Venous Thrombus Formation Research Articles

Humic Acids Inhibit Platelet Activation to Reduce Venous Thromboembolism in Mice.

We aimed to investigate the effects of the natural product humic acids (HA) on platelet activation and development of venous thromboembolism (VTE) in mice and further explore the relevant mechanism. Eight-week C57BL/6 mice were randomly assigned to three groups: sham operation group (n = 7), VTE group (n = 8), and VTE + HA group (n = 10). Thrombi were harvested to hematoxylin-eosin staining to evaluate the thrombolysis and recanalization of the thrombus. In addition, flow cytometry was performed to detect the expression levels of protein disulfide isomerase on endothelial-derived exosomes and glycoprotein IIb/IIIa on the surface of the activated platelets surface in plasma. Furthermore, the protein expression level of glycoprotein IIb/IIIa in thrombus was determined by immunohistochemistry and immunofluorescence. The length of thrombosis in the VTE + HA group was significantly shorter than that in the VTE group (P = 0.040). No significant differences were observed in thrombolysis and recanalization between the VTE + HA group and the VTE group (P > 0.05). The content of protein disulfide isomerase carried by endothelial-derived exosomes was significantly increased in the VTE group (P = 0.008) but significantly reduced by native humic acids (P = 0.012). Compared with the VTE group, the expression of glycoprotein IIb/IIIa on activated platelet surface in the VTE + HA group was significantly decreased (P = 0.002). The concentration of plasmatic P-selectin in the VTE group was significantly higher than that in the VTE + HA group (P < 0.001). We demonstrate that HA possess a pharmacological property that decreases venous thrombus formation in mice. The underlying mechanism is that HA could inhibit the expression of glycoprotein IIb/IIIa on the activated platelets surface by suppressing endothelial-derived exosomes carrying on protein disulfide isomerase, thereby blocking platelet activation.

C1 Inhibitor Deficiency Results in Increased Activation of Coagulation and Enhanced Venous Thrombosis

Introduction: C1 esterase inhibitor (C1INH) is a broad acting serine protease inhibitor that functions as a major endogenous inhibitor of activated factor (F) XII, FXI and kallikrein of the contact system. Congenital deficiency for C1INH results in hereditary angioedema (HAE) that can cause potentially life-threatening episodes of bradykinin induced swelling. Patients with HAE have increased circulating markers of activation of coagulation, however, the effect of this procoagulant state on thrombosis remains unclear. Methods: Contact pathway triggered thrombin generation was assessed in citrated plasma from patients with HAE (n=19) or age and sex matched healthy controls (n=20) using calibrated automated thrombography. The association between HAE and risk of composite arterial thromboembolism (ATE) and composite venous thromboembolism (VTE) was analyzed in a registry-based study of HAE patients (n=239) and age and sex matched controls (n=2383) with data analyzed by conditional logistic regression. Markers of activation of coagulation were measured in the plasma of C1INH deficient mice or wildtype littermate controls. C1INH deficient mice or wildtype littermate controls were evaluated in the inferior vena cava stenosis model of venous thrombosis and the carotid artery ferric chloride model of arterial thrombosis. Hemostasis was assessed in C1INH deficient mice or wildtype littermate controls using a tail transection model. Results: Plasma from patients with HAE supported significantly increased intrinsic pathway-initiated thrombin generation compared to healthy controls reflected by significantly reduced lagtime and increased peak. Furthermore, the enhanced thrombin generation negatively correlated with residual plasma levels of C1INH activity. Given that enhanced plasma thrombin generation has been associated with an increased risk of VTE in the general population the association between HAE and VTE was further explored. HAE was found to be associated with a significantly increased risk of composite VTE (OR 3.57, 95% CI 2.16-5.81, P<0.0001) but not composite ATE (OR 1.20, 95% CI 0.81-2.09, P=0.278). Consistent with this finding the cumulative incidence of composite VTE, but not ATE, was significantly higher in patients with HAE compared to controls. C1INH deficient mice had significantly increased plasma levels of coagulation markers prothrombin fragment 1+2, thrombin-antithrombin complexes and D-dimer at baseline compared to wildtype littermate controls. In addition, C1INH deficient mice exhibited enhanced venous thrombus formation in the inferior vena cava stenosis model compared to wildtype littermate controls evidenced by increased thrombus weight. Interestingly, loss of C1INH had no effect on venous thrombus composition. Arterial thrombus formation in the carotid artery ferric chloride model was not significantly different between C1INH deficient mice and wildtype controls.Further, hemostasis, assessed using a tail transection model, did not differ significantly between C1INH deficient mice and wildtype littermate controls. Conclusions: Plasma from patients with HAE supported significantly increased contact pathway-initiated thrombin generation that was dependent on the degree of C1INH deficiency. HAE was found to be associated with a selective increase in the risk of composite VTE but not composite ATE. C1INH deficient mice had evidence of increased basal activation of coagulation consistent with impaired endogenous anticoagulant activity. Further, C1INH deficient mice exhibited increased venous thrombus formation but not arterial thrombus formation phenocopying findings in humans with HAE. This work strongly suggests that plasma C1INH is an important endogenous anticoagulant regulating basal activation of coagulation and providing selective protection against venous thrombus formation.

