Arterial Thrombogenesis Research Articles

Evaluation of platelets activity and reactivity as risk factors for acute ischemic non-embolic stroke in young adults

BackgroundIschemic stroke (IS) constitutes a relevant health concern recently in younger population causing permanent cognitive and function-limiting disability and ranks as the 3rd cause of death in Egypt after cardiac and hepatic diseases. Platelet activation has a crucial mechanism in arterial thrombogenesis, thus in pathophysiology of IS. Surface expression of P-selectin (CD62P) reflects platelet activation and measured by flowcytometry. The purpose of the study is to evaluate whether platelet activity and reactivity are considered risk factors for IS so more restrict antiplatelet protocols could be implemented for management and recurrence prevention.ResultsStudy population was 60 IS patients and 60 apparently healthy age and gender-matched controls. Patients were subdivided into 37 patients without classical risk factors, aged 46.1 ± 8.2, and 23 patients with > 1 vascular risk factors, aged 52 ± 9.9. The percentage of platelets expressing CD62P reflecting ex vivo baseline activity was significantly higher in stroke patients to controls (p = 0.001), also platelet reactivity (CD62P expression after ADP provocation) was statistically significantly elevated in patients than in controls (p < 0.0001) and was significantly higher in IS patients with vascular risk factors compared to patients without risk factors (p = 0.02).ConclusionBoth baseline platelet activity and reactivity were significantly higher in IS patients, and were also higher in IS patients with other vascular risk factors than in cryptogenic stroke and considered risk factors for IS.

A holobiont view on thrombosis: unravelling the microbiota's influence on arterial thrombus growth.

The commensal microbiota has co-evolved with its host, colonizing all body surfaces. Therefore, this microbial ecosystem is intertwined with host physiology at multiple levels. While it is evident that microbes that reach the blood stream can trigger thrombus formation, it remains poorly explored if the wealth of microbes that colonize the body surfaces of the mammalian host can be regarded as a modifier of cardiovascular disease (CVD) development. To experimentally address the microbiota's role in the development of atherosclerotic lesions and arterial thrombosis, we generated a germ-free (GF) low-density lipoprotein receptor-deficient (Ldlr−/−) atherosclerosis mouse model (Kiouptsi et al., mBio, 2019) and explored the role of nutritional composition on arterial thrombogenesis.

Factor XIII deficiency does not prevent FeCl3‐induced carotid artery thrombus formation in mice

BackgroundThe compositions of venous (red blood cell–rich) and arterial (platelet‐rich) thrombi are mediated by distinct pathophysiologic processes; however, fibrin is a major structural component of both. The transglutaminase factor XIII (FXIII) stabilizes fibrin against mechanical and biochemical disruption and promotes red blood cell retention in contracted venous thrombi. Previous studies have shown factor XIII (FXIII) inhibition decreases whole blood clot mass and therefore, may be a therapeutic target for reducing venous thrombosis. The role of FXIII in arterial thrombogenesis is less studied, and the particular contribution of platelet FXIII remains unresolved. ObjectiveTo determine whether FXIII reduction prevents experimental arterial thrombogenesis. MethodsUsing wild‐type mice and mice with genetically imposed deficiency in FXIII, we measured thrombus formation and stability following ferric chloride–induced arterial thrombosis. We also determined the impact of FXIII on the mass of contracted platelet‐rich plasma clots. ResultsFollowing vessel injury, F13a+/+, F13a+/−, and F13a−/− mice developed occlusive arterial thrombi. FXIII deficiency did not significantly reduce the incidence or prolong the time to occlusion. FXIII deficiency also did not alter the timing of reflow events or decrease platelet‐rich clot mass. ConclusionsFXIII does not significantly alter the underlying pathophysiology of experimental arterial thrombus formation.

Haemotherapy with Fibrinogen for Perioperative Bleeding Prevention-A View on Arterial Thrombogenesis and Myocardial Infarction in the Rat In Vivo.

