Protective Effects of Kininogen-1 Gene Deficiency in Mouse Models of Venous Thrombosis

Background- High molecular weight Kininogen (HK) is a nonenzymatic co-factor of the contact activation system. HK binds prekallikrein (PK) and FXI to surfaces in proximity to FXII, amplifying PK activation by FXIIa and the reciprocal activation of FXII by activated PK (PKa), as well as FXI activation of FXIIa. PKa cleavage of HK also liberates bradykinin-a proinflammatory and vasoactive nanopeptide. The aim of this study was to define the pro-thrombotic role of kininogen in venous thrombosis (VT) and to use in vivo serial analysis of thrombus development to understand the recruitment and retention of platelets in the growing thrombus in the absence and presence of kininogen. Methods- The development of VT in mice deficient in kininogen (mKng1-/-) was compared to that in their wild-type littermates. A femoral-saphenous stasis VT model was prepared by ligating both saphenous and femoral veins. Next VT formation, growth, and dissolution (n=3 for each group) was monitored using intravital microscopy (IVM) via a multichannel epifluorescence microscope (Nikon Eclipse 90i). To induce stasis VT, FITC-dextran (10 mg/kg, ex/em 488/520 nm) was injected retro-orbitally, and then continuous light irradiation (20x objective, 475nm/35nm) of the saphenous vein was applied for 5 minutes. FITC-dextran fluorescence angiography monitored thrombus formation and dissolution. Immediately after VT formation, platelet accumulation at the thrombus site was monitored in the Cy5 channel (630/38 nm) via injection of a GPIbβ antibody conjugated with Dylight-649 (150nmol/kg), over time. All images were identically windowed in each channel, and thrombus area was measured using NIH ImageJ software. To corroborate IVM studies, we also evaluated a complete stasis model of inferior vena cava (IVC) ligation (n=7-8 per group). Thrombi were harvested after 48 hours and thrombus weight and length were measured to estimate thrombus mass. FXI circulates in blood as a homodimer along with HK. We determined the effect of kininogen deficiency on FXI activity. FXI activity assay used a combination of inhibitors, serially, to monitor the cleavage of substrate specific to activated FXI and release of chromogen, as a function of FXI activity. Finally, to determine the effects of Kng1 deficiency on bleeding, tail vein bleeding times were also determined (n=8 per group). Results- In femoral-saphenous stasis VT, thrombus developed in both groups immediately following FITC-channel light irradiation. However, thrombus size was smaller in Kng1-/- as compared to WT (Figure 1). Results from serial IVM of VT indicated faster thrombus dissolution in the Kng1-/- group. Lower platelet signals, as shown at 2 and 6 hours in the Kng1-/- mice may be consistent with this hypothesis. Thrombus area analysis suggested decreased thrombus formation in the Kng1-/- animals, and temporal analysis indicated faster dissolution by 6 hours (Figure 2). IVC ligation results corroborated the findings of femoral-saphenous DVT model, demonstrating that thrombus weight was significantly lower in Kng1-/- mice as compared to WT (p<0.001, Figure 3). FXI activity was also decreased in the Kng1-/- group (p<0.10). Tail vein bleeding times, however, showed no increased bleeding in Kng1-/- mice. Conclusion- These initial results suggest a pro-thrombotic role of kininogen and a protective role of kininogen deficiency in two murine venous thrombosis models, without incurring a bleeding penalty. Thrombus dissolution was faster and platelet accumulation was inhibited in Kng1-/- mice. These findings suggest that targeting kininogen may provide a new approach to prevent and treat venous thrombosis. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria. Jaffer: Mercator, Inc.: Other: Sponsred research.

Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh , inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

GRK2 Regulates ADP Signaling in Platelets Via P2Y 1 and P2Y 12

Abstract Venous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA 2, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y 1 and P2Y 12. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation. Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 -/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA 2, or convulxin. Underlying these changes in GRK2 -/- platelets is an increase in Ca 2+ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I 2. Furthermore, platelet aggregation can be restored in GRK2 -/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y 12 signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 -/- mice treated with the P2Y 12 antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 -/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y 1 and P2Y 12-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases. Disclosures No relevant conflicts of interest to declare.

