Angela L. Bergeron

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin.

Background: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)‐1 and COX‐2. Objectives: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. Methods: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325‐mg dose of aspirin. Platelet studies included light transmission aggregometry; P‐selectin and integrin αIIbβ3 expression, and serum thromboxane B2 (TxB2) levels. Reticulated platelets and platelet COX‐2 expression were measured using flow cytometry. Results: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 μg mL−1 collagen, and postaspirin aggregations to 5 μm and 20 μm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P‐selectin and integrin αIIbβ3 expression were also higher in the upper tertile both before and after aspirin. Platelet COX‐2 expression was detected in 12 ± 7% (n = 10) of platelets in the upper tertile, and in 7 ± 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB2 levels were higher in the upper (5.5 ± 4 ng mL−1) than in the lower tertile (3.2 ± 2.5 ng mL−1, P = 0.03), and decreased even further with ex vivo additional COX‐1 and COX‐2 inhibition. The incidence of aspirin resistance (≥ 70% platelet aggregation to 5 μm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). Conclusions: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX‐1 and COX‐2 activity.

Flow cytometric measurement of microparticles: Pitfalls and protocol modifications

Upon activation, many cells shed components of their plasma membranes as microparticles. Depending on the methods of preparation and analyses, microparticle counts may vary significantly between laboratories, making data analyses and clinical correlations challenging. To assess how variations in sample preparation affect microparticle measurements, blood samples from 13 healthy, adult volunteers were labeled with Annexin V, cell-specific antibodies, and antibodies against tissue factor (TF). Data were acquired and analysed using an EPICS XL-MCL flow cytometer. Annexin V+ monocyte-, platelet-, endothelial-, or erythrocyte-derived microparticles accounted for 10.4%, 38.5%, 43.8%, and 7.3% of the total number of microparticles (13.7 ± 3.0 × 103/ml of whole blood), respectively. A similar distribution of cell types was seen for TF+ microparticles (6.3 ± 2.6 × 103/ml of whole blood). No statistical difference was noted in microparticle distribution using either 19- or 21-gauge needles. Elevated levels of platelet- and erythrocyte-derived microparticles were detected in heparin and PPACK-anticoagulated samples as compared to samples anticoagulated with ACD or sodium citrate (P < 0.05, students t-test). Additional centrifugation was critical for removing platelet contamination, which significantly affected microparticle counts. Finally, Annexin V+ and TF+ microparticles were significantly reduced upon sample storage at low temperatures. Microparticle levels are significantly affected by variations in sample preparation and storage. These results illustrate the need to standardize assay protocols in order to obtain consistent measurements. Our studies further optimize sample preparation for microparticle detection.

Platelet‐derived VWF‐cleaving metalloprotease ADAMTS‐13

Summary.  The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra‐large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin‐stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation‐dependent protease. A recent study shows that platelets contain the VWF‐cleaving metalloprotease ADAMTS‐13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell‐derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS‐13 antibody from the plasma of a patient with acquired TTP. ADAMTS‐13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS‐13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor‐activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS‐13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS‐13 to encounter the increase in ULVWF release from endothelial cells.

Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs.

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.

Effects of low temperature on shear-induced platelet aggregation and activation

BACKGROUND Hemorrhage is a major complication of trauma and often becomes more severe in hypothermic patients. Although it has been known that platelets are activated in the cold, studies have been focused on platelet behavior at 4 degrees C, which is far below temperatures encountered in hypothermic trauma patients. In contrast, how platelets function at temperatures that are commonly found in hypothermic trauma patients (32-37 degrees C) remains largely unknown, especially when they are exposed to significant changes in fluid shear stress that could occur in trauma patients due to hemorrhage, vascular dilation/constriction, and fluid resuscitation. METHODS Using a cone-plate viscometer, we have examined platelet activation and aggregation in response to a wide range of fluid shear stresses at 24, 32, 35, and 37 degrees C. RESULTS We found that shear-induced platelet aggregation was significantly increased at 24, 32, and 35 degrees C as compared with 37 degrees C and the enhancement was observed in whole blood and platelet-rich plasma. In contrast to observation made at 4 degrees C, the increased shear-induced platelet aggregation at these temperatures was associated with minimal platelet activation as determined by the P-selectin expression on platelet surface. Blood viscosity was also increased at low temperature and the changes in viscosity correlated with levels of plasma total protein and fibrinogen. CONCLUSION We found that platelets are hyper-reactive to fluid shear stress at temperatures of 24, 32, and 35 degrees C as compared with at 37 degrees C. The hyperreactivity results in heightened aggregation through a platelet-activation independent mechanism. The enhanced platelet aggregation parallels with increased whole blood viscosity at these temperatures, suggesting that enhanced mechanical cross-linking may be responsible for the enhanced platelet aggregation.

Proteomic analysis of resting and thrombin-stimulated platelets reveals the translocation and functional relevance of HIP-55 in platelets

The platelet surface is a dynamic interface that changes rapidly in response to stimuli to co‐ordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin‐stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS‐PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT‐ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip‐55. HIP‐55 is an SH3‐binding protein important in T‐cell receptor signalling. Further analysis of HIP‐55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin β3 upon platelet activation. Analysis of HIP‐55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP‐55 to be an important regulator of platelet function.

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.

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling.

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin αIIbβ3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin αIIbβ3 in resting platelets and in human embryonal kidney 293 cells expressing αIIbβ3. The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin αIIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to αIIbβ3 during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Acα in 293 cells decreased αIIbβ3-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated αIIbβ3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac α-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2Acα expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac α can negatively regulate integrin αIIbβ3 signaling by suppressing the ERK1/2 signaling pathway.