Monique Freund

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

A revised model of platelet aggregation

In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin alpha(IIb)beta(3) demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin alpha(IIb)beta(3) mediated cell arrest. Platelet aggregation under flow appears to be a multistep process involving: (a) exposure of vWf on the surface of immobilized platelets; (b) a reversible phase of platelet aggregation mediated by the binding of GPIbalpha on the surface of free-flowing platelets to vWf on the surface of immobilized platelets; and (c) an irreversible phase of aggregation dependent on integrin alpha(IIb)beta(3). Studies of platelet thrombus formation in vivo demonstrate that this multistep adhesion mechanism is indispensable for platelet aggregation in arterioles and also appears to promote platelet aggregate formation in venules. Together, our studies demonstrate an important role for platelet vWf in initiating the platelet aggregation process under flow and challenge the currently accepted view that the vWf-GPIbalpha interaction is exclusively involved in initiating platelet aggregation at elevated shear rates.

A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo.

The P2X1 receptor is a fast ATP-gated cation channel expressed in blood platelets, where its role has been difficult to assess due to its rapid desensitization and the lack of pharmacological tools. In this paper, we have used P2X1 −/− and wild-type mouse platelets, treated with apyrase to prevent desensitization, to demonstrate the function of P2X1 in the response to thrombogenic stimuli. In vitro, the collagen-induced aggregation and secretion of P2X1-deficient platelets was decreased, as was adhesion and thrombus growth on a collagen-coated surface, particularly when the wall shear rate was elevated. In vivo, the functional role of P2X1 could be demonstrated using two models of platelet-dependent thrombotic occlusion of small arteries, in which blood flow is characterized by a high shear rate. The mortality of P2X1 −/− mice in a model of systemic thromboembolism was reduced and the size of mural thrombi formed after a laser-induced vessel wall injury was decreased as compared with normal mice, whereas the time for complete thrombus removal was shortened. Overall, the P2X1 receptor appears to contribute to the formation of platelet thrombi, particularly in arteries in which shear forces are high.

Differential Involvement of the P2Y1 and P2Y12 Receptors in Platelet Procoagulant Activity

Objective—In vivo, activated platelets contribute to the initiation of thrombin generation through the exposure of phosphatidylserine to form a procoagulant catalytic surface and through platelet-leukocyte interactions, which lead to the exposure of leukocyte tissue factor (TF). On the basis of observations that the platelet P2Y1 and P2Y12 receptors both contribute to thrombosis and thrombin formation in an in vivo model of TF-induced thromboembolism, we further characterized the role of these receptors in thrombin generation. Methods and Results—By using the selective P2 antagonists MRS2179 and AR-C69931MX, the P2Y12 receptor was found to be involved in thrombin-induced exposure of PS on isolated platelets and consequently in TF-induced thrombin formation in platelet-rich plasma. By contrast, the P2Y1 receptor was not involved in phosphatidylserine exposure nor in thrombin generation in platelet-rich plasma. In addition, both receptors were found to contribute to the interactions between platelets and leukocytes mediated by platelet P-selectin exposure, which result in TF exposure at the surface of leukocytes. Conclusions—Overall, these results point to a differential involvement of the 2 platelet ADP receptors in the generation of thrombin and provide further evidence for the relevance of molecules targeting these receptors as antithrombotic agents.

Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation

Mutations in the MYH9 gene encoding nonmuscle myosin IIA lead to macrothrombocytopenia as observed in MYH9-related disorders. We used mice with megakaryocyte-restricted MYH9 inactivation to explore the role of myosin in thrombopoiesis. In situ, bone marrow MYH9Delta megakaryocytes were irregularly shaped, appearing leaky with poorly defined limits. The demarcation membranes were abnormally organized and poorly developed, pointing to an insufficient reservoir for the future formation of platelets. The cytoskeletal-rich peripheral zone was lacking due to the absence of the myosin filament network that normally surrounds the granular zone in wild-type cells. In vitro studies of cultured cells showed that MYH9Delta megakaryocytes were unable to form stress fibers upon adhesion to collagen, suggesting that the leaky shape results from defects in internal tension and anchorage to the extracellular environment. Surprisingly, the proportion of cells extending proplatelets was increased in MYH9Delta megakaryocytes and the proplatelet buds were larger. Overall, this study provides evidence for a role of myosin in different steps of megakaryocyte development through its participation in the maintenance of cell shape, formation and organization of the demarcation membranes and the peripheral zone, anchorage to the extracellular matrix, and proplatelet formation.

