Platelet potential to switch to procoagulant phenotype compensates bleeding tendency in patients with hemophilia A

TO THE EDITOR:

Hemophilia A (HA) is a rare, x-linked recessive bleeding disorder that is caused by a deficiency in factor VIII (FVIII). The main clinical challenge in HA is that bleeding tendency cannot be predicted only by the FVIII plasma concentration.^1,2 Accumulating evidence suggests that procoagulant platelets (PCPs) accelerate initiation of the coagulation cascade.³ A reduced ability to generate PCPs upon platelet (PLT) stimulation has been found in individuals with bleeding disorders of unknown cause.⁴ PCPs expose phospholipids on their surface that facilitate the binding of tissue factor and FVIIa. Consequently, thrombin generation is initiated through the extrinsic tenase complex, thereby bypassing FVIII.⁵ 

We hypothesized that, in patients with HA (PwHAs), PCPs play an important compensatory role by modulating the bleeding severity independently of FVIII levels. To verify this hypothesis, we evaluated PCPs in 18 PwHAs (median FVIII activity, 7.9%; range, <1%-36%) and 6 carriers (median FVIII activity, 39%, range; 18%-76%) with significant bleeding tendency (median International Society on Thrombosis and Hemostasis – Bleeding Assessment Tool [ISTH-BAT] bleeding score, 10; range, 3-16; and 10; range, 6-34, respectively). Additional demographic and clinical data, including the annual bleeding rate (ABR), are summarized in supplemental Table 1. Citrated blood samples were collected for laboratory investigations that included light transmission aggregometry, flow cytometry to determine the potential to form PCPs and to assess calcium flux, thrombogram to measure thrombin generation, and clot retraction assay.

We observed that PLTs from PwHA and carriers have reduced aggregation only in response to low-dose adenosine 5′-diphosphate (2 μM) when compared with healthy controls (HCs) (P ≤ .0001; Figure 1A). However, no correlation was found between the PLT response to adenosine 5′-diphosphate in the light transmission aggregometry analysis and the ISTH-BAT bleeding score or the ABR (Figure 1B-C). The PLT retraction assay showed no significant difference in clot retraction was observed between the groups, suggesting that the mechanical ability of PLTs to retract fibrin clots remains intact in PwHAs (supplemental Figure 1). Next, the potential to develop a PCP phenotype upon stimulation with thrombin/convulxin was investigated. PwHAs and carriers showed higher potential to develop procoagulant phenotype than HCs (P < .0001; Figure 1D). Most importantly, a significant reverse correlation was observed between the potential to form PCP upon stimulation and the ISTH-BAT bleeding scores (r = −0.5237, P = .0086; Figure 1E), as well as the ABR (r = −0.06879, P = .0002; Figure 1F). These data may suggest a protective role of the PCP subpopulation in reducing the bleeding risk in PwHAs. Of note, no significant difference in PCP was observed before and after FVIII substitution in PwHAs (supplemental Figure 2) indicating that the formation of PCPs is largely independent of the FVIII plasma levels. This aligns with findings that the PLT surface interaction with plasma factors, rather than PLT-PLT aggregation, regulate procoagulant activity.^3,6 Of note, PLTs from PwHAs showed significantly increased GPVI (glycoprotein VI) shedding than those from HCs (supplemental Figure 3), suggesting an in vivo priming phenotype.^7,8 Next, we assessed the intracellular calcium mobilization in PLTs from PwHAs and carriers. We found faster mobilization and enhanced calcium flux upon thrombin stimulation when compared with HCs (P = .0434; Figure 2A-B). These data suggest a compensatory mechanism(s) in which higher upregulated calcium mobilization supports better PCP formation upon stimulation. A recent study⁹ emphasized the role of calcium fluxes in PCP formation. Further investigations by Zou et al¹⁰ revealed that calcium influx via the calcium release-activated calcium channel protein 1 (ORAI1)-stromal interaction molecule-1 (STIM1) pathway significantly influences the procoagulant activity of PLTs. Our findings align with these studies, indicating that the observed enhanced calcium flux in PwHA PLTs upon thrombin stimulation may facilitate the activation of signaling pathways that promote a procoagulant phenotype.

Agbani and Poole³ and Zlamal et al¹¹ showed that PCPs are essential for efficient thrombin generation on the PLT surface. In our study, the induction of a PCP phenotype by thrombin/convulxin enhanced thrombin generation and total endogenous thrombin potential in PwHAs (P < .0001; Figure 2C-D). Importantly, the proportion of PCPs correlated with endogenous thrombin potential (r = 0.5255, P = .0173; Figure 2E) and the lag time to thrombin burst (r = −0.6772, P = .0010; Figure 2F), underscoring the role of PCPs in improving coagulation efficiency. A recent study by Avery et al¹² showed that FVIII membrane binding is mediated by hydrophobic and electrostatic interactions. Notably, changes in the lipid composition, such as decreased phosphatidylserine content, reduce FVIII’s affinity for PLTs membranes, potentially impacting intrinsic tenase complex formation. In contrast with FVIII’s dependency on specific lipid binding conditions, our findings highlight that PCP-mediated procoagulant activity remains robust and independent of FVIII levels. Our data indicate that the thrombin generation on PCPs is likely independent of FVIII and, instead, is mediated by the internal potential to generate a surface that is able to initiate faster cleavage of plasmatic coagulation factors.

