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https://t.me/med1917
CHAPTER
5
https://t.me/med1917
Pathogenesis and new insights into
acute and chronic venous thrombosis
Andrea T. Obi, Peter K. Henke, and Thomas W. Wakefield
5.1 INTRODUCTION
Venous thromboembolism (VTE), which includes deep vein
thrombosis (DVT) and pulmonary embolism (PE), may affect
up to 900,000 patients per year, resulting in approximately
300,000 deaths per year in the United States.
decreasing with our improved efforts at diagnosis and treatment, this incidence has remained constant over the past
30years and appears to be even increasing. Treatment costs run
in billions of dollars per year and are increasing with the widespread adoption of the more expensive direct oral anticoagulants (DOACs).
with the long-term problems resulting from VTE, including
post-thrombotic syndrome (PTS) for DVT and chronic thromboembolic pulmonary hypertension (CTEPH) for PE.
All current and planned therapies involve targeting portions
of the coagulation system, and as such, this results in the potential complication of bleeding. The current standard of therapy,
DOACs, target either factor Xa or factor IIa. The incidence of
major bleeding with DOACs has been reported between 0.6%
and 1.4%, approximately 2% for low-molecular-weight heparin (LMWH), heparin, or vitamin K antagonists (VKAs), while
all bleeding has been seen in 4.3% to 9.4% of patients.
world comparison of bleeding among more than 60,000 nonvalvular atrial brillation patients demonstrated rates of major
bleeding of 1.4%–2.1% and any bleeding of 11%–16% for
DOACs.
rates for major bleeding may be as high as 10%.
The complications of DVT treatment extend beyond the
acute phase. Patients with signicant DVT may develop PTS
in approximately 20% to 50% of cases manifested with varicose veins, leg pain, swelling, and even venous ulceration. In
the setting of iliofemoral DVT, signicant PTS may be seen in
up to 50%–80% of patients,
treated” PE, the development of CTEPH may also occur.
2
This does not even account for costs associated
4
The reason that this is important is that case fatality
5
and in patients with “adequately
1
Rather than
3
Areal-
5.2 VENOUS THROMBOGENESIS
5.2.1 Advances in inflammation and
venous thrombosis
For over 150 years, venous thrombogenesis has been
thought to be driven by circulatory stasis, endothelial injury,
and blood hypercoagulability (known as Virchow triad).
Inammation was rst implicated in venous thrombosis by
Stewart, and in 1992, it was shown that if the glycoprotein
P-selectin was inhibited, thrombosis was decreased in a primate model.
L-selectin, are a family of calcium-dependent glycoproteins
that are expressed on the surface of platelets, endothelial
cells, and leukocytes, mediators of leukocyte and immune
cell adherence and transmigration into sites of inammation. They facilitate and augment thrombosis by modulating inammatory cell–endothelial cell and inammatory
cell–platelet interactions (Figure 5.1).
6
Selectins, including P-selectin, E-selectin, and
5.2.1.1 P-selectin
P-selectin, encoded by the selectin-P (SELP) gene in
humans, is produced in megakaryocytes and endothelial
cells. It is packaged in endothelial cell Weibel–Palade bodies
and platelet alpha granules. In response to an inammatory
stimulus, P-selectin is rapidly mobilized to the surface of the
cell and then externalized, and it can be reinternalized and
destroyed or recycled just as rapidly. P-selectin glycoprotein
ligand-1 (PSGL-1) is the main P-selectin ligand present on
most leukocytes. P-selectin also binds platelet glycoprotein
(Gp)1b, facilitating both platelet and leukocyte rolling and
adhesion. P-selectin triggers procoagulant microparticle
7
release
and increases monocyte tissue factor expression.8
PSGL-1+ macrovesicles, originating from endothelial cells
and monocytes, are elevated in patients with unprovoked
9
VTE.
P-selectin expression on platelets also stabilizes initial glycoprotein IIb/IIIa–brinogen interactions, leading to
the formation of large, stable platelet aggregates.
In a mouse model of inferior vena cava (IVC) stasis
induced by IVC ligation, animals with elevated circulating soluble P-selectin (sP-selectin) had a 50% increase
in thrombus mass (thrombus weight/IVC length), while
animals with the selectin gene deleted demonstrated a
signicant decrease in thrombus mass.
thrombus mass in mice with elevated sP-selectin was associated with the release of neutrophil extracellular traps
(NETs) and procoagulant microparticles.
Similar ndings have been noted with a model of IVC
stasis with partial IVC ligation.
ing complete IVC stasis, mice inoculated with Klebsiella
pneumoniae to induce pneumonia demonstrated increased
levels of circulating P-selectin.
13
In another study utiliz-
14
Since sepsis increases the
10
11
The increased
12
(Figure 5.1).
DOI: 10.1201/9781003328971-6
4545
46 Chapter 5 Pathogenesis and new insights into acute and chronic venous thrombosis
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5.1 E-selectin and P-selectin involvement in thrombosis and inammation.
risk for VTE and elevated sP-selectin is associated with
DVT risk, the role of P-selectin in sepsis-associated DVT
was explored. P-selectin was upregulated at the vein wall
when sepsis was induced experimentally in mice with the
15
endotoxin lipopolysaccharide.
P-selectin thus may play
an important role in VTE in the setting of infection and
sepsis. Inammatory cell extravasation into the vein wall
and surrounding tissue contributes to inammation of the
vein wall, with resultant vein wall/vein valve brosis contributing to PTS.
The soluble form of P-selectin, a monomer, has been
evaluated as a VTE biomarker. Membrane associated P-se-
among studies.
sP-selectin were 0.57 and 0.73, respectively. Anonsignificant trend to higher sP-selectin levels in proximal compared with distal DVT has also been demonstrated,
similarly, a nonsignicant trend was shown for increased
sP-selectin levels in patients with greater extent of thrombus. Additionally, patients with recurrent DVT were found
to have signicantly higher levels of sP-selectin than patients
without recurrent thrombosis (sP-selectin level greater than
the 75th percentile was 1.7 times more likely).