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Bernard-Soulier syndrome is caused by a deficiency of the platelet surface glycoprotein Ib-IX-V complex resulting in a severe platelet function defect. However this is compounded by a reduction in platelet numbers and the platelets in the circulation are abnormally large. As for many other platelet disorders, recent evidence and findings of this study suggest that the defective morphology has its origins in megakaryocyte function. See related perspective article on page 756. Background Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia. Design and Methods To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells. Results The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ−/− megakaryocytes could be differentiated in culture from Lin− fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ−/− proplatelet buds in cultured and circulating platelets. Conclusions Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.

Reduced Atherosclerotic Lesions in P2Y1/Apolipoprotein E Double-Knockout Mice: The Contribution of Non-Hematopoietic-Derived P2Y1 Receptors

Background— The P2Y1 receptor plays a key role in arterial thrombosis and is widely expressed in many cell types involved in atherosclerosis. The aim of this study was to evaluate its potential involvement in the development of atherosclerotic lesions. Methods and Results— Apolipoprotein E–deficient (ApoE−/−) and P2Y1−/−/ApoE−/− mice were maintained on regular chow for 17 or 30 weeks before analysis of atherosclerotic lesions. At 17 weeks, lesions in the aortic sinus and entire aorta were smaller in P2Y1−/−/ApoE−/− compared with those in ApoE−/− animals. At 30 weeks, the aortic sinus lesions in P2Y1−/−/ApoE−/− mice were still diminished in size and displayed reduced inflammation, reflected by decreased macrophage infiltration and diminished VCAM-1 immunostaining, compared with those in ApoE−/− mice. They also had a lower smooth muscle cell content. Unexpectedly, bone marrow transplantation showed that the absence of the P2Y1 receptor in blood cells only led to no significant modification of the lesion compared with control ApoE−/− reconstituted animals. Conversely, the absence of the P2Y1 receptor except in blood cells resulted in a reduction in lesion size similar to that in control P2Y1−/−/ApoE−/− reconstituted mice, pointing to a role of non–hematopoietic-derived P2Y1 receptors, most likely the endothelial or smooth muscle cell P2Y1 receptors. In addition, although this was not statistically significant, plasma cholesterol levels were consistently decreased in P2Y1−/− animals, suggesting that a modification of lipid metabolism could be responsible for the observed phenotype. Conclusion— The P2Y1 receptor contributes to atherosclerosis, primarily through its role in non–hematopoietic-derived cells.Background— The P2Y 1 receptor plays a key role in arterial thrombosis and is widely expressed in many cell types involved in atherosclerosis. The aim of this study was to evaluate its potential involvement in the development of atherosclerotic lesions. Methods and Results— Apolipoprotein E–deficient ( ApoE −/− ) and P2Y 1 −/− / ApoE −/− mice were maintained on regular chow for 17 or 30 weeks before analysis of atherosclerotic lesions. At 17 weeks, lesions in the aortic sinus and entire aorta were smaller in P2Y 1 −/− / ApoE −/− compared with those in ApoE −/− animals. At 30 weeks, the aortic sinus lesions in P2Y 1 −/− / ApoE −/− mice were still diminished in size and displayed reduced inflammation, reflected by decreased macrophage infiltration and diminished VCAM-1 immunostaining, compared with those in ApoE −/− mice. They also had a lower smooth muscle cell content. Unexpectedly, bone marrow transplantation showed that the absence of the P2Y 1 receptor in blood cells only led to no significant modification of the lesion compared with control ApoE −/− reconstituted animals. Conversely, the absence of the P2Y 1 receptor except in blood cells resulted in a reduction in lesion size similar to that in control P2Y 1 −/− / ApoE −/− reconstituted mice, pointing to a role of non–hematopoietic-derived P2Y 1 receptors, most likely the endothelial or smooth muscle cell P2Y 1 receptors. In addition, although this was not statistically significant, plasma cholesterol levels were consistently decreased in P2Y 1 −/− animals, suggesting that a modification of lipid metabolism could be responsible for the observed phenotype. Conclusion— The P2Y 1 receptor contributes to atherosclerosis, primarily through its role in non–hematopoietic-derived cells.