Our study provides comprehensive insights into PLT function in PwHA. In conditions of impaired plasmatic coagulation (eg, FVIII deficiency), PLTs may undergo priming, thereby predisposing them to a more rapid and efficient switch to a procoagulant phenotype. This phenomenon could serve as an adaptive mechanism to counteract the reduced clotting efficiency in bleeding disorders. The higher potential to form PCPs, along with increased GPVI shedding and accelerated calcium kinetics, indicate a compensatory mechanism by PLTs in PwHAs with increased intracellular calcium levels, playing a pivotal role in driving the transition to a procoagulant phenotype. These findings suggest that the bleeding tendencies in PwHAs is influenced not only by FVIII deficiency but also by the procoagulant properties of the PLTs. The lack of significant differences in PCP activity before and after FVIII substitution in PwHA may indicate that PCP formation occurs independent of the FVIII levels. Our novel findings identify specific molecular targets for developing targeted therapeutic strategies in PwHAs. For instance, a study by Segot et al⁴ found that low COAT (collagen- and thrombin-activated PLT) PLT levels are frequent in patients with bleeding disorders of unknown cause and can be enhanced by desmopressin. COAT PLTs are a subset of PCPs characterized by their ability to sustain thrombin generation and stabilize fibrin formation, thereby playing a key role in hemostasis and suggesting potential avenues for FVIII-independent therapies in HA as well.

In conclusion, our study provides further evidence of the significance of PLTs in modulating bleeding in PwHAs. One limitation of our study is the small sample size, which underscores the need for future studies with larger cohorts to validate our findings and further investigate the complex relationship between PCPs and bleeding risk in HA while accounting for potential confounding variables. Further research is needed to elucidate the mechanisms underlying impaired PLT response in PwHAs who present with severe bleeding symptoms despite sufficient plasmatic factor levels. A better understanding of these mechanisms will be crucial for improving the management of hemophilia and enhancing the quality of life for PwHAs. Finally, specific regulation of PCP phenotype might be a promising approach to optimize prevention or treatment of bleeding in PwHA.

The study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from all volunteers before any study-related procedures. All tests were performed with leftover blood samples from routine testing. The study protocol of patient material was approved by the institutional review board of the University of Tuebingen (838/2022/B02).

Acknowledgments: The authors thank Antonia Welker and Julian Dürr for their excellent technical support.

This work was supported by research grants from Octapharma, Bayer, and Takeda to K.A. G.U. (CS 3014-0-0) and J.Z. (CS 3015-0-0) take part in the Medical Innovation through Interdisciplinarity Clinician Scientist Program of the Medical Faculty Tuebingen, funded by the Deutsche Forschungsgemeinschaft (493665037).

Contribution: K.A., S. Halimeh, and T.B. designed the study; S.K., A.B., K.W., S. Hörber, and J.Z. performed the experiments; K.A., S. Hammer, D.G., and M.M. recruited the patients; S. Hammer, K.A., G.U., U.H., V.I., C.F., A.A., L.A., J.Z., N.W., and T.B. collected and analyzed the data, interpreted the results, and wrote the manuscript; and all authors read and approved the manuscript.

Conflict-of-interest disclosure: K.A. received honoraria that were paid to the Medical University Hospital of Tuebingen for lectures from CSL Behring, Sobi, Meet The Experts, Expanda, and Werfen; and research grants that were paid to the Medical University Hospital of Tuebingen from Octapharma, Bayer, and Takeda. T.B. has received research funding from CoaChrom Diagnostica GmbH, Deutsche Forschungsgemeinschaft, Robert Bosch GmbH, Stiftung Transfusionsmedizin und Immunhämatologie e.V., Ergomed, DRK Blutspendedienst, Deutsche Herzstiftung, Ministerium fuer Wissenschaft, and Forschung und Kunst Baden-Wuerttemberg; has received lecture honoraria from Aspen Germany GmbH, Bayer Vital GmbH, Bristol Myers Squibb GmbH & Co. KG, Doctrina Med AG, Meet The Experts Academy UG, Schoechl Medical Education GmbH, Stago GmbH, Mitsubishi Tanabe Pharma GmbH, Novo Nordisk Pharma GmbH, Leo Pharma GmbH, and Swedish Orphan Biovitrum GmbH; and has provided consulting services to Terumo, and expert witness testimony related to heparin-induced thrombocytopenia (HIT) and non-HIT thrombocytopenic and coagulopathic disorders. All of these disclosures are outside the current work (ie, not related to the current work). The remaining authors declare no competing financial interests.

Correspondence: Tamam Bakchoul, Institute for Clinical and Experimental Transfusion Medicine, Medical Faculty of Tuebingen, University Hospital of Tuebingen, Otfried-Mueller-Str 4/1, 72076 Tuebingen, Germany; email: tamam.bakchoul@med.uni-tuebingen.de.