Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

PAFR in adipose tissue macrophages is associated with anti-inflammatory phenotype and metabolic homoeostasis.

Metabolic dysfunction is associated with adipose tissue inflammation and macrophage infiltration. PAFR (platelet-activating factor receptor) is expressed in several cell types and binds to PAF (platelet-activating factor) and oxidized phospholipids. Engagement of PAFR in macrophages drives them towards the anti-inflammatory phenotype. In the present study, we investigated whether genetic deficiency of PAFR affects the phenotype of ATMs (adipose tissue macrophages) and its effect on glucose and insulin metabolism. PARFKO (PAFR-knockout) and WT (wild-type) mice were fed on an SD (standard diet) or an HFD (high-fat diet). Glucose and insulin tolerance tests were performed by blood monitoring. ATMs were evaluated by FACS for phenotypic markers. Gene and protein expression was investigated by real-time reverse transcription-quantitative PCR and Western blotting respectively. Results showed that the epididymal adipose tissue of PAFRKO mice had increased gene expression of Ccr7, Nos2, Il6 and Il12, associated with pro-inflammatory mediators, and reduced expression of the anti-inflammatory Il10. Moreover, the adipose tissue of PAFRKO mice presented more pro-inflammatory macrophages, characterized by an increased frequency of F4/80(+)CD11c(+) cells. Blood monocytes of PAFRKO mice also exhibited a pro-inflammatory phenotype (increased frequency of Ly6C(+) cells) and PAFR ligands were detected in the serum of both PAFRKO and WT mice. Regarding metabolic parameters, compared with WT, PAFRKO mice had: (i) higher weight gain and serum glucose concentration levels; (ii) decreased insulin-stimulated glucose disappearance; (iii) insulin resistance in the liver; (iv) increased expression of Ldlr in the liver. In mice fed on an HFD, some of these changes were potentiated, particularly in the liver. Thus it seems that endogenous ligands of PAFR are responsible for maintaining the anti-inflammatory profile of blood monocytes and ATMs under physiological conditions. In the absence of PAFR signalling, monocytes and macrophages acquire a pro-inflammatory phenotype, resulting in adipose tissue inflammation and metabolic dysfunction.

Platelet-activating factor receptor contributes to antileishmanial function of miltefosine.

Miltefosine [hexadecylphosphocholine (HPC)] is the only orally bioavailable drug for the disease visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani. Although miltefosine has direct leishmanicidal effects, evidence is mounting for its immune system-dependent effects. The mechanism of such indirect antileishmanial effects of miltefosine remains to be discovered. As platelet-activating factor and HPC share structural semblances and both induce killing of intracellular Leishmania, we surmised that platelet-activating factor (PAF) receptor had a significant role in the antileishmanial function of miltefosine. The proposition was supported by molecular dynamic simulation of HPC docking into PAF receptor and by comparison of its leishmanicidal function on PAF receptor-deficient macrophages and mice under HPC treatment. We observed that compared with wild-type macrophages, the PAF receptor-deficient macrophages showed 1) reduced binding of a fluorescent analog of HPC, 2) decreased TNF-α production, and 3) lower miltefosine-induced killing of L. donovani. Miltefosine exhibited significantly compromised leishmanicidal function in PAF receptor-deficient mice. An anti-PAF receptor Ab led to a significant decrease in miltefosine-induced intracellular Leishmania killing and IFN-γ production in a macrophage-T cell coculture system. These results indicate significant roles for PAF receptor in the leishmanicidal activity of HPC. The findings open new avenues for a more rational understanding of the mechanism of action of this drug as well as for improved therapeutic strategies.

Lack of platelet-activating factor receptor protects mice against diet-induced adipose inflammation and insulin-resistance despite fat pad expansion.

