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.

Platelet functions are increasingly measured under flow conditions to account for blood hydrodynamic effects. Typically, these studies involve exposing platelets to high shear stress for periods significantly longer than would occur in vivo. In the current study, we demonstrate that the platelet response to high shear depends on the duration of shear exposure. In response to a 100 dyn/cm2 shear stress for periods less than 10-20 sec, platelets in PRP or washed platelets were aggregated, but minimally activated as demonstrated by P-selectin expression and binding of the activation-dependent alphaIIbbeta3 antibody PAC-1 to sheared platelets. Furthermore, platelet aggregation under such short pulses of high shear was subjected to rapid disaggregation. The disaggregated platelets could be re-aggregated by ADP in a pattern similar to unsheared platelets. In comparison, platelets that are exposed to high shear for longer than 20 sec are activated and aggregated irreversibly. In contrast, platelet activation and aggregation were significantly greater in whole blood with significantly less disaggregation. The enhancement is likely via increased collision frequency of platelet-platelet interaction and duration of platelet-platelet association due to high cell density. It may also be attributed to the ADP release from other cells such as red blood cells because increased platelet aggregation in whole blood was partially inhibited by ADP blockage. These studies demonstrate that platelets have a higher threshold for shear stress than previously believed. In a pathologically relevant timeframe, high shear alone is likely to be insufficient in inducing platelet activation and aggregation, but acts synergistically with other stimuli.

Summary. The His131Arg polymorphism of platelet FcgRIIA affects the binding affinity of certain IgG subclasses. The Arg131 allele has been associated with (auto)immune thrombocytopenia and heparin-induced thrombocytopenia in some studies. Because FcgRIIA can transmit platelet activation signals, we studied platelet responsiveness from 73 healthy donors to determine if this polymorphism modulated platelet function. Platelet function was studied by agonist and shear-induced activation, and standard aggregation. FcgRIIA was genotyped by allele-specific PCR. Compared with His131, the Arg131 allele was associated with significantly greater binding of activation-dependent antibodies. This effect was most prominent for the receptor-induced binding site (RIBS) antibodies F26 (P < 0.0001) and RIBS1 (P ¼ 0.0057), and the ligandinduced binding site antibody LIBS1 (P ¼ 0.0367). Unexpectedly, Arg131-positive platelets did not show greater fibrinogen binding, platelet aggregation or shear-induced platelet activation. We considered whether enhanced Fc binding and FcgRIIA cross-linking were responsible for those discrepancies. The increased binding of the two RIBS antibodies to the Arg131 isoform was abolished by blocking FcgRIIA, and the FcgRIIA genotype effect on F26 IgG binding was lost when F26 F(ab 0 ) 2 fragments were used. Furthermore, intact F26 and RIBS1 IgG directly and specifically induced P-selectin expression, and this effect was greatest in Arg131-positive platelets. We concluded that (a) the His131Arg polymorphism of FcgRIIA does not affect intrinsic platelet reactivity; (b) RIBS antibodies are able to cross-link FcgRIIA and activate platelets, and this activation has a modest effect on Arg131 platelets; and (c) flow cytometric based platelet assays may need to compensate for this FcgRIIA His131Arg effect on platelet activation.

Platelet hypofunction occurs commonly in various settings and may contribute to clinically important bleeding; however, its laboratory evaluation remains a rather imprecise science. Although some platelet assays are well established in clinical practice, concerns about their reliability abound. Furthermore, few data exist on which to base laboratory criteria for identifying individuals with platelet hypofunction. To address these areas of need, we studied 449 healthy adults using assays commonly used in clinical laboratories to evaluate for impaired platelet function. These assays included optical aggregometry (using high concentrations of agonists in citrated platelet-rich plasma) and the PFA-100. In addition, we assessed the reproducibility of these assays by performing them on a subgroup of 27 individuals four times each on a weekly basis in a controlled setting. For each agonist and agonist concentration studied, we observed that a platelet aggregation response >60% clearly distinguished those subjects with complete aggregation from those who did not; we then identified minimal agonist concentrations at which ≥ 80% of healthy individuals demonstrated this degree of response (see table). Regarding reproducibility, a significant number of subjects showed widely discrepant results on repeated testing (see figure for example); the percentage of subjects who did so varied with the particular agonist employed (see table). Furthermore, our data obtained using the PFA-100 were less reproducible than has previously been reported and reproducibility varied with the cartridge employed (within-subject coefficient of variation=20.3% for epinephrine versus 8.5% for ADP). Aggregometry assays using different agonists correlated only partially with each other and with the PFA-100; the highest correlation coefficient was r=0.42 (p<.001, between ADP and epinephrine). In summary, our data provide a rational basis for specifying assay conditions and criteria (<60% aggregation in response to high concentrations of agonist) by which individuals with platelet hypofunction can be identified. However, despite studying only healthy subjects in a controlled research setting, we found the reproducibility of some commonly used platelet function tests to be rather poor; aggregometry using high concentrations of ADP or ristocetin and the PFA-100 using the ADP cartridge provided the most reliable results and are preferred assays in evaluating patients with suspected platelet hypofunction. Given the incomplete correlation between different assays, the other, less reliable assays may still provide useful information for an individual patient, but caution should be exercised in interpreting the results. Such assays may bear repeating, especially when the aggregation response to agonist is marginal (near 60%). Aggregation assay characteristics Platelet agonist Required* concentration % of subjects with discrepant** results *to yield complete aggregation in ≥ 80% of subjects; **>30% difference in assay results on separate occasions ADP 4 μ M 7 Arachidonic acid 0.5 mg/mL 25 Collagen-related peptide 0.02 μg/mL 19 Collagen 50 μ g/mL 15 Epinephrine 10 μ M 29 Ristocetin 1 mg/mL 7 Reproducibility of aggregation response in 27 subjects 50 μg/mL collagen Reproducibility of aggregation response in 27 subjects 50 μg/mL collagen