Platelet-mediated Thrombosis Research Articles

Antiphospholipid Antibodies Modify the Prognostic Value of Baseline Platelet Count for Clinical Outcomes After Ischemic Stroke.

Antiphospholipid antibodies (aPLs) have been reported to be involved in platelet-mediated thrombosis and inflammation, but the impact on the prognosis of ischemic stroke remains unclear. We aimed to examine whether the association between baseline platelet count (PLT) and long-term clinical outcomes within 2 years after ischemic stroke onset is modulated by aPLs. A total of 2938 patients with ischemic stroke were included in this prospective cohort study. Cox proportional hazards regression models were used to assess the association between the baseline PLT stratified by aPLs status and 2-year clinical outcomes after stroke onset, and an interaction effect between PLT and aPLs on clinical outcomes was tested by likelihood ratio test. There was a significant interaction effect of aPLs and PLT on recurrent stroke (Pinteraction=0.002) and cardiovascular events (Pinteraction=0.001) within 2 years after stroke onset. After multivariate adjustment, high PLT was associated with increased risks of recurrent stroke (hazard ratio [HR], 2.78 [95% CI, 1.03-7.45]; Ptrend=0.039) and cardiovascular events (HR, 2.58 [95% CI, 1.12-5.90]; Ptrend=0.024) when 2 extreme tertiles were compared among patients with aPL positive, but not among those with aPL negative. The aPLs had a modifying effect on the association between PLT and clinical outcomes within 2 years after ischemic stroke onset. Increased PLT was associated with recurrent stroke and cardiovascular events after ischemic stroke onset among patients with aPL positive, but not in those with aPL negative.

Novel Small-Molecule Atypical Chemokine Receptor 3 Agonists: Design, Synthesis, and Pharmacological Evaluation for Antiplatelet Therapy.

ACKR3, an atypical chemokine receptor, has been associated with prothrombotic events and the development of cardiovascular events. We designed, synthesized, and evaluated a series of novel small molecule ACKR3 agonists. Extensive structure-activity relationship studies resulted in several promising agonists with potencies ranging from the low micromolar to nanomolar range, for example, 23 (EC50 = 111 nM, Emax = 95%) and 27 (EC50 = 69 nM, Emax = 82%) in the β-arrestin-recruitment assay. These compounds are selective for ACKR3 versus ACKR2, CXCR3, and CXCR4. Several agonists were subjected to investigations of their P-selectin expression reduction in the flow cytometry experiments. In particular, compounds 23 and 27 showed the highest potency for platelet aggregation inhibition, up to 80% and 97%, respectively. The most promising compounds, especially 27, exhibited good solubility, metabolic stability, and no cytotoxicity, suggesting a potential tool compound for the treatment of platelet-mediated thrombosis.

"In Vitro Evaluation of the Antiplatelet Activity of Paravat Shakrita"

Platelet aggregation, a crucial process in thrombotic disorders, underscores the necessity for effective preventive and therapeutic measures. This study compares the antiplatelet effects of Aspirin, a well-established drug, with Paravat Shakrita, a lesser-known formulation from Ayurvedic texts. It underwent GCMS and microbiological analysis to determine its chemical composition and microbial load. Using an in vitro model with rat blood samples, we conducted a Platelet Aggregation Inhibition Assay. As expected, Aspirin inhibited platelet aggregation in a concentration-dependent manner, consistent with its known mechanism. Despite limited documentation, Paravat Shakrita exhibited potent antiplatelet properties, also showing dose-dependent effects. Interestingly, Paravat Shakrita demonstrated comparable or even superior effects at higher concentrations compared to Aspirin. The IC50 values revealed similar inhibitory potency between Paravat Shakrita and Aspirin. This comparison unveiled distinct mechanisms of action, providing various therapeutic options against platelet-mediated thrombosis. While promising, further investigation is crucial to understand Paravat Shakrita's mechanism, ensure its safety, and determine its clinical significance.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a β-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 μM) and C11 (EC50 = 11.4 μM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 μM) and 27 (LN6023, EC50 = 3.5 μM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Thrombo-Inflammation and Immunological Response in Ischemic Stroke: Focusing on Platelet-Tregs Interaction.

