The Covid-19 pandemic has introduced an array of organ-specific and systemic phenotypes-
some previously observed in viral infections, including severe acute respiratory syndrome
(SARS) and others that appear to be unique to SARS-coronavirus (CoV)-2. Rapidly emerging
information from clinical observations, autopsy-based findings, extrapolations from
in vitro and ex vivo studies and dynamic modeling are informing management guidelines;
however, many questions remain unanswered and clinical trials that are required to
provide evidence have not been completed in most areas. Among the many questions that
require careful thought, reflection and investigation are the mechanism(s) underlying
the development of a systemic coagulopathy and acquired thrombophilia characterized
in a majority of cases by a proclivity for venous, arterial and microvascular thrombosis.
The following review summarizes emerging insights into the pathobiology, mechanism(s),
diagnosis, management, foundations for research and either planned or ongoing clinical
trials for Covid-19-associated coagulopathy.
Venous and arterial thrombosis in Covid-19 infection
Klok et al. [1] evaluated the occurrence of venous and arterial thrombotic events,
including deep vein thrombosis (DVT), pulmonary embolism (PE), ischemic stroke, myocardial
infarction and systemic arterial events in 184 patients with Covid-19 pneumonia admitted
to the intensive care unit (ICU). All patients received standard thromboprophylaxis
(Nadroparin 2850 to 5700 IU per day based on body weight). The composite incidence
of thrombotic events was 31%. Venous thromboembolic events were the most common (27%)
and a majority were PEs. Independent predictors of thrombotic events were increased
age and evidence on screening blood tests for a coagulopathy (prothrombin time [PT] > 3 s
above the upper limit of normal [ULN]), activated partial thromboplastin time [APTT] > 5 s
above the ULN; adjusted hazard ratio 4.1, 95% CI 1.9–9.1). None of the patients experiencing
thrombotic events met strict criteria for disseminated intravascular coagulation (DIC).
Tang et al. reported on abnormal coagulation parameters and poor prognosis in 183
consecutive patients with Covid-19 pneumonia [2]. Those who did not survive their
illness compared with survivors had higher D-dimer levels, fibrin(ogen) degradation
products (FDP) and longer PT and APTT values. Abnormal coagulation parameters were
evident early after hospitalization and in some patients, fibrinogen concentrations
and antithrombin activity decreased over time.
The same investigators [3] reported in 445 patients that anticoagulant therapy, primarily
with low molecular weight heparin (LMWH) administered for 7 days or longer was associated
with a lower 28-day morality when administered to patients with a sepsis-induced coagulopathy
(SIC) score ≥ 4 or a D-dimer value greater than 6 times the ULN. The SIC score is
derived from the platelet count, PT ratio, FDPs, systemic inflammatory response syndrome
(SIRS) score and a sequential organ system failure assessment.
Helms et al. [4] reported the occurrence of thrombotic events among 150 patients with
Covid-19 and acute respiratory distress syndrome (ARDS) admitted to the ICU. Propensity
matching was undertaken to determine the risk of thromboembolic events for patients
with Covid-19 and those with non-Covid-19 infection-associated ARDS. Twenty-five patients
(16.7%) experienced a PE; among 29 patients undergoing renal replacement therapy 28
(96.6%) experienced circuit clotting and of 12 patients requiring extracorporeal membrane
oxygenation (ECMO) for refractory hypoxemia, three thrombotic circuit occlusions (in
two patients) occurred. Lupus anticoagulants were detected in 50 of 57 patients tested
(87.7%). In patients with non-Covid-19 infection-associated ARDS, 2.1% experienced
a PE. The investigators also reported that patients with Covid-19-associated ARDS
did not develop DIC and had markedly elevated circulating levels of Von Willebrand
factor (VWF) antigen, VWF activity and factor VIII [5].
Perhaps one of the least expected and most striking complications of Covid-19 is acute
large vessel occlusion with ischemic stroke in patients less than 50 years of age
[6]. Among the 5 patients reported by Oxley et al., the youngest was 33 years of age
and the mean NIHSS score was 17, consistent with severe large vessel stroke. In the
original cases reported from Wuhan China, stroke was seen in 5% of patients [7, 8];
however, the youngest patient in the Wuhan experience was 55 years.
While endocardial thrombosis has been reported in the hearts of decedents with Covid-19
representing a possible nidus for cardioembolic events [9], large vessel arterial
thrombosis in situ would support wide-scale endotheliitis involving moderate-to-large
arteries. This would have important near-term and long-term implications to include
late-onset arterial stenosis, aneurysm and pseudo-aneurysm formation with a need for
screening after recovery.
The SARS-CoV-2 virus: characteristics, unique properties and thrombosis risk
SARS-CoV-2 virus
Coronaviruses belong to the subfamily Coronavirineae in the family of coronavitidae
of the order nidovirales (reviewed in Becker) [10]. The genome is a single-stranded
positive-sense RNA (30 kb) with a 5′ cap structure and a 3′-poly-A tail. Homotrimers
of S proteins make up the spikes on the virus surface and enable binding to host receptors.
Coronaviruses contain at least 6 open-read frames (ORFs) that encode primary structural
proteins that include spike (S), membrane (M), envelope (E) and nucleocapsid (N).
