Coagulation, anticoagulation, and fibrinolysis in mammals are the cornerstones of hemostasis. The appropriate balance between these mechanisms ensures the formation of a functional thrombus, composed of fibrin, to stop bleeding from a damaged blood vessel. Fibrinolysis is the process of thrombus breakdown and aims to restore blood flow. Plasma D-dimers (DD) are breakdown products of factor XIII-activated, cross-linked fibrin, and DD can only be detected once coagulation is followed by fibrinolysis. Hemostasis is tightly regulated and orchestrated by coagulation and anticoagulation factors, as well as by fibrinolysis and antifibrinolysis factors. All factors are circulating in plasma and/or are directly derived from platelets, endothelial cells (EC), and monocytes upon activation. Similar to many mammalian species relevant systemic inflammation in the horse might alter the EC surface from a resting into a prothrombotic state, a process that is likely mediated by circulating proinflammatory cytokines.[3, 4] Although there is a continuous (low volume) production of DD during homeostasis, inflammatory conditions such as endotoxemia or neonatal septicemia in the horse have been found to cause DD product increases.[5-7]
Equid herpesvirus-associated myeloencephalopathy (EHM) is secondary disease that can follow respiratory tract infection with equid herpesvirus type-1 (EHV-1) in sporadic cases. Infectious dose, virus strain, pre-existing immunity, but also host genetic factors are thought to be responsible for differences in clinical outcome.[8, 9] In cases of EHM, lesions are localized to the central nervous system (CNS) and are commonly scattered throughout the spinal cord white and gray matter. Usually, lesions are consistent with vasculitis and thrombosis of the CNS vasculature, hemorrhage, and mononuclear cell extravasation into the neural parenchyma. Vascular lesions are thought to develop in close proximity to EHV-1-infected EC, and the virus is assumed to reach the CNS-vasculature EC exclusively by intracellular viremia in peripheral blood mononuclear cells (PBMC).[11, 12] After experimental EHV-1 infection by nasopharyngeal route there are 3 clinical phases over a 20-day postinfection (p.i.) period. Within 12–24 hours of inoculation, a primary fever spike occurs, which is typically short-lived and returns to normal by day 3 p.i. (phase 1). A secondary fever spike may begin on days 3–6 p.i. in the majority of cases; it typically lasts for 1–3 days, and commonly coincides with PBMC-associated intracellular viremia (phase 2). Phase 3 of the disease is the occurrence of EHM, and occurs from late in phase 2 or up to approximately 20 days p.i. Although the majority of EHV-1 infected horses will become viremic, only a variable but consistently lower percentage of viremic horses will develop neuropathology.
As increased DD concentrations in humans can be measured during transient ischemic attack to the brain, and because localized vasculitis and thrombosis are believed to be intrinsic components of EHM pathology, we speculated that DD concentration increases may be present as early as during viremia in the critical phase 2 of infection, consistent with a prothrombotic state. We hypothesize that phase 2 (viremia) of EHV-1 infection induces a prothrombotic state in horses leading to increased DD concentrations.
To test this hypothesis, our objective was to measure DD concentrations in plasma during experimental EHV-1 infection studies collected from yearling horses, yearling ponies, and aged horses, and determine any temporal association with viremia.
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- Materials and Methods
DD were detected during all 3 experiments in the majority of infected horses/ponies. DD increases in individual animals were associated in time with the duration of EHV-1 viremia as well as with its associated fever. In addition, high viremia scores were positively associated with high DD scores. However, DD increases during the primary fevers were absent (group 2 and 3). It is noteworthy that DD increases during viremia and viremic fevers were consistently noticed in all 3 groups that were studied, despite the use of different age groups, different breeds, and two different viral strains during the 3 different experiments.
The DD assay utilizes a mouse-derived antibody to detect specifically human DD. Although the assay has been used for a number of studies in the horse, its sensitivity and specificity have not been fully determined. However, presumed DD increases found during sepsis and endotoxemia in equids were measured in conjunction with other abnormal parameters of hemostasis, such as a prolonged PT and APTT, thrombocytopenia, decreased antithrombin activity, and others with good correlation.[5-7] Based on this validation we elected not to include more parameters of coagulation into this study.