Enhanced Thrombotic Responses Are Associated With Striatin Deficiency and Aldosterone

Background In addition to its role on blood pressure, aldosterone (ALDO) also affects the hemostatic system leading to increased experimental thrombosis. Striatin is an intermediate in the rapid, nongenomic actions of ALDO. Striatin heterozygote knockout ( Strn +/‐ ) mice have salt sensitivity of blood pressure and mildly chronically increased ALDO levels. In addition, in humans, striatin polymorphic gene variants are associated with increased salt sensitivity of blood pressure. Thus, we hypothesized that striatin deficiency would be associated with an increased prothrombotic response. Methods and Results Strn +/ ‐ mice and wild‐type littermates were maintained on a liberal sodium diet (1.6%). We measured in vivo thrombus formation following laser‐induced injury in cremaster arterioles using intravital microscopy. Mice were randomized to intravenous administration of ALDO or its vehicle. Acutely, ALDO increased thrombotic responses in wild‐type mice ( P <0.01) versus controls within minutes as determined by increased platelet accumulation and fibrin deposition at the site of laser injury. We then compared thrombus formation without ALDO administration in Strn +/‐ and wild‐type mice. Strn +/‐ mice showed highly significant increases in laser‐induced thrombosis ( P <0.001), as shown by increased platelet accumulation and fibrin deposition. Interestingly, the response in the Strn +/‐ mice basally was far greater than the wild‐type mice with ALDO administration, and ALDO administration produced no additional effect on thrombus responses in Strn +/‐ mice. Conclusions These results demonstrate a novel protective role of striatin in experimental thrombosis. Such a protective effect may be reduced in human striatin risk allele carriers, given the similar salt sensitivity of blood pressure in these individuals and Strn +/‐ mice.

Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

Background: Hepatic platelet accumulation contributes to acetaminophen (APAP)-induced liver injury (AILI). However, little is known about the molecular pathways involved in platelet recruitment to the liver and whether targeting such pathways could attenuate AILI.Methods: Mice were fasted overnight before i.p. injected with APAP at a dose of 210 mg/kg for male mice and 325 mg/kg for female mice. Platelets adherent to Kupffer cells were determined in both mice and patients overdosed with APAP. The impact of α-Chi3l1 on alleviation of AILI was determined in a therapeutic setting, and liver injury was analyzed. Results: The present study unveiled a critical role of chitinase 3-like-1 (Chi3l1) in hepatic platelet recruitment during AILI. Increased Chi3l1 and platelets in the liver were observed in patients and mice overdosed with APAP. Compared to wild-type (WT) mice, Chil1-/- mice developed attenuated AILI with markedly reduced hepatic platelet accumulation. Mechanistic studies revealed that Chi3l1 signaled through CD44 on macrophages to induce podoplanin expression, which mediated platelet recruitment through C-type lectin-like receptor 2. Moreover, APAP treatment of Cd44-/- mice resulted in much lower numbers of hepatic platelets and liver injury than WT mice, a phenotype similar to that in Chil1-/- mice. Recombinant Chi3l1 could restore hepatic platelet accumulation and AILI in Chil1-/- mice, but not in Cd44-/- mice. Importantly, we generated anti-Chi3l1 monoclonal antibodies and demonstrated that they could effectively inhibit hepatic platelet accumulation and AILI.Conclusions: we uncovered the Chi3l1/CD44 axis as a critical pathway mediating APAP-induced hepatic platelet recruitment and tissue injury. We demonstrated the feasibility and potential of targeting Chi3l1 to treat AILI. Funding: ZS received funding from NSFC (32071129). FWL received funding from NIH (GM123261). ALFSG received funding from NIDDK (DK 058369). ZA received funding from CPRIT (RP150551 and RP190561) and the Welch Foundation (AU-0042-20030616). C.J. received funding from NIH (DK122708, DK109574, DK121330, and DK122796) and support from a University of Texas System Translational STARs award. Portions of this work was supported with resources and the use of facilities of the Michael E. DeBakey VA Medical Center and funding from Department of Veterans Affairs I01 BX002551 (Equipment, Personnel, Supplies). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Analysis of microvascular thrombus mechanobiology with a novel particle-based model