Other disease states where sP-selectin has been found to
be elevated and associated with thrombosis include:
lectin primarily contributes to DVT pathogenesis, form-
•
ing dimers or oligomers that allow for leukocyte binding.
sP-selectin, as a monomer, is not able to interact with its
16,17
functional ligands.
sP-selectin is derived primarily from
the proteolytic cleavage of transmembrane P-selectin shed
from activated endothelial cells and platelets. High levels
of sP-selectin are associated with VTE, and as a biomarker
combined with the Wells Score, it functioned very well.
18,19
The combination of a high-probability Wells Score >2 and
Malignancies such as non–small cell lung cancer and
breast, lung, gastrointestinal tract, pancreas, kidney,
prostate, brain and hematological malignancies
Sickle cell disease
•
• Human immunodeciency virus
• Obesity
• Obstructive sleep apnea
• COVID-19 coagulopathy
an sP-selectin >90 ng/mL demonstrated a positive predictive value of 100% for DVT in a prospective trial. Levels
of sP-selectin <60 ng/mL combined with a low-probability
Wells Score ruled out DVT with a sensitivity of 99% and
negative predictive value of 96%. A 2014 meta-analysis
of sP-selectin that compared 586 VTE patients with 1843
controls found signicantly increased sP-selectin in patients
with VTE or with DVT alone, with minimal heterogeneity
5.2.1.2 P-selectin inhibitors
The ability of multiple P-selectin inhibitors, including small
molecule inhibitors and receptor antagonists, to limit thrombosis has been demonstrated in rodent and primate models
of IVC and iliac vein thrombosis.
published primate studies revealed that vein reopening was
signicantly greater with inhibitors to both P-selectin and
20
Pooled sensitivity and specicity for
21
and
22
23
Ameta-analysis of ve
5.2 Venous thrombogenesis 47
https://t.me/med1917
PSGL-1 compared to saline, results that were quite similar
to those obtained with LMWH.
24–29
Inammation, measured by MRI and gadolinium enhancement in the vein wall,
was signicantly decreased in the anti-P-selectin–treated
animals compared to saline controls, with no signicant
differences compared to LMWH. Importantly, there was a
lower bleeding potential as indicated by coagulation tests
(p <0.0001).
29
To further this line of inquiry, we evaluated an
anti–P-selectin aptamer (which blocks soluble and bound
P-selectin/PSGL-1 interactions) compared to an aptamer
whose function was to block von Willebrand factor (vWF)
binding and LMWH.
30
When agents were given 2 days
after thrombus formation, the anti–P-selectin aptamer
resulted in the most open iliac vein lumen as measured 21
days after thrombosis (72% vein reopening) compared to
50% for LMWH, 13% for control, and 0% for the antiVWF aptamer. When the agents were circulating at the
time of thrombosis, both aptamers produced similar good
recanalization, although neither totally prevented initial
thrombosis.
30
P-selectin inhibition resulted in less vein wall
brosis, as noted by vein wall collagen immunostaining (a
surrogate marker for PTS), with no signicant increase in
coagulation times compared to controls, suggesting a lowered bleeding potential.
Additionally, a pegylated glycomimetic of the N terminus of PSGL-1 has been developed, PEG40-GSnP-6 (P-G6),
which inhibited both mouse and human platelet–neutrophil and platelet–monocyte aggregations in vitro and platelet-leukocyte interactions in vivo.
known to be dependent on PSGL-1-P-selectin binding.
31
These interactions are
32
The agent was then tested in the IVC electrolytic injury
mouse model and found equivalent to LMWH without
an increase in bleeding time. Administration resulted in a
decrease in vein wall inltrating Ly6G+ (activated) neutrophils and CD68+ macrophages 48hours after thrombus
formation. Although this agent is very promising, a trial
in thromboprophylaxis of elective knee surgery was negative with regards to VTE and bleeding outcomes using
SELK2, a different inhibitory antibody against PSGL-1.
This data suggests perhaps inhibiting only PSGL-1 is not
as clinically effective as opposed to inhibition of the entire
P-selectin–PSGL-1 axis.
33
Additionally, as knee surgery
thrombosis is tissue factor dependent, inammation may
be less important to the initiation of thrombus after knee
surgery as compared to conditions such as infection. Thus,
P-selectin inhibition may be less important in this type of
thrombosis, and anti–P-selectin monotherapy may not be
effective in this class of thrombosis. The combination of
SelK2 plus enoxaparin appeared favorable compared to
SelK2 alone and enoxaparin alone without procedural
hemorrhage (https://clinicaltrials.gov/ct2/show/results/NCT
03812328?view=results) when used for DVT prophylaxis
in elective knee surgery.
5.2.1.3 E-selectin
E-selectin is a glycoprotein expressed from activated endothelium that facilitates thrombosis, directly modulating
neutrophil and monocyte activity (Figure5.1). E-selectin
resides primarily in the endothelium and recruits leukocytes
to sites of inammation, associating closely with P-selectin.
34
E-selectin has multiple receptors, including PSGL-1. E-selectin is upregulated later than P-selectin after cellular stimulation and is expressed experimentally approximately 2
days after DVT formation.
35
E-selectin has been shown to
be efcient at raising the afnity/avidity of CD18 macrophage-1 antigen (Mac-1) integrins, which support neutrophil trafcking to acute inammation, recruiting platelets
and red blood cells.
36
Sialyl-Lewis X (sLex), expressed on
L-selectin on leukocytes, is aggressively bound by E-selectin and, on ligation, initiates the secretion of myeloid-related protein 8 (MRP8) and myeloid-related protein 14
(MRP14). These then bind the toll-like receptor-4 (TLR4)
to elicit the extension of the β2-integrin receptor to an
intermediate afnity state.
