IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation

Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis, the major complication of atherosclerosis triggering myocardial infarction and stroke. A central regulatory pathway conveying inhibition of platelet activation/aggregation is nitric oxide (NO)/cyclic GMP (cGMP) signaling by cGMP-dependent protein kinase I (cGKI). However, the regulatory cascade downstream of cGKI mediating platelet inhibition is still unclear. Here, we show that the inositol-1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) is abundantly expressed in platelets and assembled in a macrocomplex together with cGKIbeta and the inositol-1,4,5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP3RI interaction in IRAGDelta12/Delta12 mutant mice leads to a loss of NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Intracellular calcium transients were not affected by DEA/NO or cGMP in mutant platelets. Furthermore, intravital microscopy shows that NO fails to prevent arterial thrombosis of the injured carotid artery in IRAGDelta12/Delta12 mutants. These findings reveal that interaction between IRAG and InsP3RI has a central role in NO/cGMP-dependent inhibition of platelet aggregation and in vivo thrombosis.     

The effects of PPAR-gamma ligand pioglitazone on platelet aggregation and arterial thrombus formation

BACKGROUND: It has been suggested that peroxisome proliferator-activated receptor (PPAR)-gamma ligands reduce the development of atherosclerosis and myocardial ischemia-reperfusion injury; both of these phenomena are associated with platelet activation. We postulated that PPAR-gamma activation would inhibit platelet activation and intra-arterial thrombus formation. METHODS AND RESULTS: Sprague-Dawley rats were fed chow mixed with pioglitazone (1 or 10 mg/kg/day) for 7 to 10 days. A filter soaked in 30% FeCl(3) was applied around the abdominal aorta to study the patterns of arterial thrombogenesis. The aortic blood flow was continuously monitored using an ultrasonic Doppler flow probe. ADP and arachidonic acid-induced platelet aggregation and the expression of constitutive nitric oxide synthase (cNOS) and thrombomodulin in aorta were measured. Pioglitazone feeding delayed the time to occlusive thrombus formation by 40% (P<0.01 vs. control, n=9) without affecting the weight of the thrombus. ADP- as well as arachidonic acid-induced platelet aggregation was also inhibited by pioglitazone feeding (P<0.01 vs. control, n=9). Pioglitazone feeding also upregulated the aortic expression of cNOS and thrombomodulin; both are considered important factors in platelet aggregation and thrombus formation in vivo. The effect of a high dose (10 mg/kg/day) of pioglitazone was not more potent than that of a low dose (1 mg/kg/day). CONCLUSION: These results indicate that pioglitazone administration decreases platelet aggregation and delays intra-arterial thrombus formation in rats, at least partially, by an increase in the expression of cNOS and thrombomodulin.     

Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation

Disturbances of blood flow at sites of atherosclerotic plaque rupture are one of the key pathogenic events promoting platelet activation and arterial thrombus formation. Shear effects of platelets have been extensively investigated in vitro; however, the mechanisms by which shear promotes platelet aggregation in vivo remain incompletely understood. By employing high-resolution imaging techniques to in vitro and in vivo thrombosis models, we demonstrate a unique mechanism initiating shear-dependent platelet aggregation involving aggregate formation between discoid platelets. These discoid platelet aggregates are initially unstable and result from the development of membrane tethers between coadhering platelets. Tether formation involves the adhesive function of GPIb/V/IX and integrin alphaIIbbeta3, and conversion of discoid platelet aggregates into stable aggregates requires released ADP. The efficiency of this process is regulated by 3 independent variables, including the reactivity of the adhesive substrate, the level of shear flow, and the platelet density at the adhesive surface. These studies identify a new mechanism initiating platelet aggregation that is critically influenced by shear, physical proximity between translocating platelets, and membrane tether formation. Moreover, they provide a model to explain how the discoid morphology of platelets facilitates the maintenance of adhesive interactions with thrombogenic surfaces under high shear stress conditions.     