Biomimetic Nanoplatelets to Target Delivery Hirudin for Site-Specific Photothermal/Photodynamic Thrombolysis and Preventing Venous Thrombus Formation.

Due to the high recurrence rate and mortality of venous thrombosis, there is an urgent need for research on antithrombotic strategies. Because of the short half-life, poor targeting capabilities, bleeding complications, and neurotoxic effects of conventional pharmacological thrombolysis methods, it is essential to develop an alternative strategy to noninvasive thrombolysis and decrease the recurrence rate of venous thrombosis. A platelet-mimetic porphyrin-based covalent organic framework-engineered melanin nanoplatform, to target delivery of hirudin to the vein thrombus site for noninvasive thrombolysis and effective anticoagulation, is first proposed. Owing to the thrombus-hosting properties of platelet membranes, the nanoplatform can target the thrombus site and then activate hyperthermia and reactive oxygen species for thrombolysis under near-infrared light irradiation. The photothermal therapy/photodynamic therapy combo can substantially improve the effectiveness (85.7%) of thrombolysis and prevent secondary embolism of larger fragments. Afterward, the highly loaded (97%) and slow-release hirudin (14 days) are effective in preventing the recurrence of blood clots without the danger of thrombocytopenia. The described biomimetic nanostructures offer a promising option for improving the efficacy of thrombolytic therapy and reducing the risk of bleeding complications in thrombus associated diseases.

Dynamically monitoring lymphatic and vascular systems in physiological and pathological conditions of a swine model via a portable NIR-II imaging system with ICG.

Objective: NIR-II imaging with indocyanine green (ICG) has been clinically used in liver tumor resection. However, few data are available concerning the application of ICG-NIR-II in lymphatic and vascular systems in clinic. To expand the application and promote the clinical translation of this approach, we aimed to investigate the feasibility of ICG-NIR-II imaging for monitoring both lymphatic and vascular systems in physiological and pathological conditions using a swine model and compared it to ICG-NIR-I imaging. Methods: we constructed a portable NIR-II imaging system suitable for large animals. Different simulated clinical scenarios in lymphatic and vascular systems of pigs, including lymphatic drainage, lymphorrhea, lymphatic obstruction, lymphatic reconstruction in flaps, venous thrombus formation and vascular anastomosis were modeled to evaluate the reliability of our NIR-II imaging system and the imaging quality of ICG in the NIR-I/II window. Results: Under different simulated clinical scenarios, our portable NIR-II imaging system showed good reliability for pigs. With the help of the portable imaging system, dynamical visualization of lymph vessels, lymph nodes and blood vessels of pigs in different clinical scenarios could be achieved in NIR-II imaging by using the tail fluorescence of ICG. Moreover, ICG-NIR-II imaging has lower background fluorescence and higher resolution than ICG-NIR-I imaging. Conclusions: We demonstrated the first application of a portable NIR-II imaging system for dynamically monitoring both lymphatic and vascular systems in physiological and pathological conditions using a swine model. Our study indicates that ICG-NIR-II imaging be a promising approach for the diagnosis of malfunctions in lymphatic and vascular systems and the surgical navigation of microsurgery and reconstructive surgery.