Major blood loss during cardiac surgery is associated with increased morbidity and mortality. Clinical pilot studies indicated that preoperative fibrinogen supplementation reduces postoperative blood loss without increasing thrombotic complications. However, an increase in fibrinogen concentration might rather aggravate pre-existing thrombosis than increase the incidence of thrombotic events. Therefore, we investigated, in the present study, whether fibrinogen supplementation influences (1) arterial thrombus formation, (2) the extent of myocardial infarction and (3) the cardioprotective effect of ischaemic preconditioning. Arterial thrombogenesis of the femoral artery was induced by topic FeCl3 treatment in anaesthetised Wistar rats after pretreatment with 60 mg/kg (Fiblow), 120 mg/kg (Fibhigh) or vehicle (Con). Vessel blood flow was monitored, and time to vessel occlusion was analysed as a marker for arterial thrombogenesis. In addition, regional myocardial I/R injury was induced by temporary left coronary artery occlusion in rats pretreated with or without fibrinogen supplementation. In additional groups, ischaemic preconditioning (IPC) was induced by 3 cycles of 5 min of ischaemia/reperfusion. In all groups, myocardial infarct size was determined by triphenyltetrazoliumchlorid staining. Arterial thrombogenesis was not affected by fibrinogen pretreatment. No differences in time until vessel occlusion between Con, Fiblow and Fibhigh groups were observed. In addition, fibrinogen supplementation in low and high concentrations had no effect on infarct size after regional myocardial ischaemia and reperfusion (Fiblow: 66 ± 10%, Fibhigh: 62 ± 9%; each ns vs. Con). IPC reduced infarct size from 62 ± 14% to 34 ± 12% (p < 0.05 vs. Con). Furthermore, both fibrinogen concentrations did not affect cardioprotection by ischaemic preconditioning (Fiblow + IPC: 34 ± 11%, Fibhigh + IPC: 31 ± 13%; each ns vs. IPC). Haemotherapy with fibrinogen did not affect arterial thrombogenesis, myocardial infarction and the cardioprotective effect of ischaemic preconditioning.

Relationship between interleukin‐6 and brain ischemia

Ischemic stroke is the most common disease of nervous system associated with high mortality worldwide. After the primary injury, secondary injury is caused by activation of the immune response due to an influx of neuro‐inflammatory cells increasing the production of inflammatory cytokines and edema. Inflammatory factors, such as interleukin‐6 (IL‐6), are suspected to play a role in arterial thrombogenesis. IL‐6 has a double role of pro‐inflammatory and anti‐inflammatory responding to the brain injury. Many of its effects are caused by trans‐signaling. However, the relationship between IL‐6 and brain ischemia remains unclear. Here we tried to review the role of IL‐6 in brain ischemia and summarized the underlying molecular mechanism.

Interrelationship between venous and arterial thrombosis.

In the past, arterial and venous thrombosis have been accepted as two completely different diseases. Recently, the thesis of two separate pathogenetic mechanisms of venous and arterial thrombosis has been challenged by accumulation of evidence which suggest that patients with atherothrombosis are at increased risk for venous thrombosis. Pathophysiological studies indicated that etiopathogenetic mechanisms of both diseases are similar and that coagulation and platelet activation participate in both venous and arterial thrombogenesis. Inflammation most probably represents the basic etiopathogenetic mechanism of arterial as well as venous thromboembolic disease, being a common mechanism through which different risk factors trigger thrombus formation in both veins and arteries. Atherothrombosis is accepted as a chronic inflammatory disease and the association between circulating inflammatory markers and venous thromboembolism also indicates an involvement of the inflammatory process in venous thrombosis. During inflammation, there is an increase in the production of procoagulant factors, a down-regulation of the anticoagulant mechanisms and an inhibition of the endogenic fibrinolytic activity. In line with the arguments which support the link between arterial and venous thrombosis is also the recognition that different risk factors are common in both diseases, such as age, metabolic syndrome, hypercholesterolemia and thrombophilia. A relation was also found between subjects with preclinical or clinical atherosclerotic disease and venous thromboembolism. These findings indicate close interrelationship between arterial atherothrombotic and venous thromboembolic disease. It could mean that atherosclerosis may induce venous thrombosis or that these two diseases have common risk factors which promote the development of both diseases.

Platelet function tests: why they fail to guide personalized antithrombotic medication.

Despite the essential role of platelets in arterial thrombogenesis, personalized antiplatelet therapy based on platelet function tests (PFTs) did not improve clinical outcome after coronary revascularization[1][1] and did not predict recurrent ischemic or bleeding events in individual patients.[2][2

Evaluation of antithrombotic effect: Importance of testing components and methodologies.

