Stokol Tracy, Yeo Wee Ming, Burnett Deborah, DeAngelis Nicole, Huang Teng, Osterrieder Nikolaus, Catalfamo James
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.
Institut für Virologie, Freie Universität Berlin, Berlin, Germany.
PLoS One. 2015 Apr 23;10(4):e0122640. doi: 10.1371/journal.pone.0122640. eCollection 2015.
Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.
1型马疱疹病毒（EHV-1）可引发马群的流产和神经疾病疫情。这些临床综合征的主要病因之一是胎盘和脊髓血管内形成血栓，然而血栓形成的机制尚不清楚。血小板是血栓的组成部分，可放大并促进凝血酶的生成。在此，我们检验了EHV-1激活血小板这一假说。我们发现，两种EHV-1毒株，即RacL11和Ab4，在噬斑形成单位/细胞达到0.5或更高时，可在10分钟内激活血小板，导致α颗粒分泌（表面P-选择素表达）和血小板微泡化（CD41和膜联蛋白V双阳性的小事件增加）。RacL11毒株引起的微泡化更为明显。病毒诱导的P-选择素表达需要血浆和1.0 mM的外源钙。在缺乏因子VII或X的人血浆中，P-选择素表达消失，微泡化显著减少。在缺乏因子VII的人血浆中添加纯化的人因子VIIa（1 nM）后，P-选择素表达和微泡化均可恢复。Ab4毒株的糖蛋白C缺陷型突变体激活血小板的效果与未突变的Ab4相同。用山羊多克隆抗兔组织因子抗体预孵育病毒后，P-选择素表达消失，微泡化显著减少。从经EHV-1处理的洗涤血小板中可回收感染性病毒，提示血小板与病毒存在直接相互作用。我们的结果表明，EHV-1可激活马血小板，α颗粒分泌是病毒相关组织因子触发因子X激活和凝血酶生成的结果。微泡化仅部分依赖于组织因子和凝血酶，提示病毒通过其他机制导致微泡化，可能是通过直接结合。这些发现表明，EHV-1诱导的血小板激活可能导致临床感染马匹发生血栓形成，并提供了一种病毒激活止血的新机制。