The novel and exciting research of E-selectin inhibition for deep venous thrombosis

The selectins are so important as central regulators to venous thrombus formation that new discoveries in their inhibition may have significant implications in clinical treatment and applications. A continuation of the previous work from the University of Michigan on E-selectin inhibition has now extended in the recent publication to determine if synergy exist between E-selectin inhibition and low-molecular-weight heparin (LMWH) on deep venous thrombosis (DVT) resolution and vein wall fibrosis and inflammation. 1 Myers D.D. Ning J. Lester P. Adili R. Hawley A. Durham L. et al. E-selectin inhibitor is superior to low-molecular-weight heparin for the treatment of experimental venous thrombosis. J Vasc Surg Venous Lymphat Disord. 2022; 10: 211-220 Abstract Full Text Full Text PDF Scopus (1) Google Scholar E-selectin inhibitor is superior to low-molecular-weight heparin for the treatment of experimental venous thrombosisJournal of Vascular Surgery: Venous and Lymphatic DisordersVol. 10Issue 1PreviewThis study evaluated E-selectin inhibition with GMI-1271 (Uproleselan [GMI]) alone and in combination with the standard of care low-molecular-weight heparin (LMWH) to improve vein recanalization, decrease vein wall inflammation and protect against adverse bleeding in a primate model. We sought to examine this novel treatment of venous thrombosis. Full-Text PDF

Two Stage Intravital Imaging Mouse Model to Assess Venous Thromboembolism in Sickle Cell Disease

Sickle cell disease (SCD) patients have an increased risk of venous thromboembolism (VTE). Population-based studies demonstrated that VTE has a cumulative incidence rate of approximately 25% in adult SCD patients and is associated with higher risk of mortality. VTE in SCD is most commonly manifested as deep vein thrombosis (DVT) with associated pulmonary embolism (PE). Although autopsy studies have regularly discovered pulmonary thromboembolic lesions in SCD patients, the pathophysiology of VTE in SCD remains largely unknown mostly due to the lack of relevant animal VTE model. Understanding the mechanisms that promote VTE in SCD is imperative to identify its prevention and treatment measures.To elucidate the cellular, molecular and biophysical mechanisms of VTE in SCD we developed an innovative two stage intravital imaging analysis experimental model in SCD mice. DVT in SCD mice was induced by surgical ligation of femoral vein. Venous thrombus formation was visualized using intravital multi-photon-excitation (MPE) microscopy. Venous thrombus took the form of a large mass of elliptical shape which extended in the long-axis direction of the femoral vein. It was composed of fibrin, erythrocytes and sparse platelets. Over time, thrombus was infiltrated by migrating neutrophils. To trigger acute PE, femoral vein ligation was removed and the venous thrombus was observed to spontaneously detach and travel to the SCD mouse lung. This method allowed real time visualization of acute PE in vivo using MPE microscopy of intact lung in live breathing mice. Acute PE involved embolization of the pulmonary arterioles and the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries. The embolization of arteriolar circulation led to loss of blood flow in the arterioles and the down-stream capillaries.Herein we introduce an intravital microscopy approach to probe VTE in SCD live mouse. Our model has potential application in investigating the molecular determinants of VTE associated with SCD as well as evaluating efficacy of new antithrombotic drugs. DisclosuresSundd: Bayer: Research Funding; CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Chronically Elevated Interleukin-6 Disturbs the Coagulation Cascade in Mice

Introduction: Pro-inflammatory cytokines play an essential role as activators of the hemostatic system and in the regulation of physiological antithrombotic mechanisms. Interleukin-6 (IL-6) influences platelet production and platelet activation. It was associated with accelerated clotting and intravascular coagulation in tissue factor (TF)-driven murine thrombosis models. However, the precise role of myeloid cell-derived IL-6 on thrombosis formation and the hemostatic system is still unknown.Methods and Results:To better understand the role of IL-6 in thrombosis and the hemostatic system, we developed a new mouse strain with Cre-recombinase driven constitutive IL-6 expression specifically in myeloid cells (LysM-IL-6 OE, Control mice: IL-6 OE). LysM-IL-6 OE mice had a prolonged tail bleeding time and lacked venous thrombus formation induced by inferior vena cava (IVC) stenosis. There were no differences in D-Dimer levels in LysM-IL-6 OE mice neither on baseline level nor after IVC ligation. However, we found unstoppable post-operative bleedings in LysM-IL-6 OE. They showed a prolonged aPTT, a significantly increased INR and a prolonged thrombin converting time. The Factor V and IX expression were reduced, but von Willebrand factor, antithrombin and fibrinogen expression were up-regulated and could not explain the missing thrombus formation. We found significantly elevated erythrocyte sedimentation in line with erythrocytes aggregates, which seemed to be mediated by IL-6 and α2M. Most importantly, hepatic levels of thrombin inhibitor α2 macroglobulin (α2M) mRNA and protein were increased in LysM-IL-6 OE/+ mice compared to control mice. In parallel, Platelet erythrocyte interaction seemed to be essential in the development of the bleeding phenotype.Conclusions: These findings show the role of chronically elevated IL-6 in driving the accumulation of A2m on the surface of erythrocytes, thereby mediating a phenotype of increased bleeding complications.This work was supported by the DFG KA4035/1-1 and by the German Ministry for Education and Research (BMBF 01EO1503) DisclosuresLämmle: Takeda: Membership on an entity's Board of Directors or advisory committees; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Baxter: Other: Travel Support, Speakers Bureau; Alexion: Other: Travel Support, Speakers Bureau; Siemens: Other: Travel Support, Speakers Bureau; Bayer: Other: Travel Support, Speakers Bureau; Roche: Other: Travel Support, Speakers Bureau; Sanofi: Other: Travel Support, Speakers Bureau. Ruf: ARCA bioscience: Consultancy, Patents & Royalties; ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company.

