CD147 is a Novel Interaction Partner of Integrin αMβ2 Mediating Leukocyte and Platelet Adhesion

Surface receptor-mediated adhesion is a fundamental step in the recruitment of leukocytes and platelets, as well as platelet–leukocyte interactions. The surface receptor CD147 is crucially involved in host defense against self-derived and invading targets, as well as in thrombosis. In the current study, we describe the previously unknown interaction of CD147 with integrin αMβ2 (Mac-1) in this context. Using binding assays, we were able to show a stable interaction of CD147 with Mac-1 in vitro. Leukocytes from Mac-1−/− and CD147+/− mice showed a markedly reduced static adhesion to CD147- and Mac-1-coated surfaces, respectively, compared to wild-type mice. Similarly, we observed reduced rolling and adhesion of monocytes under flow conditions when cells were pre-treated with antibodies against Mac-1 or CD147. Additionally, as assessed by antibody inhibition experiments, CD147 mediated the dynamic adhesion of platelets to Mac-1-coated surfaces. The interaction of CD147 with Mac-1 is a previously undescribed mechanism facilitating the adhesion of leukocytes and platelets.

Caplacizumab in adult patients with acquired thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is usually a fatal disease caused by a deficiency of the metalloproteinase, ADAMTS13, often due to autoimmunity. This leads to the development of pathogenic multimers of von Willebrand factor (vWF), causing an inappropriate interaction of platelets and vWF. This results in a thrombotic microangiopathy, which is treated with therapeutic plasma exchange and immune suppression. Although this treatment has reduced the mortality of TTP to only about 20%, there have been no recent significant advances in the treatment of TTP. Recently, a novel agent has been approved for use in TTP. Caplacizumab, which binds to the A1 domain of vWF, prevents the adhesion of platelets to vWF. It is a first in-class ‘nanobody’, that in clinical trials has shown marked efficacy in treating TTP and its complications. This review will discuss the development and implications of caplacizumab in the treatment of TTP.

Distinct Mechanism between Arterial and Venous Thrombosis: Impact for Clinical Manifestations

Hemostasis is a complex physiological process aiming to keep the integrity of a closed circulatory system after an occurrence of vessel wall injury. Hemostasis involving the role of circulating platelets and coagulation cascade.1 There are two major pathways that act independently to activate the platelet. The first pathway is mediated by collagen and the other by tissue factor. After intimal layer injury, platelets are recruited through the interaction between platelet’s surface glycoprotein (GPVI and GPIb/V/IX) with collagen and von Willebrand factor. This process results in adhesion of platelets in the site of injury. Further recruitment of platelets is achieved by secretion of aggregatory mediators such as thromboxane A2 and adenosine diphosphate.

VWF Mediates Platelet Activation and Secretion to Promote Osteosarcoma Metastasis

[Background] As a multimer protein that mediates the adhesion of platelets to vascular surface, VWF has been found to participate in tumor metastasis, and may also be involved in the interaction between platelets and tumor cells. We observed in our clinical studies that the expression of VWF in lung metastatic osteosarcoma tumor is significantly higher than that of in primary osteosarcoma. Based on these observations, we speculate that VWF and platelets may contribute to promoting osteosarcoma metastasis. Thus, this study is to explore the effects of VWF-mediated platelet activation and secretion on the migration and invasion ability of SAOS2 cells with VWF-expressing osteosarcoma cell line SAOS2 as a model, as well as the inhibition of the interaction between VWF and platelets by specific antibodies. [Methods] SAOS2 cells were stimulated with inflammatory factor PMA and resulted in increased expression of vWF. Then the adhesion of labeled platelets to SAOS2 cells was evaluated under static and flow conditions, respectively. The above processes were intervened with SZ-2(anti-GPIb), SZ-123(anti-VWF) and AVW3 (anti-VWF) mAbs which specifically blocked the vWF-GPIbα pathway. Transwell assay was used to explore the effect of vWF-mediated migration and invasion ability of SAOS2 cells. PDGF-BB, TGF-β and VEGF that could be secreted by platelets to promote tumor cells growth in the co-culture system of platelets and SAOS2 cells were detected by ELISA. [Results] Under static and flow conditions, SAOS2 cells treated with PMA exhibited significantly higher platelets-adhesion capacity compared to the cells untreated with PMA, and the adhesion could be partially inhibited by SZ-2 and AVW3 mAbs. Furthermore, platelets significantly promoted the migration and invasion ability of SAOS2 cells, and PDGF-BB and TGF-β secreted by platelets were also increased in these processes. Both of SZ-2 and AVW3 mAbs partially decreased the migration and invasion ability of SAOS2 cells. [Conclusions] vWF were expressed highly in some kind of non-endothelial origin cancer cells, such as SAOS2, which mediates platelet activation and secreting factors such as PDGF-BB and TGF-β to promote tumor cells growth. Activated platelets have a significant promoted effect on the migration and invasion ability of SAOS2 cells and this process can be inhibited by antibodies that specifically block the pathway of VWF-GPIbα. Disclosures No relevant conflicts of interest to declare.

