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Identification of platelet function defects by multi-parameter assessment of thrombus formation.

de Witt SM, Swieringa F, Cavill R, Lamers MM, van Kruchten R, Mastenbroek T, Baaten C, Coort S, Pugh N, Schulz A, Scharrer I, Jurk K, Zieger B, Clemetson KJ, Farndale RW, Heemskerk JW, Cosemans JM - Nat Commun (2014)

Bottom Line: Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3.Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome.We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.

ABSTRACT
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

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Effect of wall shear rate on thrombus formation on microspot surfaces.Whole blood was perfused over surfaces with or without vWF at indicated wall shear rates. (a) Measured parameters of thrombus formation at 1,600 s−1 (2 × 18 surfaces, clustering order as in Fig. 4). (b) Measurement parameters of thrombus formation at 150 s−1. (c) Linear subtraction heatmap of outcome parameters at low shear rate compared with high shear rate. P<0.01 (two-tailed Student’s t-test).
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f6: Effect of wall shear rate on thrombus formation on microspot surfaces.Whole blood was perfused over surfaces with or without vWF at indicated wall shear rates. (a) Measured parameters of thrombus formation at 1,600 s−1 (2 × 18 surfaces, clustering order as in Fig. 4). (b) Measurement parameters of thrombus formation at 150 s−1. (c) Linear subtraction heatmap of outcome parameters at low shear rate compared with high shear rate. P<0.01 (two-tailed Student’s t-test).

Mentions: To substantiate this further, we also investigated the role of vWF by comparing thrombus formation at high (1,600 s−1) and low (150 s−1) wall shear rates, using 36 surfaces. At low shear rate, all six parameters analysed contributed to formation of type I–III thrombi with high coefficients of determination (Table 1). Heatmaps indicated that, in general, many surfaces that actively supported thrombus formation at high wall shear rate performed less well at low wall shear rate (Fig. 6a,b). On the other hand, several of the surfaces that were less active at high shear rate, particularly those not containing vWF, became more active at lower shear rate. These effects were even more apparent after subtraction analysis (Fig. 6c), pointing to improved thrombus formation at high shear rate for all vWF-containing surfaces (P<0.05) on the one hand. Interestingly on the other hand, combinations of laminin (α6β1), rhodocytin (CLEC-2) and GFOGER-(GPO)n (GPVI, α2β1) provoked high and often increased thrombus formation in the absence of vWF at lower shear rate. Confirmation of these shear-dependent effects was obtained by perfusion studies with 19 surfaces at three wall shear rates of 150, 1,000 and 1,600 s−1. In general, platelet deposition at 1,000 s−1 was somewhat lower than at 1,600 s−1, the exception being laminin-containing surfaces where highest values were obtained at 1,000 s−1 (Supplementary Fig. 4).


Identification of platelet function defects by multi-parameter assessment of thrombus formation.

de Witt SM, Swieringa F, Cavill R, Lamers MM, van Kruchten R, Mastenbroek T, Baaten C, Coort S, Pugh N, Schulz A, Scharrer I, Jurk K, Zieger B, Clemetson KJ, Farndale RW, Heemskerk JW, Cosemans JM - Nat Commun (2014)

Effect of wall shear rate on thrombus formation on microspot surfaces.Whole blood was perfused over surfaces with or without vWF at indicated wall shear rates. (a) Measured parameters of thrombus formation at 1,600 s−1 (2 × 18 surfaces, clustering order as in Fig. 4). (b) Measurement parameters of thrombus formation at 150 s−1. (c) Linear subtraction heatmap of outcome parameters at low shear rate compared with high shear rate. P<0.01 (two-tailed Student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109023&req=5

f6: Effect of wall shear rate on thrombus formation on microspot surfaces.Whole blood was perfused over surfaces with or without vWF at indicated wall shear rates. (a) Measured parameters of thrombus formation at 1,600 s−1 (2 × 18 surfaces, clustering order as in Fig. 4). (b) Measurement parameters of thrombus formation at 150 s−1. (c) Linear subtraction heatmap of outcome parameters at low shear rate compared with high shear rate. P<0.01 (two-tailed Student’s t-test).
Mentions: To substantiate this further, we also investigated the role of vWF by comparing thrombus formation at high (1,600 s−1) and low (150 s−1) wall shear rates, using 36 surfaces. At low shear rate, all six parameters analysed contributed to formation of type I–III thrombi with high coefficients of determination (Table 1). Heatmaps indicated that, in general, many surfaces that actively supported thrombus formation at high wall shear rate performed less well at low wall shear rate (Fig. 6a,b). On the other hand, several of the surfaces that were less active at high shear rate, particularly those not containing vWF, became more active at lower shear rate. These effects were even more apparent after subtraction analysis (Fig. 6c), pointing to improved thrombus formation at high shear rate for all vWF-containing surfaces (P<0.05) on the one hand. Interestingly on the other hand, combinations of laminin (α6β1), rhodocytin (CLEC-2) and GFOGER-(GPO)n (GPVI, α2β1) provoked high and often increased thrombus formation in the absence of vWF at lower shear rate. Confirmation of these shear-dependent effects was obtained by perfusion studies with 19 surfaces at three wall shear rates of 150, 1,000 and 1,600 s−1. In general, platelet deposition at 1,000 s−1 was somewhat lower than at 1,600 s−1, the exception being laminin-containing surfaces where highest values were obtained at 1,000 s−1 (Supplementary Fig. 4).

Bottom Line: Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3.Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome.We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.

ABSTRACT
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Show MeSH
Related in: MedlinePlus