Reduction of perioperative venous thrombus formation by antithrombotic peripherally inserted central catheter in esophageal cancer.

Although a central venous catheter (CVC) is often needed perioperatively for intraoperative and nutritional management of esophageal cancer (EC), the catheter placement impacts the risk of venous thrombosis. We examined the risks of thrombus formation by catheter type, placement, and duration. In total, 226 patients with EC were enrolled in this retrospective study. Patients were classified into one of three groups: those with a conventional CVC (cCVC), a peripherally inserted central catheter (PICC), or an antithrombogenic agent-coated PICC (secPICC). The thrombus formation and clinicopathological features were examined. The frequency of all types of thrombosis was significantly lower in the secPICC group (p < 0.01). Although deep vein thrombosis was frequent in the cCVC group, catheter thrombosis was frequent in the PICC group. In a univariate analysis in patients with the PICC and secPICC groups, less thrombus formation was observed in the secPICC (p = 0.01), short placement time (p = 0.02), and right-sided placement (p < 0.01). Furthermore, a multivariate analysis revealed that secPICC (p = 0.049) and right-sided placement (p = 0.04) significantly reduced rates of thrombus formation. In patients with EC, secPICC and right-sided placement reduce perioperative venous thrombus formation.

PULMONARY EMBOLISM, A RARE BUT DEADLY COMPLICATION OF CO POISONING

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Carbon Monoxide (CO) is a colorless, odorless gas found in fumes produced from carbon fuels in engines, stoves, and fireplaces. CO binds to hemoglobin forming a molecule called carboxyhemoglobin (CO-Hb), which has higher affinity to O2 than hemoglobin itself which leads to symptoms of hypoxia and ischemia. Given an adequate history, clinicians can quickly initiate treatment but differentials for shortness of breath (SOB) must be sought and ruled out. Our case report will discuss the importance of CO poisoning and its involvement in thrombus formation and development of a pulmonary embolism (PE). CASE PRESENTATION: 62 year old female with history of COPD was brought to ED due to acute SOB and lethargy. Upon EMS arrival, CO level was 800. While in transport in EMS, the patient's CO-Hb level was 34 (trended to 11.4). The patient was found to have sp02 of 86% on room air (RA), and was placed on 10L nonrebreather mask. Patient was hemodynamically stable and afebrile. Labs revealed WBC 17.96, platelet 409k (clumped), troponin 0.08 ng/mL, LDH 405 units/L, and CRP 22.3 mg/L. Patient was deemed not a candidate for hyperbaric oxygen therapy. CT Head without contrast ruled out intracranial pathology and UA ruled out UTI. Chest X-ray revealed bilateral hazy opacities. CTPE revealed RUL/RML segmental and subsegmental PE as well as bilateral GGO reflecting interstitial edema in the setting of CO poisoning. Echo revealed EF of 65% with no wall motion abnormalities. COVID PCR test resulted negative making PE more likely as cause for SOB and patient was started on therapeutic Lovenox and empiric Azithromycin and Ceftriaxone. Within 24 hours, patient clinically improved and was discharged on Xarelto. DISCUSSION: PE in patients with CO poisoning should be suspected if the patient does not clinically improve with initial treatment. CO toxicity has been known to cause inflammatory and obstructive changes to arterioles and venules. Furthermore, it may induce vasospasms and increase platelet aggregation, leading to venous thrombus formation. CO poisoning may also increase oxidative stress with nitric oxide and oxygen free radicals causing further damage to endothelial lining1 and leading to further platelet dysfunction. 