37
Neutrophils rolling over
E-selectin at venous shear rates then transmit tension and
catch-bond formation, resulting in a distinct signal to move
the β2-integrin receptor to a high-afnity state, which facilitates leukocytes to stop rolling and extravasate into the
thrombus, vein wall, and local environment. Demonstrating their importance to thrombogenesis and inammation,
P/E-selectin double-knockout mice had less thrombus burden and less inammation when thrombosis was induced,
and E-selectin knockout mice had decreased thrombus
brin content, less vein wall brosis, and less vein wall
inammation.
5.2.1.4 E-selectin in human disease and as a potential
biomarker
Endotoxin-induced tissue factor–mediated coagulation is
enhanced in humans carrying the S128R E-selectin gainof-function allele.
E-selectin allele have an increased risk for VTE recurrence,
highlighting the importance of E-selectin in venous throm-
39
bosis.
E-selectin inhibition is also important in decreasing
vein wall intimal thickness, brosis, and subsequently PTS,
as reported in a recent case control study of 124 patients
with DVT. Of these patients, 31 had severe PTS, 62 mild/
no PTS, and 31 were healthy controls. Patients with severe
PTS demonstrated elevated levels of a number of biomarkers including soluble intercellular adhesion molecule-1
(sICAM-1), C-reactive protein (CRP), and sE-selectin, with
decreased matrix metalloproteinase (MMP-9) and monocyte chemoattractant protein-1 (MCP-1) levels when compared to patients with mild/no PTS.
patients with severe PTS present an altered inammatory
state many months after the acute episode. The increase in
sE-selectin levels were statistically higher in patients with
PTS compared to those with mild/no PTS.
Although the earlier discussion supports the role of
endothelial-derived E-selectin in venous thrombogenesis
and PTS, there have also been studies that have found
sE-selectin levels to be lower in DVT patients than in
controls,
41,42
not elevated in patients with PTS, although levels of
sE-selectin tend to be higher in patients with the most
severe PTS.
not dened, it is likely due to E-selectin, being an endothelial cell–associated glycoprotein, may not be readily
released into the circulation. Thus, it has been suggested
that sE-selectin is not as good a biomarker for diagnosis
of DVT as sP-selectin, which tends to be rapidly released
38
Patients homozygous for the S128R
40
This suggests that
or at least not signicantly elevated
45
Although the reasons for these ndings are
43,44
35
and
5
48 Chapter 5 Pathogenesis and new insights into acute and chronic venous thrombosis
https://t.me/med1917
into the circulation on activation of platelets and endothelial cells. However, levels of sE-selectin have recently
been suggested to be a good biomarker for patients with
COVID-19 pneumonia requiring ICU care. This likely
relates to endothelial activation and inammation with
COVID-19.
46
Other research has furthered the notion
that E-selectin plays a signicant role in VTE formation,
as 25 patients with VTE were found to have both elevations in sE-selectin (EMP
monocytes positive for E-selectin than patients without
VTE. This highlights E-selectin’s role in the inammatory
pathways signicant in VTE.
) and a greater percentage of
62E
47
VTE is a common complication in other vascular
pathologies involving E-selectin, including abdominal
malignancies, pulmonary arterial hypertension, and sex
differences. E-selectin endothelial cell–derived microparticles were higher in females than in males.
48
This higher
E-selectin level was noted during the luteal phase of the
menstrual cycle and could help explain the hypercoagulable state of premenopausal women.
5.2.1.5 E-selectin inhibitors
We studied an E-selectin inhibitor (GMI-1271) in our
mouse model of stasis with some preserved ow venous
thrombosis. This agent was equivalent to LMWH for limiting thrombosis, while at the same time, there was a signicant reduction in tail vein bleeding time.
and 2 clinical trial was then performed with GMI-1271
given to normal volunteers. In phase 1, the agent was given
as a one-time dose in a dose-dependent fashion, then as a
daily dose for 5 consecutive days and compared to enoxaparin or saline. In phase 2, the agent was used to treat
calf vein DVT. Biomarkers of inammation and coagulation were measured, along with markers of cell adhesion
and leukocyte and platelet activation.
no serious adverse events. Lower levels of sE-selectin were
found in GMI-1271–treated volunteers, the agent did not
affect thromboelastographic parameters, and lower leukocyte and platelet activation were seen in GMI-1271–
treated volunteers as noted by reduced myeloperoxidase
(MPO) and MAC-1 levels. Two patients were then able to
be treated with calf vein DVT with 5 days of GMI-1271.
Both patients had immediate relief of pain and an increase
in vein recanalization by day 19. In these patients, levels
of TNF-α decreased from baseline to day 4, while levels of
D-dimer, CRP, and tissue factor all trended lower.
Following this investigation, a primate study was performed combining E-selectin inhibition with GMI-1271
with LMWH, compared to the agent alone and LMWH
alone. This study was based on rodent studies that suggested
there might be a synergistic effect of E-selectin inhibition
along with anticoagulation with LMWH. Iliac vein thrombosis was induced by balloon occlusion. Starting 2 days
after thrombosis, nontreated controls received no agent
(n=5) compared to animals treated with GMI-1271 25
mg/kg subcutaneous (SC) once daily (n=4) for 21 days.
Other animals were treated with GMI-1271 plus a combination of LMWH 1.5 mg/kg or 40 mg (GMI + LMWHc)
SC once daily (n=8) for 19 days or animals treated with
LMWH 1.5 mg/kg or 40 mg (LMWHc) alone SC once
daily (n=6) for 19 days. The best vein recanalization was
49
Aphase 1
50
Findings included
50
51
seen in the GMI-1271-alone–treated animals followed by
GMI-1271 plus LMWHc, both signicantly different from
control. Evaluating recanalization by ultrasound examination, animals treated with GMI-1271 alone had no
decrease in open vein lumen by day 21, while decreases
were observed in combined treatment groups and controls.
Intimal brosis and intimal thickness were best preserved
in the GMI-1271-alone group, surrogate markers for PTS.
