Content uploaded by Elizabeth Gardiner
Author content
All content in this area was uploaded by Elizabeth Gardiner on May 24, 2018
Content may be subject to copyright.
A preview of the PDF is not available
Soluble Glycoprotein VI Is Raised in the Plasma of Patients With Acute Ischemic Stroke
Abstract and Figures
Ischemic stroke induced by thrombosis may be triggered by atherosclerotic plaque rupture and collagen-induced platelet activation. Collagen induces glycoprotein VI shedding.
We measured plasma-soluble glycoprotein VI (sGPVI) by enzyme-linked immunosorbent assay in 159 patients with acute (<7-day) ischemic stroke and age/sex-matched community-based control subjects.
sGPVI was elevated in stroke compared with controls (P=0.0168). ORs were higher in Quartile 4 for stroke cases (P=0.0121), and sGPVI was significantly elevated in stroke associated with large artery disease across Quartiles 2 to 4 and small artery disease in Quartile 4. sGPVI decreased 3 to 6 months after antiplatelet treatment, consistent with elevated sGPVI due to platelet activation during the thrombotic event. sGPVI correlated with P-selectin (P=0.0007) and was higher in individuals with the GPVIa haplotype (P=0.024).
Glycoprotein VI shedding is implicated in the pathology of acute ischemic stroke.
Figures - uploaded by Elizabeth Gardiner
Author content
All figure content in this area was uploaded by Elizabeth Gardiner
Content may be subject to copyright.
... Ischemic stroke, not based on cardiac embolism, is predominantly a consequence of atherothrombosis in the carotid and other cranial arteries (130). Platelet activation as measured by release markers during an acute ischemic stroke (AIS) has been demonstrated in many studies, showing elevated plasma levels of sP-selectin (131)(132)(133)(134)(135)(136)(137)(138), sCD40L (134), sGPVI (139), and sCLEC2 (140,141) in comparison to healthy volunteers ( Table 4, Supplementary Table 1). ...
... There are two studies where sGPVI was measured; elevated sGPVI levels were found in IS patients compared to healthy volunteers (139), while reduced sGPVI levels were seen in comparison to patients with non-ischemic events (145). In the latter study, the control group consisted of patients with other cerebral disorders, which might distort the interpretation of the sGPVI level in IS patients. ...
... In the latter study, the control group consisted of patients with other cerebral disorders, which might distort the interpretation of the sGPVI level in IS patients. Additionally, Wurster et al. (145) evaluated GPVI levels in chronic IS patients whereas Al-Tamimi et al. (139) investigated acute phase patients. Interestingly, Wurster et al. (145) did report increased levels of platelet-surface GPVI in IS patients. ...
Platelets are the main players in thrombotic diseases, where activated platelets not only mediate thrombus formation but also are involved in multiple interactions with vascular cells, inflammatory components, and the coagulation system. Although in vitro reactivity of platelets provides information on the function of circulating platelets, it is not a full reflection of the in vivo activation state, which may be relevant for thrombotic risk assessment in various disease conditions. Therefore, studying release markers of activated platelets in plasma is of interest. While this type of study has been done for decades, there are several new discoveries that highlight the need for a critical assessment of the available tests and indications for platelet release products. First, new insights have shown that platelets are not only prominent players in arterial vascular disease, but also in venous thromboembolism and atrial fibrillation. Second, knowledge of the platelet proteome has dramatically expanded over the past years, which contributed to an increasing array of tests for proteins released and shed from platelets upon activation. Identification of changes in the level of plasma biomarkers associated with upcoming thromboembolic events allows timely and individualized adjustment of the treatment strategy to prevent disease aggravation. Therefore, biomarkers of platelet activation may become a valuable instrument for acute event prognosis. In this narrative review based on a systematic search of the literature, we summarize the process of platelet activation and release products, discuss the clinical context in which platelet release products have been measured as well as the potential clinical relevance.