OBJECTIVE: The role of platelet-activating factor (PAF) on diet-induced inflammatory and metabolic dysfunction is unknown. The effects of diet-induced metabolic and inflammatory dysfunction in mice with deletion of the PAF receptor (PAFR(-/-) ) were evaluated in this study. METHODS: Wild-type and PAFR(-/-) mice were fed chow (WT-C and PAFR(-/-) -C) or high-refined carbohydrate-containing diet (WT-HC and PAFR(-/-) -HC). PAFR(-/-) - RESULTS: HC mice gained more weight and adiposity than PAFR(-/-) -C and WT-HC mice. Lipogenesis increased and hormone-sensitive lipase expression decreased in PAFR(-/-) -HC compared to WT-HC mice. WT-HC mice had impaired glucose tolerance and insulin sensitivity compared to WT-C mice. In contrast, glucose tolerance and insulin sensitivity in PAFR(-/-) -HC mice were similar to that of lean littermates. PAFR(-/-) -HC mice expressed significantly more peroxisome proliferator-activator receptor gamma (PPARγ) than PAFR(-/-) -C and WT-C mice. Resistin increased in WT-HC mice compared to WT-C mice. However, the levels of resistin were 35% lower in PAFR(-/-) -HC mice than WT-HC mice. PAFR(-/-) presented with less HC diet-induced adipose tissue inflammation than WT mice. Adipocytes isolated from PAFR(-/-) mice incubated in media containing normal or high levels of glucose secreted less interleukin-6 and tumor necrosis factor alpha and presented lower rate of lipolysis than WT mice. CONCLUSION: PAFR deficiency resulted in less inflammation in adipose tissue and improvement in glucose homeostasis when fed the HC diet. The higher adiposity observed in PAFR(-/-) mice fed HC diet could be owing to the maintenance of insulin sensitivity, decreased adipocyte lipolysis rate, high lipogenesis and PPARγ expression, and lower inflammatory milieu in adipose tissue.

Platelet-activating factor receptor blockade ameliorates Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice.

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by oral biofilm-producing microorganisms, such as Aggregatibacter actinomycetemcomitans. The levels of the phospholipid platelet-activating factor (PAF) in the saliva, gingival crevicular fluid, and periodontal tissues are significantly increased during inflammatory conditions, such as PD, but the exact mechanism that links PAF to alveolar bone resorption is not well understood. In the current study, alveolar bone resorption was induced by experimental PD through the oral inoculation of A. actinomycetemcomitans in wild-type (WT) and PAF receptor knockout (Pafr(-/-)) mice. In vitro experiments using A. actinomycetemcomitans lipopolysaccharide (LPS)-stimulated RAW 264.7 cells treated with a PAF receptor antagonist (UK74505) were also performed. The expression of lyso-PAF acetyltransferase in periodontal tissues was significantly increased 3 h after A. actinomycetemcomitans LPS injection in mice. WT and Pafr(-/-) mice that were subjected to oral inoculation of A. actinomycetemcomitans presented neutrophil accumulation and increased levels of CXCL-1 and tumor necrosis factor alpha (TNF-α) in periodontal tissues. However, Pafr(-/-) mice presented less alveolar bone loss than WT mice. The in vitro blockade of the PAF receptor impaired the resorptive activity of A. actinomycetemcomitans LPS-activated osteoclasts. In conclusion, this study shows for the first time that the blockade of PAF receptor may contribute to the progression of PD triggered by A. actinomycetemcomitans by directly affecting the differentiation and activity of osteoclasts.

Role of PAF receptor in proinflammatory cytokine expression in the dorsal root ganglion and tactile allodynia in a rodent model of neuropathic pain.

