Béatrice Hechler

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.

The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells

© 1997 Federation of European Biochemical Societies.

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.

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors

Summary. Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP‐induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP‐induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca2+ influx. Binding studies, using the non‐ hydrolysable ligand [33P]2MeSADP, have shown that human platelets contain about 600 high‐affinity binding sites for 2MeSADP (Kd∼ 5 niw). These sites present pharmacological characteristics of a P2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP‐induced platelet aggregation but normal shape change display very low levels of [33P]2MeSADP binding sites.The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca2+ influx and the other a Gi protein‐coupled receptor responsible for Ca2+ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP‐induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP‐induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP‐induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP‐induced shape change and aggregation in platelets from both control and clopidogrel‐treated rats. A2P5P and A3P5P (100 μM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 μM) in control and clopidogrel‐treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM). A3P5P (1 m M) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.

Preparation of washed platelet suspensions from human and rodent blood.

Citrate is the preferred anticoagulant for blood collection, as EDTA damages platelets and heparin modifies their function (1). Citrate allows the rapid generation of plateletrich plasma (PRP), with a high yield of platelets; however, this method has certain disadvantages. In particular, the PRP preparation has a limited stability (no longer than 2 h) and contains plasma proteins, including enzymes. In addition, human platelet-rich plasma (PRP) prepared from blood collected into trisodium citrate (3.8% w/v) has a depressed ionic calcium concentration, which can cause platelet aggregation and release of substances during centrifugation (2). To overcome these different problems, a centrifugation technique has been developed for the isolation and washing of platelets from human or rodent blood anticoagulated with acid-citrate-dextrose (ACD). The cells are resuspended in a physiological buffer under well-defined conditions, notably the presence of plasmatic ionic calcium concentrations (2 mM) and the absence of coagulation factors or other plasma components. The method for isolation of human platelets by centrifugation and washing described by Cazenave et al. (3) is derived directly from the technique of Mustard et al. (4). Blood collected into ACD is used to prepare PRP, from which the platelets are isolated by successive centrifugation steps and resuspended in Tyrode’s buffer, an iso-osmotic phosphate buffer at pH 7.35 containing glucose (0.1%, w/v), human serum albumin (HSA) (0.35%, w/v), calcium (2 mM), and magnesium (1 mM). Prostacyclin (PGI2) is used to prevent transitory platelet activation during the preparation. Addition of apyrase (adenosine 5′-triphosphate diphosphohydrolase, EC 3.6.1.5) to the final suspending medium prevents the cells from becoming refractory to ADP and maintains their discoid shape (5). Suspensions of washed platelets prepared by this method are stable for 5–8 h at 37°C, compared with citrated PRP preparations, which are stable for no more than 2 h.

Mechanisms underlying FeCl3-induced arterial thrombosis.

See also Brill A. A ride with ferric chloride. This issue, pp 776–8.

P2 receptors and platelet function.

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA2 and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y1 and P2Y12 receptor subtypes, while the P2X1 receptor ligand-gated cation channel is activated by ATP. The P2Y1 receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y12 receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X1 receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.

Importance of Platelet Phospholipase Cγ2 Signaling in Arterial Thrombosis as a Function of Lesion Severity

Objective—Platelet activation occurs in response to adhesion receptors for von Willebrand factor (GPIb-V-IX) and collagen (GPVI and α2β1 integrin) acting upstream of phospholipase C (PLC) γ2. However, PLCβ transduces signals from Gαq protein-coupled receptors for soluble agonists (P2y1, TxA2/TP, and thrombin/PAR). A Gi-dependent pathway amplifies most of these responses. Methods and Results—To evaluate the role of adhesion receptors signaling in arterial thrombosis, PLCγ2 knockout mice were studied in blood perfusion assays over fibrillar collagen and in a laser-induced mesenteric artery model of thrombosis. In vitro, PLCγ2-deficient platelets formed a single layer incapable of generating a thrombus on collagen, whereas Gαq-deficient platelets formed reduced size aggregates compared with wild-type cells. In the in vivo model, PLCγ2−/− mice displayed defective thrombus formation in superficial lesions but productive thrombosis after a more severe laser injury. In contrast, resistance to thrombosis was observed in Gαq−/− mice in both levels of injury. Conclusions—These results demonstrate that signaling through PLCγ2 plays an important role in arterial thrombosis, but that its contribution depends on the severity of the vascular lesion.

Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization

Summary.  Background: We previously described a model of laser‐induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb‐IIIa and P2Y12 inhibition, whereas only deep injuries were sensitive to thrombin blockade. Objective: The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition. Results: A superficial injury was found to detach the endothelial cells and expose a collagen III‐ and IV‐rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)‐collagen and GPIb–von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI‐immunodepleted mice and still strongly inhibited in VWF−/− mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury. Conclusions: This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.