Total vein wall collagen revealed a decreasing trend in all
treated groups. No clinically signicant bleeding events
were noted in any group, although the LMWH groups
tended to have prolonged coagulation test values. E-selectin inhibition did not cause clinically signicant changes in
coagulation. Thus, E-selectin inhibition alone was the best
therapy in this model and was even better than its combination with LMWH. All of these results suggest that some
type of targeted inammatory inhibition may be useful to
augment the effects of the “open vein hypothesis,” which
by itself has not been able to result in elimination of PTS
after VTE.
5.2.1.6 Other biomarkers for VTE
Plasmin cleaves brin into multiple degradation products—
the most well studied is D-dimer. D-dimer converts brinogen into a brin mesh, which is then crosslinked by factor
XIII into a scaffold for thrombus. High-sensitivity D-dimer
assays can be used to rule out, but not rule in, DVT, and it
has been suggested that duplex ultrasound imaging can be
eliminated in patients with a negative D-dimer when combined with a negative Wells clinical thrombosis prediction
score. D-dimer is thus highly sensitive but not specic, and
it can be elevated in many medical conditions besides DVT,
including COVID-19. As D-dimer increases with age, some
have proposed age-adjusted D-dimer cut-off levels.
When stimulated by proinammatory factors, neutrophils release a chromatin web known as a neutrophil
extracellular traop (NET), which both traps microbes and
promotes thrombosis. NETs are prothrombotic and induce
formation of a red blood cell–rich thrombus, while also providing a scaffold for the binding of platelets, VWF, bronectin, and brinogen.
ELISA or ow cytometry and are an independent predictor
of mortality in elderly patients. They are positive in DVT.
In the context of high thrombus concentration, brin
monomers bind together to create brin, but some brin
monomers instead bind brinogen or other brin derivatives, forming a brin monomer complex (FMC). These
complexes appear not to be affected by pregnancy (making them an attractive biomarker for VTE in pregnancy),
and they may be a good predictor in early DVT, although
they remain elevated for only a short period of time (within
24hours of the precipitating event) and may be affected by
warfarin anticoagulation, but not thrombolytic agents.
Elevated factor VIII and factor VIII:C levels are correlated with VTE risk in a linear fashion, and factor VIII
acts as a co-factor for factor IXa. Factor VIII as been
shown to correlate with an increased risk of PTS and severe
PTS in some studies,
and vascular cell adhesion molecule-1 (VCAM-1) have not
been found to correlate to DVT incidence,
has been found to be a good biomarker for PTS.
57
Circulating NETs can be detected by
62,63
but not in other studies.64 ICAM-1
64
while ICAM-1
65
52–56
58–60
5.2 Venous thrombogenesis 49
(a) (b) (c)
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Inammatory cytokines are another group of biomark-
ers that have been associated with VTE. Interleukin-1, -6,
-8, and -10 and monocyte chemotactic protein (MCP-1)
have been studied. For example, IL-6 has been associated
with an increased risk of PTS and DVT,
MCP-1 have been associated with an increased risk of VTE
as well.
tor against VTE.
67
IL-10 has been suggested to be a protective fac-
68,69
Erythrocyte sedimentation rate (ESR)
at level above 106.5mm/hr has been associated with an
increased risk of VTE in hematological malignancies
to be negatively associated with the development of PTS at
1year after DVT.
71
66,67
while IL-8 and
70
and
Fibrinogen (in the upper tertial of controls) and hs-CRP
levels (>3 mg/L) have been observed to be higher in patients
with unprovoked VTE compared with risk-associated
72
and absolute admission leukocyte counts (>13,000/
VTE,
mm) have also been associated with an increased risk of
in-hospital VTE despite thromboprophylaxis.
73
Elevated
neutrophil levels [>9.0 × 10(9)/L] have been associated
with an increased risk of venous thrombosis (even when
not due to cancer, infection, or steroids),
74
while hypereosinophilia (both higher peak absolute eosinophil count and
longer duration) has been associated with an increased risk
of VTE.
an increase in early mortality after VTE
in the development of both PTS (venous dysfunction) after
DVT and right ventricular dysfunction after PE.
75
Finally, admission CRP has been associated with
76
and an elevation
77,78
5.2.2 Advances in coagulation and VT
5.2.2.1 Von Willebrand factor and VT
Multiple clinical studies dating back to 1995 have implicated vWF as a risk factor for VT (Figure5.2).
in a 2021 prospective study, a dose-dependent association
between vWF concentration and incident VTE risk was
identied in a Norwegian population.
ing vWF have also been linked to VTE recurrence: vWF
levels >200% are associated with an HR of ≥3.7.
79,80
Recently,
81
Elevated circulat-
82
Biologically, vWF is an acute-phase, multimeric glycoprotein that mediates platelet adhesion and stabilizes procoagulant factor VIII, functionally promoting the initiation
and formation of a stable thrombus at the site of vascular injury.
83
vWF is found in the Weibel–Palade bodies of
endothelial cells and α granules of platelets. In hemostasis,
exposure of subendothelial collagen serves as a binding site
for globular vWF, which is then elongated by shear forces
of uid to expose the A2 domain, the location of the vWF
binding site for platelet receptor Gp1bα.
84
Platelet adhesion and activation in response to Gp1bα binding result
in release of α granule and exposure of platelet receptor
αIIbβ3, capable of binding vWF and brin.
85
Platelets and
endothelial cells release vWF in ultra-large (UL-vWF) form.
UL-vWF more readily binds platelets than lower-molecular-weight vWF and therefore can create a procoagulant
environment. ADAMST13, a circulating disintegrin and
metalloproteinase with vWF as its only known substrate,
cleaves vWF under shear stress.
86
Partial proteolysis of
UL-vWF to high-molecular-weight fraction by ADAMST13
is essential for maintaining efcient hemostasis and avoiding creation of a prothrombotic state.
