Fig 2.
Revised flow cytometry gating strategy for quantification of platelet-derived microparticles in equine platelet samples.
A: Non-virus events in samples containing virus only (RacL11 shown) were identified and gated as CD41-positive events (R1 region) in a CD41 fluorescence versus forward scatter dotplot. Virus events were defined as CD41-negative events (R2, left panel). Large numbers of events (>100,000) were counted in the virus alone sample to optimize establishment of the gate. Quadrant regions of an Annexin V fluorescence versus forward scatter plot showed few Annexin V-positive events (middle panel, R1 gate), whereas many (57%) of the CD41-negative virus events were positive for Annexin V, with about 80% being small events (<101 log forward scatter units, right panel, R2 gate). B: Representative images of platelets in citrated platelet-rich plasma exposed to the RacL11 strain of EHV-1 at 1 plaque forming unit (PFU)/cell. Platelet-derived microparticles (PDMPs) were defined as small events (<101 log forward scatter units) that were double positive for CD41 and Annexin V. CD41-positive events were first defined as above (R1 gate, left panel), then the PDMP percentage was obtained from the lower right quadrant of an Annexin V fluorescence dotplot of the R1 gate, with positive fluorescence for Annexin V being defined on a sample with no added Annexin V. In this sample, there are 8.7% PDMPs. The events in the upper left and right quadrants are platelets that are negative (58%) and positive (25%) for Annexin V, respectively. Note, that the Annexin V-positive platelet events (upper right quadrant) could reflect virus bound to platelets (since virus alone binds Annexin V) versus phosphatidylserine exteriorization on the platelet surface. C: Representative image of PDMP quantification in platelets exposed to rabbit kidney (RK) cell lysate at an equivalent volume to 1 PFU/cell (mock-infected control). In this sample, there are 0.1% PDMPs (lower right quadrant) and 1% of platelets are weakly positive for Annexin V (upper right quadrant).
Fig 3.
EHV-1 induces platelet P-selectin expression and shedding of platelet-derived microparticles in equine platelet-rich plasma.
Platelets were exposed for 10 minutes to RacL11 or Ab4 EHV-1 strains at increasing PFU/cell (0.01, 0.1, 0.5, or 1) with PBS and RK lysate as negative controls and thrombin-convulxin (TC, 0.15 U/mL-0.05 ug/mL) as a positive control. Then the mean percentage ± SD of platelets positive for P-selectin (A, n = 8) and PDMPs (B, n = 4) were quantified. At the higher PFU/cell of 0.5 and 1, both strains induced P-selectin expression and microvesiculation. Dotplots of forward versus side scatter (C, left panels) and CD41 fluorescence versus forward scatter (C, right panels) for RK lysate, TC and both viruses show that, at the higher PFU/cell of 0.5 and 1, both strains caused compaction and narrowing of the platelet event cloud and increased CD41-positive small events (arrowheads). At these higher PFUs/cell, the RacL11 strain caused more vesiculation (note fewer events in platelet cloud), with more CD41-negative smaller events (arrows) than the Ab4 strain at equivalent PFUs/cell. CD41-negative events include virus aggregates, which are difficult to distinguish from small platelet events on the forward versus side scatter plot. Exposure to both viruses at 5 PFU/cell replicated these findings, demonstrating dose-dependent microvesiculation (S1 Fig). * p ≤ 0.001 versus PBS or RK negative controls for each virus strain, ** p < 0.001 versus both viral strains at higher PFU/cell (0.5, 1) for P-selectin and p < 0.001 versus Ab4 at the higher PFU/cell (0.5, 1) and versus RacL11 at 0.5 PFU/cell for PDMPs, *** p < 0.001 versus Ab4 at the higher PFU/cell (0.5, 1).
Fig 4.
10 minutes to RacL11 at 1 PFU/cell with PBS and RK lysate negative controls and a thrombin (T, 0.15 U/mL) positive control.
Washed platelets did not express P-selectin when exposed to virus unless plasma was present. In contrast, thrombin-induced P-selectin expression was independent of plasma (n = 5). Data represents mean ± SD. * p < 0.001 versus PRP or WP + MDP for RacL11-exposed platelets. B: Effect of calcium: Equine citrate-anticoagulated PRP was exposed to RacL11 at 1 PFU/cell for 10 minutes with increasing calcium concentrations (0 to 2.5 mM), with the above controls. EHV-1-induced P-selectin expression required at least 1 mM of exogenous calcium (n = 3). Data represents mean ± SD. * p < 0.001 versus 1.0, 2.0 or 2.5 mM calcium.
Fig 5.
However, supplementation of FVII-deficient MDP with purified human FVIIa (1 nM, FVII- + FVIIa) re-established P-selectin expression induced by RacL11, indicating FX generation was secondary to extrinsic pathway activation (E, n = 3 to 7). Microvesiculation was also significantly reduced in FVII- and FX-deficient MDP, with supplemental FVIIa boosting the response in FVII-deficient MDP (F, n = 4). * p <0.05 versus Full MDP. In contrast, addition of human FIX-, FXI- or FXII-deficient MDP to washed platelets did not significantly affect P selectin expression (G) or PDMP release (H) (n = 3 to 7). Similar results were seen with Ab4 (S2 Fig).
Fig 6.
Tissue factor mediates EHV-1-induced platelet activation, with no role for virus glycoprotein C.
A goat polyclonal anti-rabbit tissue factor (TF) antibody (anti-TF, 152 ug/mL) abolished P-selectin expression (A) and significantly decreased the numbers of platelet-derived microparticles (PDMP) (B) in equine platelets exposed to RacL11 and Ab4 EHV-1 strains at 1 PFU/cell (n = 6). Data shown is mean ± SD. * p < 0.001 for P-selectin and p = 0.025 for PDMPs versus goat IgG controls. In contrast, platelets still expressed P-selectin (C) and shed PDMPs (D) when exposed to an Ab4-based envelope glycoprotein C deletion mutant (ΔgC) at 1 PFU/cell (n = 8). Data shown is mean ± SD.
Fig 8.
Model for EHV-1-induced activation of platelets.
In our model of EHV-1-induced platelet activation, we propose that the virus first binds to platelets. Concurrently, factor VII (FVII) forms a complex with TF in the viral envelope and activates factor X (FX), with FXa generating thrombin in the vicinity of or on the surface of platelets. Thrombin generation may be propagated by phosphatidylserine (PS) in the viral envelope. Thrombin binds to protease-activated receptors (PAR) on platelet surfaces, inducing release of α-granules with subsequent surface P-selectin and PS expression and microvesiculation. If marked, microvesiculation results in loss of membrane-anchored P-selectin on microparticles. Depending on the viral strain or load, thrombin may be generated on the surface of virus not attached to platelets.