Differences In Platelet Response Research Articles

Extrapolation and Justification of Nplate Dosing to Improve Overall Survival in Acute Radiation Syndrome

Background: Acute Radiation Syndrome (ARS) is an acute illness caused by exposure to a high dose of penetrating radiation over a short period of time. Hematopoietic subsyndrome of ARS (HS-ARS) is characterized by dose dependent bone marrow depression leading to lymphopenia, neutropenia, thrombocytopenia, and anemia. Death due to HS-ARS from infection or excessive bleeding usually occurs within 2 to 3 weeks. Duration of thrombocytopenia is a predictor of overall survival (OS) in irradiated animal models, suggesting that a treatment for thrombocytopenia may increase survival in humans with HS-ARS. Romiplostim, a thrombopoietin receptor agonist, treatment resulted in prevention of severe thrombocytopenia and increased OS in irradiated animals. As human clinical trials for HS-ARS are not feasible or ethical, a romiplostim pharmacokinetic/pharmacodynamic (PKPD)-OS model for irradiated humans was developed. The model, informed by PKPD data in healthy/irradiated rhesus monkeys (RM) and healthy volunteers (HV), was subsequently used to predict the survival benefit of romiplostim relative to placebo in humans with HS-ARS. Methods: A PKPD model of romiplostim exposure-platelet response in healthy RM was developed and updated with radiation parameters to estimate the radiation effects on thrombopoiesis, on PKPD of romiplostim, and on differences in platelet response due to sex and body weight (Pritchard-Bell, ACoP11, 2020). A parametric time-to-event model relating platelet time course to OS in irradiated RM with/without romiplostim treatment was developed to quantify the impact of platelet response on OS (Jones, ACoP11, 2020). To extrapolate radiation effects to humans, romiplostim PKPD radiation parameters and radiation effects estimated from irradiated RM were applied to a romiplostim HV PKPD model. The extrapolated irradiated human PKPD model and irradiated RM OS model were combined and calibrated against published historical mortality data of humans exposed to acute radiation (Scott BR 1990). Following calibration, the extrapolated irradiated human PKPD and OS models were used to conduct simulations of OS in irradiated humans receiving romiplostim or placebo treatment. Relative survival benefit (RSB) of romiplostim, proportion of romiplostim treated humans surviving relative to placebo treated humans post radiation exposure, was summarized for various romiplostim doses (1 [initiation dose per label], 3, or 10 µg/kg [maximum dose per label]) and treatment scenarios (time after irradiation, adult vs pediatric) based on simulations of 10,000 humans/scenario randomized 1:1 to receive romiplostim or placebo. Results: An HV PKPD model of thrombopoiesis was updated to incorporate radiation specific scaling factors estimated from irradiated RM on romiplostim pharmacokinetic parameters and platelet lifespan, nonlinear inhibitory effect of radiation dose on megakaryocyte production, treatment effect of romiplostim on platelet lifespan, radiation sensitivity and radiation intensity. The extrapolated PKPD model for irradiated humans combined with the OS model from irradiated RM was calibrated with a scaling factor of 1.24 on radiation parameters to result in 50% survival 60 days post radiation exposure for humans exposed to a 3.07 Gy radiation dose (1 Gy/hr over 3.07 hr) consistent with published data. The calibrated OS model was used to simulate OS following romiplostim treatment 24 hours post irradiation and predicted 75% (RSB: 1.5), 80% (RSB: 1.6), and 87% (RSB: 1.7) survival on day 60, for romiplostim doses of at 1, 3, and 10 µg/kg, respectively compared to 50% survival for placebo. Percent survival was >75% when 10 µg/kg romiplostim was administered 24, 48, or 72 hours post irradiation and when administered to pediatric sub-groups (0-2, >2-6, >6-12, >12-18). Conclusions: A PKPD model of romiplostim exposure-response in irradiated humans was used to predict platelet response in humans with HS-ARS with/without treatment with romiplostim and was combined with an OS model to simulate 60-day survival in humans with HS-ARS. Simulations demonstrated the robust survival benefit of a single 10 µg/kg romiplostim dose compared with placebo in both adult and pediatric humans with HS-ARS following an acute radiation event. Selection of a romiplostim dose for treatment of HS-ARS is pending review of the irradiated RM studies and subsequent modeling and simulation analyses by the FDA. Disclosures Doshi: Amgen Inc: Current Employment. Jones:Amgen Inc: Current Employment. Pritchard-Bell:Amgen Inc: Current Employment. Park:Amgen Inc: Current Employment. Olsson Gisleskog:POG Pharmacometrics Ltd: Current Employment.