Experimentally, the rst study on vWF in VT was performed in 1995 by two vascular surgeons, using an ex vivo
ow system to assess for venous thrombosis. Antibody inhibition of vWF impaired thrombus formation.
87
The development of physiologically relevant animal models of DVT
have greatly assisted our understanding of vWF’s role in
DVT pathophysiology. Using a ferric chloride venous injury
model, Chauhan et al. reported that occlusive thrombus
formation is dependent upon vWF and not GPIbα, indicating that vWF uses other adhesion molecules under venous
ow conditions.
88
In murine models of IVC stenosis, vWF
depletion via genetic deletion or pharmacologic inhibition
was associated with protection against the development of
DVT in wild-type and obese mice.
79,89
In a nonhuman primate model of DVT, an inhibitor of vWF was used as treatment and prophylaxis (on board at the time of thrombus
initiation).
30
Only those animals receiving vWF inhibitor as
5
ExtrinsicIntrinsic
Plasminogen
Fibrinolysis
5.2 Coagulation, brinolysis, and venous thrombosis (VT). The coagulation cascade results in the formation of the thrombus, and
its size will depend on several mechanisms, including brinolysis (a) (schematic representation). Considering these two variables,
increases in coagulation or decreases in brinolysis will result in large thrombi (b), and decreases in coagulation or increases in
brinolysis will result in small thrombi (c). t-PA: tissue plasminogen activator; uPA: urokinase-type plasminogen activator; PAI-1:
plasminogen activator inhibitor-1.
t-PA/uPA
PAI-1
Plasmin
Degradation products
(including D-dimer)
Coagulation
Fibrinolysis
Coagulation
Large thrombus Small thrombus
Coagulation
Fibrinolysis
50 Chapter 5 Pathogenesis and new insights into acute and chronic venous thrombosis
https://t.me/med1917
prophylaxis demonstrated improved vein recanalization by
magnetic resonance venography versus controls.
These data suggest the participation of platelets in the
VT initiation process and play a less important role in the
later events of VT. Clinically, vWF interactions with platelets, collagen, and factor VIII are well established. Experimentally, vWF interactions with inammatory cells, NETs,
and erythrocytes highlight additional, less understood interactions that may be productive future routes of research.
90
5.2.2.2 Tissue factor and VTE
TF and VTF are transmembrane glycoproteins (47 kDa)
that play an essential role in hemostasis.
trauma, TF forms a complex with FVIIa, activating blood
coagulation via cleavage of FX and FIX to active forms.
TF forms a hemostatic “envelope” around the body: it is
constitutively expressed in cells that envelope blood vessels (adventitial pericytes), organs (epithelial cells), and the
body (keratinocytes).
93
Mice decient in TF die in utero,
and those with very low (~1%–2%) TF expression have
prolonged bleeding time after hemostatic challenges.
TF expression can also be dynamic; some cells increase
TF production upon exogenous or endogenous stimulation, such as smooth muscle cells, endothelial cells, and
monocytes.
95,96
TF expression on monocyte surfaces promotes monocyte interactions with activated platelets and
endothelial cells, leading to brin formation and deposition
into the developing thrombus. TF can also be found in procoagulant microvesicles derived from circulating platelets
and leukocytes in the plasma. Increased induced expression
of TF is proposed to contribute to VTE pathophysiology.
Among patients with pancreatic cancer, elevated levels of
TF-positive microvesicles are associated with VTE.
from animal thrombosis models have suggested that ow
dynamics may play a key role in cellular TF activation:
blood ow stasis models demonstrate that endothelial TF
plays a predominant role in thrombus formation, whereas
in continuous blood ow models (IVC stenosis), leukocyte-derived TF predominates.
98–100
TF inhibitors have been
studied clinically, however, primarily in the area of sepsis
and acute arterial syndromes, and no clinical study has
been performed in the context of VTE.
91,92
After vessel
94
97
Data
5.2.3 Advances in fibrinolysis and VT
Fibrinolysis is produced by the brinolytic system, which is
critical for regulating hemostasis and comprises an inactive
proenzyme, plasminogen, which can be converted to the
active enzyme, plasmin (Figure5.2). The fate of a forming thrombus is dependent upon the equilibrium between
coagulation and brinolytic factors. Recent data have
demonstrated that beyond thrombus formation, the brinolytic system can play an important role in the fate of the
associated vessel wall.
5.2.3.1 PAI-1 and VT
Under normal conditions, the brinolytic system acts as
a balance to the coagulation system in order to prevent
vascular thrombosis via brinolysis. Plasminogen activator inhibitor-1 (PAI-1) is the pivotal, fast-acting enzyme
responsible for regulating brinolysis. PAI-1 constrains
brin degradation by quenching the enzymatic activity of
plasmin activators urokinase-type plasminogen activator
(uPA) and tissue-type plasminogen activator (Figure5.2a).
Plasmin, a serine protease inhibitor, is the primary enzyme
responsible for cleaving brin and brinogen during brinolysis. The end result of this process is the formation of
fragment E and two molecules of fragment D, which exist
as a covalently linked dimer (D-dimer) (Figure5.2a).
101
The
size of the thrombus within a vein results from the balance between the coagulation cascade (forming the thrombus) and the brinolytic system (dissolving the thrombus).
Increases in coagulation activity and/or decreases in brinolytic activity result in a large thrombus (Figure 5.2b).
Decreases in coagulation activity and/or increases in brinolytic activity result in a small thrombus (Figure5.2c).
Circulating PAI-1 levels are under genetic control of
a polymorphism (4G/5G) at the PAI-1 promoter. The 4G
allele is associated with elevated circulating PAI-1 levels.
PAI-1 levels also increase with age and vary according
to gender and circadian rhythm.
source of PAI-1 is platelets.
has been described in multiple populations harboring the
4G polymorphism.
104,105
Elevated PAI-1 levels have been
associated with postoperative VTE.