2 This combination of vessel wall injury and stasis in blood flow are important for development of thrombus. Treatment of given thrombus will lead to moderate resolution of symptoms. CONCLUSIONS: Treatment may be delayed in patients with coexisting PE and CO poisoning as it may be misdiagnosed. It is especially difficult to identify PE in patients with stable O2 saturation. However, the combination of endothelial damage and eventual increased platelet aggregation plays a critical role in thrombus formation in patients with CO toxicity. As a result, it is an important differential for patients with CO poisoning especially when symptoms do not respond to initial treatment. REFERENCE #1: Ruth-Sahd LA, Zulkosky K, Fetter ME. Carbon monoxide poisoning: case studies and review. Dimens Crit Care Nurs. 2011 Nov-Dec;30(6):303-14. doi: 10.1097/DCC.0b013e31822fb017. PMID: 21983502. REFERENCE #2: Ikeda H, Koga Y, Oda T, Kuwano K, Nakayama H, Ueno T, Toshima H, Michael LH, Entman ML. Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries. J Am Coll Cardiol. 1994 Dec;24(7):1749-56. doi: 10.1016/0735-1097(94)90183-x. PMID: 7963124. REFERENCE #3: Sevinc A, Savli H, Atmaca H. An interesting cause of pulmonary emboli: acute carbon monoxide poisoning. Clin Appl Thromb Hemost. 2005 Jul;11(3):353-7. doi: 10.1177/107602960501100317. PMID: 16015424. DISCLOSURES: no disclosure on file for Jagadish Akella; No relevant relationships by Javed Iqbal, source=Web Response No relevant relationships by Vaishali Mehta, source=Web Response No relevant relationships by Najia Sayedy, source=Web Response

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Platelet GPVI (Glycoprotein VI) and Thrombotic Complications in the Venous System

The immunoglobulin receptor GPVI (glycoprotein VI) is selectively expressed on megakaryocytes and platelets and is currently recognized as a receptor for not only collagen but also a variety of plasma and vascular proteins, including fibrin, fibrinogen, laminin, fibronectin, and galectin-3. Deficiency of GPVI is protective in mouse models of experimental thrombosis, pulmonary thromboembolism as well as in thromboinflammation, suggesting a role of GPVI in arterial and venous thrombus formation. In humans, platelet GPVI deficiency is associated with a mild bleeding phenotype, whereas a common variant rs1613662 in the GP6 gene is considered a risk factor for venous thromboembolism. However, preclinical studies on the inhibition of GPVI-ligand interactions are focused on arterial thrombotic complications. In this review we discuss the emerging evidence for GPVI in venous thrombus formation and leukocyte-dependent thromboinflammation, extending to venous thromboembolism, pulmonary thromboembolism, and cancer metastasis. We also recapitulate indications for circulating soluble GPVI as a biomarker of thrombosis-related complications. Collectively, we conclude that the current evidence suggests that platelet GPVI is also a suitable cotarget in the prevention of venous thrombosis due to its role in thrombus consolidation and platelet-leukocyte complex formation.

Abstract 110: C1 Esterase Inhibitor Functions As An Anticoagulant In Human And Murine Venous Thrombosis