TNF-Induced Vaso-Occlusive and Inflammatory Processes in Mice with Sickle Cell Anemia Are Abrogated By the Platelet Activation Inhibitor, Prasugrel

Background: Vaso-occlusive processes in sickle cell anemia (SCA) are triggered by inflammatory molecules, along with pancellular activation and the recruitment and adhesion of leukocytes to the endothelium. Interactions between platelets, neutrophils, and erythrocytes are observed in the SCA circulation, but the exact role of platelets in the initiation and propagation of vaso-occlusion is not well understood. Aim: We, herein, evaluated the effects of the inhibition of platelet activation on TNF-α (TNF)-triggered vaso-occlusive processes in the microcirculation of mice with SCA, using prasugrel, a platelet ADP P2Y12 receptor inhibitor. Methods: Chimeric male control (CON) and SCA (SCA) mice received prasugrel (1 mg/kg, by gavage) or vehicle (saline) in a single administration, 1h prior to TNF (0.5 μg/g, i.p). In some experiments, 1 mg/kg prasugrel was administered chronically for 5x per week for 4 weeks. Following i.v. injection of anti-CD41 PE, anti-CD45 PeCy-5 and anti-Ly6G Alexa 488 antibodies for labeling platelets, leukocytes (in general) and neutrophils respectively, the cremaster muscle was exposed and the microcirculation was observed by conventional and confocal intravital microscopy. Results: While the acute administration of prasugrel to CON and SCA mice did not alter the quantity of platelets in the microcirculation (data not shown), it significantly reduced the adhesion of platelets to the vascular wall in both CON animals (12±1.6 vs 5.7±1.4 % adhered of total platelets, n=3; p<0.05) and SCA mice after TNF (36.2±7.1 vs 4.8±2.5 % platelets adhered for w/out and with prasugrel, respectively; n=3; p<0.0001) (see Figure). Furthermore, prasugrel significantly reduced the number of platelet/neutrophil aggregates in TNF-treated CON (2.7±0.4 vs 0.53±0.3 aggregates adhered 100 µm−1, p<0.001 n=3) and SCA mice (3.1±0.7 vs 0.76±0.3 aggregates adhered 100 µm−1, p<0.01 n=3), and also platelet/non-neutrophil leukocytes in CON (1.3±0.7 vs 0.2±0.1 aggregates adhered 100 µm−1, p<0.01 n=3) and SCA mice (2.0±0.3 vs 0.2±0.1 aggregates adhered 100 µm−1, p<0.001 n=3). Alterations in platelet adhesion to the vascular wall and platelet-leukocyte aggregate formation were accompanied by reductions in the recruitment of leukocytes to the venule walls, after prasugrel administration. Prasugrel significantly decreased the rolling, adhesion and extravasation of leukocytes in both CON (4.9±2.7 vs 1.14±0.5 min−1; 9.2±0.8 vs 2.7±0.5 100 µm−1; 1.9±0.3 vs 0.4±0.1 100x50 µm-2; for w/out and with prasugrel, respectively; n=3; p<0.001) and SCA mice (36.0±3.0 vs 3.0±0.6 min−1; 16.6±1.5 vs 6.4±1.2 100 µm−1; 6.9±0.8 vs 1.7±0.4 100x50 µm-2; for w/out and with prasugrel, respectively; n=3; p<0.0001). Acute prasugrel treatment also increased blood flow velocity after TNF stimulation in CON mice (37±10 vs 110±35 mm3/s, p<0.01, n=3) and substantially augmented flow in SCA mice (13±2 vs 94±12 mm3/s, p<0.0001, n=3), approaching flow velocities similar to those observed in control animals that had not received TNF (Data not shown). The survival of SCA animals treated with a single dose of prasugrel increased by 1.3 hours compared to the SCA animals that received the vehicle (n=3, p<0.05). Complimentary experiments demonstrated a similar effect of the chronic administration of prasugrel, which significantly reduced leukocyte recruitment to the vascular wall after TNF in both CON and SCA mice (data not shown, P<0.05). Conclusions: Our findings show that the inhibition of platelet activation by prasugrel, while significantly decreasing the adhesion of platelets to venules walls and the formation of heterocellular aggregates, was able to almost abolish the recruitment of leukocytes to the microcirculation of SCA mice after an inflammatory stimulus. This reduction in platelet and leukocyte vascular recruitment was associated with major improvements in blood flow and in the survival of animals with SCA. Thus, our data suggest that platelet activation and the consequent adhesion of platelets to the vascular wall is a fundamental step in the initiation of the vaso-occlusive process in response to inflammatory stimuli. Our findings support further investigations into the use of drugs that inhibit platelet activation (including prasugrel itself) as a therapeutic approach for SCA. Disclosures No relevant conflicts of interest to declare.

