Aggregometry is widely used to assess platelet function, but its use in identifying platelet hyperreactivity is poorly defined. We studied platelet aggregation in 359 healthy individuals using the agonists adenosine diphosphate (ADP), epinephrine, collagen, collagen-related peptide, and ristocetin. We also assessed the reproducibility of these assays in 27 subjects by studying them repeatedly on at least 4 separate occasions. Healthy subjects exhibited considerable interindividual variability in aggregation response to agonists, especially at concentrations lower than those typically used in clinical laboratories. For each agonist tested at these submaximal concentrations, a small proportion of individuals demonstrated an unusually robust aggregation response. Subjects who exhibited such in vitro hyperreactivity to one agonist tended to demonstrate a similar response to others, suggesting that hyperreactivity is a global characteristic of platelets. Epinephrine and collagen-related peptide were especially reliable and efficient in detecting hyperreactivity. For epinephrine, excellent reproducibility persisted for up to 3 years, and hyperreactivity was associated with female sex and higher fibrinogen levels (P < .02). We recommend these assays as appropriate candidates for future studies requiring accurate assessment of increased platelet reactivity. These include clinical studies to improve risk assessment for arterial thrombosis, as well as genetic studies to establish determinants of the hyperreactive platelet phenotype. (Blood. 2005;106:2723-2729)

Summary. Background: Although platelet hyperreactivity constitutes an important cardiovascular risk factor, standardized methods for its measurement are lacking. We recently reported that aggregometry using a submaximal concentration of epinephrine identifies individuals with in vitro platelet hyperreactivity; this hyperreactivity was reproducible on multiple occasions over long periods of time. Objective and methods: To better understand this aberrant reactivity, we studied in a large group of subjects (n ¼ 386) the relationship between healthy individualsÕ platelet reactivity to epinephrine and their platelet phenotype as measured by other functional assays. Results: Subjects with hyperreactivity to epinephrine were more likely to exhibit hyperfunction in each major aspect of platelet activity, including adhesion (response to low-dose ristocetin; P < 0.001), activation (surface P-selectin expression and PAC-1 binding after stimulation; P £ 0.003) and aggregation to other agonists [no agonist, adenosine diphosphate (ADP), arachidonic acid, collagen, collagen-related peptide and ristocetin; P £ 0.025] and to applied shear stress (PFA-100 and cone-and-plate viscometer; P < 0.05). These differences persisted after adjusting for demographic and hematologic differences between groups. We studied candidate genes relevant to epinephrine-mediated platelet activation and found that hyperreactivity to epinephrine was associated with a polymorphism on the gene (GNB3) encoding the beta-3 subunit of G proteins (P ¼ 0.03). Conclusions: Robust aggregation to a submaximal concentration of epinephrine establishes a true hyperreactive platelet phenotype that is ÔglobalÕ as opposed to agonist specific; detection of this phenotype could be useful for studying patients at risk for arterial thrombosis. The mechanisms underlying hyperreactivity to different types of platelet stimulation may share common signaling pathways, some of which may involve specific G protein subunits.

SummaryArterial stenosis results in a complex pattern of blood flow containing an extremely fast flow in the throat of stenosis and a post-stenosis low flow. The fast flow generates high shear stress that has been demonstrated in vitro to activate and aggregate platelets. One potential problem of these in vitro studies is that platelets are invariably exposed to a high shear stress for a period that is significantly longer than they would have experienced in vivo. More importantly, the role of the poststenosis low flow in platelet activation and aggregation has not been determined. By exposing platelets to a shear profile that contains both high and low shear segments, we found that platelets aggregate when they are exposed to a high shear stress of 100 dyn/cm2 for as short as 2.5 s, a period that is significantly shorter than those previously reported (30–120 s). Platelet aggregation under this condition requires a low shear exposure immediately after a high shear pulse, suggesting that post-stenosis low flow enhances platelet aggregation. Furthermore, platelet aggregation under this condition is not activation-dependent because the CD62P expression of sheared platelets is significantly less than that of platelets treated with ADP. Based on these findings, we propose that shear-induced platelet aggregation may be a process of mechanical crosslinking of platelets, requiring minimal platelet activation. This process may function as a protective mechanism to prevent in vivo irreversible platelet activation and aggregation under temporary high shear.

Summary. We examined the basis of the differences observed between different collagen preparations in their ability to aggregate platelets and support their adhesion under flow. As in previous studies, we found fibrillar collagen to be 10-fold more potent than acid-soluble collagen in inducing platelet aggregation and found that acid-soluble collagen did not support the adhesion of washed platelets under flow. Further, platelets in whole blood adhered to surfaces coated with either fibrillar or acid-soluble collagen, but thrombi formed faster and grew larger on fibrillar collagen. As a possible basis for this difference, we found that fibrillar collagen, but not acid-soluble collagen, contains a substantial quantity of von Willebrand factor (VWF), as demonstrated by enzyme-linked immunosorbent assay and by the ability of fibrillar collagen to support the adhesion of VWF antibody-coated beads and to agglutinate GPIb-IX-V complex-expressing Chinese hamster ovary cells. Supporting a role for VWF in collagen-induced platelet aggregation, aggregation induced by acid-soluble collagen was greatly enhanced by added VWF. Further, platelet aggregation by fibrillar collagen was partially blocked by a GPIba antibody that inhibits the GPIb-VWF interaction. Taken together, these results suggest that much of the difference in prothrombotic potency of different collagens is directly related to their differences in VWF content. This probably accounts for the different conclusions made regarding the relative importance of different direct and indirect collagen receptors in collagendependent platelet functions and further emphasizes the close synergistic roles of the GPIb-IX-V complex and the collagen receptors GPVI and a 2 b 1 in supporting platelet adhesion.

In this initial prevalence study of a clinically diverse group of pediatric patients, frequencies of AR were assay-dependent; however, the prevalence of true AR is likely low in children (2.3%; 95% CI 0.1-10.7%), in agreement with adult studies. To better define the clinical relevance of AR in children, multicenter, prospective cohort studies are imperative.