Factor (F) XII and FXI play a central role in venous thrombus formation in preclinical disease models and contributes to venous thromboembolism (VTE) in humans. Agents targeting FXIa have been developed and effectively prevent VTE in humans. C1 esterase inhibitor (C1INH) is a serine protease inhibitor that inhibits several proteases, including FXII and FXI. In this study, we investigated the anticoagulant activity of C1INH in humans and mice. First, plasma C1INH levels were determined in a population-based nested case-control study consisting of 405 VTE patients and 829 age- and sex-matched controls derived from the Tromsø Study. Participants with plasma C1INH levels in the highest quartile had a significantly lower risk of VTE (odds ratio [OR] of 0.68, confidence interval 0.49-0.96, P&lt;0.05), unprovoked VTE (OR 0.59, confidence interval 0.39-0.89) and pulmonary embolism (OR 0.57, confidence interval 0.34-0.92) compared to participants with C1INH levels in the lowest quartile after adjustment for age and sex. Secondly, plasma-based thrombin generation studies were conducted to assess the anticoagulant function of C1INH. Supplementation of normal human pooled plasma with exogenous human purified C1INH (0.2 and 0.4mg/ml) significantly inhibited thrombin generation initiated with silica (P&lt;0.05) and significantly prolonged the activated partial thromboplastin assay clotting time (P&lt;0.001). Thirdly, administration of a clinically approved human purified C1INH product to mice significantly reduced thrombus weight (P&lt;0.05) in the murine inferior vena cava stenosis model of venous thrombosis. Studies with C1 inhibitor deficient mice and substrate selective C1INH variants are currently ongoing. Our results indicate that C1INH serves as an endogenous anticoagulant with higher plasma levels of C1INH associated with a decreased future risk of VTE. Further, exogenous C1INH effectively inhibits thrombin generation in vitro and venous thrombus formation in a mouse model. Our findings suggest that C1INH may prevent thrombosis caused by activation of the intrinsic coagulation pathway.

Extracellular Vesicles and Thrombosis: Update on the Clinical and Experimental Evidence.

Extracellular vesicles (EVs) compose a heterogenous group of membrane-derived particles, including exosomes, microvesicles and apoptotic bodies, which are released into the extracellular environment in response to proinflammatory or proapoptotic stimuli. From earlier studies suggesting that EV shedding constitutes a cellular clearance mechanism, it has become evident that EV formation, secretion and uptake represent important mechanisms of intercellular communication and exchange of a wide variety of molecules, with relevance in both physiological and pathological situations. The putative role of EVs in hemostasis and thrombosis is supported by clinical and experimental studies unraveling how these cell-derived structures affect clot formation (and resolution). From those studies, it has become clear that the prothrombotic effects of EVs are not restricted to the exposure of tissue factor (TF) and phosphatidylserines (PS), but also involve multiplication of procoagulant surfaces, cross-linking of different cellular players at the site of injury and transfer of activation signals to other cell types. Here, we summarize the existing and novel clinical and experimental evidence on the role and function of EVs during arterial and venous thrombus formation and how they may be used as biomarkers as well as therapeutic vectors.

Matrine Impairs Platelet Function and Thrombosis and Inhibits ROS Production.

Matrine is a naturally occurring alkaloid and possesses a wide range of pharmacological properties, such as anti-cancer, anti-oxidant, anti-inflammatory effects. However, whether it affects platelet function and thrombosis remains unclear. This study aims to evaluate the effect of matrine on platelet function and thrombus formation. Human platelets were treated with matrine (0–1 mg/ml) for 1 h at 37°C followed by measuring platelet aggregation, granule secretion, receptor expression by flow cytometry, spreading and clot retraction. In addition, matrine (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time, arterial and venous thrombus formation. Matrine dose-dependently inhibited platelet aggregation and ATP release in response to either collagen-related peptide (Collagen-related peptide, 0.1 μg/ml) or thrombin (0.04 U/mL) stimulation without altering the expression of P-selectin, glycoprotein Ibα, GPVI, or αIIbβ3. In addition, matrine-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction along with reduced phosphorylation of c-Src. Moreover, matrine administration significantly impaired the in vivo hemostatic function of platelets, arterial and venous thrombus formation. Furthermore, in platelets stimulated with CRP or thrombin, matrine significantly reduced Reactive oxygen species generation, inhibited the phosphorylation level of ERK1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182) and AKT (Thr308/Ser473) as well as increased VASP phosphorylation (Ser239) and intracellular cGMP level. In conclusion, matrine inhibits platelet function, arterial and venous thrombosis, possibly involving inhibition of ROS generation, suggesting that matrine might be used as an antiplatelet agent for treating thrombotic or cardiovascular diseases.

Management and Perspective of Coronavirus Disease 2019 (COVID-19), Pregnancy, and Hypercoagulability.