The beneficial antithrombotic effect of some dietary components may offer the most promising approach of prevention of cardiovascular diseases and arterial thrombosis. The major stumbling block in finding effective dietary components is the lack of physiologically relevant techniques which can detect potential antithrombotic effect in humans. The presently used platelet function and coagulation tests do not allow the assessment of global thrombotic status and their value in screening dietary components for antithrombotic effect is questionable. Most of these in vitro tests ignore the effect of flow and shear stress, thrombin generation and vascular endothelium, the major contributors to arterial thrombogenesis in humans. As a gold standard, we employed the helium-neon (He-Ne) laser-induced thrombosis test in murine carotid artery and mesenteric microvessels, as the pathomechanism of this test closely reflects arterial thrombogenesis in humans. Results obtained with laser thrombosis test were compared with various shear-induced in vitro platelet function tests which use native blood (Haemostatometry, Thrombotic Status Analyser, Global Thrombosis Test-GTT). Contribution of vascular endothelium to thrombogenesis was assessed by measuring flow-mediated vasodilation (FMV) in vivo. The combination of the two shear-induced ex vivo thrombosis tests (Haemostatometry and GTT) with FMV correlated most closely with the laser-thrombosis test. Our findings suggest that combining the commercially available point-of-care GTT with the FMV test could provide a better assessment of the overall thrombotic status than either of the two tests alone.

Human Mannose-Binding Lectin Inhibitor Prevents Myocardial Injury and Arterial Thrombogenesis in a Novel Animal Model

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 μg/mL in MBL2(+/+)Mbl1(-/-)Mbl2(-/-) [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 μg/mL) or 14 (9 μg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride-induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.

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The antithrombotic activity of mini-type tomatoes is dependent on the particular variety and the stage of harvest. Lycopene content does not contribute to antithrombotic activity

The prevention of arterial thrombotic disease has a high priority in developed countries. We have focused our studies on the antithrombotic activity of those fruits and vegetables with the potential to prevent the disease, and the present study was undertaken as part of a series of investigations to examine beneficial fruits and vegetables. For this purpose, suitable laboratory tests as well as diets have been devised. In the current investigation, we have classified various tomato varieties with antithrombotic properties, and we now have extended our overall data to include more than ten antithrombotic varieties of fruits and vegetables. A method designed to measure shear-induced platelet activity (the Global Thrombosis Test, GTT) was used to assess haemostasis in vitro and a He-Ne laser-induced thrombosis technique was utilized to examine arterial thrombogenesis in vivo. Concentrations of the antioxidant, lycopene, were also measured. Three mini-type tomato varieties, coded “Cin”, “Pik” and “Caec”, and one mediumtype variety, coded “K”, were harvested at different stages of maturity. All mini-type varieties demonstrated antithrombotic activity at an early (green) stage. The antithrombotic activity decreased with the maturation of “Cin” and “Caec” but remained constant at all stages of maturity with “Pik”. The medium variety, “K”, did not possess antithrombotic activity. Lycopene was not detected at any stage in any of the tomato varieties, suggesting that this antioxidant did not contribute to antithrombotic activity. The present results indicated that the antithrombotic activity of tomatoes is dependent on the particular variety and stage of maturity, and that this activity is not due to lycopene.

Endogenous and natural complement inhibitor attenuates myocardial injury and arterial thrombogenesis

Endogenous and natural complement inhibitor attenuates myocardial injury and arterial thrombogenesis

Recurrent Arterial Thrombosis Associated With the Antithrombin Basel Variant and Elevated Lipoprotein(a) Plasma Level in an Adolescent Patient

Both myocardial infarction and ischemic stroke are rare in the young. Yet a 15-year-old male patient suffered a myocardial infarction and later an ischemic stroke despite uninterrupted antiplatelet therapy. His medical history involved the surgical correction of an incomplete atrioventricular canal defect at the age of 13 years. No cardiovascular risk factors other than elevated lipoprotein(a) level could be identified. His antithrombin (AT) activity was decreased and DNA sequence analysis revealed heterozygosity for AT Basel (p.Pro41Leu), a variant with impaired heparin binding. This report supports a possible additional pathophysiological role for AT Basel and elevated lipoprotein(a) level in arterial thrombogenesis.