102
103
The primary cellular
A higher instance of VTE
106
The hypobrinolytic
state induced by PAI-1 is thought to be the reason for VTE
predisposition.
Data from genetically altered mice have conrmed the
essential role of PAI-1 in brinolysis. Mice globally decient
in PAI-1 demonstrate accelerated brinolysis, whereas those
with overexpressing PAI-1 demonstrate a marked inability
to resolve a venous thrombus.
107,108
Experimentally, PAI-1
inhibition signicantly reduced thrombus weight in rats
undergoing IVC stenosis compared to controls. Although
in this work enoxaparin-treated animals showed similar
thrombus weight reductions to the PAI-1 inhibitor group,
the coagulation parameters were signicantly altered in the
enoxaparin group compared to the PAI-1 inhibitor–treated
109
group.
Interestingly, the effect of PAI-1 extends beyond
the thrombus to the vein wall, with less vein wall brosis seen in mice with PAI-1 overexpression. Two possible
explanations for this nding revolve around other functions of PAI-1. PAI-1 can serve as an MMP activator and
also can prevent inammatory cell inux via binding of
the uPAR receptor, preventing engagement with glycoproteins present on the extracellular membrane
107
: it remains
unclear which is the dominant mechanism in vascular
brosis. While PAI-1 inhibition has been trialed in other
disease states, none have been attempted in VTE.
5.3 ESTABLISHED VENOUS
THROMBOSIS
5.3.1 Early thrombus resolution
The in vivo thrombus is not inert, but biologically active,
with specic cellular types and matrix components orchestrated in a temporal fashion. The normal thrombus (even
without anticoagulation treatment) does lyse over time,
presumably through the plasminogen system, activated by
5.3 Established venous thrombosis 51
Stiffness
Stiffness
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110,111
uPA.
It is likely that uPA is produced from leukocytes
that have inuxed into the thrombus as well as resident vein
wall cells that produce tPA. At the current time it is not
known what specic cellular signals modulate this process,
but these probably include the natural anticoagulant factors
of antithrombin, protein C, protein S, and thrombin.
VT resolution resembles wound healing and involves
probrotic growth factors, collagen deposition, and MMP
expression and activation.
58,59,61,112
In the rodent models of
IVC stasis-induced VT and the electrolytic IVC model, we
have found an acute to chronic inammatory response in
the vein wall and thrombus in response to the IVC insult
and thrombosis induction.
62,63,113–115
In the vein wall, PMNs
are signicantly elevated above sham control animals at
day 2 after thrombosis, and monocytes are signicantly
elevated above sham controls at day 6 after thrombosis.
Total inammatory cell counts are signicantly elevated at
both time points.
To eliminate the role of stasis but assess the contribution of the thrombus to the injury, a transvenous chemical
injury was induced with a 3-minute application of 10%
on the exposed IVC.
FeCL
3
duces a thrombus in the IVC for ≥24hours. Preliminary
112,116
This consistently pro-
studies with these models suggest that nonstasis thrombosis causes lesser injury than stasis VT (e.g., decreased vein
wall stiffness and no alteration in collagen levels, with less
activation of MMP-9), and it seems that the longer a stasis
thrombus is in contact with the vein wall, the greater the
injury (Figure5.3).
Depending on the rodent model, PMNs may be essential for early thrombus resolution by promoting both
brinolysis and collagenolysis.
63,117,118
We have found that
neutropenia in a rat model of stasis VT is associated with
larger thrombi at 2 and 7 days, increased thrombus brosis
(larger and fewer cellular thrombi), and signicantly lower
63,117
thrombus levels of both uPA and MMP-9.
in a nonstasis rodent model, PMN depletion was associated with smaller VT.
99
Conversely,
Stimulating the proinammatory PMN response
with exogenous administration of the chemotactic peptide IL-8 can accelerate experimental VT resolution.
68
It is speculated that IL-8 increases intrathrombus PMN
activation and release of plasminogen activators. To further investigate the role of the chemokines involved in
PMN inux into the resolving VT, we utilized mice with
targeted gene deletion of the CXC receptor (CXCR2
KO) whose ligands include KC and MIP-2, analogs of
human IL-8.
62
The CXCR2 KO mice had larger, less organized early thrombi; fewer intrathrombus PMNs; and
fewer monocytes (over the rst 8 days). Decreased late
(day 12 and 21) thrombus neovascularization was also
observed, as well as impaired brinolysis. Taken together,
PMNs play a role in early thrombus resolution, whereas
monocytes predominate later; both are mediated by CXC
chemokine activity.
The monocyte is probably the most important cell for
VT resolution, as it is multifunctional and directs resident
cell activation through multiple signals. Monocyte inux
into the thrombus peaks at 8 days after thrombogenesis
and correlates with elevated MCP-1 levels. This is one of
the primary CC chemokines that directs monocyte chemotaxis and activation
with VT resolution.
61,69
and has also been associated
119
Targeted deletion of CC receptor-2
(CCR-2 KO) in the mouse model of stasis thrombosis was
associated with early and late impairment of thrombus
resolution, probably via impaired early interferon-γ (IFNγ)–mediated MMP-2 and -9 activity. Indeed, CCR-2 KO
mice with stasis thrombosis supplemented with exogenous
IFN-γ had full restoration of thrombus resolution, in part
due to recovery of MMP-2 and -9 activities, without an
5
5.3 The distribution of the thrombus is not homogeneous, and there are areas of total occlusion combined with areas of partial
occlusion. The main parameters for tissue remodeling that have been explored are presented for conditions with and without the
presence of blood ow. Note that enlarged vein diameters occur in order to host the thrombus. MMP: matrix metalloproteinase.
Thrombus
Large occlusive thrombus
Stretch
No blood flow
MMP 2
MMP 9
Collagen:elastin
Large diameter
Thrombus
Small nonocclusive thrombus
Less stretch
Blood flow present
MMP 2
MMP 9
Collagen:elastin
52 Chapter 5 Pathogenesis and new insights into acute and chronic venous thrombosis
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increase in thrombus monocytes or brinolytic activity.
