[Show abstract][Hide abstract]ABSTRACT: Platelet glycoprotein Ibalpha (GPIb alpha) is part of the receptor complex GPIb-V-IX, which has a critical role in hemostasis, especially through interactions with the subendothelial von Willebrand factor. As there is accumulating evidence for a contribution of platelet receptors to hematogenous tumor metastasis, GPIb alpha is an interesting molecule to study in this context. We have investigated the effect of GPIb alpha inhibition by monovalent Fab fragments on experimental pulmonary metastasis in a syngeneic mouse model using C57BL/6 mice and B16F10 melanoma cells. The early fate of green fluorescent protein (GFP)-transfected melanoma cells under GPIb alpha blockade was also assessed, as was the effect of GPIb alpha inhibition on pulmonary metastasis in mice lacking P-selectin. Surprisingly and, to our knowledge previously unreported, GPIb alpha inhibition led to a significant increase in pulmonary metastasis, and assessment of the early fate of circulating GFP-labeled B16F10 showed improved survival and pulmonary arrest of tumor cells shortly after GPIb alpha inhibition, indicating that inhibition of a platelet protein can, in some cases, promote metastasis of a malignant tumor. In contrast, GPIb alpha blockade in P-selectin-deficient mice had no enhancing effect on metastasis, suggesting the involvement of GPIb alpha in the initial, P-selectin-dependent steps of metastasis. These findings suggest that GPIb alpha contributes to the control of tumor metastasis, in addition to its role in hemostasis.
Full-text Article · Oct 2009 · Journal of Investigative Dermatology
[Show abstract][Hide abstract]ABSTRACT: Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.
Article · Sep 2008 · Pflügers Archiv - European Journal of Physiology
[Show abstract][Hide abstract]ABSTRACT: Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke1. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel2. The shift is mediated through binding of the cytoskeletal protein Talin to the subunit cytoplasmic tail3, 4, 5. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of -integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.
[Show abstract][Hide abstract]ABSTRACT: Platelets stably interact with collagen via glycoprotein (GP)VI and alpha2beta1integrin. With alpha2-null mice, we investigated the role of alpha2beta1 in thrombus formation and stability in vivo and in vitro. Using a FeCl(3)-induced thrombosis model, in arteries from alpha2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A(2) analogue U46619 was borderline effective in suppressing the embolisation in alpha2-null mice. In vitro, perfusion of alpha2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from alpha2-null mice. We conclude that integrin alpha2beta1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A(2) release. The increased embolization in alpha2-null mice may argue against the use of alpha2beta1 integrin inhibitors for antithrombotic therapy.
Full-text Article · Dec 2007 · Thrombosis and Haemostasis
[Show abstract][Hide abstract]ABSTRACT: Platelets stably interact with collagen via glycoprotein (GP)VI and alpha 2 beta l integrin.With alpha 2-null mice, we investigated the role of alpha 2 beta l in thrombus formation and stability in vivo and in vitro. Using a FeCl3-incluced thrombosis model, in arteries from alpha 2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects,while the thromboxaneA(2) analogue U46619 was borderline effective in suppressing the embolisation in alpha 2-null mice. In vitro, perfusion of alpha 2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from alpha 2-null mice. We conclude that integrin alpha 2 beta l plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A(2) release. The increased embolization in alpha 2-null mice may argue against the use of alpha 2 beta l integrin inhibitors for antithrombotic therapy.
[Show abstract][Hide abstract]ABSTRACT: Ischemic stroke is a frequent and serious disease with limited treatment options. Platelets can adhere to hypoxic cerebral endothelial cells by binding of their glycoprotein (GP) Ib receptor to von Willebrand factor. Exposure of subendothelial matrix proteins further facilitates firm attachment of platelets to the vessel wall by binding of collagen to their GPVI receptor. In the present study, we addressed the pathogenic role of GPIb, GPVI, and the aggregation receptor GPIIb/IIIa in experimental stroke in mice.