How Nanotopography-Induced Conformational Changes of Fibrinogen Affect Platelet Adhesion and Activation.

The conformational state of adsorbed human plasma fibrinogen (HPF) has been recognized as the determinant factor in platelet adhesion and thrombus formation on blood-contacting biomaterials. Studies have highlighted the ability to control the HPF conformation merely by tailoring surface nanotopographical features. However, a clear relationship between the conformational changes of adsorbed HPF and the degree of platelet adhesion and activation achieved with different surface nanotopographies is still unclear. Here, we examined HPF assembly characteristics on nanostructured polybutene-1 (PB-1) surfaces with nanosized lamellar crystals (LCs), needle-like crystals (NLCs), and a nanostructured high-density polyethylene (HDPE) surface with shish-kebab crystals (SKCs), at a biologically relevant HPF concentration. By exposing the nanostructured surfaces with preadsorbed HPF to human platelets, significant differences in platelet response on LCs/SKCs and NLCs were identified. The former presented a uniform monolayer in the advanced stage of activation, whereas the latter exhibited minimal adhesion and the early stage of activation. Distinct platelet response was related to the postadsorption conformational changes in HPF, which were confirmed by topography-dependent shifts of the amide I band in attenuated total reflection-Fourier transform infrared (ATR-FTIR) analysis. Supported by atomic force microscopy (AFM) characterization, we propose that the mechanism behind the nanotopography-induced HPF conformation is driven by the interplay between the aspect ratios of polymeric crystals and HPF. From the biomedical perspective, our work reveals that surface structuring in a nanoscale size regime can provide a fine-tuning mechanism to manipulate HPF conformation, which can be exploited for the design of thromboresistant biomaterials surfaces.

Influence of Sex on Platelet Reactivity in Response to Aspirin.

BackgroundThere are sex differences in the efficacy and safety of aspirin for the prevention of myocardial infarction and stroke. Whether this is explained by underlying differences in platelet reactivity and aspirin response remains poorly understood.Methods and ResultsHealthy volunteers (n=378 208 women) and patients with coronary artery disease or coronary artery disease risk factors (n=217 112 women) took aspirin for 4 weeks. Light transmittance aggregometry using platelet‐rich plasma was used to measure platelet reactivity in response to epinephrine, collagen, and ADP at baseline, 3 hours after the first aspirin dose, and after 4 weeks of daily aspirin therapy. A subset of patients underwent pharmacokinetic and pharmacodynamic assessment with levels of salicylate and cyclooxygenase‐1–derived prostaglandin metabolites and light transmittance aggregometry in response to arachidonic acid and after ex vivo exposure to aspirin. At baseline, women had increased platelet aggregation in response to ADP and collagen. Innate platelet response to aspirin, assessed with ex vivo aspirin exposure of baseline platelets, did not differ by sex. Three hours after the first oral aspirin dose, platelet aggregation was inhibited in women to a greater degree in response to epinephrine and to a lesser degree with collagen. After 4 weeks of daily therapy, despite higher salicylate concentrations and greater cyclooxygenase‐1 inhibition, women exhibited an attenuation of platelet inhibition in response to epinephrine and ADP.ConclusionsWe observed agonist‐dependent sex differences in platelet responses to aspirin. Despite higher cyclooxygenase‐1 inhibition, daily aspirin exposure resulted in a paradoxical attenuation of platelet inhibition in response to epinephrine and ADP over time in women but not in men.

Diabetes and Platelet Response to Low-dose Aspirin

Previous studies have suggested less cardioprotective benefit of aspirin in adults with diabetes, raising concerns about "aspirin resistance" and potentially reduced effectiveness for prevention of cardiovascular disease (CVD). To examine differences in platelet response to aspirin by diabetes status. We examined platelet response before and after aspirin (81 mg/day for 14 days) in 2113 adults (175 with diabetes, 1,938 without diabetes), in the Genetic Study of Aspirin Responsiveness cohort, who had family history of early-onset CVD. In vivo platelet activation (urinary thromboxane B2), in vitro platelet aggregation to agonists (arachidonic acid, adenosine diphosphate, collagen), and platelet function analyzer-100 closure time. Although adults with diabetes had higher in vivo platelet activation before aspirin, the reduction in in vivo platelet activation after aspirin was similar in those with vs without diabetes. Likewise, the reduction in multiple in vitro platelet measures was similar after aspirin by diabetes status. In regression analyses adjusted for age, sex, race, BMI, smoking, platelet counts, and fibrinogen levels, in vivo platelet activation remained higher in adults with vs without diabetes after aspirin (P = 0.04), but this difference was attenuated after additional adjustment for preaspirin levels (P = 0.10). No differences by diabetes status were noted for any of the in vitro platelet measures after aspirin in fully adjusted models that also accounted for preaspirin levels. In vitro platelet response to aspirin does not differ by diabetes status, suggesting no intrinsic differences in platelet response to aspirin. Instead, factors extrinsic to platelet function should be investigated to give further insights into aspirin use for primary prevention in diabetes.

Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia.

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.

Abstract WP263: Mutagenesis Studies Revealed Minimal Impact of Human A120T Variant of Protease-activated Receptor 4 on Receptor function or Pharmacological Response to a Potent and Selective Antagonist

Introduction: Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor and a novel target for ischemic stroke treatment. Recent reports suggested a subtle racial difference in platelet responses to submaximal concentrations of PAR4 agonist peptide (PAR4-AP). One of the PAR4 variants, A120T, is more common in African American than white (63% vs. 19%, Edelstein et al., Blood 2014). It was suggested that this variant contributes to the difference in response to PAR4-AP and impacts the in vitro response to YD-3, a PAR4 antagonist. In this study, site-directed mutagenesis was used to evaluate the effect of the A120T variant on PAR4 function and response to a newly discovered potent and selective PAR4 antagonist, UDM-001651. Unlike YD-3, UDM-001651 inhibited thrombin-induced platelet aggregation and prevented thrombosis in a monkey model and thus served as a relevant PAR4 pharmacology tool. Methods: Human PAR4 cDNAs expressing A120 and T120 variants were stably expressed in HEK293 cells. Cell surface expression of PAR4 was analyzed by FACS. Functional responses of cells were evaluated by monitoring calcium mobilization induced by BMS PAR4-AP, which was optimized based on AYPGKF. The potency of UDM-001651 was derived from an 11-point concentration response curve in the calcium assay using PAR4-AP at the EC80 concentration. Transient transfection studies were also performed to confirm the results. Results: Comparable levels of expression and functional responses were observed between A120 and T120 expressing cells. Results from side-by-side comparison between the two cell lines demonstrated no detectable difference in the mean EC50 values (0.42±0.054 versus 0.46±0.051 uM standard error, n=9) in calcium responses to PAR4-AP stimulation. Similarly, the potency of UDM-001651 in the same assay were similar between these two cell lines. Moreover, preliminary clinical results did not show differences in PAR4-mediated platelet response between African-American and white subjects. Conclusion: In contrast to the previous report, the results reported herein from site directed mutagenesis studies indicate that the A120T variant of PAR4 has no apparent impact on calcium signaling in response to agonist stimulation or response to a PAR4 antagonist.

Transcription Factor RUNX1 Regulates Pctp (Phosphatidylcholine Transfer Protein) in Platelets: A Potential Role in Regulating Platelet Function

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a critical hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, numerous platelet function defects, and increased leukemia risk. We have previously described a patient with a heterozygous RUNX1 nonsense mutation in the conserved runt domain necessary for DNA binding (Sun et al Blood 2004;103;948-54). The patient's platelet abnormalities included impaired aggregation and secretion in response to multiple agonists, granule deficiency, and decreased phosphorylation of myosin light chain and pleckstrin, activation of GPIIb-IIIa, 12-HETE production, and platelet protein kinase C-θ. Transcript expression profiling of patient platelets (Sun et al, J Thromb Haemost 2007;5:146-54) showed numerous genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP is a member of the START (Steroidogenic Acute Regulatory Protein-Related Transfer) domain superfamily and is responsible for the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Several findings indicate that PCTP is important for platelet function. PC is the main fraction of platelet phospholipids and source of arachidonic acid (AA) upon activation. Release of free AA from PC is the rate-limiting step in thromboxane production. PC is also a substrate for phospholipase D, which yields phosphatidic acid that can generate the second messenger diacylglycerol. Importantly, platelet PCTP has recently been demonstrated to have a major role in the racial differences in platelet responses - increased PCTP expression has been linked to the higher platelet aggregation and calcium mobilization induced by thrombin receptor protease-activated receptor 4 (PAR4) in blacks as compared to whites (Edelstein et al Nat Med 2013;19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to 995 bp of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding sites 1 (-232/-227) and 2 (-345/-340), at site 3 (-519/-514), and encompassing sites 4 and 5 (-861/-856, -884/-879). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-232/-227) and both sites 4 and 5 (-861/-856, -884/-879). To assess the effect of modulating RUNX1 expression on PCTP expression, PMA-treated HEL cells were transfected with RUNX1 overexpression plasmid or siRNA. PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown, suggesting that PCTP is regulated by RUNX1.Conclusions: Our results provide evidence that PCTP is a direct transcriptional target of and regulated by RUNX1, and a cogent molecular mechanism for downregulation of platelet PCTP in our patient with RUNX1 haplodeficiency. Regulation of PCTP by RUNX1 may be relevant to the platelet dysfunction in RUNX1 haplodeficiency as well as to racial differences in platelet responses linked to the differential platelet expression of PCTP. DisclosuresNo relevant conflicts of interest to declare.