A PLCγ2-independent platelet collagen aggregation requiring functional association of GPVI and integrin α2β1

The role of the phospholipase C (PLC)γ2 isotype in platelet activation was evaluated by studying PLCγ2 −/− mice. These mice have a prolonged bleeding time but their platelets respond normally to non‐collagenous agonists. PLCγ2‐null platelets show residual aggregation response to collagen fibres (6% versus 74% for wild‐type) with minimal granule secretion and no shape change. A delayed shape change is observed at later aggregation times. Specific activation by glycoprotein (GP)VI agonists (convulxin, collagen‐related peptide and GPVI crosslinking) is, however, abolished. Antibodies against integrin α2β1 and GPVI each inhibit the residual collagen response, implying a role of α2β1 in platelet activation and a functional association with GPVI. These responses are also prevented by blocking integrin αIIbβ3 and phosphoinositide 3‐kinase, whereas aspirin treatment and ADP receptor blockade only inhibit shape change. These results provide evidence for a PLCγ2‐independent collagen activation pathway requiring cooperation between GPVI and α2β1 leading to αIIbβ3‐dependent aggregation and shape change by released ADP and thromboxane A2.

Decreased Thrombotic Tendency in Mouse Models of the Bernard-Soulier Syndrome

Objective—The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIb&bgr; subunit. Methods and Results—Complete deletion (GPIb&bgr;−/−) or an intracellular truncation (GPIb&bgr;&Dgr;IC−/−) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIb&bgr;&Dgr;IC−/−. Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl3-induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIb&bgr;−/− mice, while a more modest effect was observed in GPIb&bgr;&Dgr;IC−/− animals. Finally, the two strains were protected against death in a model of systemic thromboembolism. Conclusions—This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.

A Humanized Glycoprotein VI (GPVI) Mouse Model to Assess the Antithrombotic Efficacies of Anti-GPVI Agents

Glycoprotein VI (GPVI) has been proposed as a promising antiplatelet target, because its blockade prevents experimental thrombosis without impairing hemostasis. The objective of this study was to develop a preclinical tool to evaluate the role of human GPVI (hGPVI) in various models of thrombosis and to screen anti-GPVI compounds. A genetically modified mouse strain expressing hGPVI has been developed using a knockin strategy. The mice were viable and fertile and did not present any hematological defects. Approximately 3700 copies of human GPVI were detected at the platelet surface. Platelet aggregation, fibrinogen binding, and P-selectin exposure were normal in response to various agonists. The 9O12.2 Fab fragment directed against human GPVI bound to hGPVI platelets in vitro and ex vivo and markedly reduced collagen- and collagen-related peptide-induced responses. Injection of 9O12.2 into hGPVI animals did not prolong the tail bleeding time but provided protection against lethal thromboembolism induced by a collagen/adrenaline mixture. In addition, 9O12.2 reduced arterial thrombus growth by 44% after superficial laser injury, 43% after deep laser injury in mice pretreated with hirudin, and 48% after mechanical injury. In conclusion, we have developed a humanized mouse model that could be used in preclinical studies to evaluate the effects of anti-GPVI compounds.