120
These experiments suggest a broader and intriguing role
of early Th1 lymphokine activity (e.g., IFN-γ) in thrombus
resolution, probably mediated by CCR2
ers have also shown a similar dependence of VT resolution
on CCR2 cellular signaling activity.
+
monocytes. Oth-
121
Proresolving Mo/MΦ cells directly contribute to the
process of DVT resolution through a variety of mechanisms. They phagocytize erythrocytes, platelets, matrix
debris, and other cellular remains.
the thrombus iron, as measured by MRI.
122–125
They also process
126
Proresolving
Mo/MΦ cells directly express brinolytic and collagenolytic enzymes that allow them to invade the thrombus tissue, particularly uPA and MMP9.
or angiogenesis is also promoted by proresolving Mo/MΦ
131–133
cells,
and although evidence is mixed whether neovascularization directly speeds DVT resolution,
phenomenon has been shown in PTS in humans.
127–130
Neovascularization
134,135
136
this
Monocytes/macrophages may be enhanced to speed VT
resolution. Although the monocyte phenotypes were not
dened, peritoneal macrophage injection decreased experimental thrombus size by vefold and exogenous MCP-1
decreased size by sixfold,
137
a strong effect. IFN-γ–decient
mice displayed a phenotype characteristic of proresolving Mo/MØ activation, with enhanced MM9 and VGEF
expression.
MΦ cells signicantly impairs DVT resolution.
138
Consistently, depletion of proresolving Mo/
139
Recent data suggest that stimulating the Nr4a1 pathway with cytosporin B (CsnB), which drives the prohealing Mo/MΦ phenotype, can both pre-emptively and
post-thrombotically accelerate VT resolution, as dened
by duplex ultrasonography in a stenosis model of VT.
140
Similarly, a proinammation resolution pathway can affect
VT resolution. For example, Resolvin D4 reduces PMN
inltration and antagonizes NET formation, in addition
to recruiting pro-resolving monocytes and reducing the
thrombus size.
141
Thus, directly modulating the many Mo/
MØ functions to accelerate VT resolution is ripe for further investigation.
Several toll-like receptors modulate sterile processing
of cell turnover. TLR-9 signaling on thrombus resolution
was investigated using the IVC stasis mouse model of VT.
The thrombi were signicantly larger in TLR9
−/–
mice
compared with WT mice, whereas thrombus collagen and
neovascularization were 55% and 37% less, respectively,
at 8 days after thrombosis was initiated.
ulation with a TLR9 agonist was associated with smaller
142
VT.
142
Finally, stim-
Healing tissue depends on physiologic neovascularization, and a thrombus is similar to a wound-healing milieu.
The aforementioned experiments with chemokine receptor–deleted mice have also conrmed a strong association
between thrombus resolution and neovascularization.
However, neovascularization may reect thrombus organization and not impact thrombolysis. For example, we
have administered exogenous pro-angiogenic agents in the
rat model of stasis VT, and despite documenting increased
thrombus microvascular blood ow, no signicant decrease
in thrombus size was found.
143
However, other investigators have found a potential role of vascular endothelial
growth factor in accelerating thrombus resolution when
administered exogenously.
144
5.3.2 Later thrombus resolution and vein
wall damage
As the thrombus resolves, numerous proinammatory factors are released in the local thrombus environment. These
include IL-1, IL-6, TNF-α, and transforming growth factor-β
(TGF-β), which are present in the thrombus at differing times
and may have direct effects on the vein wall.
ple, elevated circulating IL-6 correlates with incident DVT
in humans.
148,149
sis
resolving Mo/MΦs.
a signicant difference in early thrombus size between control and IL-6
146,147
In mice, IL-6 contributes to thrombogene-
and is expressed on both proinammatory and pro-
–/–
mice,
150–152
This may partly explain the lack of
153
as both inammatory and prohealing
Mo/MΦ function is impaired. Mice treated with anti–IL-6
antibodies have decreased Mo/MΦ inux and accelerated
DVT resolution, which is tied to reduced CCL2 expression,
the primary chemoattractant for Mo/MØ.
Late brosis has been observed in the mouse model of
VT, with a signicant increase in total vein wall collagen after
stasis thrombosis.
155
However, early vein wall collagenolysis
(rather than collagen production) seems to occur within the
rst 7 days in stasis VT in the rat model, representing an
acute response to injury. Interestingly, P-selectin inhibition
has been found to be associated with a decrease in thrombus
collagen content and vein wall brotic injury in our mice, rat,
and baboon models,
30,156,157
suggesting that such inhibition
may be protective against late vein wall damage.
The plasmin activator–plasmin activator inhibitor axis
is critical for VT resolution. Using the mouse IVC ligation model in uPA
counterparts, VT were signicantly larger in both 8-day
and 21-day uPA
were signicantly smaller in both 8-day and 21-day PAI-
–/–
mice as compared with WT mice.
1
8-day plasmin levels were reduced by half in uPA
and increased threefold in PAI-1
–/–
or PAI-1
–/–
mice as compared with WT mice and
–/–
mice and their genetic WT
–/–
mice when compared
with respective WT thrombi. The endothelial cell marker
CD31 was elevated twofold in PAI-1
reduced 2.5-fold at 21 days in uPA
with WT mice, suggesting less endothelial preservation.
Collagen was twofold greater at 8 days in PAI-1
as compared with WT mice, with no differences observed
–/–
in uPA
mice. This work supports the notion that in stasis
VT, plasmin activity is critical for thrombus resolution.
In another recent study focused on thrombus resolution
and vein wall remodeling, deletion of MMP-2 was associated with less midterm vein wall brosis and inammation. Consideration that VT resolution was impaired with
MMP-2 (and MMP-2/9) deletion suggests that direct inhibition will likely also require anticoagulant therapy.