Distinct Genetic Loci Are Associated with Platelet Aggregation Responses to Collagen and Collagen Synthetic Related Peptide (SRP) in Mice

There is substantial inter-individual variation in responsiveness of human platelets to different agonists in platelet aggregation studies. Much of this variation is likely heritable, and some of this variation can be attributed to genetic variants in platelet agonist receptors. However, beyond variants in known candidate receptor genes, the genetic factors underlying variation in platelet aggregation are undefined. In this study, we are taking a genome wide approach to identify genetic factors associated with platelet aggregation responses to different agonists in mice. We developed a method to quantify platelet aggregation responses to multiple agonists in single mouse donors and tested platelets from C57BL6/J (B6) and FVB/NJ (FVB) inbred mouse strains for their patterns of response to collagen (type I), collagen synthetic reactive peptide (SRP), the thromboxane A2 (TxA2) mimetic U44069, and PAR-4 activating peptide (AYPGKF) (B6 and FVB, n=9 for each strain). The platelet aggregation response was then quantified as an area under the aggregation curve (AUC) for each agonist. There was a significant difference between B6 and FVB in platelet responsiveness to collagen (9.06 x 103 ± 1.44 x 103 vs 3.7 x 103 ± 1.77 x 103. p<0.018), collagen SRP (2.04 x 103 ± 1.31 x 103 vs 1.25 x 104 ± 1.55 x 103. p<5 x 10-5), and U44069 (1.2 x 104 ± 2.19 x 103 vs 6.47 x 103 ± 1.85 x 103. p<0.05), respectively. No difference was observed in response to PAR-4 AP (7.07 x 103 ± 6.92 x 102 vs 8.24 x 103 ± 1.25 x 103. p=0.2). We then performed a B6 x FVB F2 intercross which generated 152 mice which were phenotyped for platelet aggregation responsiveness to collagen, collagen SRP, and U44069, using AYPGKF as a positive control. Thus far, we have performed genome-wide association studies in 54 mice using a 1449 SNP Illumina BeadChip platform. Quantitative trait loci (QTL) analysis was performed to identify QTLs associated with platelet responsiveness to collagen, SRP, and U44069. In this interim analysis, we have identified several significant QTL for platelet aggregation response to collagen and SRP. A single highly significant QTL for collagen SRP was identified on proximal chromosome 7 (LOD = 8.26) that spans ~23 Mb (5’ end to rs6239372), termed Plsrp1. Additionally, statistically significant but broad QTLs were identified for collagen on chromosome 14 (Plcoll1 and Plcoll2, peak LOD scores 3.78 and 3.87, respectively), with additional suggestive regions on chromosome 10 and chromosome 7. Intriguingly, the QTL with the strongest associations for collagen on chromosome 14 (Plcoll1 and Plcoll2) are necessarily distinct from the single collagen SRP QTL (Plsrp1) on chromosome 7. Interestingly, Plcoll1 and Plcoll2 would have been suspected from previous studies of collagen responsiveness in mice as chromosome 14 does not harbor the major candidate genes thought to interact with collagen, in contrast to Plsrp1 which contains Gp6. These findings indicate distinct and previously unsuspected genetic elements underlie the differences in platelet responsiveness to collagen and collagen SRP in mice. DisclosuresNo relevant conflicts of interest to declare.

Reference range determination for whole-blood platelet aggregation using the Multiplate analyzer.