Inverse Effects of EDTA on αIIbβ3-Mediated Adhesion to Immobilized Fibrinogen Versus the D98 Plasmin Fragment of Fibrinogen

αIIbβ3 is known to mediate adhesion of platelets to immobilized fibrinogen through its interaction with the C-terminal γ chain dodecapeptide (γ12) and EDTA inhibits the adhesion by binding divalent cations required for ligand interaction with the β3 metal ion-dependent adhesion site (MIDAS) cation. Studies by several groups, however, suggest that αIIβb3 can also interact with other sites on fibrin(ogen). To identify potential additional sites of interaction between fibrinogen and αIIbβ3, we studied the adhesion of HEK293 cells expressing αIIbβ3 (αIIbβ3-HEK) to the D98 plasmin fragment of fibrinogen, which lacks the γ12 peptide. The D98 fragment did not contain the γ12 peptide as judged by both immunoblotting with mAb 7E9 (anti-γ12) and mass spectroscopy. αIIbβ3-HEK did not bind to immobilized D98 (10 µg/ml coating concentration) in the presence of 2 mM Ca2+/1 mM Mg2+, but they did bind to immobilized intact fibrinogen (10 µg/ml coating concentration) and the adhesion was inhibited by mAbs 10E5 (anti-αIIbβ3), 7E3 (anti-αIIbβ3 + αVβ3), and 7E9. Adhesion of αIIbβ3-HEK to fibrinogen was nearly eliminated by 10 mM EDTA [13,007 ± 3,676 vs 304 ± 331 arbitrary fluorescence intensity units (AFU); n=9; p<0.001]. Unexpectedly, and in dramatic contrast, 10 mM EDTA increased adhesion of αIIbβ3-HEK to D98 nearly 25-fold, from 458 ± 601 to 10,718 ± 3,106 AFU (n=9; p=0.001). The adhesion to D98 in the presence of EDTA was not inhibited by mAb 7E9 or mAb LM609 (anti-αVβ3), and was inhibited by mAb 7E3 by less than 10%. EDTA-dependent adhesion was, however, inhibited by mAb 10E5, which binds to the αIIb cap domain and inhibits fibrinogen binding to αIIbβ3, by 85% ± 4% (n=7; p=0.003). Dose-response experiments demonstrated that 3 mM EDTA was sufficient to block adhesion to fibrinogen and 3-4 mM EDTA was required to enhance adhesion to D98. Adding the β3 D119 mutation to αIIbβ3-HEK (αIIbβ3-D119-HEK), which disrupts the MIDAS, eliminated adhesion to fibrinogen (αIIbβ3-HEK: 17,342 ± 6,148 vs. αIIbβ3-D119-HEK: 0 ± 241 AFU; n=3; p=0.006), but had little or no effect on the binding to D98 in the presence of EDTA (αIIbβ3-HEK: 11,363 ± 3,700 vs. αIIbβ3-D119-HEK: 9,026 ± 3,252 AFU; n=3; p=0.054). However, unlike EDTA-dependent adhesion of αIIbβ3-HEK to D98, the adhesion of αIIbβ3-D119-HEK was inhibited by mAb 10E5 by only 20% ± 19% (n=3; p=0.247). We conclude that EDTA exposes one or more sites on αIIbβ3 that bind(s) to a site(s) on immobilized D98 that is either not accessible or not expressed on intact fibrinogen. These data are consistent with the known effect of EDTA in altering the conformation of αIIbβ3 as judged by its exposing "ligand-induced" binding sites recognized by mAbs such as AP5 and PMI-1, even in the absence of ligand, and the ability of αIIbβ3 to mediate interactions with fibrin to support clot retraction even in the presence of EDTA. EDTA-treated αIIbβ3 may, therefore, provide insights into potential ancillary sites of interaction between αIIbβ3 and fibrin(ogen). Disclosures No relevant conflicts of interest to declare.