S proteins are responsible for attachment to host receptors. M proteins contain transmembrane
domains that contribute to virus shape and binding to the nucleocapsid. The E protein
is involved with virus assembly and pathogenesis. The N protein packages and encapsulates
the genome into virions and also antagonizes silencing RNA (reviewed in Becker) [10].
Binding to Host Cells
The available information suggests that SARS-CoV-2 binds to host cells via the angiotensin
converting-enzyme (ACE) 2 receptor (R)—a metallopeptidase (reviewed in Becker) [10].
ACE2 mRNA is present in all major organs, however, its protein expression is greatest
in several key organs and locations that play important roles in the initiation of
infection and its phenotypic expression, including venous, arterial and microvascular
thrombosis. The major organs include the nasopharynx, oropharynx, lungs, stomach,
small intestine, spleen, liver, kidney and brain with binding to ACE2 receptors on
epithelial, endothelial and enterocytic cells. The density of ACE2R is particularly
high in the lungs, heart, veins and arteries [11].
Hamming et al. [11] used human tissues obtained from patients undergoing diagnostic
biopsies or surgery, unused organs procured for transplantation purposes and during
autopsies. They found a high density of ACE2R expression in endothelial cells from
small and large arteries and veins in all tissues. They also found ACE2R expression
in arterial smooth muscle cells in some organs, specifically the brain. This observation
may have relevance in the complication of acute ischemic stroke discussed previously.
Chen et al. (Chen, 2020 #9069}) developed a single cell atlas of the adult human heart
to determine the distribution and density of ACE2R expression. They identified a high
level of expression (RNA and protein) in pericytes and postulated that viral entry
and injury may in turn cause capillary endothelial cell injury and microvascular dysfunction.
ACE2R expression in human hearts was identified across eight major cell types, including
cardiomyocytes, endothelial cells, macrophages, fibroblasts, pericytes, smooth muscle
cells, T cells and neuron-like cells. The potential role of pericytes in the Covid-19-associated
thrombosis will be discussed in greater detail in a section to follow.
While the question remains whether direct cellular invasion is both necessary and
sufficient for end-organ injury, observations derived from hospital based and epidemiologic
data show phenotypic variability, ranging from complete absence of symptoms (asymptomatic
carriers) to cardiogenic shock, refractory respiratory failure and death. Viral load,
immunologic response and pre-existing conditions likely contribute to organ damage
and overall outcomes.
Viremia and viral load
The original patient cohort of what ultimately was discovered to be SARS-CoV-2 cited
a 15% incidence of viremia-RNAemia [7]. Prior experiences with highly pathogenic virus
outbreaks may offer insights into the distribution of Covid-19 infections. The Avian
influenza H7N9 virus caused human viremias and in a murine model of virus infection,
live virus was detectable in serum. Infected A549 cells secreted exosomes containing
the entire viral genome representing a potential mechanism for extrapulmonary infection.
High serum levels of cytokines were detectable in patients and test animals [12, 13].
Viral load dynamics and disease severity were reported by Zheng et al. [14]. From
96 consecutive patients, RNA viral load was measured in respiratory, stool, serum
and urine samples (total number of samples 3497). RNA was detected in the stool of
55 (59%) and in the serum of 39 (41%) of patients. All patients had SARS-CoV-2 detected
in sputum and saliva samples. Only one patient had a positive urine sample. The median
duration of RNA detection in the stool was 22 days (IQR 17–31 days); in the sputum
and saliva, 28 days (IQR 13–29 days) and in the serum 16 days (IQR 18–21 days). Virus
persistence correlated with disease severity.
Hu et al. determined the time to negative conversion of viral RNA in patients hospitalized
with Covid-19 infection [15]. The median time from the first day of a positive RNA
test to consecutive negative tests was 14 (IQR 10–18) days. From a patient cohort
of 59 patients, 10 patients had intermittent negative culture results from the same
site(s).
SARS-CoV-2 viremia correlates with Covid-19 infection severity and acuity [16].
Potential mechanism(s) for Covid-19-associated thrombosis
Virchow’s triad that includes abnormalities of blood flow, vascular injury and abnormalities
within the circulating blood is a time-honored pathological construct that provides
a foundation for understanding and investigating thrombosis whether it occurs in arteries,
veins or the microvascular circulation. While each of the three conditions are met
in a majority of conditions, it is important to recognize that the contributions can
vary by conditions and across vascular beds. This construct will be discussed in further
detail in subsequent sections.
Autopsy series in decedents with Covid-19 infection
The Covid-19 pandemic has impacted people worldwide. Several cities have been particularly
hard-hit in the United States. Autopsies were performed on four decedents from New
Orleans, LA. Each tested positive for SARS-CoV-2 by real-time polymerase chain reaction
(RT-PCR) and had laboratory values notable for elevated fibrinogen, ferritin, PT and
D-dimer levels at the time of hospitalization [9]. The following observations were
made at autopsy: the pulmonary arteries at the hilum were free of thromboembolism,
there was diffusely edematous lung parenchyma, hemorrhage in the peripheral parenchyma
(3 of 4 decedents), small and firm thrombi in sections of the peripheral parenchyma
and absence of gross inflammation. Microscopic findings on histology included: mild-to-moderate
lymphatic infiltrates (predominately in the interstitial space), CD4+lymphocytes aggregated
around small blood vessels that contained platelets and small thrombi, desquamated
type-2 pneumatocytes within the alveolar spaces, small vesicles believed to represent
viral inclusions, thickening of alveolar capillaries with fibrin thrombi, entrapped
neutrophils and CD61+megakaryocytes (Fig. 1). The hearts were notable for cardiomegaly
and right ventricular dilation. Coronary artery thrombosis was not seen in any of
the four decedents on histologic examination, however, there was scattered individual
myocyte necrosis with adjacent lymphocytes.