To develop reference ranges for platelet aggregation using the Multiplate analyzer (Roche Diagnostics, Mannheim, Germany) in blood anticoagulated with sodium citrate (Na-citrate), lithium heparin (Li-heparin), or hirudin. The study was performed at three sites on consented, healthy adults (n = 193) not taking antiplatelet medication. Platelet aggregation was evaluated in response to adenosine-5'-diphosphate, arachidonic acid, collagen, thrombin receptor activating peptide, ristocetin, and adenosine-5'-diphosphate combined with prostaglandin E1. Precision testing was conducted using healthy donors and donors taking aspirin. Whole-blood platelet aggregation showed anticoagulant-dependent differences in platelet responses to all agonists. Samples collected in Na-citrate demonstrated the lowest responses to all agonists. The highest responses were obtained using Li-heparin. Precision testing revealed high variability in platelet aggregation at lower agonist doses, regardless of anticoagulant. Highest platelet response variations occurred in response to arachidonic acid in blood anticoagulated with hirudin from participants taking aspirin. These data demonstrate the importance of establishing locally relevant reference ranges.

Platelet function and HIV: a case–control study

Cardiovascular disease and myocardial infarction are of increasing concern in HIV-infected populations. Although platelets mediate arterial thrombosis, central to myocardial infarction, data on platelet function in HIV infection are lacking. We hypothesized that HIV-infected patients would have altered platelet reactivity. A case-control study of platelet reactivity in 20 HIV-infected (HIVpos) and 20 age and sex-matched HIV-negative (HIVneg) individuals. Time-dependent platelet aggregation was measured in response to increasing concentrations of platelet agonists: epinephrine, collagen, thrombin receptor-activating peptide and ADP using light absorbance. In both groups, mean age was 34 years, and 65% were men. Sixteen out of 20 (80%) of the HIVpos patients were on antiretroviral therapy with 12 out of 20 (60%) patients having HIV RNA less than 50 copies/ml. There were significant between-group differences in platelet reactivity across all four agonists. Platelets from HIVpos patients were more reactive to epinephrine [mean (SD) log concentration required to induce 50% maximal aggregation, 1.9 (1.2) versus 3.0 (1.7) mumol/l in HIVneg individuals, P = 0.028], whereas less platelet aggregation was observed in response to submaximal concentrations of the other agonists [thrombin receptor-activating peptide 72.5 (14.5)% versus 82.2 (7.6)% at 10 mumol/l, P = 0.011; ADP 67.3 (12.1)% versus 75.2 (8.8)% at 10 mumol/l, P = 0.035; collagen 16.6 (25.1)% versus 35.4 (31.5)% at 71.25 microg/ml, P = 0.007]. Between-group differences in platelet responses to all agonists suggest multiple underlying defects in platelet function in HIV infection. Further research is required to determine the contribution of antiretroviral therapy and relationships between platelet function and the increased cardiovascular disease observed in HIV-infected populations.

PAR1 antagonist mediated antithrombotic activity in extracorporeal arterio-venous shunt in the rat

The activation of platelets by thrombin is mediated at least in part by cleavage of protease-activated receptors (PARs) [ [1] Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057-1068 Abstract Full Text PDF PubMed Scopus (2641) Google Scholar ]. Four distinct PARs have been cloned to date, with PAR1, PAR3 and PAR4 being activated by thrombin, whereas PAR2 is sensitive to trypsin [ [2] Ossovskaya V.S. Bunnett N.W. Protease-activated receptors: contribution to physiology and disease. Physiol Rev. 2004; 84: 579-621 Crossref PubMed Scopus (908) Google Scholar ]. Human platelets express PAR1 and PAR4 and activation of either is sufficient to trigger platelet aggregation and secretion [ 1 Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057-1068 Abstract Full Text PDF PubMed Scopus (2641) Google Scholar , 3 Kahn M.L. Nakanishi-Matsui M. Shapiro M.J. Ishihara H. Coughlin S.R. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999; 103: 879-887 Crossref PubMed Scopus (680) Google Scholar ]. Studies with selective neutralizing antibodies have indicated that PAR1 mediates activation of human platelets at low thrombin concentrations and that, in the absence of PAR1 function, PAR4 can mediate platelet activation but only at high thrombin concentrations [ [4] Coughlin S.R. Thrombin signalling and protease-activated receptors. Nature. 2000; 407: 258-264 Crossref PubMed Scopus (2083) Google Scholar ]. Curiously, PAR1 appears not to play a role in rat platelets. PAR1-activating peptides do not activate rat platelet aggregation [ 5 Kinlough-Rathbone R.L. Rand M.L. Packham M.A. Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets. Blood. 1993; 82: 103-106 PubMed Google Scholar , 6 Derian C.K. Santulli R.J. Tomko K.A. Haertlein B.J. Andrande-Gordon P. Species differences in platelet responses to thrombin and SFLLRN receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases. Thromb Res. 1995; 78: 505-519 Abstract Full Text PDF PubMed Scopus (65) Google Scholar ]. In contrast to this, PAR3 is not expressed by human or guinea-pig platelets, but by mouse and rat platelets [ [7] Hishihara H. Connolly A.J. Zeng D. Kahn M.L. Zheng Y.W. Timmons C. et al. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997; 386: 502-506 Crossref PubMed Scopus (794) Google Scholar ]. Recently, Leger et al. [ [8] Leger A.J. Jacques S.L. Badar J. Kaneider N.C. Derian C.K. Andrade-Gordon P. et al. Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis. Circulation. 2006; 113: 1244-1254 Crossref PubMed Scopus (187) Google Scholar ] elegantly showed that in human platelets PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. Collectively, all these studies reveal the complexity of the PAR-mediated platelet activation in different animal species. Thus, the aim of the present study was to investigate the putative in vivo role of PAR1 in a thrombosis model conducted in anesthetized rats. We have investigated the effects of selective PAR4 antagonist, YD-3 [ [9] Wu C.C. Hwang T.L. Liao C.H. Kuo S.C. Lee F.Y. Lee C.Y. et al. Selective inhibition of protease-activated receptor 4-dependent platelet activation by YD-3. Thromb Haemost. 2002; 87: 1026-1033 PubMed Google Scholar ] but also relatively selective PAR1 antagonists ER-121958 and SCH-203099 [ 10 Ahn H.S. Foster C. Boykow G. Stamford A. Manna M. Graziano M. Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist. Biochem Pharmacol. 2000; 60: 1425-1434 Crossref PubMed Scopus (138) Google Scholar , 11 Ahn H.S. Chackalamannil S. Nonpeptide thrombin receptor antagonists. Drugs Future. 2001; 26: 1065-1085 Crossref Scopus (13) Google Scholar , 12 Chackalamannil S. Xia Y. Thrombin receptor (PAR-1) antagonists as novel antithrombotic agents. Expert Opin Ther Patents. 2006; 16: 493-505 Crossref PubMed Scopus (27) Google Scholar ] and conventional antithrombotic agents on an extracorporeal arteriovenous shunt model in the rat.