... Previous studies investigating GPVI in thrombotic disease have focussed on either quantifying soluble GPVI (sGPVI), the metalloproteinase-cleaved ectodomain of GPVI shed from the platelet upon activation [21,22], or on platelet surface expression of total GPVI (the sum of monomeric and dimeric GPVI) [23]. In these studies, stroke and TIA patients consistently demonstrated higher platelet expression of total GPVI [23]. ...
... At this time, there is still a low but significant level of platelet activation and the P-selectin exposure is still linearly dependent on the patients' GPVI-dimer level, although this is less dramatic than day-0 (Fig 3D, P� 0.0001, for both day-0 and day-90). Al-Tamimi et al. [21] reported that sGPVI was elevated in the plasma of ischemic stroke patients. To reconcile those observations with our own, we suggest that under the acute stroke conditions of day-0, patients' platelets are highly activated and have marked increases in both total GPVI and GPVI-dimers, compared to control platelets. ...
Objectives
Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure.
Methods
129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301).
Results
The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer ( P <0.0001) as well as demonstrating higher resting P-selectin exposure ( P <0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r ² = 0.88, P <0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission ( P <0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls ( P <0.0001).
Conclusions
Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.
... Consequently, sGPVI acts as a platelet-specific marker of activation. Elevated plasma sGPVI is observed in prothrombotic scenarios such as stroke, 25,26 myocardial infarction, and other coronary pathologies, 27 deep vein thrombosis, 28 disseminated and inducing platelet signaling, leading to platelet degranulation, release of second messengers (ADP, thromboxane [TxA]) and Ca 2+ , exposure of negatively charged phosphatidylserine on the platelet surface, which coordinates and accelerates local thrombin generation, and upregulation of active αIIbβ3, which binds fibrinogen, enabling formation of a molecular bridge with other platelets and establishment of a stable thrombus. Figure created intravascular coagulation, 24 and in microangiopathies. ...
Platelet adhesion and activation is fundamental to the formation of a hemostatic response to limit loss of blood and instigate wound repair to seal a site of vascular injury. The process of platelet aggregate formation is supported by the coagulation system driving injury-proximal formation of thrombin, which converts fibrinogen to insoluble fibrin. This highly coordinated series of molecular and membranous events must be routinely achieved in flowing blood, at vascular fluid shear rates that place significant strain on molecular and cellular interactions. Platelets have long been recognized to be able to slow down and adhere to sites of vascular injury and then activate and recruit more platelets that forge and strengthen adhesive ties with the vascular wall under these conditions. It has been a major challenge for the Platelet Research Community to construct experimental conditions that allow precise definition of the molecular steps occurring under flow. This brief review will discuss work to date from our group, as well as others that has furthered our understanding of platelet function in flowing blood.
... Cleavage of GPVI by metalloproteinases is a feature of collagen-mediated platelet activation (50). This generates soluble GPVI (sGPVI), and increased levels of sGPVI have been demonstrated in stroke (51). Revacept, a GPVI-Fc fusion protein, has shown some promise in reducing cerebral infarct volume and improving functional outcomes in a stroke model without bleeding complications (36). ...
Colchicine has been demonstrated to reduce cardiovascular death, myocardial infarction (MI), ischemic stroke, and ischemia-driven coronary revascularization in people with coronary artery disease (CAD). These reductions were observed even in patients already taking antiplatelet therapy. As well as having anti-inflammatory effects, colchicine demonstrates antiplatelet effects. We propose that colchicine's antiplatelet effects primarily target collagen-induced platelet activation via the collagen receptor, glycoprotein (GP)VI, which is critical for arterial thrombosis formation. In settings such as stroke and MI, GPVI signaling is upregulated. We have demonstrated in vitro that therapeutic concentrations of colchicine lead to a decrease in collagen-induced platelet aggregation and alter GPVI signaling. Clinical studies of colchicine given for 6 months lead to a significant reduction in serum GPVI levels in CAD patients, which may ameliorate thrombotic risk. Future evaluation of the effects of colchicine in clinical trials should include assessment of its effects on collagen-mediated platelet activation, and consideration be given to quantifying the contribution of such antiplatelet effects additional to the known anti-inflammatory effects of colchicine.