Two other studies have explored the link between PAI-1
and vein wall damage.
107,159
In the rst one, the authors
observed that the absence of vitronectin increases circulating PAI-1, which positively modulates vein wall brosis
in a dose-dependent manner.
107
This occurred despite the
fact that in animals with elevations in PAI-1, the thrombus was larger. Another work evaluated the effect of PAI-1
and LMWH on vein wall injury after thrombosis.
authors showed that LMWH is protective against vein wall
brosis, but that this is abrogated in PAI-1–deleted mice
and correlated with monocyte vein wall inux.
62,145
For exam-
154
108
Correspondingly,
–/–
mice at 8 days, but
–/–
mice, as compared
–/–
mice IVC
–/–
mice
158
159
159
These
108
108
The
5.5 Conclusion 53
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data support the clinical observation that LMWH may be
protective against post-thrombotic vein wall injury in a
PAI-1–dependent manner.
159
In human and experimental studies, circulating bone
marrow endothelial progenitor cells have been shown to be
important in the repair of arterial injury. Intriguing work
from Modarai and colleagues
160
has shown these cells also
play a signicant role in VT resolution. We have found
evidence of these circulating cells in the resolving thrombus and also in the expression of CCR7. This chemokine
receptor is involved in lymphocyte hemostasis and also
confers brogenesis in models of pulmonary inamma-
161
Interestingly, post-thrombotic vein wall remodeling
tion.
is impaired in CCR7
is dependent on the thrombotic mechanism, and is mediated by circulating CCR7
brotic responses, CCR7
for positive vein wall remodeling.
−/–
mice with a probrotic phenotype,
+
cells. Unlike other postinjury
+
cell signaling may be important
162
The resolving thrombus becomes covered with endothelium by day 4 in a murine model of VT, as measured by
intravital imaging on the abluminal side. The endothelialization can be measured indirectly by exposure of brin FTPIII
binding, and thus exogenous brinolysis is more effective
prior to complete abluminal endothelization.
has also shown a very time-dependent nature of lysis using
a clip–unclip IVC thrombus model in mice.
progenitor cells accelerate DVT resolution,
tion and migration are promoted by VEGF, angiopoietins,
LMWH,
167
and various endogenous micro-RNAs.
163
Recent work
164
Endothelial
165
and prolifera-
168–171
166
In addition, gene expression of PAI-1 was decreased in the
IVC and signicantly decreased in the liver in the rosuvastatin group at the same time point. These results suggest
that rosuvastatin decreased PAI-1 and ultimately improved
the brinolytic system in hyperlipidemic mice. This was
further supported by another work demonstrating that
statins improve VT resolution via probrinolytic, anticoagulant, antiplatelet, and anti–vein wall scarring effects.
Statins may offer a new pharmacotherapeutic approach
to improving VT resolution and reducing vein wall injury
post-VT
174
and are being tested in humans.
5.4.2 Galectins and VT
Galectin3 (gal3) and gal3 binding protein (gal3 bp) play
important roles in a number of pathologies, including
cancer, infections, and rheumatoid arthritis, but their role
in VT has not been dened.
be upregulated in microparticles collected from human
patients diagnosed with deep VT.
of the lectin family and is associated with integrin-mediated cell adhesion.
185
gal3 and gal3 bp are associated with murine thrombogenesis and colocalization and that thrombogenesis is
in part gal3 dependent. We also showed that gal3 could
be a potential biomarker in patients with acute VT.
However, despite the fact that the concentration levels
of gal3 bp exceeded gal3 levels, our data showed that the
increased levels of gal3 were higher in VT compared to
the non-VT condition.
175–183
Gal3 bp was found to
184
Gal3 bp is a member
Our laboratory discovered that
174
186
5
5.4 CURRENT TRANSLATIONAL
5.5 CONCLUSION
POTENTIAL THERAPIES IN
VENOUS THROMBOSIS
5.4.1 Statins, hyperlipidemia, and VT
Statins are associated in humans with decreased incident
172
VTE.
and the IVC ligation model to be consistent with our previous work.
Our laboratory used ApoE
173
We demonstrated that rosuvastatin lessens
VT due to the following: (1) Signicantly decreased soluble P-selectin at all time points compared to controls. It is
known that activated endothelial cells and platelets are the
main source of soluble P-selectin in VT initiation. (2) Signicant decreases in circulating active and total PAI-1 were
found 6hours after thrombosis in the rosuvastatin group.
Consensus Statements 5.0 of the American Venous Forum on Acute and Chronic Venous Thrombosis: Pathogenesis
and New Insights
No. Consensus Statements
5.1 Acute venous thrombosis causes an acute-to-chronic inammatory response in both the vein wall and the thrombus. This
leads to thrombus amplication, organization, and recanalization and damage to the wall and the valves.
5.2 D-dimer, platelet-derived microparticles, NETs, and soluble P-selectin are markers of thrombosis, and they are increased in
patients with acute venous thromboembolism.
5.3 Resolution of the thrombus is modulated by the plasmin axis, MMPs, and the natural anticoagulants Antithrombin, Protein C
5.4 Polymorphonuclear cells promote both brinolysis and collagenolysis, and they play a key role in thrombus resolution. Mono-
and Protein S, and thrombin.
cytes are essential in late thrombus resolution.
–/–
mice on a normal diet
It is an exciting time to study venous thrombogenesis and
the pathophysiology of post-thrombotic vein wall damage, as it has been relatively neglected compared with
arterial disease. Adjuncts to or replacement therapies for
anticoagulants hold tremendous promise and will hopefully decrease the early risk of PE and the late complications
of PTS for the benet of the patient. More scientists and
clinicians investigating the pathobiology of VT resolution
are greatly needed. We acknowledge the thrombus models
are not perfect, but these do provide histologically similar
specimens to late human vein post-thrombotic specimens.
We also acknowledge that late-stage postinammation
brosis is challenging, but strides have been made in liver,
kidney, and lung brosis.
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