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High-throughput elucidation of thrombus formation
expected. For the two patients with storage pool disease, these values appeared to be in the lower ranges of normal. To further assess the subject-dependent component of this thrombus signature, we evaluated the intra- and inter- individual variations in Σ(P2-5, P8). This analysis indicated 2 to 5 times higher inter-individual coefficients of varia- tion (Figure 6B). Interestingly, this measure of the throm- bus signature was not related to GPVI expression of GPVI- induced platelet responses, thus pointing to other factors determining the platelet aggregation profile under flow
conditions.
Discussion
Multiparameter assessment of thrombus formation using microfluidic assays has proven to be relevant for the assessment of platelet dysfunction in patients with differ- ent bleeding diatheses.7-9 In the present study, we further developed this technique to assess the sources of variabil- ity in thrombus formation between individuals with nor- mal hemostasis. We evaluated platelet quantitative traits as well as thrombus formation in a cohort of 94 genetical- ly defined healthy subjects. Regression analysis identified an overall strong correlation between most parameters per microspot surface. When comparing different microspots, especially parameters of platelet aggregation and throm-
bus morphology (P2-5) correlated with each other for GPVI- and CLEC-2-activating surfaces (M1-2, M4-5). These parameters appear to describe subject-dependent differences in thrombus formation or thrombus signature, such as was also deduced from strong alterations in these parameters in blood from patients with Glanzmann thrombasthenia. Comparative matrices of other traits did not reveal age or sex as determinants of thrombus forma- tion, in accordance with previous analyses.20,25,26 A clear relation was however observed between GPVI expression levels and CRP-XL-induced platelet activation markers, which is in agreement with an early report.27
Several PCA were applied to compare the matrix of thrombus formation parameters with other platelet traits. An effect of platelet count and crit was seen for thrombus formation on collagen-containing microspots (M1-2), in agreement with an earlier conclusion that platelet count is a regulatory determinant of collagen-dependent thrombus formation.9 Regression analysis confirmed the predicted associations between mean platelet volume and expres- sion levels of most platelet membrane glycoproteins. In addition, associations were seen between platelet activa- tion markers after ADP, CRP-XL or TRAP activation and glycoprotein expression levels.
A PCA of thrombus parameters plus glycoprotein expression levels revealed marked contributions of most CD markers to the first component (95.5%) for M1, M2
Figure 6. Identification of thrombus signatures across microspots. (A) Supervised clustering of thrombus parameters P2-5 and P8 for microspots M1-6, aligned as indicated. Ranking of data from 94 subjects (cohort 2), patients with storage pool disease (SPD1-2) or Glanzmann thrombasthenia (GT1-3) and day-controls (C1-3) was according to the sum of normalized thrombus parameters of all surfaces Σ(P2-5, P8). Order of subjects (compare Figure 1B): 92, 49, 33, 4, 57, 35, 9, 19, 6, 8, 15, 24, 78, 50, 16, 42, 20, 65, C3, 72, 41, 44, 76, 71, 18, 25, 26, 52, 40, 10, C2, 51, 5, 12, 28, 29, 36, 67, 23, 32, 38, 2, 88, 17, 54, 59, 89, 81, 60, 80, 11, 82, 73, 79, 69, 55, 87, 48, 47, 66, 53, 7, 70, 93, 64, 1, 45, 46, 30, 91, 3, 94, 77, 68, 43, 34, 86, 62, 58, 37, 75, 63, 27, 39, 83, C1, 22, 85, 90, 21, 13, 31, 61, 74, 84, 56, SPD1, SPD2, GT1, GT2, GT3. Note the overall consistency of the platelet aggregation-linked parameters (P2-5, P8) per subject. (B) Intra- and inter-individual coeffi- cients of variance (CV) of summative value Σ(P2- 5, P8) per microspot (cohort 2), together with ratios indicating high subject-dependency of this thrombus signature.
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