Fig. 1
Shows a damaged alveolus with enlarged nuclei and cytologic atypia (a) with abundant
DNA (red) and RNA (green) within tissue sections and virally infected cells with an
abundance of extracellular DNA and RNA in the cytoplasm (b) co-localized with fibrin
and extracellular nucleic acids (c). d is a control lung specimen. Panels e and f
show focal degeneration of cardiomyocytes. A pathology hallmark of Covid-19 infection
is diffuse small vessel (venule, arteriole and capillary) platelet–fibrin thrombosis
and intravascular megakaryocytes in all major organs, including the heart, lungs,
kidneys, liver and mesenteric fat. From [9] with permission
More on pericytes
Pericytes and perivascular cells are present on the abluminal surface of micro vessels
where they are embedded in the basement membrane [17]. Gap junctions provide a portal
of direct communication between endothelial cells and pericytes through which ions,
paracrine proteins and small molecules can be exchanged for the purpose of signaling
and maintaining vascular integrity. Pericytes play many important roles in vascular
homeostasis, ranging from vascular repair to regulation of vascular tone; however,
they play a particularly important role in states of inflammation where they cover
gap junctions (reviewed in Sims [18]. Abnormalities within pericytes or degeneration
cause tissue injury and metabolic changes that can also be detrimental to vital organs.
Magro et al. [19] reported on the autopsy findings from five decedents with severe
Covid-19 infections and ARDS. They identified a pattern of tissue damage involving
the lung and skin consistent with complement-mediated microvascular injury. There
was marked deposition of C5b-9, C4d and Mannan-binding lectin serine protease (MASP)-2
supporting a generalized activation of alternative and lectin-based pathways. Similar
to other autopsy studies, they described pauci-inflammatory capillary injury with
mural and luminal fibrin deposition. In addition, hallmarks of classic ARDS with diffuse
alveolar damage, hyaline membranes, inflammation and type II pneumocyte hyperplasia
were not prominent findings. The skin lesions were characterized as a pauci-inflammatory
thrombogenic vasculopathy.
The contribution of neutrophil extracellular traps (NETs) to the phenotypic expression
and end-organ injury among patients with COVID-19 is an important area for consideration.
Barnes et al. summarized their findings from an autopsy series and developed a working
hypothesis to integrate pulmonary infection, cytokines and thrombosis [20]. They observed
the following pathological features in three descendants: neutrophil infiltration
in pulmonary capillaries, acute capillaritis within fibrin deposition, extravasation
of neutrophils into the alveolar space and neutrophilic mucositis.
Observations shared by pathologists around the world point to a very unique picture
in decedents with Covid-19 infection: macro and microvascular thrombosis with the
former consisting of both red (erythrocytes, leukocytes, fibrin) and white (platelets
and fibrin) and the latter platelet–fibrin micro-thrombi in venules, arterioles and
capillaries in all major organs including mesenteric fat, minimal evidence of microangiopathy,
intravascular megakaryocytes, endocardial thrombi, viral particles in adipocytes and
an unusual abundance of platelets in the spleen.
Endotheliitis
The widescale expression of ACE2 receptors within endothelial cells raises a question
of its vulnerability to SARS-CoV-2 binding, membrane fusion and viral entry causing
infection and attendant vascular injury and dysfunction. Engineered human blood vessel
organoids can be infected with SARS-CoV-2 [21]. This can be blocked by human recombinant
soluble ACE2. Varga et al. describe endothelial cell involvement across vascular beds
in a small series of descendants with Covid-19 [22]. Accumulation of inflammatory
cells and viral inclusions by histology and electron microscopy, respectively, were
identified within the endothelium of the heart, small bowel, kidneys, and lungs. In
autopsy and surgical tissue specimens there was diffuse lymphocytic endotheliitis
and apoptotic bodies (Fig. 2). It is important to consider that apoptosis may not
require host cell viral entry, but rather binding to the cell surface and subsequent
apoptotic pathway signaling [23].
Fig. 2
The mechanism(s) underlying a prolonged thrombophilic state following Covid-19 infection
are unknown, but may be related to diffuse vascular endothelial cell infection, injury
and dysfunction. Viral particles and inclusion bodies are seen within endothelial
cells of the glomerular capillary loops (a and b); in panel c, inflammatory cells
are observed within capillaries serving the small intestine, while panel d demonstrates
apoptotic endothelial cells and mononuclear cells. The available clinical and pathological
evidence, coupled with strong and mature data that supports a relationship between
CFRNA, polyphosphate and factor XI activation, raises a logical scientific premise
that requires investigation. From [23] with permission
Reticular inclusions, primarily within vascular endothelial cells are composed of
glycoproteins and phospholipids that originate within the rough endoplasmic reticulum in
response to interferon (IFN)-1 expression [24]. Viral infections and autoimmune diseases
are the most common causes of high IFN-1 expression and reticular inclusions that,
in turn, cause endothelial cell injury, dysfunction and prothrombotic gene expression.
