Abstract 5451: Deficiency of Glutathione Peroxidase-3 Promotes Platelet Activation
            in vivo

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 plays an important role in plasma against oxidant stress by scavenging reactive oxygen species. A deficiency of this enzyme has been associated with platelet dependent thrombosis. We recently developed an animal model to assess platelet function in GPx-3 deficient mice. We hypothesized that GPx-3 deficiency induces platelet activation in vivo . GPx-3 (−/−) mice showed an attenuated bleeding time compared with wild-type mice (94.5 ± 28.8 s versus 153.4 ± 32.3, P<0.05). We also noted an increase in the plasma levels of soluble P-selectin, a marker of platelet activation and prothrombotic activity, in GPx-3 (−/−) mice compared with wild-type mice (137.8 ± 12.3 ng/ml plasma versus 101.5 ± 8.8, P<0.05). Cyclic GMP, a key intracellular second messenger molecule and marker for activation of soluble guanylyl cyclase by nitric oxide, was decreased in the plasma of GPx-3 (−/−) mice compared with wild-type mice (5.38 ± 1.75 pmol/ml plasma versus 23.67 ± 3.59, P<0.001), consistent with less bioactive NO in GPx-3 (−/−) mice. ADP was infused into the right ventricle of mice to induce platelet aggregation in the pulmonary vasculature; this assay resulted in higher pulmonary artery pressure in GPx-3 (−/−) compared with wild-type mice suggesting a more robust platelet activation response in the GPx-3 (−/−) mice. To confirm this interpretation, histological sections from the pulmonary vasculature of GPx-3 (−/−) compared with wild-type mice showed increased thrombi per 7.5 mm 2 section normalized to wild-type mice based on staining intensity for P-selectin (1.7 ± 0.4 versus 1.0 ± 0.1, P<0.001), as well as a higher percentage of occluded vessels (0.82 ± 0.16 % versus 0.54 ± 0.21, P<0.05). These findings demonstrate that GPx-3 deficiency causes platelet activation resulting in a prothrombotic state. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity and platelet-dependent thrombosis.

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Biphasic Roles for the Soluble Guanylyl Cyclase (sGC) In Platelet Activation In Mice

Blood ◽

10.1182/blood.v116.21.486.486 ◽

2010 ◽

Vol 116 (21) ◽

pp. 486-486

Author(s):

Guoying Zhang ◽

Binggang Xiang ◽

Radek C. Skoda ◽

Susan S. Smyth ◽

Xiaoping Du ◽

...

Keyword(s):

Platelet Activation ◽

Guanylyl Cyclase ◽

Conflicts Of Interest ◽

Soluble Guanylyl Cyclase ◽

Wild Type ◽

No Donor ◽

Cgmp Production ◽

Deficient Mice

Abstract Abstract 486 The role of intracellular secondary messenger cGMP in platelet activation has been controversial, with both stimulatory and inhibitory roles reported. The platelet cGMP is believed to be predominantly synthesized by soluble guanylyl cyclase (sGC), which is activated by nitric oxide (NO). To specifically determine the role of sGC-dependent cGMP synthesis in platelet function and in vivo thrombosis and hemostasis, we produced mice harboring a “floxed” sGC beta1 allele. In the “floxed” sGC beta1 mice (sGC beta1fl/fl), the exons 7 and 8 of sGC beta1 gene and an inserted Neo cassette were flanked with three LoxP sites. Platelet-specific deletion of sGC beta1fl/fl allele was accomplished through breeding of the sGC beta1fl/fl mice with pf4-Cre recombinase transgenic mice. Immunoblotting showed the complete absence of this protein in sGC beta1fl/fl/Cre platelets. Mice lacking sGC beta1 in platelets appeared to develop normally and had normal blood counts, including platelets. Blood pressure of platelet-specific sGC deficient mice was comparable to that of wild-type littermates. Inactivating the sGC beta1 gene in platelets abolished cGMP production induced by either NO donors or platelet agonists that are known to activate endogenous NO synthesis, confirming that both the platelet agonist-induced and NO donor-induced platelet cGMP production are predominantly mediated by sGC. Platelets lacking sGC exhibit a marked defect in aggregation and secretion in response to low doses of platelet agonists, collagen and thrombin. Importantly, tail-bleeding times were significantly prolonged in the platelet-specific sGC deficient mice compared with the wild-type littermates. In a FeCl3-induced carotid artery thrombosis model, time to occlusive thrombosis was prolonged in the platelet-specific sGC deficient mice, compared to wild type littermates. Thus, the agonist-stimulated sGC activation is important in promoting platelet granule secretion and aggregation. On the other hand, NO donor SNP-induced inhibition of platelet activation was abolished in sGC-deficient platelets. However, at high concentrations (>100μM), SNP inhibited platelet activation in both wild type and sGC deficient mice, indicating that both cGMP-dependent and -independent mechanisms are involved in NO donor-induced inhibition of platelet activation. Together, our data demonstrate that sGC contributes to both agonist-induced platelet activation and NO donor-induced platelet inhibition. Disclosures: No relevant conflicts of interest to declare.