Effect of the Thromboxane Prostaglandin Receptor Antagonist Terutroban on Arterial Thrombogenesis after Repeated Administration in Patients Treated for the Prevention of Ischemic Stroke

Background: The antithrombotic, antiplatelet and endothelial activity of terutroban, a specific thromboxane prostaglandin receptor antagonist, was assessed in patients previously treated with aspirin for the prevention of ischemic stroke. Methods: This double-blind, parallel-group, 10-day study included 48 patients (age = 70.5 ± 9.5 years) with cerebral ischemic event and/or carotid stenosis in 4 groups: terutroban 10 mg/day (n = 13), aspirin 300 mg/day (n = 12), terutroban 10 mg/day + aspirin 300 mg/day (n = 11) or clopidogrel 75 mg/day + aspirin 300 mg/day (n = 12). The measurements included parameters from an ex vivo model of thrombosis, platelet aggregation in platelet-rich plasma and plasma biomarkers of endothelial/platelet activation. Results: Between days 0 and 10, the mean cross-sectional surface of dense thrombus significantly decreased with terutroban (58%, p = 0.001), terutroban + aspirin (63%, p = 0.005) and clopidogrel + aspirin (61%, p < 0.05). On day 10, the value for terutroban was significantly lower than that for aspirin (p < 0.01) and was comparable to the dual therapy terutroban + aspirin or clopidogrel + aspirin. Similar results were found for total thrombus surface and platelet adhesion. Platelet aggregation induced by the specific thromboxane prostaglandin receptor agonist U46619 was almost completely inhibited on day 10 in both terutroban groups but not in the others. As regards markers of endothelial/platelet activation or lesions, thrombomodulin significantly increased and plasma soluble P selectin significantly decreased by day 10 in both terutroban groups, whereas the von Willebrand factor did not change significantly. Terutroban was found to be safe and well tolerated. Conclusions: Terutroban has demonstrated an antithrombotic activity that is superior to aspirin and similar to clopidogrel + aspirin; it induces a significant in vivo reduction in endothelial/platelet activation.

Effect of Pharmaceutical Interventions Targeting Thromboxane Receptors and Thromboxane Synthase in Cardiovascular and Renal Diseases

The present review focuses on the roles of thromboxane A2 (TxA2) in arterial thrombosis, atherogenesis, vascular stent-related ischemic events and renal proteinuria. Particular emphasis is laid on therapeutic interventions targeting the TxA2 (TP) receptors and TxA2 synthase (TS), including dual TP-receptor antagonists and TS inhibitors. Their significant inhibitory efficacies on arterial thrombogenesis, atherogenesis, restenosis after stent placement, vasoconstriction and proteinuria indicate novel and improved treatments for cardiovascular and selected renal diseases. New therapeutic interventions of the TxA2 pathway may also be beneficial for patients with poor biological antiplatelet drug response, for example, to aspirin and/or clopidogrel. These new TP/TS agents offer novel improved treatments to efficiently and simultaneously interfere with thrombogenesis and atherogenesis, and to enlarge the existing panel of platelet inhibitors for efficient prophylaxis and treatment of arterial thrombosis and renal proteinuria.

Abstract 3218: Pharmacokinetics, Pharmacodynamics, and Safety of an Anti-von Willebrand Factor Therapeutic Aptamer, ARC1779, in Healthy Volunteers

Background: The prominent role played by vWF in arterial thrombogenesis suggests that vWF inhibition may offer an effective adjunct therapy to PCI in ACS patients. ARC1779 is a PEG-conjugated aptamer that blocks platelet activation through inhibition of vWF A1 domain binding to platelet receptor GPIb. Design: This was an ascending-dose, double-blind, placebo-controlled study in 47 healthy volunteers at doses of 0 (placebo, n = 6) or 0.05 to 1.0 mg/kg ARC1779 (n = 41) given via IV push, “slow bolus” IV infusion over 15 minutes, or “slow bolus” followed by 4-hour IV infusion. PK parameters were estimated from plasma ARC1779 concentrations determined with a validated assay. PD effects were measured by an ELISA for free vWF A1 binding sites and by a platelet function analyzer, the PFA-100 ® . PK: The concentration-time profiles for ARC1779 after IV push or slow bolus appeared monophasic, though the terminal phase may not have been fully captured. The C max and AUC values were dose-proportional. The highest exposure was observed after 1.0 mg/kg slow bolus, with mean C max of 21.15 μg/mL and AUC (0-∞) of 80.92 μ g·hr/mL. The mean apparent elimination half-life (t 1/2β ) was ~2 hours and mean residence time (MRT) was ~3 hours. The mean apparent volumes of distribution (V z and V ss ) were ~1/2 of the blood volume, suggesting that ARC1779 distribution is in the central compartment. The mean clearance (CL) values ranged from ~10% to 21% of GRF, suggesting that renal filtration may not be a major mechanism of clearance of ARC1779. PD: Inhibition of vWF A1 binding was achieved in a dose- and concentration-dependent manner, with respective EC 50 and EC 90 values of 0.22 μ g/mL (17 nM) and 1.98 μg/mL (151 nM). Platelet function inhibition (PFA-100 ® closure time) was achieved, with respective EC 50 and EC 90 values of 0.75 μ g/mL (57 nM) and 2.57 μg/mL (196 nM). vWF activity returned in a dose- and concentration-dependent manner. Safety: ARC1779 was generally well tolerated and no bleeding was observed. Adverse events tended to be minor and not dose related. One volunteer had a hypersensitivity reaction to IV push administration, but no such reactions occurred at higher doses given by slow bolus or infusion. Conclusion: The PK, PD and safety profile of ARC1779 supports its therapeutic potential for use in ACS.