Hyperinflammation
The profound increase of inflammatory biomarkers in patients with Covid-19 infection
and their association with poor clinical outcome has been attributed to an unregulated
immune response to a new pathogen-antigen in the form of SARS-CoV-2. While inflammation
has been observed in the lungs and other organs, as previously discussed, the pathology
community has commented on the relative paucity of inflammation that appears to be
less than anticipated for the degree of tissue damage and circulating levels of many
cytokines. The specific tissues with inflammation is likely pivotal to understanding
clinical phenotypes, including thrombosis and will be discussed in greater detail
in a section to follow.
Some clinicians and investigators have raised the question of a hemophagocytic lymphohistiocytosis
(HLH)-like syndrome in Covid-19 pneumonia. In classic HLH, there is excessive inflammation
often in response to infection and-or malignancy resulting from impaired down-regulation
of activated macrophages and lymphocytes (reviewed in Jumic [25]. While patients with
Covid-19 meet some of the criteria for HLH, there is wide variability that likely
reflects a combination of its protean nature, secondary infections and end-organ failure
[26]. Similarly, the broad category of macrophage activation syndrome or secondary
HLH has been applied to acknowledge some of the pulmonary findings of Covid-19 pneumonia
and concomitant suppression of CD4+T cell interferon-γ production that collectively
yield a hyper-inflammatory immunosuppressive state [27].
Platelets in Covid-19-associated thrombosis
Platelets can either protect or promote immune-mediated responses to pathogens [28]
(reviewed in Becker) [29]. Platelets bind to a number of different microbes, either
through direct interactions, often mediated by platelet Fc receptors, or indirectly
via plasma protein bridges [30]. Similar to agonist-activation, the binding of pathogens
can trigger granule cargo release [31] and liberation of “platelet microbial” proteins
and peptides, including platelet factor (PF)-4, regulated on activation, normal T
cell expressed and secreted (RANTES), and fibrinopeptide B. Platelets may also play
an important role in the clearance of viral pathogens. Platelet interactions with
leukocytes trigger recruitment and tissue infiltration necessary for pathogen clearance
(reviewed in Guo [32]. In critically ill patients, thrombocytopenia correlates with
multi-organ failure and death, and a decline in platelet count, even in the absence
of overt thrombocytopenia, portends a worse outcome [33]. As mentioned previously,
a unique feature of Covid-19 infection is the presence of extramedullary megakaryocytes
that actively produce platelets.
NETs as a unifying theme in Covid-19 infection and thrombosis phenotypes
In response to strong stimulation, neutrophils, and to a lesser degree monocytes and
eosinophils, release extracellular traps (ETs), consisting of DNA and histones (Fig. 3)
in a process known as NETosis. The process involves histone (H) citrullination (Cit)
by peptidylarginine deiminase (PAD)-4, chromatin unwinding, breakdown of nuclear membranes
and cytolysis [34, 35]. There is also a vital or “non-lytic” NETosis, wherein nuclear
materials (eg. DNA and histones) are released without cellular membrane disruption
[36, 37].
Fig. 3
Neutrophil extracellular traps (NETs) consist of extracellular chromatin strands (nucleic
acids, DNA) wrapped around histones (nucleosomes) and inter-woven with fibrin strands.
NETs are an ideal foundation or template for binding activated platelets, erythrocytes
and leukocytes, activating factor XI and generating thrombin for fibrin production
Circulating cell-free (cf) nucleic acids are DNA and RNA species present in either
serum or plasma. Circulating cf-DNA in healthy individuals originates primarily from
apoptotic cells and is truncated to small and uniform DNA fragments of 188–200 base
pairs. By contrast, cf-DNA released in the setting of infection is the result of cellular
and tissue necrosis, apoptosis, autophagy or mitotic catastrophe [38] (Fig. 4).
Fig. 4
D-dimer is a fibrin degradation product or small protein fragment present in the peripheral
blood after fibrin is formed from fibrinogen (in the presence of thrombin) and subsequently
degraded by plasminogen activators. Its name is derived from having D fragments of
the fibrin protein joined by a covalently bound cross-link (factor XIII)
SARS-CoV-2 is an RNA virus. This may be pathologically and clinically relevant for
several reasons. Nakazawa et al. first identified cf-RNA that could initiate coagulation
by serving as a cofactor for the auto-activation of factor VII-activating protease
[39]. Kannemeier et al. performed a series of experiments to determine the functional
significance of intracellular material exposed to blood following tissue injury. Extracellular
RNA was found to activate proteases of the contact system of coagulation, including
factors XI and XII—both of which exhibited strong RNA binding. Administration of RNA
provoked a thrombotic response and RNA exposed following vascular injury with ferric
chloride (FeCl3) was less prothrombotic in mice pre-treated with RNAase. Thus, under
conditions characterized by tissue injury, extracellular RNA serves as a template
for contact activation–dependent thrombosis [40].