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Compensatory mechanisms influence hemostasis in setting of eNOS deficiency

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00819.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. H1627-H1632 ◽

Cited By ~ 40

Author(s):

Mark D. Iafrati ◽

Olga Vitseva ◽

Kahraman Tanriverdi ◽

Price Blair ◽

Sybille Rex ◽

...

Keyword(s):

Vascular Reactivity ◽

Vascular Tissue ◽

Arterial Injury ◽

Tissue Expression ◽

Activity Levels ◽

Wild Type ◽

Platelet Activity ◽

Compensatory Mechanisms ◽

Normal Life Span

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice ( P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of t-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel-Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency.

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IWR-1 Inhibits Collagen-Induced Platelet Activation and Protects against Thrombogenesis

Hämostaseologie ◽

10.1055/s-0038-1676822 ◽

2019 ◽

Vol 39 (04) ◽

pp. 392-397

Author(s):

Wei Wang ◽

Songqing Lai ◽

ZiJin Xiao ◽

Haiyue Yan ◽

Yongxi Li ◽

...

Keyword(s):

Platelet Activation ◽

Antiplatelet Agent ◽

Wnt Signalling ◽

In Vivo Studies ◽

Dependent Manner ◽

Platelet Activity ◽

Occlusion Time ◽

Negative Effect

AbstractPlatelets play a crucial role in haemostasis and several pathophysiological processes. Collagen is a main initiator for platelet activation and aggregation. Given that Wnt signalling negatively regulates platelet function, and IWR-1 (a small molecule inhibitor for Wnt signalling) has the potential of inhibiting collagen synthesis, it is essential to investigate whether IWR-1 regulates collagen-induced platelet activation and protects against thrombogenesis. In the present study we found that IWR-1 pretreatment effectively suppressed collagen-induced platelet aggregation in a dose-dependent manner. In addition, IWR-1 also resulted in a decrease of P-selectin and phosphatidylserine surface exposure using fluorescence-activated cell sorting analysis. In vitro studies further revealed that IWR-1 had a negative effect on integrin a2β1 activation and platelet spreading. More importantly, the results from in vivo studies showed that IWR-1 exhibited a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Taken together, current results demonstrate that IWR-1 could effectively block collagen-induced platelet activity in vitro and in vivo, and suggest its candidacy as a new antiplatelet agent.

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The Variability of β Thromboglobulin and Platelet Factor 4 in Healthy Subjects

10.1055/s-0039-1687207 ◽

1979 ◽

Author(s):

J. Zahavi ◽

N.A.G. Jones ◽

M. Dubiel ◽

J. Leyton ◽

V.V. Kakkar

Keyword(s):