5-Fluorouracil induces defects in platelet function

Platelet factor-3 (PF₃) availability and platelet aggregation to ADP (4 μmol/l) and adrenaline (1.2 μmol/1) were evaluated in patients being treated with 5-fluorouracil (5-FU) for gastrointestinal malignancy. It produced a significant reduction in platelet aggregation and platelet factor-3 (PF₃) availability (P <0.001) without being associated with thrombocytopenia. The changes in platelet aggregation occurred during the first week of chemotherapy and continued with subsequent courses. This acquired abnormality may be responsible for a haemorrhagic diathesis even without obvious thrombocytopenia. Study of platelet function is important and may be considered to assess the haematological toxicity in patients being treated with systemic 5-FU therapy.     

5-Fluorouracil induces defects in platelet function

Platelet factor-3 (PF(3)) availability and platelet aggregation to ADP (4 micromol/l) and adrenaline (1.2 micromol/l) were evaluated in patients being treated with 5-fluorouracil (5-FU) for gastrointestinal malignancy. It produced a significant reduction in platelet aggregation and platelet factor-3 (PF(3)) availability ( P < 0.001) without being associated with thrombocytopenia. The changes in platelet aggregation occurred during the first week of chemotherapy and continued with subsequent courses. This acquired abnormality may be responsible for a haemorrhagic diathesis even without obvious thrombocytopenia. Study of platelet function is important and may be considered to assess the haematological toxicity in patients being treated with systemic 5-FU therapy.     

The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collagen-induced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VInull platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VInull or FcR-gammanull animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VInull platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils.     

Anagrelide metabolite induces thrombocytopenia in mice by inhibiting megakaryocyte maturation without inducing platelet aggregation

OBJECTIVE: The mechanism for anagrelide's potent platelet lowering activity in human subjects is not well defined. Studies related to anagrelide function have been hampered by its lack of activity in nonhuman primates and water insolubility. In an effort to define the mechanism whereby anagrelide exerts its therapeutic effect, we identified a water-soluble metabolite (anagrelide.met). The availability of anagrelide.met allowed, for the first time, parallel in vitro and in vivo animal studies centered on the mechanisms by which anagrelide lowers platelet levels. MATERIALS AND METHODS: The effects of anagrelide.met on proliferation and maturation of mega-karyocytes (MKs) as well as platelet production were studied both in vitro and in vivo. RESULTS: Anagrelide.met is capable of blocking in vitro MK migration by 20% to 40%. At 100 ng/mL, anagrelide.met selectively blocked in vitro MK maturation, resulting in a 50% decrease in the total number of CD41a(+) MKs, corresponding with a 30% decrease in MK ploidy by day 10 and a 60% decrease by day 20. Daily intraperitoneal injections of anagrelide.met 100 microg into BALB/c mice was sufficient to significantly decrease platelet counts within 24 to 48 hours, stabilizing to 40 to 50% of normal levels by day 5. This was associated with a 45% decrease in the number of developing MKs and an increase in thrombopoietin levels. Anagrelide.met did not alter WBC counts, hematocrit, or bleeding time, or lead to any apparent signs of toxicity. Furthermore, unlike the parent anagrelide compound, anagrelide.met did not inhibit ADP-induced platelet aggregation even at high concentrations (10 microg/mL). CONCLUSIONS: We describe a cross-species reactive anagrelide metabolite that selectively inhibits MK maturation and migration, lowering platelet levels without influencing platelet aggregation.     

Clinical aspects of platelet inhibitors and thrombus formation

The platelet, once thought to be solely involved in clot formation, is now known to be a key mediator in various others processes such as inflammation, thrombosis, and atherosclerosis. Supported by the wealth of evidence from clinical trials demonstrating their benefits in patient outcomes, antiplatelet agents have become paramount in the prevention and management of various diseases involving the cardiovascular, cerebrovascular, and peripheral arterial systems. Despite being among the most widely used and studied classes of medical therapies, new discoveries regarding important clinical aspects and properties of these agents continue to be made. As our understanding of platelet biology expands, more effective and safer novel therapies continue to be developed. The use of more refined agents in conjunction with a better understanding of their effects will further the ability to provide more optimized care on an individual basis.     

