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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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Stimulating effect of co-coating of microspots with vWF-BP or vWF.Whole blood from control subjects was perfused over arrays of microspots for 3.5 min at 1.600 s−1, and analysed for thrombus formation as in Fig. 1. (a) Sub-heatmaps of thrombus formation parameters of single coatings (left panel), co-coatings with vWF-BP (middle panel) or co-coatings with vWF (right panel). Formation of type I, II and III thrombi is represented by colour bars from grey to black. (b) Subtraction heatmaps, indicating the effects of co-coating with vWF-BP (middle) or with vWF (right). Colour code is from −1 to 10. *P<0.05 (two-tailed Student’s t-test) compared with single coating, per row or column.
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f5: Stimulating effect of co-coating of microspots with vWF-BP or vWF.Whole blood from control subjects was perfused over arrays of microspots for 3.5 min at 1.600 s−1, and analysed for thrombus formation as in Fig. 1. (a) Sub-heatmaps of thrombus formation parameters of single coatings (left panel), co-coatings with vWF-BP (middle panel) or co-coatings with vWF (right panel). Formation of type I, II and III thrombi is represented by colour bars from grey to black. (b) Subtraction heatmaps, indicating the effects of co-coating with vWF-BP (middle) or with vWF (right). Colour code is from −1 to 10. *P<0.05 (two-tailed Student’s t-test) compared with single coating, per row or column.

Mentions: Given the established role of vWF/GPIb in platelet adhesion at high-shear blood flow221, we performed a sub-analysis of thrombi formed on microspots co-coated with indirectly or directly GPIb-binding substances, that is, vWF-BP or vWF, respectively. The resulting heatmaps of Fig. 5a show that, for the majority of microspots, the presence of vWF-BP or vWF increased thrombus formation, as assessed from the morphological score, integrated feature size, stable platelet adhesion, platelet deposition and thrombus volume. Values of these parameters significantly increased (P<0.05, Student's t-test) compared with the surfaces without vWF (-BP), with the exception of microspots containing α6β1 ligand, laminin (Fig. 5b). Other parameters, such as fibrinogen binding and P-selectin expression, increased to a lesser extent, still significant with co-coated vWF but not vWF-BP. Procoagulant activity was not increased. Overall, the analysis identified most prominent roles of vWF (-BP) on thrombus formation co-coated with, in decreasing order, (assigned receptors in brackets): rhodocytin (CLEC-2)>(GPO)n (GPVI), GFOGER-(GPP)n (α2β1)>decorin, osteopontin, fibrinogen, fibronectin, vitronectin (integrins α2β1, α5β1, αIIbβ3, αvβ3)>laminin, thrombospondin-1 (α6β1, CD36).


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)

Stimulating effect of co-coating of microspots with vWF-BP or vWF.Whole blood from control subjects was perfused over arrays of microspots for 3.5 min at 1.600 s−1, and analysed for thrombus formation as in Fig. 1. (a) Sub-heatmaps of thrombus formation parameters of single coatings (left panel), co-coatings with vWF-BP (middle panel) or co-coatings with vWF (right panel). Formation of type I, II and III thrombi is represented by colour bars from grey to black. (b) Subtraction heatmaps, indicating the effects of co-coating with vWF-BP (middle) or with vWF (right). Colour code is from −1 to 10. *P<0.05 (two-tailed Student’s t-test) compared with single coating, per row or column.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Stimulating effect of co-coating of microspots with vWF-BP or vWF.Whole blood from control subjects was perfused over arrays of microspots for 3.5 min at 1.600 s−1, and analysed for thrombus formation as in Fig. 1. (a) Sub-heatmaps of thrombus formation parameters of single coatings (left panel), co-coatings with vWF-BP (middle panel) or co-coatings with vWF (right panel). Formation of type I, II and III thrombi is represented by colour bars from grey to black. (b) Subtraction heatmaps, indicating the effects of co-coating with vWF-BP (middle) or with vWF (right). Colour code is from −1 to 10. *P<0.05 (two-tailed Student’s t-test) compared with single coating, per row or column.
Mentions: Given the established role of vWF/GPIb in platelet adhesion at high-shear blood flow221, we performed a sub-analysis of thrombi formed on microspots co-coated with indirectly or directly GPIb-binding substances, that is, vWF-BP or vWF, respectively. The resulting heatmaps of Fig. 5a show that, for the majority of microspots, the presence of vWF-BP or vWF increased thrombus formation, as assessed from the morphological score, integrated feature size, stable platelet adhesion, platelet deposition and thrombus volume. Values of these parameters significantly increased (P<0.05, Student's t-test) compared with the surfaces without vWF (-BP), with the exception of microspots containing α6β1 ligand, laminin (Fig. 5b). Other parameters, such as fibrinogen binding and P-selectin expression, increased to a lesser extent, still significant with co-coated vWF but not vWF-BP. Procoagulant activity was not increased. Overall, the analysis identified most prominent roles of vWF (-BP) on thrombus formation co-coated with, in decreasing order, (assigned receptors in brackets): rhodocytin (CLEC-2)>(GPO)n (GPVI), GFOGER-(GPP)n (α2β1)>decorin, osteopontin, fibrinogen, fibronectin, vitronectin (integrins α2β1, α5β1, αIIbβ3, αvβ3)>laminin, thrombospondin-1 (α6β1, CD36).

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