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P-selectin glycoprotein ligand-1 (PSGL-1) interacts with P-selectin expressed on endothelial cells and platelets. PSGL-1 extracellular mucin-like domain displays a variable number of tandem repeats (VNTRs) polymorphism. The wildtype consists of 16 decameric repeats (designated A isoforms) and variants with 15 (B allele) and 14 (C allele) repeats that are assumed to be associated with reduced risk of vascular disease. We investigated the adhesion of these natural variants to P-selectin in native human neutrophils. Healthy volunteers were genotyped and the adhesion of neutrophils expressing the PSGL-1 isoforms A/A, A/B and B/B were studied under static and physiologic flow conditions. Homozygous B/B neutrophils attached significantly weaker to P-selectin at elevated shear rates from 24 up to 64 dyn/cm(2) than A/A and A/B neutrophils. No difference in adhesion rate was found under static conditions and shear stress below 24 dyn/cm(2), but B/B neutrophils rolled significantly faster than A/A neutrophils at shear stress ≥ 12 dyn/cm(2). There was no difference in the adhesive capacity between A/A an A/B neutrophils. These data support the view that the role of the decamers is to extend the ligand binding domain far above the cell surface to support stable leukocyte adhesion and rolling., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: In pharmacodynamic studies with antiplatelet agents, platelets are usually activated in vitro with single agonists (e.g., ADP) solely. We questioned whether differences occur between single and combined stimulation of platelets [involving the major thrombin-receptors, protease-activated receptors (PAR)1 and PAR4], and whether the pharmacodynamic response to common antiplatelet drugs vary when a combined stimulus is applied instead of a single agonist., Methods: We investigated the influence of different antiplatelet agents (aspirin [500 mg]) in vivo, the P2Y12-antagonist AR-C 69931MX (4 nM) and the GPII/IIIa-antagonist (abciximab ([5 microg/ml] in vitro) on the degranulation response (CD62) and expression of the activated GPIIb/IIIa-receptor (PAC-1) after stimulation with ADP (2 microM), collagen (4 microg/ml), a PAR1-activating peptide (3 microM TRAP) and a PAR4-activating peptide (200 microM AYPGKF) alone or in a combination of each two agonists by flow cytometry in healthy subjects., Results: (1) Combined activation of TRAP with AYPGKF resulted in synergistic CD62 and PAC-1 expression. Only AYPGKF but neither TRAP nor ADP acted synergistically with collagen. (2) AR-C 69931MX inhibited platelet degranulation (CD62) in all inducer combinations with ADP or the combination TRAP with AYPGKF. The effect was considerably smaller or absent for the combination of collagen with a second inducer. (3) Aspirin intake reduced platelet degranulation and PAC-1 expression only for AYPGKF costimulation with collagen., Conclusion: Because a variety of different agonists influence platelet activation and its distinct functions at a time, investigations which regard the concert of these agonists might be closer to the in vivo situation and better reflect the pharmacodynamic profile of an antiplatelet agent than using one single inducing agent.

The sodium-hydrogen exchanger isoform 1 (NHE-1) contributes to platelet activation at elevated pH. Effects of NHE-1 inhibitors on platelet degranulation and formation of proinflammatory and procoagulatory platelet-leukocyte aggregates (PLA) and possible interactions with P2Y(12) inhibitors--which also affect platelet degranulation--have not been investigated. Whole blood from healthy human subjects was incubated with the NHE-1 inhibitor cariporide and the P2Y(12) inhibitor AR-C 69331 MX at clinically reasonable concentrations, in the presence of normal pH or in a propionate model to activate the NHE-1 (approximately pH 7.0). The degranulation marker CD62p, the expression of the activated GPIIb/IIIa receptor (PAC-1), and formation of platelet-leukocyte (monocyte) aggregates (PLA) was assessed by flow cytometry. Cariporide at concentrations up to 20 microg/ml had no effects on ADP- (5 microM) or TRAP- (2 microM) induced CD62p expression or PLA formation at normal pH. At pH 7.0 and stimulation with ADP, PLA decreased from 64+/-24% (control) to 47+/-23% under cariporide at 2 microg/ml (p<0.05), and the MFI of PLA (i.e. the platelet mass attached at monocytes) decreased from 547+/-203 to 360+/-96 units (p<0.05). PAC-1 MFI decreased from 66+/-23 to 34+/-18 units (p<0.05) after ADP and from 74+/-29 to 42+/-17 units (p<0.05) after TRAP, respectively. AR-C 69331 MX (10 nM) had inhibitory effects on all parameters irrespectively of the pH, and the combination of both agents at pH 7.0 shows additive effects. In conclusion, our investigation points to-perhaps clinically relevant-effects of NHE-1 inhibition on the degranulation of platelets and formation of platelet-leukocyte aggregates.