... Furthermore, soluble GPVI (inactive form) is highly detected in plasma of patients with AIS, which might suggest the compensatory mechanism to reduce platelet reactivity to collagen. This elevated level of soluble GPVI is significantly decreased after 3 to 6 months of aspirin treatment 87 . Remarkably, the in vitro study has demonstrated that rosiglitazone inhibits collagen-mediated platelet aggregation by reducing the phosphorylation of several GPVI-associated signaling molecules (e.g. ...
Platelets, the cytoplasmic fragments of megakaryocytes, play a primary role in hemostasis. In addition, current evidence demonstrates the contribution of platelets in inflammation. Platelet-associated cell surface proteins (e.g. CD40L, P-selectin, GPVI and CLEC-2) and secretory molecules (e.g. PF4 and RANTES) regulate inflammatory response in various conditions, including cardio-cerebrovascular diseases, inflammatory bowel disease, rheumatoid arthritis and infection/sepsis. Anti-platelets and anti-inflammatory drugs have been shown to reduce the expression and/or function of these platelet-derived molecules, in association with the improved clinical outcomes in patients with inflammatory diseases. Moreover, the novel anti-inflammatory agents that act against platelet-specific targets have been being developed, which might be a potential therapeutic approach in thrombo-inflammatory diseases.
Introduction Atrial fibrillation (AF) increases the risk of ischemic stroke (IS). We hypothesized that the functional form of platelet receptor glycoprotein (GP) VI, GPVI-dimer, which binds to collagen and fibrin causing platelet activation, is overexpressed in patients with AF who have not had a stroke.
Methods A total of 75 inpatients with AF were recruited. None were admitted with or had previously had thrombotic events, including IS or myocardial infarction. Platelet surface expression of total GPVI, GPVI-dimer, and the platelet activation marker P-selectin were quantitated by whole blood flow cytometry. Serum biomarkers were collected in AF patients. Results were compared against patients contemporaneously admitted to hospital with similar age and vascular risk-factor profiles without AF (noAF, n = 30).
Results Patients with AF have similar total GPVI surface expression (p = 0.58) and P-selectin exposure (p = 0.73) on their platelets compared with noAF patients but demonstrate significantly higher GPVI-dimer expression (p = 0.02). Patients with paroxysmal AF express similar GPVI-dimer levels compared with permanent AF and GPVI-dimer levels were not different between anticoagulated groups. Serum N-terminal pro b-type natriuretic peptide (p < 0.0001) and high sensitivity C-reactive protein (p < 0.0001) were significantly correlated with GPVI-dimer expression in AF platelets. AF was the only vascular risk factor that was independently associated with higher GPVI-dimer expression in the whole population (p = 0.02).
Conclusion GPVI inhibition is being explored in clinical trials as a novel target for IS treatment. As GPVI-dimer is elevated in AF patients' platelets, the exploration of targeted GPVI-dimer inhibition for stroke prevention in patients at high risk of IS due to AF is supported.
Background and aims:
Glycoprotein VI (GPVI) is the major platelet-specific collagen receptor. GPVI shedding with generation of soluble GPVI (sGPVI) is an endogenous feedback mechanism preventing platelet overstimulation. sGPVI has not been investigated in patients with chronic coronary syndrome (CCS) undergoing percutaneous coronary intervention (PCI), especially regarding its potential value as a predictor of ischemic and bleeding risk.
Methods:
Baseline plasma sGPVI levels were available in 318 patients with CCS undergoing PCI. Platelet function was assessed by measuring both adenosine diphosphate (ADP) and collagen-induced platelet aggregation. Co-primary endpoints were a composite of death or myocardial injury at 48 hours after PCI, and Bleeding Academic Research Consortium (BARC) type 1 to 5 bleeding at 30 days.
Results:
There was no significant correlation between sGPVI and platelet function at baseline or at 48 hours after PCI and loading with antiplatelet drugs. Baseline plasma sGPVI levels were not associated with the ischemic risk: the incidence of the ischemic endpoint was 25.0% in the lower, 22.9% in the middle, and 26.7% in the upper sGPVI tertile (p = 0.82). There was a significant nonlinear relationship between sGPVI and the risk of bleeding: the incidence of the bleeding endpoint was 11.8% in the lower, 12.6% in the middle, and 26.4% in the upper sGPVI tertile (p = 0.006).
