Carol Sun

Platelet hyperreactivity generalizes to multiple forms of stimulation

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 = 386) the relationship between healthy individuals’ 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 = 0.03). Conclusions: Robust aggregation to a submaximal concentration of epinephrine establishes a true hyperreactive platelet phenotype that is ‘global’ 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.

Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation.

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.

Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation.

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 GPIbα 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 collagen‐dependent platelet functions and further emphasizes the close synergistic roles of the GPIb–IX–V complex and the collagen receptors GPVI and α2β1 in supporting platelet adhesion.

Platelet FcgammaRIIA His131Arg polymorphism and platelet function: antibodies to platelet-bound fibrinogen induce platelet activation.

Summary.  The His131Arg polymorphism of platelet FcγRIIA 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 FcγRIIA 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. FcγRIIA 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 = 0.0057), and the ligand‐induced binding site antibody LIBS1 (P = 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 FcγRIIA cross‐linking were responsible for those discrepancies. The increased binding of the two RIBS antibodies to the Arg131 isoform was abolished by blocking FcγRIIA, and the FcγRIIA genotype effect on F26 IgG binding was lost when F26 F(ab′)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 FcγRIIA does not affect intrinsic platelet reactivity; (b) RIBS antibodies are able to cross‐link FcγRIIA 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 FcγRIIA His131Arg effect on platelet activation.

Low prevalence and assay discordance of “aspirin resistance” in children

Although “aspirin resistance” (AR‐inadequate platelet inhibition as suggested by in vitro testing of aspirin‐treated patients) has been widely studied in adults and linked to increased risk of adverse outcomes, its prevalence and clinical significance are largely unknown in children.