BACKGROUND: Neuropathic pain is a highly debilitating chronic pain following damage to peripheral sensory neurons and is often resistant to all treatments currently available, including opioids. We have previously shown that peripheral nerve injury induces activation of cytosolic phospholipase A(2) (cPLA(2)) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia, a hallmark of neuropathic pain. However, lipid mediators downstream of cPLA(2) activation to produce tactile allodynia remain to be determined. PRINCIPAL FINDINGS: Here we provide evidence that platelet-activating factor (PAF) is a potential candidate. Pharmacological blockade of PAF receptors (PAFRs) reduced the development and expression of tactile allodynia following nerve injury. The expression of PAFR mRNA was increased in the DRG ipsilateral to nerve injury, which was seen mainly in macrophages. Furthermore, mice lacking PAFRs showed a reduction of nerve injury-induced tactile allodynia and, interestingly, a marked suppression of upregulation of tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) expression in the injured DRG, crucial proinflammatory cytokines involved in pain hypersensitivity. Conversely, a single injection of PAF near the DRG of naïve rats caused a decrease in the paw withdrawal threshold to mechanical stimulation in a dose-dependent manner and an increase in the expression of mRNAs for TNFalpha and IL-1beta, both of which were inhibited by pretreatment with a PAFR antagonist. CONCLUSIONS: Our results indicate that the PAF/PAFR system has an important role in production of TNFalpha and IL-1beta in the DRG and tactile allodynia following peripheral nerve injury and suggest that blocking PAFRs may be a viable therapeutic strategy for treating neuropathic pain.

Platelet glycoprotein VI facilitates experimental lung metastasis in syngenic mouse models.

BACKGROUND: Glycoprotein (GP)VI is a key receptor for collagen on the platelet surface. It is a member of the immunoglobulin superfamily and is uniquely expressed on the surface of platelets, where it is assembled with the immunoreceptor tyrosine activation motif subunit, FcR-gamma. We have previously reported the generation of a murine model of GPVI deficiency that revealed profound defects in collagen-induced platelet aggregation and in platelet activation following adhesion to collagen. Beyond the hemostasis/thrombosis paradigm, platelet receptors are emerging as significant participants in tumorigenesis and inflammation. OBJECTIVE: In the current study, we have evaluated a role for platelet GPVI in primary tumor growth and experimental metastasis. METHODS: Primary tumor induction and experimental metastasis assays were performed using syngenic immunocompetent animals and tumor cells derived from the C57BL/6J mouse strain in wild-type (C57BL/6J) and N10 C57BL/6J congenic GPVI-deficient mice. RESULTS: Using either a Lewis lung carcinoma (D121) or melanoma (B16F10.1) cell line, we observed an approximately 50% reduction in the number of visible tumor foci in GPVI-deficient mice as compared with control C57BL/6J mice. Additional studies were performed to compare the size of subcutaneously implanted tumor cells, that is, primary tumor growth. Here, we observed no noticeable size difference when comparing the presence or absence of platelet GPVI. CONCLUSIONS: The results demonstrate that the presence of platelet GPVI facilitates experimental tumor metastasis but does not contribute to the growth of primary tumors.

Negative regulation of activated alpha-2 integrins during thrombopoiesis.

Circulating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, alpha2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface alpha2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, alpha2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated alpha2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated alpha2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoietic cells during migration between the adhesive environment of the bone marrow and the nonadhesive environment of the circulating blood.

Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis.

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

Platelet activating factor receptor-deficient mice present delayed interferon-gamma upregulation and high susceptibility to Leishmania amazonensis infection.

We investigated the role of the platelet activation factor (PAF) receptor (PAFR) in the outcome of infection with Leishmania amazonensis. PAFR deficient (PAFR(-/-)) mice were infected with L. amazonensis and the course of infection was followed. We found that PAFR(-/-) mice in the C57BL/6 background were more susceptible to infection with L. amazonensis than the wild-type controls, as seen both by lesion size and parasite number at the site of infection. Interferon (IFN)-gamma production was delayed in PAFR(-/-) mice, and lower levels of Ccl5 were found in lesions. Expression of nitric oxide synthase-2 mRNA was found impaired in PAFR(-/-) associated with higher levels of arginase-1 mRNA. Moreover, higher levels of antibodies were produced in response to L. amazonensis by PAFR(-/-) mice. We conclude that signaling through the PAFR is essential for the ability of the murine host to control L. amazonensis infection by driving an adequate immune response.