The modern-day pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread rapidly. There is limited data about the effects of the virus on pregnant women, even in women who were infected by other strains of coronavirus such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). After reviewing numerous articles published in the peer-reviewed journals and other authentic sources, in this mini-review, we evaluated various key clinical and laboratory aspects of coronavirus disease 2019 (COVID-19) in relation to pregnancy. Eligibility criteria included the patient being pregnant upon admission to the hospital, clinically diagnosed, and/or laboratory-confirmed COVID-19. Taking a comprehensive approach by reviewing numerous studies, it is safe to say that there is no concrete evidence of intrauterine transmission. With adequate infection control measures, breastfeeding in neonates of mothers with COVID-19 is safe postpartum. A disruption of Virchow’s triad by COVID-19 and the normal physiologic changes of pregnancy put the expectant mothers at great risk of arterial, venous, and placental thrombus formation, which can be managed by antithrombotic and related pharmacologic agents including antiviral and anti-inflammatory drugs.

The role of platelets in thrombus fibrosis and vessel wall remodeling after venous thrombosis

PurposePlatelets are known to play an important role in venous thrombogenesis, but their role in thrombus maturation, resolution, and postthrombotic vein wall remodeling is unclear. The purpose of this study was to determine the role that circulating platelets play in the later phases of venous thrombosis. MethodsWe used a murine inferior vena cava (IVC) stenosis model. Baseline studies in untreated mice were performed to determine an optimal postthrombotic time point for tissue harvest that would capture both thrombus maturation/resolution and postthrombotic vein wall remodeling. This time point was found to be postoperative day 10. After undergoing IVC ultrasound on day 2 to confirm venous thrombus formation, mice were treated with a daily injection of platelet‐depleting antibody (anti‐GP1bα) to maintain thrombocytopenia or with control IgG until postoperative day 10, at which time IVC and thrombi were harvested and thrombus length, volume, fibrosis, neovascularization, and smooth muscle cell invasion analyzed. Vein wall fibrosis and intimal thickening were also determined. ResultsMice that were made thrombocytopenic after venous thrombogenesis had thrombi that were less fibrotic, with fewer invading smooth muscle cells. Furthermore, thrombocytopenia in the setting of venous thrombosis resulted in less postthrombotic vein wall intimal thickening. Thrombus volume did not differ between thrombocytopenic mice and their control peers. ConclusionsThis work suggests that circulating platelets contribute to venous thrombus maturation, fibrosis, and adverse vein wall remodeling, and that that inhibition of platelet recruitment may decrease thrombus and vein wall fibrosis, thus helping thrombolysis and preventing postthrombotic syndrome.

COVID-19 vasculitis and novel vasculitis mimics.

COVID-19 has been occasionally linked to histologically confirmed cutaneous vasculitis and a Kawasaki-like vasculitis, with these entities generally having minimal or no lung involvement and a good prognosis. Unlike these vasculitis types, patients with severe COVID-19 pneumonia can develop cutaneous vasculitis-like lesions and systemic arterial and venous thromboemboli, including cryptogenic strokes and other vasculopathy features. Proposed underlying mechanisms for these severe manifestations have encompassed immune dysregulation, including an anti-phospholipid syndrome-like state, complement activation, viral dissemination with direct systemic endothelial infection, viral RNAaemia with immunothrombosis, clotting pathway activation mediated by hypoxaemia, and immobility. In this Viewpoint, we highlight how imaging and post-mortem findings from patients with COVID-19 indicate a novel thrombosis in the pulmonary venous territory distal to the alveolar capillary bed, a territory that normally acts as a clot filtration system, which might represent an unappreciated nidus for systemic microembolism. Additionally, we suggest that this mechanism represents a novel vasculitis mimic related to COVID-19 that might lead to cryptogenic strokes across multivessel territories, acute kidney injury with haematuria, a skin vasculitis mimic, intestinal ischaemia, and other organ ischaemic manifestations. This finding is supported by pathological reports of extensive pulmonary venular thrombosis and peripheral organ thrombosis with pauci-immune cellular infiltrates. Therefore, severe COVID-19 pneumonia with extensive pulmonary intravascular coagulopathy might help to explain the numerous systemic complications of COVID-19, in which the demonstration of direct organ infection has not adequately explained the pathology.

A mathematical model to quantify the effects of platelet count, shear rate, and injury size on the initiation of blood coagulation under venous flow conditions.