Gajsiewicz et al. [41] investigated the ability of polyphosphates to modulate the
contact-mediated pathway of coagulation. They observed that polyphosphates facilitated
factor XI activation. Secondary structures of RNA, particularly hair-pin forming oligomers
are highly procoagulant. There is an RNA length-contact activation relationship (reviewed
in Baker [42], however, even relatively short polyphosphates released from activated
platelets accelerate factor V activation, inhibit the anticoagulant activity of tissue
factor pathway inhibitor (TFPI), promote factor XI activation by thrombin, and contribute
to the synthesis of thicker fibrin strands that are resistant in fibrinolysis. Extracellular
polyphosphates and nucleic acids (RNA and DNA) often co-localize following cellular
injury and in highly inflammatory environments.
Tissue NETs cause platelet activation and thrombosis, possibly from NET-associated
histones that can induce platelet aggregation through toll-like receptors (TLRs) on
platelets and other cells. Platelet signaling activates the major platelet adhesion
receptor, integrin αIIbβ3, which mediates platelet aggregation, as well granule release,
phosphatidylserine exposure, FV/Va expression and thrombin generation [43–45]. NETs
are recognized as linking inflammation, coagulation and thrombosis both locally and
systemically in multiple conditions [46].
Zuo et al. [47] reported high levels of circulating cf-DNA and DNA-myeloperoxidase
complexes in patients with Covid-19. The levels correlated with the acuity of illness,
inflammatory response and need for mechanical ventilation.
More on the platelet-NET interface
Platelets play a pivotal role in the recruitment of neutrophils to sites of inflammation
as well as their subsequent trans-endothelial migration. Von Willebrand Factor (VWF)
is actively involved in this process. The interaction of platelets and neutrophils,
to include NET formation, occurs through several signaling pathways independent of
platelet aggregation and thrombosis (reviewed in Pitchford) [48]. Accumulation of
platelets and VWF within microvessels is a unifying step for endothelial cell activation,
impaired vascular integrity, leukocyte recruitment, trans-endothelial migration, tissue
inflammation, and target organ injury [49].
While platelets dissociate from leukocytes during trans-endothelial migration in high
shear stress conditions, platelet-leukocyte complexes can remain intact under low
mechanical stress as well [50]. Inflammation and its triggers stimulate the formation
of ultra-large VWF fibers that become immobilized on the endothelial cell surface
where they are transformed to highly adhesive strings under shear conditions [51].
Platelets contain functional RNA that can be transferred to other cells in a process
referred to as horizontal transfer (reviewed in Clancy) [52]. The transfer of platelet
cytosolic RNA to nucleated cells increases protein translation and biological effects
at the vascular level and, if the recipient cell undergoes trans-endothelial migration,
at the tissue level [53]. Platelet micro-vesicles are also an important source of
RNA that can be transferred to a variety of cells, including neutrophils, T lymphocytes,
monocytes, macrophages and smooth muscle cells (reviewed in Edelstein) [54].
NETs and microvascular thrombosis
NETs represent part of a continuum of sterile inflammation and thrombosis that can
involve all vascular beds, including the microvascular circulation [55–66].
The contribution of Von Willebrand factor
While NETs “trap” bacteria and other pathogens [67], they can also injure host tissues
through the release of proteolytic proteins [68]. Similar processes have been described
in the heart. For example, ischemia–reperfusion injury of the myocardium causes an
increase in plasma nucleosomes, but in addition, there is abundant neutrophil infiltration
at the tissue level and citrullinated histone H3 at the site of injury. [69, 70].
Von Willebrand Factor Kinetics in Covid-19 infection
Escher et al. identified several unique features in a patient with Covid-19 pneumonia
and ARDS [71]. In addition to a continued rise in D-dimer levels, as others have reported,
the patient had anti-cardiolipin antibodies (IgM) and IgM anti-β2-GPI, a marked increase
in VWF antigen, VWF activity (~ fourfold above the ULN) and factor VIII levels. The
findings are consistent with wide-scale systemic vascular endothelial cell activation.
Covid-19-associated coagulopathy: are the lungs a potential site of origin?
Covid-19 begins in a majority of symptomatic persons as an upper respiratory tract
illness with rhinorrhea, anosmia, cough and fever. In those in whom clinical progression
takes place, there is involvement of the lower respiratory tract. The typical features
that in parallel include chest CT findings of bilateral patchy infiltrates and a “ground
glass” pattern, coupled with biopsy and autopsy findings raise an important question
about the lungs as a primary source of Covid-19- associated coagulopathy.
Consider the following well recognized properties and characteristics of the lungs
under normal and pathological conditions (reviewed in Moldoveanu [72]. First, being
a primary portal for entry of pathogens the lungs have robust innate (non-specific)
and adaptive (specific) immunity potential and reserve. Epithelial cells secrete mucins,
defensins, lactoferrin and nitric oxide as an early defense. They also secrete IL-1β,
tumor necrosis factor (TNF)-α, granulocyte–macrophage colony stimulating factor (GM-CSF)
and platelet activating factor to recruit inflammatory cells. Second, dendritic cells
and macrophages line the respiratory tree where they present and phagocytose pathogens
and also secrete chemokines, cytokines and other inflammatory mediators. Third, lymphocytes
are present throughout the airways and lung parenchyma. T cells provide cell-mediated
immunity and β-cells are responsible for human immune responses by synthesizing antibodies.
