Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline.

Light transmission aggregometry (LTA) is considered the gold standard method for evaluation of platelet function. However, there are a lot of variation in protocols (pre-analytical procedures and agonist concentrations) and results. The aim of our study was to establish a national LTA protocol, to investigate the effect of standardization and to define national reference values for LTA. The SSC guideline was used as base for a national procedure. Almost all recommendations of the SSC were followed e.g. no adjustment of PRP, citrate concentration of 109 mM, 21 needle gauge, fasting, resting time for whole blood and PRP, centrifugation time, speed and agonists concentrations. LTA of healthy volunteers was measured in a total of 16 hospitals with 5 hospitals before and after standardization. Results of more than 120 healthy volunteers (maximum aggregation %) were collected, with participating laboratories using 4 different analyzers with different reagents. Use of low agonist concentrations showed high variation before and after standardization, with the exception of collagen. For most high agonist concentrations (ADP, collagen, ristocetin, epinephrine and arachidonic acid) variability in healthy subjects decreased after standardization. We can conclude that a standardized Dutch protocol for LTA, based on the SSC guideline, does not result in smaller variability in healthy volunteers for all agonist concentrations.

Potentiating role of Gas6 and Tyro3, Axl and Mer (TAM) receptors in human and murine platelet activation and thrombus stabilization.

BACKGROUND: Interaction of murine Gas6 with the platelet Gas6 receptors Tyro3, Axl and Mer (TAM) plays an important role in arterial thrombus formation. However, a role for Gas6 in human platelet activation has been questioned. OBJECTIVE: To determine the role of Gas6 in human and murine platelet activation and thrombus formation. METHODS AND RESULTS: Gas6 levels appeared to be 20-fold higher in human plasma than in platelets, suggesting a predominant role of plasma-derived Gas6. Human Gas6 synergizes with ADP-P2Y(12) by enhancing and prolonging the phosphorylation of Akt. Removal of Gas6 from plasma impaired ADP-induced platelet aggregation. Under flow conditions, absence of human Gas6 provoked gradual platelet disaggregation and integrin α(IIb) ß(3) inactivation. Recombinant human Gas6 reversed the effects of Gas6 removal. In mouse blood, deficiency in Gas6 or in one of the TAM receptors led to reduced thrombus formation and increased disaggregation, which was completely antagonized by external ADP. In contrast, collagen-induced platelet responses were unchanged by the absence of Gas6 in both human and mouse systems. CONCLUSIONS: The ADP-P2Y(12) and Gas6-TAM activation pathways synergize to achieve persistent α(IIb) ß(3) activation and platelet aggregation. We postulate a model of thrombus stabilization in which plasma Gas6, by signaling via the TAM receptors, extends and enhances the platelet-stabilizing effect of autocrine ADP, particularly when secretion becomes limited.

Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin alpha2beta1 and glycoprotein VI.

BACKGROUND: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). OBJECTIVE: The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation. RESULTS: At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n). CONCLUSIONS: Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.