Anti-protein C monoclonal antibody induces thrombus in mice

Protein C (PC) is considered to be an important regulator of blood coagulation and fibrinolysis. During the production of monoclonal antibodies (MoAbs) against human PC in mouse ascitic fluid, one hybridoma was found to induce heavy thrombus in mice, resulting in severe hemorrhage. Intravenous infusion of the purified MoAb (PC01) from this hybridoma also caused thrombosis in mice. The crossreacting substance was then isolated from mouse plasma with PC01 immunoaffinity column, which was identified as mouse PC by several criteria. Mouse PC prolonged the activated partial thromboplastin time of mouse plasma, and PC01 neutralized this in vitro anti-coagulant activity. Therefore, heavy thrombosis observed in PC01-treated mice is likely to be ascribed to the defect of PC caused by PC01.     

Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow.

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann's thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s(-1)). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (>/=1, 210 s(-1)) but normal thrombus formation under low shear (相似文献   

Ristocetin induces platelet aggregation: a morphological demonstration

Changes in the morphology of human platelets induced by ristocetin in platelet-rich plasma (PRP) have been analysed at the ultrastructural level by means of a tannic acid procedure. Studies were also undertaken to measure the release of serotonin. Modifications of the aggregation tests induced by apyrase, a monoclonal antibody (Mab) to GPIIb/IIIa and by EDTA were also investigated. Transmission electron microscopy revealed that ristocetin precipitated adhesive proteins on the platelet membrane. An electron-dense deposit was seen within 20 s after ristocetin was added. When experiments were carried out in the aggregometer cuvette during stirring, groups of platelets became activated, changed their shape, and finally aggregated releasing part of their contents. The morphology of aggregates did not differ from those formed in the presence of ADP. Aggregation studies demonstrated that a Mab to GPIIb/IIIa modified the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet-rich plasma (c-PRP), while apyrase modifies the extent, but not the slope, of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in the presence of EDTA. All this evidence suggests that RIPA in c-PRP, besides reflecting the interaction of GPIb with vWF, may also test other mechanisms of the platelet function including: assembly of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein complex, and possibly the release reaction.     

The influence of platelet aggregation inhibitors on metastasis formation in mice (3LL)

Summary Platelets were suggested to play a specific role in the haematogenous spread of experimental tumours. To test this hypothesis mice were treated with various inhibitors of platelet function (acetyl-salicylic acid, RA 233, ben-cyclan-, cyproheptadine). The effect of treatment on the development of lung colonies after i.v. tumour cell injection as well as on the formation of spontaneous metastases from the solid Lewis-lung carcinoma was evaluated. A significant increase of lung colonies after pretreatment with the platelet aggregation inhibitors was found. The effect of long term treatment on spontaneous metastasis formation gave equivocal results. The present investigations do not support the importance of the integrity of platelet function as a prerequisite for metastasis formation.

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Der Einfluß von Plättchenaggregationshemmern auf die hämatogene Metastasierung bei Mäusen (3LL)

Zusammenfassung Von verschiedenen Autoren wurde eine spezifische Rolle der Blutplättchen bei der hämatogenen Metastasierung angenommen. Zur Prüfung dieser Hypothese wurden Mäuse mit verschiedenen thrombocytenaktiven Pharmaka behandelt (Azetylsalicylsäure, RA 233, Benzyclan und Cyproheptadin), und der Effekt dieser Behandlung sowohl auf die Ausbildung von Lungenkolonien nach i.v. Tumorzellinjektion als auch auf die Spontanmetastasierung des Lewis-lung-Carcinoms wurde gemessen. Die Behandlung mit Plättchenaggregationshemmern führte zu einer signifikanten Zunahme der Lungenkolonien, während kein wesentlicher Einfluß auf die Spontanmetastasierung nachweisbar war. Die vorliegenden Ergebnisse sprechen nicht für eine pathogenetische Bedeutung der Thrombocyten bei der experimentellen Metastasierung.

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Supported by a grant from the 'Deutsche Forschungsgemeinschaft (Hi 213/2).