Forth, neutrophils are recruited rapidly to sites of infection or injury where they
migrate from capillaries into alveolar and interstitial spaces. NETs, as previously
described have platelet and contact protease activating potential, complex with histones
that have cytotoxic effects and, in addition, produce DNA-myeloperoxidase complexes
that can enter the circulation and exert proinflammatory and prothrombotic effects
“at a distance” from the initial cluster [46]. Viral infections typically activate
the innate immune system through toll-like receptors (TLRs) that recognize molecular
patterns (pathogen-associated molecular patterns or PAMPs) [73]. T helper cells produce
IF-γ.
Among the most important functional roll held by the lungs during infection is regulation
of inflammatory responses and maintenance of systemic homeostasis. Continued activation
of TLRs exerts a negative feedback loop through ILs and TGF- β that down-regulates
proinflammatory cytokine production and resulting inflammatory responses [74]. In
addition, neural-immune interactions may contribute to suppressing inflammatory signals
[75]. A failure to down-regulate or control the needed intensity of inflammation is
a common observation in fatal Covid-19 infection.
Among the most consistent, yet unexpected findings at autopsy among decedents of COVID-19
is extramedullary megakaryocytes within the microvessels serving most major organs,
including the lungs. Might there be a connection to Covid-19 pneumonia? Is there a
possible connection to Covid-19-associated coagulopathy?
Megakaryocytes circulate through the pulmonary microcirculation and release platelets
in a dynamic fashion [76]. The bone marrow is the site of origin for pulmonary megakaryocites
and, while anchored within the pulmonary vascular can contribute substantially to
platelet biogenesis. Pulmonary megakaryocytes and haematopoietic progenitor cells
can migrate to and repopulate bone marrow stores.
The density of pulmonary megakaryocytes increases with infection, impaired lung function,
cardiovascular disease and circulatory compromise [77]. Increased pulmonary thrombopoies
is observed in patients with acute lung injury and ARDS (reviewed in Weyrich) [78]
and activated platelets themselves can contribute to further injury. The number of
circulating megakaryocytes is determined by pulmonary and systemic conditions. The
available evidence suggests that ~ 90% of intact megakaryocytes of pulmonary origin
remain in the microcirculation of the lungs; however, an increased proportion can
leave and enter the arterial circulation in the presence of lung infection and inflammation
[79]. Platelet production from megakaryocytes in the peripheral circulation can occur.
The high density of entrapped neutrophils in the lungs of Covid-19 decedents described
by Fox et al. [9] could represent a proinflammatory and prothrombotic manufacturing
plant that produces in a poorly regulated state the conditions necessary and sufficient
for Covid-19-associated coagulopathy.
The profound cytokine response observed in critically ill patients with Covid-19 and
similarities with secondary HLH or macrophage activation syndrome raise the possibility
that drugs designed to inhibit one or more pathogenic cytokines in the lungs may have
both local and systemic benefit. Several targets for treatment include IL1β (canakinumab),
IL6 (tocilizumab), (TNF)-α (infliximab) and (GM-CSF) (lanzilumab) to name a few. While
Covid-19 infection involves multiple organs, the initial infection in a majority of
cases is pulmonary in origin. Accordingly, therapies that target early lung infections
with the goal to minimize accelerating or escalating disease acuity, excessive immune
response, hyper-inflammation, cytokine storm syndrome and systemic pathological effects
may have a favorable effect on the initiation and steady progression of Covid-19-associated
coagulopathy. This hypothesis will require testing-ideally, in the form of prospectively
designed substudies of ongoing clinical trials targeting SARS-CoV-2 and its associated
cytotoxic and heightened inflammatory properties.
Distinguishing laboratory features of Covid-19-associated Coagulopathy
A consistent observation among patients with Covid-19, particularly those with severe
illness is an elevation of D-dimer in the peripheral blood (reviewed in Becker) [80].
The large case series of patients with COVID-19 (n = 5700) in the New York City area
included baseline measures of D-dimer [81]. The median level was 438 ng/ml (IQR: 262–872 ng/ml)
(Reference normal range [0–229 ng/ml]).
D-dimer is a degradation product of fibrin, formed as a result of the conversion of
fibrinogen to fibrin employing thrombin as a catalyst. The presence of D-dimer in
the circulation signals the breakdown of fibrin polymers by plasmin. The terminology
of D-dimer is based on its containment of two D-fragments of fibrin joined by a cross-link
(factor XIII). While the presence of D-dimer within the peripheral circulation supports
existing thrombus and correlates directly with the burden of fibrin that subsequently
undergoes lysis, it does not specify the site(s) of thrombus.
The well-characterized pathogenesis and diagnosis of disseminated intravascular coagulation
(DIC) are relevant for a discussion of Covid-19-associated coagulopathy [82]. DIC
is recognized for its contribution to multi-organ system failure caused by platelet–fibrin
thrombi in the microvasculature and concomitant bleeding phenotype caused by consumption
of coagulation factors and thrombocytopenia. A common underlying theme that is believed
to be responsible for DIC is systemic inflammation, the presence of and exposure of
circulating coagulation proteins to tissue factor and diffuse vascular endothelial
cell injury/dysfunction with critical loss of physiologic anticoagulants and fibrinolytic
proteins, including tissue plasminogen activator and urokinase-like plasminogen activator.