Diagnostic Evaluation of Platelet Function Disorders in Neonates and Children: An Update

Investigation of platelet function disorders in infants and small children requires the collaborative efforts of clinicians and clinical laboratories. A detailed personal, family, and medication history, and a search for additional clinical phenomena may help to direct diagnostic laboratory investigations. Testing for these disorders in young children presents several challenges: the requirement of relatively large volumes of blood, lack of standardization, and the absence of well-established age-specific reference ranges. Neonates show the most notable differences in platelet function compared to older children and adults; the decreased platelet activation responses persist for the first 2 to 4 weeks after delivery. Small studies of normal postneonatal pediatric populations have provided data on platelet function assays, including bleeding times, PFA-100 closure times, thromboelastography, aggregation, secretion, and flow cytometry. The majority of these studies, comparing normal children with adults, found only minor differences in platelet responses measured by these assays.

Differences in responses of platelets to fluid shear stress in patients with peripheral artery disease (PAD) and coronary artery disease (CAD)

Information on differences in platelet function between patients with peripheral arterial disease (PAD) and patients with coronary artery disease (CAD) is limited. We sought to examine the differences in the platelets response to shear stress in patients with PAD compared to those with CAD. Men with symptomatic PAD (ankle brachial index [ABI] < 0.9; n = 29) were compared with similarly aged men with CAD (post coronary artery bypass grafting; n = 40) but without PAD. All participants were on aspirin, and none were on clopidogrel. We measured changes in shear-induced platelet aggregation (SIPA) and shear-induced P-selectin expression (SIPE) under fluid shear rates of 5000 and 10,000 s−1which are typically found in arterioles and stenosed arteries, respectively. Aggregation was also induced by a combined stimulation of collagen, fluid shear stress, and adenosine diphosphate (ADP) or epinephrine using a platelet function analyzer (PFA-100) as well as optical aggregometry (arachidonic acid, collagen and epinephrine). Analyses of covariance adjusted for age, aspirin dose, and statin use were used to estimate differences between the groups. Values of SIPA at fluid shear rates of 5000 and 10,000 s−1 were significantly higher in the PAD group, while there were no differences between the PAD and CAD groups in SIPE at both fluid shear rates. However, baseline shear-induced P-selectin expression was higher in patients with PAD than CAD (mean fluorescence intensity [MFI] = 2.93 ± 1.37 vs.1.94 ± 0.67; p = 0.01), while the percentage increases in SIPA and SIPE at fluid shear rates of 5000 and 10,000 s−1 were significantly higher in patients with CAD when compared to PAD (p < 0.001 for all comparisons). Although there were several similarities in platelet function between men with PAD and men with CAD, significant differences in platelet responses to shear stress were observed in men with PAD when compared to those with CAD. Although the mechanism for these observed differences are not clear, we hypothesize that in vivo platelet activation in PAD patients may contribute to the differences and will need to be further investigated.