Conclusion:
In patients with CCS undergoing PCI, plasma levels of sGPVI did not correlate with ADP- or collagen-induced platelet aggregation. Patients with higher baseline levels of sGPVI may carry an increased risk of bleeding at 30 days after PCI but no excess risk of ischemic events.
Objective: To observe the effect of moxibustion at Danzhong (CV17) at different time scales on the levels of molecular markers of platelet activation in ApoE-/- mice with atherosclerosis by tail vein injection of GP6 overexpression lentivirus, so as to explore whether moxibustion can improve atherosclerosis by reducing the level of platelet activation.
Methods: A total of 63 ApoE-/- mice aged 8 weeks were randomly divided into model, moxibustion and clopidogrel groups, with 21 mice in each group. Another 21 8-week-old C57BL/6 mice with the same genetic background were used as the control group and fed with normal diet. The mice in the moxibustion group were treated with moxibustion at Danzhong (CV 17) for 20min/ day, the mice in the medication group were treated with clopidogrel solution 14mg/kg by gavage once a day, and the mice in the model group were treated with sham moxibustion. The intervention lasted for 5 days/week. The blank group received no additional intervention. We collected samples from five mice after 4, 8, and 12 weeks of intervention. One week before sampling, ApoE-/- mice were injected with 100μl GP6 lentivirus at a titer of 1.27×10⁹ V.G./ml at 4, 8 and 12 weeks, and C57BL/6 mice were injected with 100μl EGFP fluorescent expression plasmid at 4,8 and 12 weeks. After 48h of injection, the intervention was continued for 5 days, after which the mice were sacrificed. The heart and thoracic aorta were taken from the sacrificed animals, and were stained by HE staining and Oil red "O" staining. Then, the pathological tissue were used for quantitative analysis of aortic plaque. The fluorescence transfection of bone marrow cells was observed under a fluorescence microscope to indirectly evaluate the success of lentivirus transfection in vivo. The platelet-rich blood were detected by flow cytometry for observing the expression levels of platelet activation molecular markers CD63, CD62p and CD154.
Results: After 4 weeks of moxibustion intervention, the levels of CD63 and CD154 were down-regulated, and the levels of CD63 and CD154 in the moxibustion group were significantly lower than those in the clopidogrel group (P < 0.0001), and the level of CD63 in the moxibustion group was lower than that in the control group (P > 0.05). After 8 weeks of moxibustion intervention, the levels of CD63, CD62P and CD154 were down-regulated, and the levels of CD63 and CD62P were significantly lower than those in the clopidogrel group, and were close to the levels in the control group (P > 0.05). The levels of CD63, CD62P and CD154 in the 12-week moxibustion group were higher than those in the clopidogrel group, but there was no statistically significant difference (P > 0.05), suggesting that after over-expression of GPVI injection in vivo and continuous intervention for 12 weeks, the down-regulation effect of moxibustion on platelets was less than that of clopidogrel group. Conclusion: Moxibustion therapy has a certain inhibitory effect on platelet activation, which can effectively slow down the progress of atherosclerosis by reducing the platelet activation rate. The intervention effect has the characteristics of a time scale.
Conclusion: Moxibustion therapy has a definite inhibitory effect on platelet activation, which can effectively slow down the progression of arteriosclerosis by reducing the platelet activation level, and the intervention has time-scale characteristics. The effect of moxibustion for the treatment of atherosclerosis by inhibiting platelet activation is more obvious in the early stage of the disease.
Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species.
The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.
Glycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseased vessels in the setting of myocardial infarction or stroke. GPVI is an attractive target for antithrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and/or ectodomain shedding. Metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family have been proposed to mediate this ectodomain shedding, but direct evidence for this is lacking. Here, we studied GPVI shedding in vitro and in vivo in newly generated mice with a megakaryocyte-specific ADAM10 deficiency and in Adam17(ex/ex) mice, which lack functional ADAM17. We demonstrate that GPVI cleavage in vitro can occur independently through either ADAM10 or ADAM17 in response to distinct stimuli. In contrast, antibody (JAQ1)-induced GPVI shedding in vivo occurred in mice lacking both ADAM10/ADAM17 in their platelets, suggesting the existence of a third GPVI cleaving platelet enzyme. This was supported by in vitro studies on ADAM10/ADAM17 double-deficient platelets. These results reveal that ectodomain shedding of GPVI can be mediated through multiple differentially regulated platelet-expressed proteinases with obvious therapeutic implications.
The 2 most common haplotypes of human GP6, GP6a and GP6b, generate the allelic isoforms glycoprotein VI (GPVI)a and GPVIb that differ by 5 amino acids: S219P, K237E, and T249A in the ectodomains, and Q317L and H322N in the cytoplasmic domain. By quantitative Western blot, we found no association between GP6 genotype and total platelet GPVI content among 132 normal subjects. When expressed as soluble products or as membrane-associated receptors, GPVIa and GPVIb have identical affinities for type I collagen, collagen-related peptide, or convulxin. However, the cytoplasmic domain substitutions in GPVIb have a significant effect on GPVI-dependent subcellular associations and ligand-induced signal transduction. L317 increases binding to calmodulin, whereas N322 attenuates binding to Fyn/Lyn. Consistent with the latter finding, convulxin-induced Syk phosphorylation is significantly attenuated in Dami cells stably transfected with GPVIb, relative to GPVIa. This represents direct evidence that haplotype-related GPVI functional differences are inherent in the cytoplasmic domain substitutions, whereby GPVIb binds less strongly to Fyn/Lyn and attenuates the rate and extent of Syk phosphorylation. These allelic differences in GP6a and GP6b explain functional differences in the respective isoforms, but the molecular basis for the several-fold range in GPVI levels of human platelets remains to be determined.
Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcgammaRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of approximately 1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 +/- 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to approximately 7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was approximately 10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.
In ischemic stroke, treatment options are limited. Therapeutic thrombolysis is restricted to the first few hours after stroke, and the utility of current platelet aggregation inhibitors, including GPIIb/IIIa receptor antagonists, and anticoagulants is counterbalanced by the risk of intracerebral bleeding complications. Numerous attempts to establish neuroprotection in ischemic stroke have been unfruitful. Thus, there is strong demand for novel treatment strategies. Major advances have been made in understanding the molecular functions of platelet receptors such as glycoprotein Ib (GPIb) and GPVI and their downstream signaling pathways that allow interference with their function. Inhibition of these receptors in the mouse stroke model of transient middle cerebral artery occlusion prevented infarctions without increasing the risk of intracerebral bleeding. Similarly, it is now clear that the intrinsic coagulation factor XII (FXII) and FXI play a functional role in thrombus formation and stabilization during stroke: their deficiency or blockade protects from cerebral ischemia without overtly affecting hemostasis. Based on the accumulating evidence that thrombus formation and hemostasis are not inevitably linked, new concepts for prevention and treatment of ischemic stroke may eventually emerge without the hazard of severe bleeding complications. This review discusses recent advances related to antithrombotic strategies in experimental stroke research.
Cardiovascular and cerebrovascular diseases combined are the leading killers worldwide today. Mortality related to chronic atherothrombotic disease is projected to grow disproportionately, with the focus being in low- and middle-income countries. In the developed world, although we have gained successes with regard to reduction in mortality attributable to a mix of preventive and treatment strategies, we have largely shifted the curve of the disease to the right, turning these conditions into chronic morbidities. In turn, this has reflected in the 368 billion dollars spent by the United States for these illnesses, a 3-fold increase in the span of a decade1 (Figure 1). In this article, we discuss some of the emerging mechanisms that simultaneously affect the atherosclerotic process in the cardiac and cerebrovascular regions and touch on management issues relevant to the clinician and population health researcher.