Fibrin(ogen) degradation products, including D-dimer cause platelet activation [83,
84]. There is a direct correlation between the mass of FDPs and the degree of platelet
activation. Platelet glycoprotein VI, in its dimeric form, binds to both collagen
(in the early stage of thrombosis), fibrin D fragment and D-dimer facilitating platelet
aggregation at sites of fibrin formation [85]. In fact, platelet GPVI may serve as
receptor for polymerized fibrin that amplifies thrombin generation and recruits additional
circulating platelets to the site of thrombus development.
The unique nature of Covid-19-associated coagulopathy and thrombophilia was underscored
in a small case series by Panigada et al. [86]. A total of 24 laboratory-confirmed
patients was included. Employing whole blood thromboelastography (TEG) features of
heightened coagulation parameters were identified (decreased R [time to fibrin formation]
and K [time to 20 mm clot] values and increased K angle [speed to clot, 20 mm] and
MA [clot strength]).
The available evidence derived from clinical observations and autopsy series distinguish
Covid-19-associated coagulopathy from thrombotic microangiopathy and DIC. Potential
overlaps can be observed in critically ill patients in whom circulatory collapse,
multi-organ system failure, refractory hypoxemia and ARDS cause full-blown DIC.
Thrombotic microangiopathy
The prototypical features of thrombotic microangiopathy are Coomb’s negative hemolytic
anemia, thrombocytopenia and microvascular platelet thrombi. The most common disorders
associated with thrombotic microangiopathy are thrombotic thrombocytopenia purpura
(TTP) and hemolytic uremia syndrome (HUS). Organ dysfunction involving kidneys, brain
and gastrointestinal tract is the result of impaired perfusion. There are primary
and secondary causes of TTP and HUS. Secondary causes of TTP include infections (viral,
bacterial), pregnancy, collagen-vascular diseases and drugs. Secondary causes of HUS
include infections (most commonly enteric pathogens), solid organ and bone marrow
transplant recipients, drugs and pregnancy. Thrombotic microangiopathy is a well-described
complication of preeclampsia and eclampsia. The typical laboratory features of thrombotic
microangiopathy include anemia (with schistocytes, reticulocytosis, plasma free hemoglobin,
elevated LDH and decreased haptoglobin), and thrombocytopenia. In TTP, VWF cleaving
protease levels are low. The pathological features include disseminated arteriolar
and capillary thrombi consisting of aggregated platelets, VWF and fibrin with adjacent
vascular endothelial cell swelling. Bleeding is not common in thrombotic microangiopathy.
Disseminated intravascular coagulation
DIC is recognized as a syndrome that complicates a variety of diseases and conditions
with systemic activation of coagulation leading to thrombotic obstruction of small
and less commonly medium-sized blood vessels. Unlike microangiopathies and Covid-19-associated
coagulopathy, bleeding can dominate the clinical phenotype of DIC. In addition to
activation of coagulation proteins, tissue factor and vascular endothelial cells,
DIC is associated with activation of the fibrinolytic system, reduced endothelial
cell surface proteases (antithrombin, protein C) and thrombocytopenia. The most common
causes of DIC are severe infection, sepsis, major trauma, malignancy (acute or chronic
DIC), complications of pregnancy, toxin exposures, severe allergic reactions and immunologic
reactions (e.g. blood product transfusion).
The laboratory features of DIC vary widely depending on the stage encountered. In
early DIC, there is compensated activation of the hemostatic system, however with
progression to decompensated hemostatic activation, characteristic findings are observed.
These include thrombocytopenia, increased PT and PTT, elevated fibrin(ogen) degradation
products and decreased protease inhibition. Fibrinogen levels vary, however, in advanced
stages of DIC fibrinogen levels decrease. VWF and factor VIII levels are typically
increased from endothelial cell activation, but historically they are not elevated
to the degree currently being observed in Covid-19-associated coagulopathy (Table
1).
Table 1
Distinguishing laboratory features of disseminated intravascular coagulation, thrombotic
microangiopathy and Covid-19- associated coagulopathy
DIC
Microangiopathy
Covid-19
PT
↑ ↑
↔
↑ ↑
PTT
↑ ↑
↔
↑
Fibrinogen
↓
↔
↑ ↑
FDPs
↑ ↑
↔
↑ ↑
D-dimer
↑
↔
↑ ↑ or ↑ +
Platelet count
↓↓
↓
↑ or ↔
Peripheral blood Smear + +
+
+ +
+
VWF
↑ ↑
↔
↑ ↑
ADAMTS 13
↓
↔
AT
↓
↓
↑
ACA
↔
↔
+
PC
↓
↔
+
+ ≥ 6 times the ULN
++ peripheral blood smear containing fragmented red blood cells
PT prothrombin time, APTT activated partial thromboplastin time, FDPs fibrin(ogen)
degradation products, VWF von Willebrand Factor, ADAMTS-13 a disintegrin and metalloproteinase
with a thrombospondin type 1 motif, member, AT antithrombin, ACA anticardiolipin antibodies,
PC protein C
Guidelines and consensus statements
The International Society on Thrombosis and Haemostasis published an interim guidance
statement for the recognition and management of coagulopathy in Covid-19 [87]. The
document highlights several key factors, including an elevated D-dimer and its association
with poor clinical outcomes, lack of thrombocytopenia, and late onset DIC in some
patients. The recommendation for treatment calls for LMWH administered at prophylaxis
doses pending the emergence of additional data.