Platelets upregulate the generation of thrombin and reinforce the fibrin clot which increases the incidence risk of venous thromboembolism (VTE). However, the role of platelets in the pathogenesis of venous cardiovascular diseases remains hard to quantify. An experimentally validated model of thrombin generation dynamics is formulated. The model predicts that a high platelet count increases the peak value of generated thrombin as well as the endogenous thrombin potential (ETP) as reported in experimental data. To investigate the effects of platelets density, shear rate, and wound size on the initiation of blood coagulation, we calibrate a previously developed model of venous thrombus formation and implement it in 3D using a novel cell-centered finite-volume solver. We conduct numerical simulations to reproduce in vitro experiments of blood coagulation in microfluidic capillaries. Then, we derive a reduced one-equation model of thrombin distribution from the previous model under simplifying hypotheses and we use it to determine the conditions of clotting initiation on the platelet count, the shear rate, and the plasma composition. The initiation of clotting also exhibits a threshold response to the size of the wounded region in good agreement with the reported experimental findings.

P47phox deficiency impairs platelet function and protects mice against arterial and venous thrombosis

NADPH oxidase-derived reactive oxygen species (ROS) regulates platelet function and thrombosis. It remains controversial regarding NOX2’s role in platelet function. As a regulatory subunit for NOX2, whether p47phox regulates platelet function remains unclear. Our study intends to evaluate p47phox’s role in platelet function. Platelets were isolated from wild-type or p47phox-/- mice followed by analysis of platelet aggregation, granule secretion, surface receptors expression, spreading, clot retraction and ROS generation. Additionally, in vivo hemostasis, arterial and venous thrombosis was assessed. Moreover, human platelets were treated with PR-39 to inhibit p47phox activity followed by analysis of platelet function. p47phox deficiency significantly prolonged tail-bleeding time, delayed arterial and venous thrombus formation in vivo as well as reduced platelet aggregation, ATP release and αIIbβ3 activation. In addition, p47phox-/- platelets presented impaired spreading on fibrinogen or collagen and defective clot retraction concomitant with decreased phosphorylation of Syk and PLCγ2. Moreover, CRP or thrombin-stimulated p47phox-/- platelets displayed reduced intracellular ROS generation which was further decreased after inhibition of NOX1. Meanwhile, p47phox deficiency increased VASP phosphorylation and decreased phosphorylation of ERK1/2, p38, ERK5 and JNK without affecting AKT and c-PLA2 phosphorylation. Furthermore, p47phox translocates to membrane to interact with both NOX1 and NOX2 after stimulation with CRP or thrombin. Finally, inhibition of p47phox activity by PR-39 reduced ROS generation, platelet aggregation and clot retraction in human platelets. In conclusion, p47phox regulates platelet function, arterial and venous thrombus formation and ROS generation, indicating that p47phox might be a novel therapeutic target for treating thrombotic or cardiovascular diseases.

AKT2 regulates endothelial-mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis.

Venous thromboembolism (VTE) carries a high risk of morbidity and mortality. Understanding the mechanisms of venous thrombus formation and resolution is critical for improving VTE management. AKT2 kinase is essential for platelet activation and arterial thrombosis. In this study, we examined the role of AKT2 in venous thrombosis in a mouse model of venous thrombosis induced by inferior vena cava (IVC) ligation. We observed an induction of AKT2 expression in the ligated IVC of wild-type (WT) mice. Interestingly, although the initial thrombus size of the ligated IVC was similar between Akt2-/- mice and WT mice, thrombus resolution was delayed in the ligated IVC of Akt2-/- mice. Compared with the ligated IVC of WT mice, the ligated IVC of Akt2-/- mice displayed decreased levels of thrombomodulin (TM) and increased levels of tissue factor (TF), apoptosis, and necroptosis. In addition, intrathrombotic endothelial cells in the ligated IVC of Akt2-/- mice failed to form small vessels, resulting in impaired recanalization and thrombus resolution. TGF-β signaling activation and fibrotic remodeling were increased in the thrombus and vein wall of the ligated IVC of Akt2-/-mice. We further investigated the AKT2-mediated regulation of coagulation factors in endothelial cells and found that forkhead box protein O1 (FOXO1), a target of AKT, enhanced TF and inhibited TM expression. By inhibiting FOXO1, AKT2 suppressed TF expression while increasing TM expression. Our findings indicate that AKT2 may protect endothelial cells against cell death, regulate endothelial-mediated coagulation homeostasis, and promote intrathrombotic recanalization and thrombus resolution in venous thrombosis. These observations suggest dynamic roles of AKT2 in venous thrombus formation and resolution.