Complete blockade of GPIb alpha was achieved by intravenous injection of 100 microg Fab fragments of the monoclonal antibody p0p/B to mice undergoing 1 hour of transient middle cerebral artery occlusion. At 24 hours after transient middle cerebral artery occlusion, cerebral infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride staining. In mice treated with anti-GPIb alpha Fab 1 hour before middle cerebral artery occlusion, ischemic lesions were reduced to approximately 40% compared with controls (28.5+/-12.7 versus 73.9+/-17.4 mm3, respectively; P<0.001). Application of anti-GPIb alpha Fab 1 hour after middle cerebral artery occlusion likewise reduced brain infarct volumes (24.5+/-7.7 mm3; P<0.001) and improved the neurological status. Similarly, depletion of GPVI significantly diminished the infarct volume but to a lesser extent (49.4+/-19.1 mm3; P<0.05). Importantly, the disruption of early steps of platelet activation was not accompanied by an increase in bleeding complications as revealed by serial magnetic resonance imaging. In contrast, blockade of the final common pathway of platelet aggregation with anti-GPIIb/IIIa F(ab)2 fragments had no positive effect on stroke size and functional outcome but increased the incidence of intracerebral hemorrhage and mortality after transient middle cerebral artery occlusion in a dose-dependent manner.
Our data indicate that the selective blockade of key signaling pathways of platelet adhesion and aggregation has a different impact on stroke outcome and bleeding complications. Inhibition of early steps of platelet adhesion to the ischemic endothelium and the subendothelial matrix may offer a novel and safe treatment strategy in acute stroke.
[Show abstract][Hide abstract]ABSTRACT: P-selectin ctin has been implicated in important platelet functions. However, neither its role in thrombus formation and cardiovascular disorders nor its suitability as a therapeutic target structure is entirely clear.
Platelet aggregation was assessed in complementary in vitro settings by measurements of static aggregation, standardized aggregometry and dynamic flow chamber assays. Degradation of aggregates was also analyzed under flow conditions using video microscopy. In vivo, platelet rolling in cutaneous venules was assessed by intravital microscopy in wild-type mice treated with selectin-blocking compounds as well as in P-selectin-deficient mice. FeCl3-induced arterial thrombosis was studied by intravital microscopy in untreated mice or mice treated with an inhibitor of selectin functions. Finally, inhibition of selectin functions was studied in an ischemia/reperfusion injury model in rats.
Antibody- or small-molecule-mediated inhibition of P-selectin functions significantly diminished platelet aggregation (p<0.03) and platelet-neutrophil adhesion in vitro (p<0.01) as well as platelet aggregate sizes under flow (p<0.03). Established aggregates were degraded, either via detachment of single platelets following addition of efomycine M, or via detachment of multicellular clumps when P-selectin-directed Fab-fragments were used. In vivo, selectin inhibition resulted in a greater than 50% reduction of platelet rolling in cutaneous venules (p<0.01), producing rolling fractions similar to those observed in P-selectin-deficient mice (p<0.05). Moreover, inhibition of selectin functions significantly decreased the thrombus size in FeCl3-induced arterial thrombosis in mice (p<0.05). In an ischemia/reperfusion injury model in rats, small-molecule-mediated selectin inhibition significantly reduced myocardial infarct size from 18.9% to 9.42% (p<0.001) and reperfusion injury (p<0.001).
Inhibition of P-selectin functions reduces platelet aggregation and can alleviate platelet-related disorders in disease-relevant preclinical settings.
[Show abstract][Hide abstract]ABSTRACT: Platelet activation plays a central role in hemostasis and thrombosis. Many platelet agonists function through G-protein-coupled receptors. Epinephrine activates the alpha(2A)-adrenergic receptor (alpha(2A)) that couples to G(z) in platelets. Although alpha(2A) was originally cloned from platelets, its role in thrombosis and hemostasis is still unclear. Through analysis of alpha(2A)-deficient mice, variable tail bleeding times were observed. In vitro, epinephrine potentiated activation/aggregation responses of wild-type but not alpha(2A)-deficient platelets as determined by flow cytometry and aggregometry, whereas perfusion studies showed no differences in platelet adhesion and thrombus formation on collagen. To test the in vivo relevance of alpha(2A) deficiency, mice were subjected to 3 different thrombosis models. As expected, alpha(2A)-deficient mice were largely protected from lethal pulmonary thromboembolism induced by the infusion of collagen/epinephrine. In a model of FeCl(3)-induced injury in mesenteric arterioles, alpha(2A)(-/-) mice displayed a 2-fold increase in embolus formation, suggesting thrombus instability. In a third model, the aorta was mechanically injured, and blood flow was measured with an ultrasonic flow probe. In wild-type mice, all vessels occluded irreversibly, whereas in 24% of alpha(2A)-deficient mice, the initially formed thrombi embolized and blood flow was reestablished. These results demonstrate that alpha(2A) plays a significant role in thrombus stabilization.