Increased platelet aggregation in vivo in the Zucker Diabetic Fatty rat: differences from the streptozotocin diabetic rat

Diabetes mellitus, especially type 2, is associated with increased arterial thrombosis. Our aims were (i) to characterize and compare platelet aggregation in vivo and in vitro in a type 2 diabetes model; and (ii) to determine whether these results differ from those in a type 1 diabetes model. Platelet aggregation to ADP in lean or obese Zucker Diabetic Fatty (ZDF) rats and in streptozotocin (STZ)-treated or control Wistar rats was measured in vitro, using Born aggregometry, and in vivo, by (111)Indium-labelled pulmonary platelet accumulation. In vivo, ADP responses were higher in obese (type 2 model) than lean ZDF rats. However, in vitro, ADP aggregation did not differ between platelet-rich plasma from ZDF lean or obese rats; nor was any difference seen in ADP responses when platelets from either lean or obese ZDF rats were suspended in plasma from obese or lean ZDF rats, respectively. In vivo, ADP responses were similar in STZ treated (type 1 model) and control rats whereas, in vitro, isolated platelets from STZ diabetic rats were more responsive to ADP aggregation than controls. Platelets from control or STZ-treated rats suspended in plasma from STZ-treated rats exhibited reduced ADP aggregation, compared to when suspended in plasma from control rats. The platelet aggregation results obtained in vitro do not reflect those in vivo, therefore in vitro aggregation data should be interpreted with caution. Moreover, both in vitro and in vivo, different diabetic models exhibit important differences in platelet responsiveness.

Platelet activation in a circulating flow loop: combined effects of shear stress and exposure time

Measurement of small changes in platelet activation state (PAS) in circulating stenotic systems in vitro has been problematic because of a paucity of real-time assay methods and circulation systems of low platelet-activating potential. PAS was measured by a modified prothrombinase assay in which activated platelets provide the essential cofactors in the activation of prothrombin by factor Xa. Chemical modification of the prothrombin ensures that the thrombin produced, while assayable, does not activate platelets. Human platelets were circulated in loops in which exposure to shear stress was adjusted by independently varying flow rate, viscosity, and the time of exposure to shear. Although with some differences in platelet response to different conditions of stress, the PAS directly increased with time of circulation, shear stress, and time of exposure to shear. The results show that low-level platelet activation caused by shear stress in a circulation loop can be quantitatively assessed in near-real time in a system of tube geometry. They confirm previous results obtained in non-circulating systems that exposure of platelets to shear conditions on the same order as found in the vasculature causes significant platelet activation, and that this activation is dependent on both shear stress and time of exposure.

A comparison of human and killer whale platelet fatty acid composition

Activation of blood platelets and their subsequent aggregation results from the interactions of several complex metabolic pathways. Considered to be of critical importance are the platelet lipids. Subsequent to platelet activation, several membrane lipids undergo hydrolysis and the free fatty acids are metabolized to prostanoids which mediate platelet function in response to vascular injury. It is conceivable then, that differences in platelet membrane fatty acid content could result in significant differences in platelet responses to aggregatory stimuli, especially between species. The objective of this study was to identify specific differences in fatty acid content between human and killer whale platelets. Blood was collected, washed platelets were prepared, and platelet fatty acids were extracted. Methyl esters of the extracted fatty acids were analyzed by gas chromatography and reported as relative concentrations. Analysis of the data revealed significant differences between the two species for several relevant fatty acids, i.e. 16:0 ( P<0.05), and 18:0, 18:1, 18:2, and 20:4 ( P<0.001). The differences in platelet fatty acid composition and concentration may explain at least some of the differences in platelet function which have previously been identified between these species.

Sol Sherry lecture in thrombosis: how thrombin 'talks' to cells: molecular mechanisms and roles in vivo.

