Abstract
Many platelet function disorders (PFD) present as uncharacterized disorders that impair aggregation responses to multiple agonists and/or cause non-syndromic dense granule deficiency (DGD). We postulated that some of these commonly encountered PFD might also impair the ability of platelets to support thrombin generation, given that an important subgroup are caused by mutations in transcription factors, such as RUNX1, that impair multiple aspects of platelet function. Accordingly, we initiated a study of thrombin generation (TG) in a prospective cohort of individuals presenting with an uncharacterized PFD. In addition, we reassessed how Quebec platelet disorder (QPD, previously called Factor V Quebec) affects coagulation as this disorder (which is caused by a duplication mutation of PLAU) triggers intraplatelet (but not systemic) plasmin generation and proteolysis of platelet but not plasma factor V (FV), and the normal plasma FV might potentially compensate for the loss of platelet FV in QPD.
The study was conducted with written informed consent of participants and with the approval of the Hamilton Integrated Research Ethics Board and the Research Ethics Board of Centre Hospitalier Universitaire Sainte Justine. Participants included: 1) five individuals with QPD; 2) eighteen individuals presenting with an uncharacterized PFD and confirmed reduced maximal aggregation responses to ≥2 agonists and/or confirmed DGD, including five that had a pathogenic RUNX1 mutation; and 3) eighteen similarly-aged general population controls. TG was assessed by the calibrated automated thrombogram (CAT) procedure on a Fluoroskan (Thermo Fisher Scientific AG, Reinach, Switzerland) using Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), manufacturer- and ISTH-recommended protocols and reagents for testing platelet poor plasma (PPP) and platelet rich plasma (PRP). Endpoints included: endogenous thrombin potential (ETP, nM·min), peak thrombin concentration (nM), time-to-peak (min) and lag time (min). Platelet lysate and plasma FV concentrations were determined by enzyme-linked immunoassay. Data were analyzed using Mann-Whitney tests with Bonferroni correction for multiple comparisons.
All TGA endpoints for PPP were comparable for controls and participants with PFD, including QPD (p values ≥0.10). In TGA with PRP, most PFD participants had findings similar to controls, however, the subgroups with pathogenic RUNX1 mutations or QPD had significantly reduced ETP and peak thrombin concentration compared to controls (data as median [range]: ETP: controls: 1860 [1530-2630]; RUNX1 mutation subgroup: 1520 [805-1640], p=0.004; QPD: 1370 [981-2010], p=0.01; peak thrombin concentration: controls: 111 [65-152]; RUNX1 mutations: 66 [32-93], p=0.006; QPD: 59 [41-91], p=0.005). Plasma FV levels were similar in all subjects (µg/ml PPP, median [range]: controls: 7.7 [6.3-10.7]; QPD: 8.4 [7.0-10.2], p=0.33; other PFD: 7.5 [5.4-12], p=0.74) and showed no significant association to TG endpoints for PPP or PRP (p values ≥0.14). Only QPD subjects had platelet FV deficiency (µg FV/mg platelet protein, median [range]: controls: 0.89 [0.63-1.54]; QPD: 0.35 [0.18-0.46], p<0.001; other PFD: 0.83 [0.47-1.75], p=0.48; RUNX1 mutation subgroup: 0.73 [0.50-1.53], p=0.41). In QPD, but not other participants, platelet FV showed a significant association to ETP (R2=0.81, p=0.04) and peak TG endpoints (R2=0.88, p=0.01).
Our study illustrates that platelet-dependent thrombin generation, evaluated by CAT, is abnormal in QPD but normal in many uncharacterized PFD with defective aggregation and/or DGD except for the important subgroup with pathogenic RUNX1 mutations, which impair TG but do not reduce platelet FV (unlike QPD). In QPD, the platelet-dependent TG defect shows a unique relationship to the platelet FV deficiency, which suggests that the normal levels of plasma FV are insufficient to compensate for the platelet procoagulant abnormalities in this PFD.
Disclosures
No relevant conflicts of interest to declare.
Thrombin generation abnormalities in commonly encountered platelet function disorders