Platelet Activation ◽

Healthy Subjects ◽

Platelet Factor 4 ◽

Vascular Changes ◽

Platelet Activity ◽

Platelet Factor ◽

Platelet Concentration ◽

The Mean ◽

Age Range

Plasma β TC was measured by radioimmunoassay (RIA)in 202 healthy subjects (age range 12-103); 111 young (mean age 25.2) 34 middle aged (MA) (mean age 55.6) and 57 old (mean age 82.2). Their mean ±1SE plasma β TG levels in ng/ml were 28.3 ± 1.5 (range 3-74), 31.9-2-70 (range 7-65) and 49.99 ± 2.9 (range 14-95) respectively. Plasma βTG level was significantly raised in the old subjects compared to young or MA (p ⩽ 0.0005). Furthermore the ratio of plasma β TG to platelet concentration in whole blood (PC) was higher in the MA subjects compared to the young (p ⩽ 0.009). Plasma platelet factor 4 (PF4) was measured by RIA in 4l healthy subjects, 11 young and 30 old and correlated to plasma βTG. A significant correlation between the 2 proteins was found in the 2 groups (r = 0.8337 in the young and r = 0.0602 in the old subjects), indicating that both proteins are released in-vivo from the same pool and presumably at the same rate. The mean plasma PF4 level in ng/ml was 14.6 (range 6-48) in the young and 18.2 (range 7.7-50) in the old and the ratio of the plasma PF4 to PC was higher in the old subjects (p ⩽ 0.04), These results suggest that in-vivo platelet activation and “release reaction” are increased in old and MA subjects compared to young, presumably due to atherosclerotic vascular changes. This enhanced platelet activity may reflect a pre-thtombotic state.

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Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.

Blood ◽

10.1182/blood.v108.11.1508.1508 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1508-1508 ◽

Cited By ~ 1

Author(s):

Shawn M. Jobe ◽

Katina M. Wilson ◽

Lori Leo ◽

Jeffery D. Molkentin ◽

Steven R. Lentz ◽

...

Keyword(s):

Platelet Activation ◽

Mitochondrial Permeability Transition ◽

Critical Role ◽

In Vitro Tests ◽

Cyclophilin D ◽

Wild Type ◽

Glycoprotein Vi ◽

Stimulation Of

Abstract Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation includeincreased surface retention of procoagulant alpha granule proteins,high-level phosphatidylserine (PS) externalization, andmodulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A. Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨm), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H2O2 (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05). Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses. We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.

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The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v112.11.112.112 ◽

2008 ◽

Vol 112 (11) ◽

pp. 112-112

Author(s):

Aleksandra Stojanovic ◽

Matvey Gorovoy ◽

Tatyana Voyno-Yasenetskaya ◽

Xiaoping Du

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Wild Type ◽

Glycoprotein Ib ◽

Lim Kinase ◽

And Function

Abstract LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.

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P-Selectin and Platelet Clearance

Blood ◽

10.1182/blood.v92.11.4446.423k19_4446_4452 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4446-4452 ◽

Cited By ~ 3

Author(s):

Gaëtan Berger ◽

Daqing W. Hartwell ◽

Denisa D. Wagner

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Platelet Activation ◽

Soluble Form ◽

Wild Type ◽

Adhesion Receptor ◽

Activated Platelets ◽

Deficient Mice

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

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In VivoAntioxidant Activity of Deacetylasperulosidic Acid in Noni

Journal of Analytical Methods in Chemistry ◽

10.1155/2013/804504 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 9

Author(s):

De-Lu Ma ◽

Mai Chen ◽

Chen X. Su ◽

Brett J. West

Keyword(s):

