baseline (4.8 vs. 0.3 ng/mL and 23.2 vs. 2.8 ng/mL), findings were consistent when platelets were isolated and stimulated on a separate occasion (5.5 vs. 1.3 ng/mL and 10.2 vs. 0.2 ng/mL). Taken together, agonist-induced platelet PDI expression was significantly increased in patients with congenital hemophilia A. Furthermore, release of PDI antigen into supernatants of TRAP-6-activated platelets was significantly increased in patients compared to healthy controls. Up-regulation of platelet PDI may thus represent a compensatory mechanism under conditions of defective thrombin generation and fibrin deposition, and variations in platelet PDI expression and release could at least partially explain the heterogeneity in bleeding severity among patients with congenital hemophilia A and similar FVIII:C plasma levels.


Langer:Baxalta: Consultancy, Other: Travel support; Pfizer: Research Funding; CSL Behring: Consultancy, Other: Travel support, Research Funding. Voigtländer:CSL Behring: Other: Travel support. Holstein:CSL Behring: Consultancy, Other: Travel support, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.