Strokes are mainly caused by thromboembolic obstruction of a major cerebral artery. Major clinical manifestations include paralysis hemiplegia, aphasia, memory, and learning disorders. In the case of ischemic stroke (IS), hyperactive platelets contribute to advancing an acute thrombotic event progression. Therefore, the principal goal of treatment is to recanalize the occluded vessel and restore cerebral blood flow by thrombolysis or mechanical thrombectomy. However, antiplatelets or thrombolytic therapy may increase the risk of bleeding. Beyond the involvement in thrombosis, platelets also contribute to the inflammatory process induced by cerebral ischemia. Platelet-mediated thrombosis and inflammation in IS lie primarily in the interaction of platelet receptors with endothelial cells and immune cells, including T-cells, monocytes/macrophages, and neutrophils. Following revascularization, intervention with conventional antiplatelet medicines such as aspirin or clopidogrel does not substantially diminish infarct development, most likely due to the limited effects on the thrombo-inflammation process. Emerging evidence has shown that T cells, especially regulatory T cells (Tregs), maintain immune homeostasis and suppress immune responses, playing a critical immunomodulatory role in ischemia-reperfusion injury. Hence, considering the deleterious effects of inflammatory and immune responses, there is an urgent need for more targeted agents to limit the thrombotic-inflammatory activity of platelets and minimize the risk of a cerebral hemorrhage. This review highlights the involvement of platelets in neuroinflammation and the evolving role of Tregs and platelets in IS. In response to all issues, preclinical and clinical strategies should generate more viable therapeutics for preventing and managing IS with immunotherapy targeting platelets and Tregs.

A Continuum Model for the Unfolding of von Willebrand Factor.

von Willebrand Factor is a mechano-sensitive protein circulating in blood that mediates platelet adhesion to subendothelial collagen and platelet aggregation at high shear rates. Its hemostatic function andthrombogenic effect, as well as susceptibility to enzymatic cleavage, are regulated by a conformational change from a collapsed globular state to a stretched state. Therefore, it is essential to account for the conformation of the vWF multimers when modeling vWF-mediated thrombosis or vWF degradation. We introduce a continuum model of vWF unfoldingthat is developed within the framework of our multi-constituent model of platelet-mediated thrombosis. The model considers two interconvertible vWF species corresponding to the collapsed and stretched conformational states. vWF unfolding takes place via two regimes: tumbling in simple shear and strong unfolding in flows with dominant extensional component. These two regimes were demonstrated in a Couette flowbetween parallel plates and anextensional flow in a cross-slotgeometry. The vWF unfolding model was then verified in several microfluidic systems designed for inducing high-shear vWF-mediated thrombosis and screening for von Willebrand Disease. The model predicted high concentration of stretched vWF in key regions where occlusive thrombosis was observed experimentally. Strong unfolding caused by the extensional flow was limited to the center axis or middle plane of the channels, whereas vWF unfolding near the channel walls relied upon the shear tumbling mechanism. The continuum model of vWF unfolding presented in this work can be employed in numerical simulations of vWF-mediated thrombosis or vWF degradation in complex geometries. However, extending the model to 3-D arbitrary flows and turbulent flows will pose considerable challenges.

Aberrant glycosylation of anti-SARS-CoV-2 spike IgG is a prothrombotic stimulus for platelets

AbstractA subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in severely ill patients with COVID-19 are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesized that platelets might be susceptible to activation by anti–severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies and might contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike immunoglobulin G enhanced platelet-mediated thrombosis on von Willebrand factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcγRIIA, and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by the therapeutic small molecules R406 (fostamatinib) and ibrutinib, which inhibit tyrosine kinases Syk and Btk, respectively, or by the P2Y12 antagonist cangrelor.

In Vitro Antiplatelet Activity of Mulberroside C through the Up-Regulation of Cyclic Nucleotide Signaling Pathways and Down-Regulation of Phosphoproteins.

Physiological agonists trigger signaling cascades, called “inside-out signaling”, and activated platelets facilitate adhesion, shape change, granule release, and structural change of glycoprotein IIb/IIIa (αIIb/β3). Activated αIIb/β3 interacts with fibrinogen and begins second signaling cascades called “outside-in signaling”. These two signaling pathways can lead to hemostasis or thrombosis. Thrombosis can occur in arterial and venous blood vessels and is a major medical problem. Platelet-mediated thrombosis is a major cause of cardiovascular disease (CVD). Therefore, controlling platelet activity is important for platelet-mediated thrombosis and cardiovascular diseases. In this study, focus on Morus Alba Linn, a popular medicinal plant, to inhibit the function of platelets and found the containing component mulberroside C. We examine the effect of mulberroside C on the regulation of phosphoproteins, platelet-activating factors, and binding molecules. Agonist-induced human platelet aggregation is dose-dependently inhibited by mulberroside C without cytotoxicity, and it decreased Ca2+ mobilization and p-selectin expression through the upregulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756), and downregulation of extracellular signal-regulated kinase (ERK). In addition, mulberroside C inhibited thromboxane A2 production, fibrinogen binding, and clot retraction. Our results show antiplatelet effects and antithrombus formation of mulberroside C in human platelets. Thus, we confirm that mulberroside C could be a potential phytochemical for the prevention of thrombosis-mediated CVDs.