Platelet accumulation at the site of vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood. Here we describe a novel computational model of microvascular thrombus formation for detailed analysis of thrombus mechanics. Adopting a previously developed two-dimensional particle-based model focused on the thrombus shell formation, we revise it to introduce platelet agonists. Blood flow is simulated via computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process. The new model qualitatively reproduces enhanced thrombus formation due to granule secretion in line with in vivo findings and provides a mechanism for thrombin confinement at the early stages of aggregate formation. Our calculations also predict that release of dense granules results in additional mechanical stabilization of the inner layers of the thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of thrombus, which are expected to result in mechanical disruptions at the later stages of thrombus formation.

Chitinase 3-like-1 Contributes to Acetaminophen-induced Liver Injury by Promoting Hepatic Platelet Recruitment

Hepatic platelet accumulation contributes to acetaminophen (APAP)-induced liver injury (AILI). However, little is known about the molecular pathways involved in platelet recruitment to the liver and whether targeting such pathways could attenuate AILI. The present study unveiled a critical role of chitinase 3-like-1 (Chi3l1) in hepatic platelet recruitment during AILI. Increased Chi3l1 and platelets in the liver were observed in patients and mice overdosed with APAP. Compared to wild-type (WT) mice, Chi3l1-/- mice developed attenuated AILI with markedly reduced hepatic platelet accumulation. Mechanistic studies revealed that Chi3l1 signaled through CD44 on macrophages to induce podoplanin expression, which mediated platelet recruitment through C-type lectin-like receptor 2. Moreover, APAP treatment of CD44-/- mice resulted in much lower numbers of hepatic platelets and liver injury than WT mice, a phenotype similar to that in Chi3l1-/- mice. Recombinant Chi3l1 could restore hepatic platelet accumulation and AILI in Chi3l1-/- mice, but not in CD44-/- mice. Importantly, we generated anti-Chi3l1 monoclonal antibodies and demonstrated that they could effectively inhibit hepatic platelet accumulation and AILI. Overall, we uncovered the Chi3l1/CD44 axis as a critical pathway mediating APAP-induced hepatic platelet recruitment and tissue injury. We demonstrated the feasibility and potential of targeting Chi3l1 to treat AILI.

Computer Simulation of Platelet Adhesion around Stent Struts in the Presence and Absence of Tissue Defects around Them

Aim. To predict platelet accumulation around stent struts in the presence or absence of tissue defects around them. Methods. Computer simulations were performed using virtual platelets implementing the function of the three membrane proteins: glycoprotein (GP) Ibα, GPIIb/IIIa, and GPVI. These platelets were perfused around the stent struts implanted into the vessel wall in the presence or absence of tissue defects around them using within the simulation platform. The number of platelets that adhered around stent struts was calculated by solving the blood flow using Navier–Stokes equation along with the adhesion of membrane protein modeled within the platform. Results. Platelet accumulation around stent struts occurred mostly at the downstream region of the stent strut array. The majority of platelets adhered at the downstream of the first bend regardless of the tissue defect status. Platelet adhesion around stent struts occurred more rapidly in the presence of tissue defects. Conclusion. Computer simulation using virtual platelets suggested a higher rate of platelet adhesion in the presence of tissue defects around stent struts.

A conformally adapted all-in-one hydrogel coating: towards robust hemocompatibility and bactericidal activity

Hospital-acquired infections and thrombosis caused by bacteria attached to the device surface, or fibrin crosslinking owing to platelet accumulation/activation, are major healthcare challenges that cause morbidity and mortality. To prevent...