Electric injury model of murine arterial thrombosis

Background and Purpose Murine models of arterial thrombosis have gained utility with applications in genetically manipulated mice. Implementation of current models requires specialized equipment and provides limited outcome measures. A new murine model of continuously monitored arterial thrombosis was created. Methods An electric injury was delivered to the exterior surface of the common carotid artery using the flat end of a 140-μm steel microsurgery needle connected to the anode of a 3-V battery source. Direct current was delivered for 30 s. The developing thrombus was apparent as a white, platelet-dominated region at the site of injury. This region was continuously monitored and recorded by videotape for 30 min. Subsequently, the thrombus area was measured directly on the TV monitor, generating a time course for thrombogenesis. In a further evaluation of the model, three pharmacologically treated groups of mice were evaluated, with drug infusion immediately before thrombus induction: (1) saline (control), (2) heparin (60 units/kg), and (3) GR144053, a GPIIb/IIIa-specific antagonist (10 mg/kg). Results The basic model showed consistent thrombus development by 7–9 min, occasionally forming an occlusive thrombus. Most of the thrombi underwent one or more cycles of embolization and thrombus regrowth. In the experimental series, the heparin-treated group had a significantly decreased thrombus area versus controls ( p < 0.0001); the GR144053-treated mice had no apparent thrombus, supporting a dominant role of platelet aggregation in arterial thrombogenesis. Conclusion This new model is simple to do, uses readily available instrumentation, and provides a continuously recorded quantifiable measure of thrombogenesis.

HPA-1 polymorphism of αIIbβ3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system

BackgroundPlatelet adhesion and subsequent thrombus formation on a subendothelial matrix at the site of vascular damage play a crucial role in the arrest of posttraumatic bleeding but also in different pathological thrombotic events, such as acute coronary syndrome and stroke. Recently published studies have clearly demonstrated that platelet integri αIIbβ3 is intimately involved in the occlusive thrombus formation at the site of endothelial damage. Therefore, any genetic variation in the expression of this receptor may lead to an excessive bleeding or excessive thrombus formation. In this study, we evaluated the influence of HPA-1 polymorphism of integrin αIIbβ3 on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating blood flow.MethodsPlatelets in anticoagulated whole blood [49 healthy previously genotyped blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 sec, 1 and 5 minutes after start of perfusion.ResultsDuring perfusion, the platelet adhesion linearly increased with regard to exposition time and shear rate. Perfusion of blood preincubated with Abciximab over fibrinogen-coated cover-slips showed reduced platelet adherence (absolute fluorescence: 168 ± 35 U vs. 53000 ± 19000 at control experiments, p < 0.05), as well as by perfusion over BSA-coated glass coverslips. Platelet with HPA-1a/1a genotype exhibited initial better adhesion but they also exhibited higher detachment under arterial flow conditions compared to the HPA-1b/1b platelets. Analysis of stable adhesion rate indicate that the platelets carrying the HPA-1b/1b genotype have a higher reactivity threshold for initial interaction with fibrinogen but under the higher shear rate (in regard to time of perfusion) also realize more stable bonds with fibrinogen than platelets with the HPA-1a/1a genotype.ConclusionOur data support the contention that genetically determined variants of platelet integrins αIIbβ3 could play a role in arterial thrombogenesis and thus confirm the hypothesis derived from epidemiological studies.

Platelet count and platelet activation: Impact of a fat meal and day time

Platelet activation plays a pivotal role in arterial thrombogenesis and in cardiovascular diseas [[1], [2]]. It has been suggested that there is circadian rhythm of platelet function and that incre...

Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ deficiency

AbstractFibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRγ-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRγ-chain complex (FcRγ–/–). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRγ is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRγ was associated with a 30% reduction in thrombus growth. Analysis of FcRγ–/– platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRγ deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRγ compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRγ–/– mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRγ deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.