Ginsenoside Rk1 suppresses platelet mediated thrombus formation by downregulation of granule release and αIIbβ3 activation

Background and objectiveSynthetic ginsenoside compounds G-Rp (1,3, and 4) and natural ginsenosides in Panax ginseng 20(S)-Rg3, Rg6, F4 and Ro have inhibitory actions on human platelets. However, the inhibitory mechanism of ginsenoside Rk1 (G-Rk1) is still unclear thus, we initiated investigation of the anti-platelet mechanism by G-Rk1 from Panax ginseng. MethodologyOur study focused to investigate the action of G-Rk1 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin αIIbβ3 using flow cytometry, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane B2 secretion. Thrombin-induced clot retraction was also observed in human platelets. Key ResultsCollagen, thrombin, and U46619-stimulated human platelet aggregation were dose-dependently inhibited by G-Rk1, while it demonstrated a more effective suppression on collagen-stimulated platelet aggregation using human platelets. Moreover, G-Rk1 suppressed collagen-induced elevation of Ca2+ release from endoplasmic reticulum, granule release, and αIIbβ3 activity without any cytotoxicity. Conclusions and implicationsThese results indicate that G-Rk1 possess strong anti-platelet effect, proposing a new drug candidate for treatment and prevention of platelet-mediated thrombosis in cardiovascular disease.

Platelets as Mediators of Neuroinflammation and Thrombosis.

Beyond platelets function in hemostasis, there is emerging evidence to suggest that platelets contribute crucially to inflammation and immune responses. Therefore, considering the detrimental role of inflammatory conditions in severe neurological disorders such as multiple sclerosis or stroke, this review outlines platelets involvement in neuroinflammation. For this, distinct mechanisms of platelet-mediated thrombosis and inflammation are portrayed, focusing on the interaction of platelet receptors with other immune cells as well as brain endothelial cells. Furthermore, we draw attention to the intimate interplay between platelets and the complement system as well as between platelets and plasmatic coagulation factors in the course of neuroinflammation. Following the thorough exposition of preclinical approaches which aim at ameliorating disease severity after inducing experimental autoimmune encephalomyelitis (a counterpart of multiple sclerosis in mice) or brain ischemia-reperfusion injury, the clinical relevance of platelet-mediated neuroinflammation is addressed. Thus, current as well as future propitious translational and clinical strategies for the treatment of neuro-inflammatory diseases by affecting platelet function are illustrated, emphasizing that targeting platelet-mediated neuroinflammation could become an efficient adjunct therapy to mitigate disease severity of multiple sclerosis or stroke associated brain injury.

Prdx2 Deficiency Exacerbates Platelet-mediated Thrombosis in the Hyperlipidemic State

Prdx2 Deficiency Exacerbates Platelet-mediated Thrombosis in the Hyperlipidemic State

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbβ3, TXA2, and cAMP levels

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbβ3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule.In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbβ3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25–100 μM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbβ3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbβ3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.

A Novel Anti-Glycoprotein (GP) Iba-Targeting Chimeric Fibrinolytic Agent Demonstrates In Vivo Efficacy Against Human Platelet-Enriched Thrombi in Mice

