Platelet-Dependent Thrombin Generation Is Correlated with Phenotypic Severity in Severe Hemophilia a

Hemophilias A and B are classified into mild (>5-40%), moderate (2-5%) and severe (<1%) disease based upon plasma factor activity levels. Severity of bleeding is commensurate with the baseline factor levels in general. However, heterogeneity of bleeding pattern and severity in patients with severe disease is well described. The biologic mechanism responsible for this phenotypic heterogeneity has yet to be thoroughly explained. A role of platelets, as key contributors to hemostatic plug formation and coagulation, has been investigated in previous small cohorts of patients with severe hemophilia with variable results.

A single-center cohort of 94 patients with severe hemophilia A were enrolled into an IRB approved study. Patients with concomitant bleeding disorders or thrombocytopenia (<100K/μL) were excluded. Flow cytometry was utilized to assess multiple aspects of platelet function, including 1) granule release (P-selectin) and integrin α_IIbβ₃ activation (PAC-1 high) at low and high doses of thrombin and the GPVI agonist convulxin and 2) procoagulant platelet formation (Annexin V high, PAC-1 low) with combined thrombin and convulxin stimulation. In 46 patients who had not received factor prophylaxis in the 96 hours prior to laboratory assessment, a thrombin generation profile, initiated by low concentration tissue factor, was obtained in both platelet-rich (PRP) and platelet-poor (PPP) plasma. To assess phenotypic severity, the Hemophilia Severity Score (Schulman S et al, 2008) was determined for each patient. The HSS score integrates bleeding rate, joint damage, and factor usage to obtain a composite measure of disease severity. Regression analysis was utilized to assess association (significance = p<0.05).

No association with HSS was found with either granule release or integrin activation. This lack of association with bleeding phenotype in severe hemophilia is consistent with previously reported results. The formation of coated, or procoagulant platelets, has previously been linked with phenotype in hemophilia A. In this large single-center cohort, no association of procoagulant platelet formation with HSS was noted, either in the whole population, or when analyzed by genotype or prophylaxis versus on-demand therapy. Measures of thrombin generation in PRP have been weakly linked with phenotype in severe hemophilia A. In our single-center cohort, in severe hemophilia A patients with large genetic mutations a strong association was noted between HSS and both time to peak (r = .410) and lag time (r=.450). This association persisted in PPP for time to peak, but was lost in PPP for lag time demonstrating the critical modulatory role of platelets in this association. No significant association was noted with other measures of thrombin generation. The results presented here indicate a critical modulatory role for platelet-dependent initiation of thrombin generation in determining phenotypic severity in patients with severe hemophilia A. Confirmation of these results would identify a potentially early biologic signal that could be used to differentiate phenotypic severity in young patients with severe hemophilia A and suggest the potential utility of modulation of platelet function in severe hemophilia A patients, especially those with large genetic mutations.