Antioxidant Activity ◽

Superoxide Dismutase ◽

Glutathione Peroxidase ◽

Catalase Activity ◽

Antioxidant Properties ◽

Superoxide Dismutase Activity ◽

Morinda Citrifolia ◽

Control Group ◽

Dose Dependent

Deacetylasperulosidic acid (DAA) is a major phytochemical constituent ofMorinda citrifolia(noni) fruit. Noni juice has demonstrated antioxidant activityin vivoand in human trials. To evaluate the role of DAA in this antioxidant activity, Wistar rats were fed 0 (control group), 15, 30, or 60 mg/kg body weight per day for 7 days. Afterwards, serum malondialdehyde concentration and superoxide dismutase and glutathione peroxidase activities were measured and compared among groups. A dose-dependent reduction in malondialdehyde was evident as well as a dose-dependent increase in superoxide dismutase activity. DAA ingestion did not influence serum glutathione peroxidase activity. These results suggest that DAA contributes to the antioxidant activity of noni juice by increasing superoxide dismutase activity. The fact that malondialdehyde concentrations declined with increased DAA dose, despite the lack of glutathione peroxidase-inducing activity, suggests that DAA may also increase catalase activity. It has been previously reported that noni juice increases catalase activityin vivobut additional research is required to confirm the effect of DAA on catalase. Even so, the current findings do explain a possible mechanism of action for the antioxidant properties of noni juice that have been observed in human clinical trials.

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TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Blood ◽

10.1182/blood-2021-152975 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1050-1050

Author(s):

Angela Doerr ◽

Denise Pedrosa ◽

Maria Schander ◽

Yotis A. Senis ◽

Alexandra Mazharian ◽

...

Keyword(s):

Platelet Activation ◽

Type I Collagen ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Type I ◽

Wild Type ◽

Platelet Surface ◽

Knock Out ◽

Integrin Αiibβ3

Abstract Background Thrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma. Results VWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1. Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice. Conclusions This study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

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Novel Antibodies Against Glycoprotein Ibα Inhibit Pulmonary Metastasis By Affecting Vwf-Gpibα Interaction

Blood ◽

10.1182/blood-2018-99-117613 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1133-1133

Author(s):

Qi Yingxue ◽

Wenchun Chen ◽

Ke Xu ◽

Fengying Wu ◽

Xuemei Fan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Binding Sites ◽

Tumor Metastasis ◽

Cancer Metastasis ◽

Platelet Glycoprotein ◽

Induce Platelet Aggregation ◽

Metastasis Model

Abstract Background: Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy. Methods and Results: To generate antibodies that bind to mouse platelet GPIbα, washed mouse platelet lysate was used as the antigen for rat immunization. Obtained hybridoma clones were screened in ELISA for binding affinity to the GPIb-IX complex. Positive clones were further screened for their abilities to inhibit platelet-cancer cell adhesion. Finally, at static condition, two antibodies, 2B4 and 1D12, had virtually no effect on the activation of integrin αIIbβ3, which is used to indicate platelet activation. Then, we characterized the binding sites of 2B4 and 1D12 by 20 purified recombinant GPIbα fragments binding. Results showed that 2B4 and 1D12 shared the same binding sites with vWF. To determine whether 2B4 and 1D12 affect vWF binding, we tested the binding by flow cytometry using recombined mouse vWF, and then, we investigated platelet aggregation induced by several agonists, including vWF binding agonist ristocetin. Our data demonstrated clearly that 2B4 and 1D12 could inhibit vWF binding. To investigate whether the inhibition of vWF-GPIbα interaction was associated with tumor metastasis, we examined the effect of 2B4 and 1D12 in each of the following interactions in vitro: between activated platelets and tumor cells, platelets and endothelial cells. Meanwhile, We further investigated the inhibitory effect of these antibodies in vivo using the experimental metastasis model and the spontaneous metastasis model. Results showed that 2B4 and 1D12 could potently inhibit the adhesion of cancer cells in vitro, and metastasisin vivo. We next investigated whether 2B4 and 1D12 could affect platelet activation and/or induce thrombocytopenia in vivo. Results showed that the addition of 2B4 or 1D12 to PRP did not induce platelet aggregation and injection of 2B4 or 1D12 Fab at appropriate dose did not affect tail-bleeding time and platelet count. Based on the above findings, we obtained anti-human platelet GPIbα monoclonal antibody YQ3 using the same approach to explore the role of human GPIbα in cancer metastasis. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia. Conclusion: Our results therefore supported the hypothesis that GPIbα contributes to tumor metastasis, and suggested potential value of using anti-GPIbα mAb to suppress cancer metastasis. Disclosures Li: Neoletix: Consultancy, Equity Ownership.

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