We previously described (Fuentes, J Clin Invest 2016) a chimeric protein, GPIIb/uPA-T, that provided effective thromboprophylaxis composed of an N-terminal scFv specifically to human (h) GPIIb linked to a C-terminal variant of low molecular weight urokinase containing a thrombin cleavable activating site. uPA-T prevented FeCl3-induced carotid arterial thrombosis in immunocompromized NOD/SCID, IL2 receptor γ-chain deficient (NSG) mice that had been pre-infused with human platelets (hPlts) without causing rebleeding from pre-existing tail-clips clots. However, this construct and subsequent hGPIIb-targeting scFv uPA-T chimeric constructs caused significant thrombocytopenia in hPlt-infused NSG (hPlt/NSG) mice and in hGPIIb+ mice. Therefore, we developed a new chimeric uPA-T targeting hGPIbα based on 5G6, a monoclonal antibody that binds to the hGPIbα juxtamembrane ADAM17 cleavage site and is known to not cause thrombocytopenia. The chimera 5G6/uPA-T binds to the surface of hPlts and hGPIbα-expressing mouse platelets at levels ~30% of those seen with hGPIIb/uPA-T, consistent with the difference in expression of GPIIb vs. GPIbα. 5G6/uPA-T did not reduce platelet counts in hPlt/NSG or hGPIα+ mice at doses that caused significant thrombocytopenia after infusion of GPIIb/uPA-T (1 mg/kg each, bolus + 30-minute infusion). We then tested the efficacy of 5G6/uPA-T in hPlt/NSG mice that that also carried a single mouse-to-human arginine (R) to histidine (H) substitution at position 1326 in von Willebrand factor, VWFH, created using CRISPR/Cas9 technology in NSG mice. This substitution, described by T. Diacovo (Megalion, Circulation 2011), markedly impairs binding of mouse Plts that express murine GPIbα, but increases the affinity for hPlt expressing hGPIbα. We hypothesized that such mice would allow studies of hPlt-enriched thrombi in mice. To test this hypothesis, hPlts were infused into NSG mice to achieve a circulating level of 10-20% of the total platelet pool. Following FeCl3-induced carotid arterial injury, similar numbers of hPlts were incorporated into the thrombi in wildtype VWFR/R NSG and heterozygous VWFR/H littermate NSG mice, whereas 1.4-1.8-fold more hPlts were incorporated in VWFH/H NSG littermates over the 3-minute observation period (n = 12-17, p<0.04 between VWFH/H NSG mice vs. VWFR/R and VWFR/H mice.Infusion of 5G6/uPA-T (1 mg/kg bolus+infusion) into hPlts-infused VWFH/H NSG mice prevented carotid artery thrombosis induced by FeCl3 and observed for 30 minutes as follows: In the absence of infused hPlts, 2/5 injured carotid arteries in the VWFH/H NSG mice completely occluded with a total flow volume in all vessels measured by Doppler of 1158±377 ml/min.s area under the curve (AUC). In contrast, 5/5 vessels occluded hPlts-infused VWFH/H NSG mice with an average time to occlusion of 13.0±3.4 mins and an AUC reduced to 692±75 ml/min.s (p<0.04 compared to no infused hPlts). In contrast, only 1/5 vessels occluded in hPlts-infused VWFH/H NSG mice given 5G6/uPA-T prior to injury, with an AUC of 1487±190 ml/min.s (p<0.006 compared to hPlt-infused VWFh/h NSG mice). None of the mice given 5G6/uPA-T exhibited untoward bleeding at the surgical site. 5G6/uPA-T did not induce thrombocytopenia, visible bleeding elsewhere, rebleeding in hPlt+/VWFH/H NSG mice when given 10 minutes following tail vein clipping, nor were other overt toxicities observed.In summary, we believe that 5G6/uPA-T combines a number of desirable features for a drug designed to prevent untoward platelet-mediated thrombosis: i. 5G6/uPA-T binds specifically to a hPlt-specific receptor, allowing targeted delivery to evolving thrombi while sparing established clots where it likely poorly penetrates into the thrombi. ii. The drug is effective even while binding to the base of the GPIbα chain near the platelet surface, where it also likely maintains GPIb/IX biology by limiting GPIbα cleavage, and does not cause thrombocytopenia. iii. Finally, 5G6/uPA-T delivers a form of urokinase that needs the co-presence of thrombin to become activated further limiting its activity to sites of evolving thrombi. These properties were demonstrated in an in vivo model that selectively incorporates hPlts. We believe that 5G6 may have clinical application in settings where there is a high risk for thrombosis, such as maintaining stent patency, while avoiding untoward lysis of previously established clots. DisclosuresLi:Neoletix: Consultancy, Equity Ownership. Poncz:Incyte Corporation: Consultancy, Research Funding.

GLS-409, an Antagonist of Both P2Y1 and P2Y12, Potently Inhibits Canine Coronary Artery Thrombosis and Reversibly Inhibits Human Platelet Activation

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12 ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes.