Absence of platelet-activating factor receptor protects mice from osteoporosis following ovariectomy.

While platelet-activating factor (PAF) is produced in various diseases associated with bone resorption, its functions in bone metabolism remain unknown. Using PAF receptor-deficient mice, we evaluated the role of PAF in the development of bone resorption following ovariectomy, a model of postmenopausal osteoporosis. Through observations of bone mineral density and histomorphometric parameters, it was found that bone resorption was markedly attenuated in PAF receptor-deficient mice, indicating that PAF links estrogen depletion and osteoporosis in vivo. Osteoclasts expressed higher amounts of the enzymes required for PAF biosynthesis than osteoblasts. TNF-alpha and IL-1beta increased the acetyl-coenzyme A:lyso-PAF acetyltransferase activity in osteoclasts. Osteoclasts, but not osteoblasts, expressed the functional PAF receptor. PAF receptor stimulation prolonged the survival of osteoclasts in vitro. Furthermore, osteoclasts treated with a PAF receptor antagonist, and also those from PAF receptor-deficient mice, showed reductions in survival rate and Ca resorption activity. Consistently, in organ cultures, bone resorption was significantly suppressed by a PAF receptor antagonist treatment or genetic PAF receptor deficiency. Thus, these results suggest that, through the inflammatory cytokines, estrogen depletion enhances PAF production as a unique autocrine factor for osteoclast functions. Inhibition of PAF function might pave the way for a new strategy to prevent postmenopausal bone loss without disturbing osteoblast functions.

Augmentation of chemotherapy-induced cytokine production by expression of the platelet-activating factor receptor in a human epithelial carcinoma cell line.

In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-alpha induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-kappaB were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NFkappaB pathway using a super-repressor IkappaBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-kappaB-dependent process.

Sponge-induced angiogenesis and inflammation in PAF receptor-deficient mice (PAFR-KO).

1. To determine biological functions of platelet-activating factor (PAF) in chronic inflammation, we have investigated the kinetics of angiogenesis, inflammatory cells recruitment and cytokine production in sponge-induced granuloma in wild type and PAF receptor-deficient mice (PAFR-KO). 2. Angiogenesis as determined by morphometric analysis and hemoglobin content was significantly higher in the implants of PAFR-KO mice at all time points. Treatment with PAF receptor antagonist UK74505 (30 mg kg(-1)) also increased angiogenesis in sponge implants. 3. Neutrophils and macrophages accumulation, as determined by myeloperoxidase and N-acetylglucosaminidase activities in the supernatant of implanted sponges were markedly decreased in PAFR-KO mice. Surprisingly, the levels of the proinflammatory chemokines, keratinocyte-derived chemokine and chemokine monocyte chemoattractant protein 1 were higher in the implants of the transgenic animals. 4. We have shown that angiogenesis was stimulated in PAFR-KO mice whereas inflammation was decreased, indicating that PAF is an endogenous regulator of new blood vessels formation in the inflammatory microenvironment induced by the sponge implant.

Platelet-activating factor receptor-deficient mice are protected from experimental sleep apnea-induced learning deficits.

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet-activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR-/-), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR-/- exhibit attenuated elevations in inflammatory signaling (cyclo-oxygenase-2 and inducible nitric oxide synthase activities), degradation of the ubiquitin-proteasome pathway and apoptosis observed in wild-type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.

Platelet-activating factor receptor deficiency delays elimination of adult worms but reduces fecundity in Strongyloides venezuelensis-infected mice.

We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR(-/-)) and wild-type mice after a single Strongyloides venezuelensis infection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR(-/-) and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR(-/-) mice. Although PAFR(-/-) infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR(-/-) mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR(-/-) mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR(-/-) mice than in the corresponding preparations from wild-type mice. Thus, in S. venezuelensis-infected PAFR(-/-) mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.

Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2.

Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet-collagen interaction, adhesion is mediated via the inside-out regulation of integrin alpha2beta1 and activation through the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. The present study extends this model by demonstrating that engagement of alpha2beta1 by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase-based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI-FcR gamma-chain complex. The same events do not occur in platelet suspensions. alpha2beta1 activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI-FcR gamma-chain cascade, including Src, Syk, SLP-76, and PLCgamma2 as well as plasma membrane calcium ATPase and focal adhesion kinase. alpha2beta1-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLCgamma2-deficient platelets. Spreading is abolished by chelation of intracellular Ca2+. Demonstration that adhesion of platelets to collagen via alpha2beta1 generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of beta1-deficient thrombi and why loss of the GPVI-FcR gamma-chain complex has a relatively minor effect on bleeding.

Distinct roles of GPVI and integrin alpha(2)beta(1) in platelet shape change and aggregation induced by different collagens.

1. Various platelet membrane glycoproteins have been proposed as receptors for collagen, in some cases as receptors for specific collagen types. In this study we have compared the ability of a range of collagen types to activate platelets. 2. Bovine collagen types I-V, native equine tendon collagen fibrils and collagen-related peptide (CRP) all induced platelet aggregation and shape change. 3. Responses were abolished in FcRgamma chain-deficient platelets, which also lack GPVI, indicating a critical dependence on the GPVI/FcRgamma chain complex. 4. Responses to all collagens were unaffected in CD36-deficient platelets. 5. A monoclonal antibody (6F1) which binds to the alpha(2) integrin subunit of human platelets had a minimal effect on the rate and extent of aggregation induced by the collagens; however, it delayed the onset of aggregation following addition of all collagens. For shape change, 6F1 abolished the response induced by collagen types I and IV, substantially attenuated that to collagen types II, III and V, but only partially inhibited Horm collagen. 6. Simultaneous blockade of the P2Y(1) and P2Y(12) receptors, and inhibition of cyclo-oxygenase demonstrated that CRP can activate platelets independently of ADP and TxA(2); however, responses to the collagens were dependent on these mediators. 7. This study confirms the importance of the GPVI/FcRgamma chain complex in platelet responses induced by a range of collagen agonists, while providing no evidence for collagen type-specific receptors. It also provides evidence for a modulatory role of alpha(2)beta(1), the significance of which depends on the collagen preparation.

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.

Variable protection of beta 3-integrin--deficient mice from thrombosis initiated by different mechanisms.

Platelet integrin alpha IIb beta 3 (GPIIb/IIIa) plays a central role in the initiation of arterial thrombosis, but its contribution to disseminated microvascular thrombosis is less well defined. Therefore, wild-type mice (beta 3(+/+)), beta 3-integrin-deficient mice (beta 3(-/-)), and wild-type mice treated with a hamster monoclonal antibody (1B5) that blocks murine alpha IIb beta 3 function were tested in models of large-vessel and microvascular thrombosis. In the large-vessel model, ferric chloride was used to injure the carotid artery, and the time to thrombosis was measured. In beta 3(+/+) mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the beta 3(-/-) mice tested (P <.001). Fab and F(ab')(2) fragments of 1B5 increased the median time to occlusion. To initiate systemic intravascular thrombosis, prothrombotic agents were administered intravenously, and platelet thrombus formation was monitored by the decrease in circulating platelet count. Three minutes after the injection of adenosine diphosphate (ADP), collagen + epinephrine, or tissue factor, the platelet counts in beta 3(+/+) mice decreased by 289, 424, and 429 x 10(3)/microL, respectively. beta 3(-/-) mice and wild-type mice pretreated with 1B5 Fab (1 mg/kg, IP) were nearly completely protected from the effects of ADP. In contrast, beta 3(-/-) mice were only partially protected from the effects of collagen + epinephrine and minimally protected from the effects of tissue factor. In all cases, less fibrin became deposited in the lungs of beta 3(-/-) mice than in wild-type mice. These results suggest that though alpha IIb beta 3 plays a dominant role in large-vessel thrombosis, it plays a variable role in systemic intravascular thrombosis. (Blood. 2001;98:1055-1062)