Figure 1. Increasing costs of cardiovascular and cerebrovascular disease. Reprinted with permission from Nature Reviews Cardiology. 1
### Defense Mechanisms
Basic science animal and human autopsy studies have defined the role of the endothelium, vasa–vasorum, and lipids in the regulation of the atherothrombotic plaque.2 The vulnerable plaque is believed to have a thin fibrotic cap, a lipid-rich necrotic core, neovascularization, and inflammation. The body has its own defense mechanisms to maintain the plaque homeostasis. Endothelial progenitor cells have been shown to play an important role in the regulation of endothelium and abort the progression of endothelial dysfunction to plaque rupture. Similarly, the vasa–vasorum regulated in turn by extracellular matrix, fibroblasts, smooth muscle cells, and growth factors also is a critical player in the process of plaque regression. In addition, high-density lipoprotein cholesterol is believed to regulate the lipid content of the plaque preventing formation and expansion of the core of the plaque. Finally, vascular inflammation regulated by the monocyte–macrophage system plays a role in …
Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation. We aimed to investigate GPVI in patients presenting with symptoms of acute cerebrovascular disease and to define GPVI as biomarker for acute stroke.
We consecutively evaluated 205 patients, who admitted the stroke unit with symptoms for stroke. Surface expression of the platelet activation markers (GPVI, CD62P, GPIb) was determined by two-color whole blood flow cytometry.
Patients with transient ischemic attack (TIA) (n = 18; 8.8%) as well as with stroke (n = 133; 64.9%) showed a significantly enhanced GPVI expression (mean fluorescence intensity +/- SD) on admission compared to patients with non-ischemic (NI) events (n = 54; 26.3%) (TIA: 20.9 +/- 7.1 vs. NI: 16.2 +/- 3.9; P = 0.002; stroke: 20.4 +/- 5.7 vs. NI; P = 0.002). Neither CD62P nor GPIb surface expression showed a significant difference. Logistic regression analysis revealed that on admission GPVI was associated with stroke independent of conventional laboratory markers such as C-reactive protein, blood glucose, and creatine kinase. Using a receiver operating characteristic curve on GPVI, we have determined the cut off value of 18.2 for stroke. Thus, patients with enhanced GPVI expression levels (>or=18.2) had a 2.4-fold relative risk for stroke. Patients with elevated platelet GPVI expression level had a poorer clinical outcome in cumulative event-free survival for stroke, myocardial infarction, and cerebro-/cardiovascular death at 3-month follow-up (log rank; P = 0.045).
These findings indicate that platelet GPVI surface expression is significantly enhanced in patients with TIA and stroke compared to patients with NI events. Determination of platelet-specific GPVI may be useful as an early biomarker for cerebral ischemia.
Activation of endothelial cells and platelets is an important mediator of atherothrombosis. Markers of endothelial cell and platelet activation such as soluble adhesion molecules can be measured in plasma. We hypothesized that patients with acute ischemic stroke would have increased blood concentrations of soluble E-selectin and von Willebrand factor (vWF), primarily reflecting activation of endothelial cells, and increased concentrations of soluble P-selectin and platelet-derived microvesicles (PDM), primarily reflecting activation of platelets, compared with healthy controls. We also hypothesized that these markers would be differentially elevated in ischemic stroke caused by large- and small-artery atherothrombosis compared with cardiogenic embolism.
We conducted a case-control study of 200 hospital-referred cases of first-ever ischemic stroke and 205 randomly selected community controls stratified by age, sex, and postal code. Using established criteria, we classified cases of stroke by etiological subtype in a blinded fashion. The prevalence of vascular risk factors and blood concentrations of E-selectin, P-selectin, vWF antigen, and PDM were determined in stroke cases within 7 days and at 3 to 6 months after stroke and in controls.
Mean blood concentrations of soluble E-selectin, P-selectin, and PDM within 7 days of stroke onset were all significantly higher in cases compared with controls. At 3 to 6 months after stroke, the mean blood concentrations of E-selectin and P-selectin fell significantly below that of controls, and PDM concentrations remained elevated. There was a strong, graded, and independent (of age, sex, and vascular risk factors) association between increasing blood concentrations of E-selectin during the acute phase and all etiological subtypes of ischemic stroke, particularly ischemic stroke caused by large-artery atherothrombosis. There was also a significant, graded, and independent association between increasing blood concentrations of vWF during the acute phase and ischemic stroke caused by large-artery atherothrombosis.