The Antithrombotic Effects of 12‐LOX Derived Metabolites of DPA, ω‐6

IntroductionPlatelet‐mediated thrombosis is the primary underlying mechanism leading to cardiovascular life‐threatening clinical events. Regulating excessive platelet reactivity is an essential aspect of antithrombotic therapy. A number of anti‐platelet drugs have been developed to target specific signaling pathways or endpoints involved in platelet activation. Despite the effectiveness of current anti‐platelet therapies, uncontrolled thrombosis or bleeding complications still persist, warranting the development of novel anti‐thrombotic strategies. Previously, we had presented that 12‐HETrE, an oxylipin generated by 12‐lipoxygenase (12‐LOX) oxidation of ω‐6 polyunsaturated fatty acid (PUFA), dihomo‐g‐linolenic acid (DGLA), could be an approach to modulate platelet reactivity through the prostacyclin receptor. Here, we demonstrate that the elongated form of DGLA, docosapentaenoic acid (DPA; 22:5n‐6) can also be endogenously oxidized by platelet 12‐LOX to generate two distinct oxylipins, 11‐oxy‐DPA and 14‐oxy‐DPA, to regulate platelet reactivity.ObjectiveTo determine the efficacy of and the mechanistic target of 12‐LOX derived metabolites of DPA 11‐ and 14‐oxy‐DPA, ω‐6 in the regulation of platelet function.ResultsBoth 11‐ and 14‐oxy‐DPA potently inhibited platelet aggregation in an aggregometer measuring optical density. Treatment of whole blood with 11‐ and 14‐oxy‐DPA, ω‐6 also significantly reduced platelet adhesion as well as thrombus growth on an ex vivo collagen‐coated perfusion chamber under arterial shear. In support of these findings, using flow cytometry, calcium mobilization was attenuated in platelets treated with 11‐ and 14‐oxy‐DPA, ω‐6. Interestingly, both 11‐ and 14‐oxy‐DPA inhibited platelet activation in a Gas independent manner. Instead, 11‐oxy‐DPA and 14‐oxy‐DPA exerted their anti‐platelet effects through a peroxisome proliferated‐activated receptor (PPAR), by which pharmacological antagonism of the PPARα isoform reversed the ability of the metabolites to inhibit platelet aggregation. In supplement of these ex vivo findings, ongoing experiments are being performed to measure laser‐induced thrombus formation (platelet and fibrin) in the cremaster vessel of wild‐type mice intravenously administered with either 11‐ and 14‐oxy‐DPA.ConclusionThis is the first study to demonstrate the novel metabolites of DPA, ω‐6 generated by 12‐LOX also have a role in regulating platelet activation through the PPAR pathway. Our findings suggest that 11‐ and 14‐oxy‐DPA, ω‐6 shows great promise as a potential anti‐platelet therapeutic for the treatment for cardiovascular‐related diseases.Support or Funding InformationThis work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ischemic Conditioning Attenuates Platelet-Mediated Thrombosis: A Tale of Reverse Translation.

Data obtained in both preclinical models and humans have revealed that the favorable cardiac consequences of ischemic conditioning extend beyond a direct effect on the cardiomyocyte. In the current review, we summarize our as-yet limited understanding of the complex relationships between ischemic conditioning, platelet activation-aggregation, and cardioprotection.

Computational fluid dynamics characterization of pulsatile flow in central and Sano shunts connected to the pulmonary arteries: importance of graft angulation on shear stress-induced, platelet-mediated thrombosis

Central (aorta) and Sano (right ventricle)-to-pulmonary artery (PA) shunts, palliative operations for infants with complex heart defects, can develop life-threatening thrombosis. We employed computational fluid dynamics (CFD) to study pulsatile flow in these shunts, with the goal to identify haemodynamic characteristics conducive to thrombus formation. CFD, using the finite volume method with cardiac catheterization data, and computer simulations, based on angiography, were employed to determine flow-velocity field, wall shear stress (WSS) profile and oscillatory shear index (OSI). At prominent angulation, in central shunts (4 and 3.5 mm), WSS reached 245 and 123 (Pascal-Pa), peak systole and 137 and 46 Pa, end diastole; and, in Sano shunts (5 and 6 mm), WSS attained 203 and 133 Pa, peak systole and 1.6 and 1.5 Pa, end diastole. Counter-rotating flow vortices augmented WSS. These high WSSs can promote platelet aggregation, leading to thrombus formation. The OSIs averaged 0.39, indicative of multidirectional shearing forces. Shunt burden was assessed by averaging WSS, over its luminal area and the cardiac cycle. For the central shunts, these WSSs were 73.0 and 67.2 Pa; whereas, for the Sano shunts, 34.9 and 19.6 Pa. For modified Blalock-Taussig shunts (4 and 3.5 mm), the averaged WSSs were significantly lower at 26.0 and 27.5 Pa, respectively. CFD modelling is an important tool to determine blood flow behaviour in shunts. Graft angulation presents a risk for shear stress-induced, platelet- mediated thrombosis, which is more likely to occur in elongated central than in Sano shunts.