[Show abstract][Hide abstract]ABSTRACT: Collagen and thrombin are the strongest physiological platelet agonists, acting through different receptors, among which glycoprotein VI (GPVI) and protease-activated receptors, respectively, are the essential ones. In mice, targeting of GPVI with the monoclonal antibody JAQ1 induces depletion of the receptor from circulating platelets, resulting in abolished collagen responses and long-lasting antithrombotic protection.
Mice were treated with JAQ1, and the early effects of this treatment were analyzed. In addition to the known abolition of the collagen reactivity, this treatment also affected platelet response to thrombin but not other agonists. In platelets from JAQ1-treated mice, thrombin-induced activation of integrin alphaIIbbeta3, the surface expression of P-selectin, and the procoagulant activity were decreased on days 1 and 2, then progressively recovered and returned to normal on day 5. In parallel, the mice were transiently protected from lethal tissue factor-induced pulmonary thromboembolism (100% survivors versus 40% in control group), which appeared to be based on a decreased generation and activity of intravascular thrombin.
Anti-GPVI treatment induces 2-phase antithrombotic protection in mice consisting of a partial and transient inhibition of thrombin responses in platelets and a prolonged and complete loss of the collagen response.
Full-text Article · Aug 2006 · Arteriosclerosis Thrombosis and Vascular Biology
[Show abstract][Hide abstract]ABSTRACT: Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.
[Show abstract][Hide abstract]ABSTRACT: Aspirin is effective in the therapy of cardiovascular diseases, because it causes acetylation of cyclooxygenase 1 (COX-1) leading to irreversible inhibition of platelets. Additional mechanisms can be suspected, because patients treated with other platelet COX inhibitors such as indomethacin do not display an increased bleeding tendency as observed for aspirin-treated patients. Recently, aspirin and other anti-inflammatory drugs were shown to induce shedding of L-selectin in neutrophils in a metalloproteinase-dependent manner. Therefore, we investigated the effects of aspirin on the von Willebrand Factor receptor complex glycoprotein (GP) Ib-V-IX, whose lack or dysfunction causes bleeding in patients. As quantified by fluorescence-activated cell sorting analysis in whole blood, aspirin, but not its metabolite salicylic acid, induced dose-dependent shedding of human and murine GPIbalpha and GPV from the platelet surface, whereas other glycoproteins remained unaffected by this treatment. Biotinylated fragments of GPV were detected by immunoprecipitation in the supernatant of washed mouse platelets, and the expression level of GPIbalpha was decreased in these platelets as measured by Western blot analysis. Although shedding occurred normally in COX-1-deficient murine platelets, shedding was completely blocked by a broad-range metalloproteinase inhibitor and, more importantly, in mouse platelets expressing an inactive form of ADAM17. Shed fragments of GPIbalpha and GPV were elevated in the plasma of aspirin-injected mice compared with animals injected with control buffer. These data demonstrate that aspirin at high concentrations induces shedding of GPIbalpha and GPV by an ADAM17-dependent mechanism and that this process can occur in vivo.
Article · Jan 2006 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected alphaIIbbeta3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin alphaIIbbeta3 activation was significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors (diphenylene iodonium [DPI] [45% +/- 9%] and apocynin [43% +/- 11%]) and superoxide scavengers (tiron [60% +/- 9%] and Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin [MnTMPyP] [70% +/- 6%]). These inhibitors also reduced platelet aggregation and thrombus formation on collagen under high shear and achieved their effects independent of the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway.
[Show abstract][Hide abstract]ABSTRACT: Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)-mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy.
Full-text Article · Aug 2005 · Journal of Experimental Medicine