This article is a summary of the Sol Sherry Lecture of the Council on Arteriosclerosis, Thrombosis, and Vascular Biology, which was presented at the 70th Scientific Sessions of the American Heart Association in November 1997. It highlights work from our laboratory addressing the molecular mechanisms by which the coagulation protease thrombin elicits cellular responses, notes some of the novel issues that protease signaling raises, and cites recent work on the role of thrombin signaling in vivo. Thrombin is a multifunctional serine protease. In adult animals, active thrombin is generated in the context of vascular injury when activation of the coagulation cascade triggers conversion of the circulating zymogen prothrombin to active protease. Thrombin generation may also be important in other contexts, as will be shown below. Several of thrombin’s functions involve cleavage of circulating protein substrates, eg, conversion of fibrinogen to fibrin monomer or activation of protein C. However, thrombin also has important actions on cells. It is the most potent activator of platelets.1 It causes endothelial cells to deliver the leukocyte adhesion molecule P-selectin to their surfaces,2 to secrete von Willebrand factor,2 and to elaborate growth factors and cytokines.3 4 It is also a mitogen for fibroblasts and vascular smooth muscle cells.5 Such cellular actions of thrombin raised several important questions. How does thrombin, a protease, act like a hormone to control cellular behaviors? And what are the roles of thrombin-regulated cellular events in vivo? Thrombin’s actions on platelets are of particular interest. Arterial thrombosis underlies most cases of unstable angina and myocardial infarction. Studies in animal models and clinical trials suggest that these events are both platelet dependent and thrombin dependent, but the relative contributions of and interactions between thrombin-induced platelet activation and fibrin formation in acute coronary syndromes are not known. By elucidating …

Species differences in platelet responses to thrombin and SFLLRN. Receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca 2+] i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca 2+] i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca 2+] i studies. The kinetic profile for [Ca 2+] i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca 2+] i in guinea pigs, had no effect in rat, and increased [Ca 2+] i in all other species. Staurosporine inhibited SFLLRN-induced [Ca 2+] i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca 2+] i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.

Effects of Acetylsalicylic Acid on Inhibition of Ex Vivo Platelet Aggregation and Secretion by SKF 107260, a Novel GPIIb/IIIa Receptor Antagonist

SKF 107260 is a potent pentapeptide antagonist of the platelet membrane glycoprotein receptor GP IIb/IIIa. The in vitro platelet inhibitory effects of SKF 107260, acetylsalicylic acid (ASA), and their combination, on collagen-induced platelet aggregation and secretion (ATP release) were assessed in human whole blood. Additionally, the concentration-response relationships for these inhibitors were compared for males and females in order to explore gender differences in platelet responsiveness. SKF 107260 caused a concentration-dependent inhibition of platelet aggregation which was significant at concentrations > or = 30 nM. ASA also caused a concentration-dependent inhibition of platelet aggregation which was significant at concentrations > or = 1 mg/dl. The addition of ASA 1 mg/dl to increasing concentrations of SKF 107260 resulted in a more pronounced inhibition of platelet aggregation than when either agent was used alone. These data suggest a pharmacologic interaction, especially at SKF 107260 concentrations < or = 30 nM. Since ATP release was significantly inhibited at concentrations > or = 1 nM, platelet secretion appears to be more sensitive than aggregation to inhibition by SKF 107260. These data suggest that platelet secretion in response to collagen is dependent on the aggregation response mediated by GP IIb/IIIa. In conclusion, SKF 107260 is a potent inhibitor of both whole blood platelet aggregation and secretion and these anti-aggregatory effects may be augmented by concomitant ASA administration.

Effects of low dose aspirin on platelet function in patients with recent cerebral ischemia.

We tested the antiplatelet effects of low-dose aspirin in patients with occlusive cerebrovascular disease, because conventional dosage aspirin inhibits vascular synthesis of prostacyclin at the same time that it inhibits platelets. The effects on platelet function and thromboxane A2 synthesis of 40 mg of aspirin daily or 40 mg aspirin plus dipyridamole were measured in 23 patients starting within a week after the onset of cerebral ischemia. All patients had normal baseline platelet aggregation responses to four stimuli: arachidonate, epinephrine, adenosine diphosphate and collagen. The generation of thromboxane A2 by platelets, measured as serum thromboxane B2, was also normal. After 3 to 7 days of low dose aspirin therapy, platelet aggregation responses were suppressed to the extent observed with higher dosage aspirin. Serotonin release during platelet aggregation was inhibited by more than 95% and thromboxane B2 levels in clotted blood fell by more than 95%. Responses to aspirin treatment were similar in patients with transient ischemic attacks and in those with stroke and were also similar in both sexes. No differences in platelet responses were observed between patients receiving aspirin alone and aspirin plus dipyridamole. Thus 40 mg aspirin daily inhibited platelet responses as effectively as higher doses of aspirin in patients who had recent cerebral ischemia and showed a cumulative antiplatelet effect.