The chimeric monoclonal antibody MHCSZ-123 against human von Willebrand factor A3 domain inhibits high-shear arterial thrombosis in a Rhesus monkey model

BackgroundSZ-123, a murine monoclonal antibody that targets the human von Willebrand factor (VWF) A3 domain and blocks the binding of collagen, is a powerful antithrombotic. In a Rhesus monkey model of thrombosis, SZ-123 had no side effects, such as bleeding or thrombocytopenia.MethodsThe mouse/human chimeric version of SZ-123, MHCSZ-123, was developed and maintained inhibitory capacities in vitro and ex vivo after injection into monkeys. CHO-S cells were selected for stable expression of MHCSZ-123. Cell clones with high levels of MHCSZ-123 expression were screened with G418 then adapted to serum-free suspension culture. The antithrombotic effect of MHCSZ-123 on acute platelet-mediated thrombosis was studied in monkeys where thrombus formation was induced by injury and stenosis of the femoral artery, which allowed for cyclic flow reductions (CFRs). CFRs were measured in the femoral artery of anesthetized Rhesus monkeys before and after intravenous administration of MHCSZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet counts, and template bleeding time were used as measurements of antithrombotic activity. In addition, plasma VWF and VWF occupancy were measured by ELISA.ResultsInjection of 0.1, 0.3, and 0.6 mg/kg MHCSZ-123 significantly reduced CFRs by 29.4%, 57.9%, and 73.1%, respectively. When 0.3 and 0.6 mg/kg MHCSZ-123 were administered, 46.6%–65.8% inhibition of ristocetin-induced platelet aggregation was observed between 15 and 30 min after injection. We observed minimal effects on bleeding time, minimal blood loss, and no spontaneous bleeding or thrombocytopenia.ConclusionsThe VWF-A3 inhibitor MHCSZ-123 significantly reduced thrombosis in Rhesus monkeys and appeared to be safe and well tolerated.

Abstract 11277: Computational Fluid Dynamics Characterization of Pulsatile Flow in Patient Specific Central and Sano Shunts: Relevance to Shear-Stress Induced, Platelet-Mediated Thrombosis

Introduction: Central shunts (aorta) and Sano shunts (right ventricle) connected to the pulmonary arteries (PAs) are vital operations for infants with complex heart defects. However, they can become thrombosed. Hypothesis: At sites of prominent angulation, flow augments wall shear stress (WSS), a determinate of platelet-mediated thrombosis. Methods: We employed a patient-based computational model (Fluent) of pulsatile flow (viscosity 5 cPa-s), using a finite volume method and cardiac catheterization data, and geometric simulations (Autodesk Inventor), using angiography, to study flow behavior and WSS. Two central (4.0 and 3.5 mm) and Sano (5 and 6 mm) shunts were assessed. At the inlet to the central and Sano shunts, ascending aorta (AAo, 87/40 and 93/35 mmHg) and right ventricle (RV, 99/10 and 84/10 mmHg) pressures were used, respectively. At the outlets, measured PA pressures were used. Results: For central shunts, Fig 1, WSS reached 245 Pa and 123 Pa, peak systole. For Sano shunts, Fig 2, WSS attained 203 Pa and 133 Pa, peak systole. At sites of angulation, we identified flow vortices augmenting WSS (&gt;100 Pa), and downstream regions of flow stagnation and recirculation, which are conducive to platelet aggregation and thrombus formation. Shunt burden, assessed by averaging WSS over the luminal surface of the shunts and the cardiac cycle, was 73.0 Pa and 67.2 Pa for central shunts, and 34.9 Pa and 19.6 Pa for Sano shunts. Conclusions: Our results indicate central shunts present a higher risk for thrombosis than Sano shunts. Importantly, WSS induced, platelet-mediated thrombosis is insensitive to aspirin, as the process does not involve the cyclooxygenase pathway.

Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.