The British Society of Hematology has recommended use of the ISTH DIC score [88, 89]
as a prognostic indicator in patients with Covid-19 to guide treatment. Specifically
in the absence of a bleeding phenotype, therapeutic doses of anticoagulants should
be considered; however, prophylactic doses of either unfractionated heparin or LMWH
are recommended (https://b-s-h.org.uk).
The American Society of Hematology has recommended thromboprophylaxis with either
LMWH or fondaparinux (suggested over UFH to reduce patient contact) unless the risk
of bleeding exceeds the risk of thrombosis. Dose adjustment for obesity should be
considered. In patients with a contraindication for anticoagulation, pneumatic compression
devices should be used (April 17, 2020 www.ash.com). Post-discharge thromboprophylaxis
in patients with Covid-19 using a regulatory agency approved regimen (betrixaban 160 mg
as a first dose, followed by 80 mg daily for 35–42 days or rivaroxaban 10 mg daily
for 31–39 days) is favored.
The American College of Cardiology [90] recommended pharmacological VTE prophylaxis
in Covid-19 patients requiring ICU-level care as well as those with pneumonia, respiratory
failure or other comorbid factors such as heart failure, cancer, prolonged periods
of immobility, and possibly pregnant women who are hospitalized. Extended post-discharge
prophylaxis was considered reasonable for high risk patients (reduced mobility, co-morbid
factors such as active cancer and possibly an elevated D-dimer at the time of discharge).
A consensus statement from several national Chinese societies and working groups [91]
highlighted the importance of thromboprophylaxis and vigilant monitoring for thrombotic
complications among patients with Covid-19 infection.
The Anticoagulation Forum (ACF) has recently drafted a guidance document for anticoagulation
management employing case-based scenarios for patients with Covid-19 requiring hospitalization
as well as for patients with indications for anticoagulation who are at risk for infection.
Clinical trials of anticoagulant therapy in patients with Covid-19 infection
At the time of this writing, there were eight clinical trials of anticoagulation in
patients with Covid-19 registered on Clinicaltrials.gov. Two of the trials are actively
recruiting. A majority of the trials are designed to compare traditional prophylactic
VTE doses of LMWH with higher doses or treatment doses. One of the trials is designed
to study patients with acute coronary syndrome as a complication of Covid-19 infection
and includes aspirin, clopidogrel and rivaroxaban (2.5 mg twice daily). The proposed
sample size is 3170 participants.
Anticoagulation and hemostasis agent decisions
The acuity of illness in some patients with Covid-19 pneumonia complicated by end-organ
injury (liver, kidneys), coupled by the administration of a wide-variety of medications
that may include anti-viral and anti-inflammatory agents should alert clinicians,
pharmacists and other health care providers to potential drug-drug interactions and
drug-related adverse effects (reviewed in Hermans) [92]. Careful consideration of
drug and dose selection and close observation is particularly important for patients
that have inherited disorders of hemostasis, hemophilia A, hemophilia B and Von Willebrand
disease who require regular replacement therapy. In addition to developing specific
management algorithms for patients with Covid-19, the ever-changing landscape created
by the pandemic has and will likely continue to impact the drug and equipment supply
chain, access to coagulation monitoring, laboratories and pharmacies. Under these
circumstances, anticoagulants that do not require regular laboratory monitoring or
the use of point-of-care testing and remote coagulation management are favored. Disorders
of hemostasis, under ideal circumstances, would be treated with longer half-life replacement
products, including those that can be administered subcutaneously.
Conclusions and future directions
Covid-19 infections are characterized by widely variable phonotypic expressions that
involve most major organs and organ systems. An acquired syndrome known as Covid-19-associated
coagulopathy has emerged and proven itself to be common, multifactorial with involvement
of the venous, arterial and microcirculatory systems and distinct from other viral
illnesses. The available information distinguishes Covid-19-associated coagulopathy
from DIC and thrombotic microangiopathy in its early stages and while anticoagulant
therapy for thromboprophylaxis has been recommended by all major societies in the
fields of cardiology, hematology and thrombosis optimal treatment has not yet been
established through rigorously conducted clinical trials.
While many unanswered questions remain, the etiology of Covid-19-associated coagulopathy
appears to follow Virchow’s Triad (Fig. 5). The inclusion of thrombosis substudies
in ongoing clinical trials of anti-viral, anti-inflammatory and immune-modulating
therapies is strongly encouraged as are dedicated studies targeting pathobiology-based
targets that could include NETs, VWF, platelets and factor XI among others.
Fig. 5
Virchow’s Triad represents a fundamental construct in which three components interact
to establish an environment favoring or provoking thrombosis. They include abnormalities
of the blood vessel wall or endothelial surface, altered blood flow and prothrombotic
constituents within the circulating blood. In Covid-19-associated coagulopathy there
is wide-scale endothelial cell inflammation and dysfunction, abnormal flow dynamics
and activated platelets, high concentrations of von Willebrand Factor, cell free DNA,
histones and viral RNA that collectively cause factor XI activation, thrombin generation
and fibrin formation