Although a number of publications on experimental EHV-1 infections have shown the association between a secondary fever and cell-associated viremia, DD concentration increases have, so far, only been measured in horses with other significant systemic disease.[4, 5] We showed that DD production, as an indicator of an activated coagulation, appears to be linked to EHV-1 viremia; however, at this stage, we can only speculate about possible pathways that lead to DD increases. Potential pathways can be 2-fold. One possibility could be an indirect pathway where viremia induces proinflammatory cytokine production from infected and uninfected PBMC, which induces a prothrombotic state with, subsequently, an increased fibrin generation and break-down. Circulating cytokines TNF-α, IL-1, IL-6, IL-8, and MCP-1 (CCL2) in humans are associated with a prothrombotic state,[8-10] and whereas an activated coagulation and/or increased DD concentrations were found during severe gastrointestinal, ischemic disorders in horses, Lopes et al successfully detected mRNA up-regulation of TNF-α, IL-1RA, IL-6, and IL-8 in PBMC under similar conditions of gastrointestinal disease.[6, 21, 22] Furthermore, TNF-α concentration increases in plasma and abdominal fluids from horses with these conditions have also been detected.[23, 24] Although these results suggest that there is evidence of concurrent cytokine and DD production, it is possible that DD production in horses/ponies during EHV-1 viremia is also cytokine-driven. A mainly proinflammatory cytokinemia could also provide a good explanation of the viremia-associated fevers during EHV-1 infections. Few studies, in vitro or in vivo, have focused on cytokine and chemokine production during EHV-1 infection mainly using PBMC.[25-27] However, none of the cytokines/chemokines identified in these studies could be linked to DD production as it occurs in other species; neither was there significant overlap with viremia. This was unexpected when we searched for possible explanations; however, as cytokine production and breakdown is rapid and usually arranged in cascade-like pathways, a low sampling frequency, as is typical during EHV-1 infection experiments, may have caused a lack of cytokine detection. Furthermore, all studies used PBMC, whereas other cell populations, eg, EC, pericytes, myocytes, osteoblasts, or fibroblast with a potential of inflammatory cytokine production could not be included in these studies.
An alternative possible explanation of DD-production during EHV-1 viremia has been recently suggested by Yeo et al. They describe significantly increased release of tissue factor from EHV-1-infected equine monocytes in vitro when compared to uninfected monocytes. As the percentage of infected PBMC is very low during EHV-1 viremia and the monocyte is not the predominant virus carrier during viremia, these interesting findings warrant further investigation.[30, 31]
Typically, there is a biphasic fever in most animals during experimental EHV-1 infection. The first fever is noticed within 24–36 hours of infection and is believed to be caused by viral replication and tissue destruction within the upper respiratory tract. Although the second fever is associated with EHV-1 viremia it is also the phase where DD production was consistently increased. The start of EHV-1 viremia also corresponded to the initiation of DD production. Although the locality, respiratory epithelium versus blood stream, and potential causes for the 2 distinctly separate periods of fevers are different, there may be another reason for the lack of DD production during the primary fever period. A prozone effect could have occurred which can be detected when an overwhelming amount of antigen is present in the test sample. A high DD concentration, produced in the first phase of the infection, could have blocked the agglutination reaction of the assay, resulting in false negative results. However, the prozone effect can be overcome by careful sample dilution to decrease the amount of antigen, which was performed. Still, after dilution of the samples none of the previously negative samples turned positive for DD, and previously measured low DD-concentration results did not increase after dilutions. This strengthens the idea that DD-production is linked to viremia and its associated fever, but not to the primary fever.
In individual animals, on occasions, we noticed discrepancies between simultaneous presence of fever, viremia, and DD concentrations in same-day samples. An explanation may be the once-a-day sampling technique that is typical for experimental EHV-1 infection studies, which may not be sensitive enough to detect fluctuations of parameters of interest within a 24 hour period. This has to be addressed in future studies.
DD research in humans showed that increases, in combination with other biomarkers, help the emergency room clinician to identify transient ischemic attack or cerebral stroke. This is an intriguing finding as EHM pathology has similarities with transient ischemic attack or cerebral stroke in humans. However, our studies were not designed to evaluate correlations between DD production and the development of (stages of) myelopathy or EHM, as we used yearling horses (group 1) and ponies (group 2), which are known to be least likely to develop EHM under experimental conditions. This was different in group 3 where aged horses were selected because of an increased likelihood to develop EHM; however, some of these horses were also simultaneously treated with virustatics during phase II-fevers to evaluate the drugs' effects on EHM outcome and severity.[9, 12, 33] EHM most often follows the last day of viremia, and thrombosis of spinal cord vessels is a feature of EHM, but it is not the predominant feature. High DD concentrations are therefore more likely to be part of a systemic response and not associated with the spinal cord vasculature. Furthermore, DD concentrations started to rise immediately with the beginning of viremia, and not toward the end of viremia.
In a previous article we showed that in vitro infection of EC with EHV-1 only occurs in a contact model between the virus-infected PBMC and the EC. We further presented in vitro data that demonstrate significantly decreased EC infection in our established contact model when PBMC and EC were pretreated with anti-inflammatory drugs. We postulate that the same or simultaneously produced inflammatory stimuli that are capable of systemic DD production may also play a role during EC infection with EHV-1 by inducing contact molecules on EC and PBMC. Contact between a virus-infected PBMC and an EC would then be facilitated and EC infection may be increased. DD increases are markers of inflammation and associated with increased proinflammatory cytokine production in many species. With this in mind, DD may be a surrogate marker for inflammation and the prudent use of anti-inflammatory drugs early on during the course of an EHV-1 infection may be warranted to decrease the rate of EC infection.
This study suggests that mechanisms to cause a fever during viremia are different from those responsible for the primary fever during an EHV-1 infection. Only during viremia there is substantial inflammation and activated coagulation leading to a detectable DD increase. DD increases alert us to the extent of inflammation and the possible effects this inflammation might have on vasculature surfaces and the infection of EC with EHV-1. Whether there is a role for coagulation during the early stages of EHM, however, needs to be determined.