We have demonstrated significant associations between acute elevation of blood markers of endothelial cell and platelet activation and ischemic stroke and between acute elevation of blood markers of endothelial cell activation and ischemic stroke caused by large-artery atherothrombosis. Persistent elevated blood concentrations of PDM may be a marker of increased risk of ischemic stroke.
We investigated the plasma levels of D-dimer, fibrinogen, beta-thromboglobulin (BTG) and platelet factor-4 (PF-4), indices of the occurrence of platelet activation in vivo, to find out their role in pathophysiology of ischemic stroke and whether or not such a role has any effect on the disability and the prognosis of stroke patients. A total of 76 patients with AIS aged from 26 to 85 (32 men, 44 women) and 30 cases as controls with similar age (18 men, 12 women) were included in the study. The plasma levels of D-dimer, BTG and PF-4 were measured by ELISA method using a special commercial kit. The cases were allocated into two groups as non-embolic (NEI) and cardioembolic stroke (CEI). The D-dimer levels in 76% of 42 patients in NEI group (p<0.05) and 85.2% of 34 patients in CEI group (p<0.05) were outside the confidence interval (CI) defined for the control group. The levels of BTG were elevated in 81% of 42 cases with NEI (p<0.05) and in 76% of 34 cases with CEI, with reference to CI of control group. The levels of PF-4 were significantly increased in 86% of cases with NEI (p<0.05) and in 88% of cases with CEI than controls (p<0.05). It was observed that the cases with high Rankin scores had higher levels of D-dimer (p<0.005), BTG (p<0.01) and PF-4 (p<0.01) than those with lower scores. There was a correlation between hemostatic markers, platelet activation and functional disability. D-dimer levels were an important marker that determined to degree of the activation of hemostatic system, especially in CEI subtype. The platelet aggregation had an important role in pathophysiology of ischemic stroke and this condition is significant in NEI subgroup and subjects with large infarcts and high disability scores.
Platelet-mediated thrombus formation at the site of vascular injury is a major trigger for thrombo-ischemic complications after coronary interventions. The platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in the initiation of arterial thrombus formation. Endothelial denudation of the right carotid artery in rabbits was induced through balloon injury. Subsequently, local delivery of soluble, dimeric fusion protein of GPVI (GPVI-Fc) (n = 7) or control Fc (n = 7) at the site of vascular injury was performed with a modified double-balloon drug-delivery catheter. Thrombus area within the injured carotid artery was quantified using a computer-assisted image analysis and was used as index of thrombus formation. The extent of thrombus formation was significantly reduced in GPVI-Fc- compared with control Fc-treated carotid arteries (relative thrombus area, GPVI-Fc vs. Fc: 9.3 +/- 4.2 vs. 2.3 +/- 1.7, p < 0.001). Local delivery of soluble GPVI resulted in reduced thrombus formation after catheter-induced vascular injury. These data suggest a selective pharmacological modulation of GPVI-collagen interactions to be important for controlling onset and progression of pathological arterial thrombosis, predominantly or even exclusively at sites of injured carotid arteries in the absence of systemic platelet therapy.
Several polymorphisms of integrin alpha2beta1 and glycoprotein (GP) VI that may modify platelet-collagen interactions or subsequent signaling have been described. We conducted a case-control study involving 180 stroke patients and 172 controls to determine whether the alpha2 C807T and GPVI Q317L polymorphisms were associated with an increased risk of ischemic stroke. We found no statistically significant differences in the distribution of alpha2 C807T and GPVI Q317L in patients and controls overall or after stratification by etiological subtype. The GPVI 317QQ genotype was found to be over-represented in a subgroup of patients >/=60 years compared to corresponding controls. However, this association did not remain significant after adjustment for other cardiovascular risk factors. Our results do not support a role for the integrin alpha2 C807T and GPVI Q317L polymorphisms in the development of first-ever ischemic stroke. However, larger studies are required to confirm this.