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Parts of the present paper are submitted by H.H. to the Faculty of Medicine (Essen) as M.D. thesis.     

The influence of platelet aggregation inhibitors on metastasis formation in mice (3LL).

Platelets were suggested to play a specific role in the haematogenous spread of experimental tumours. To test this hypothesis mice were treated with various inhibitors or platelet function (acetyl-salicylic acid, RA 233, bencyclan-, cyproheptadine). The effect of treatment on the development of lung colonies after i.v. tumour cell injection as well as on the formation of spontaneous metastases from the solid Lewis-lung carcinoma was evaluated. A significant increase of lung colonies after pretreatment with the platelet aggregation inhibitors was found. The effect of long term treatment on spontaneous metastasis formation gave equivocal results. The present investigations do not support the importance of the integrity of platelet function as a prerequisite for metastasis formation.     

Correlation of plasma serotonin changes with platelet aggregation in an in vivo dog model of spontaneous occlusive coronary thrombus formation

The role of platelets in contributing to occlusive coronary artery thrombus formation remains unresolved. A large number of studies have utilized in vitro techniques to study platelet aggregation. This report describes a model of spontaneous in vivo thrombus formation which involves application of current in the left circumflex coronary artery of the dog. Changes in mean coronary blood flow velocity (50% above control) are used to predict the point at which current can be discontinued without interrupting the ongoing process of thrombus formation. Thrombus formation proceeds to total vessel occlusion within 62 +/- 18 minutes after discontinuation of current. Coronary sinus plasma serotonin concentrations are used as an in vivo index of platelet aggregation during thrombus formation. Plasma serotonin levels increased only slightly above baseline levels during initial thrombus formation. Coronary sinus serotonin levels rose markedly after cessation of current, reaching a peak just prior to total vessel occlusion. The marked increase in serotonin concentration observed in the latter stages of thrombus formation strongly suggests that platelet aggregation is a significant factor in the evolution of an occlusive coronary thrombus.     

A monoclonal antibody to platelet glycoprotein IIb induces aggregation of platelet rich plasma

The monoclonal antibody (MAb) GAP 5.9 directed against platelet glycoprotein IIb is described. This MAb is able to induce aggregation of platelet rich plasma in the absence of further stimuli and likely recognizes a glycoprotein epitope not closely related to calcium binding sites on the molecule.     

Streptokinase inhibits acute platelet thrombus formation in stenosed dog coronary arteries

Previous evidence has suggested that plasmin, in addition to its proteolytic action on fibrin, may affect platelet function. To test the effects of plasmin generated in vivo by the thrombolytic agent streptokinase (SK) on platelet-dependent vascular occlusion, we have used a well-established canine model of experimental coronary artery stenosis which produces platelet aggregate-dependent cyclical variations in coronary blood flow. Infusion of SK into 22 dogs at doses sufficient to cause a systemic lytic state led to complete abolition of cyclical blood flow reductions (CFR's) at sites of coronary artery injury. Inhibition of coronary platelet occlusion was associated with marked prolongations of the bleeding time (from 3.2 ± 0.6 min before to 14 ± 5 min after SK infusion, mean ± SD, n = 22). Despite the striking effects of SK on in vivo platelet-vessel wall interactions, only platelet aggregation in response to collagen was diminished among the ex vivo parameters of platelet function that were studied simultaneously. Platelet aggregation in response to other agonists, thromboxane A2 production, monoclonal antibody binding to platelet membrane glycoprotein (Gp) IIb-IIIa, Gp Ib-dependent botrocetin-induced platelet aggregation and platelet levels of cyclic AMP were not significantly altered. Therefore the thrombolytic agent streptokinase appears to cause important inhibitory effects on in vivo platelet reactivity with injured vascular intimal surfaces, possibly due to localised changes in platelet aggregate formation in the microenvironment of exposed collagen. These findings suggest that plasmin generated by thrombolytic agents may exhibit platelet inhibitory activity, and that this effect may be important in reestablishing blood flow in certain forms of platelet-mediated arterial thromboses.