Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases.

Interindividual variability of the inhibitory effect of clopidogrel on platelet functions leading to clopidogrel resistance has been described in some patients with ischemic cardiovascular disease. A reliable laboratory test is therefore needed to identify patients insufficiently protected by this antiplatelet treatment. The phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an intraplatelet actin regulatory protein, is dependent on the level of activation of the platelet P2Y12 receptor, which is targeted by clopidogrel. The aim of this study was to use a flow cytometric VASP phosphorylation assay to evaluate the efficacy of clopidogrel therapy. The platelet reactivity index (PRI), expressed as a percentage, is the difference in VASP fluorescence intensity between resting (+PGE1) and activated (+ADP) platelets. In vitro, the PRI was strongly correlated with the inhibition of platelet aggregation induced by specific blockade of the P2Y12 receptor by the competitive antagonist AR-C69931MX (R = 0.72, P < 0.0001). Ex vivo, the PRI was 78.3 +/- 4.6% in 47 healthy donors, 79.0 +/- 4.1% in 34 patients not receiving clopidogrel and 61.1 +/- 17.0% in 33 patients treated with clopidogrel (P < 0.0001). In the clopidogrel group, the PRI values were widely dispersed (from 6.6 to 85.8%) and more than 30% of these patients had a PRI equivalent of values in patients not receiving clopidogrel. The flow cytometric analysis of VASP phosphorylation seems to be a suitable test to evaluate the efficacy of clopidogrel treatment. This assay demonstrated a wide interindividual variability of the inhibitory response of platelets to clopidogrel and showed that one-third of the patients treated appeared to be 'unprotected' by this therapy.

Mpl ligand increases P2Y1 receptor gene expression in megakaryocytes with no concomitant change in platelet response to ADP.

The P2Y(1) receptor is responsible for the initiation of platelet aggregation in response to ADP and plays a key role in thrombosis. Although this receptor is expressed early in the platelet lineage, the regulation of its expression during megakaryocyte differentiation is unknown. In the mouse megakaryocytic cell line Y10/L8057, we detected P2Y(1) mRNA of three sizes (2.5, 4.4, and 7.4 kb). These cells have previously been shown to respond to Mpl ligand, the pivotal regulator of megakaryocytopoiesis, by increasing their expression of differentiation markers. Mpl ligand enhanced levels of P2Y(1) mRNAs in Y10/L8057 cells and this effect was selective: the same cytokine did not increase levels of A2a adenosine receptor mRNA. Although Mpl ligand did not affect the short half-lives of the P2Y(1) mRNAs, it enhanced transcription of the P2Y(1) gene. It also increased cell size and the number of cell surface P2Y(1) receptors, but not P2Y(1) receptor density. Injection of Mpl ligand into mice up-regulated P2Y(1) receptor mRNAs in megakaryocytes, as shown by in situ hybridization. However, platelets isolated from these mice did not exhibit a higher P2Y(1) receptor density or increased reactivity to ADP. This correlates with the finding that Mpl ligand increases GPIIb mRNA in megakaryocytes but not the density of the protein per platelet. Thus, the enhancement of P2Y(1) receptor expression induced by Mpl ligand in megakaryocytes may be an integral feature of their differentiation, whereas clinical use of this compound might not be associated with platelet hyper-reactivity to ADP.

Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. (Blood. 2001;98:1038-1046)

Key role of the P2Y(1) receptor in tissue factor-induced thrombin-dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.

BACKGROUND: ADP plays a key role in hemostasis, acting through 2 platelet receptors: the P2Y(1) receptor and an unidentified P2 receptor, called P2cyc, coupled to adenylyl cyclase inhibition, which is the target of the antiplatelet drug clopidogrel. We showed that the P2Y(1) receptor is an essential cofactor in thrombotic states induced by intravenous infusion of collagen and epinephrine. The aim of the present study was to assess the role of this receptor in thrombin-dependent tissue factor-induced thromboembolism. METHODS AND RESULTS: Human thromboplastin was injected intravenously into wild-type or P2Y(1)-deficient mice, and the effects on platelet count and mortality were determined and plasma thrombin-antithrombin III (TAT) complexes were quantified. P2Y(1)-deficient mice were resistant to the thromboembolism induced by injection of thromboplastin. Whereas the platelet count decreased sharply in wild-type mice, there was no significant drop in platelets in P2Y(1)-knockout mice. The platelet consumption in wild-type mice was probably due to thrombin generation, because it was abolished by hirudin. Thromboplastin also led to a rise in TAT complexes in plasma, again reflecting thrombin formation. This effect, however, was less important in P2Y(1)-knockout mice than in wild-type mice, indicating that less thrombin was generated in the absence of P2Y(1). Similar results were obtained after intravenous administration of N:(6)-methyl-2'-deoxyadenosine-3':5'-bisphosphate, a selective antagonist of the P2Y(1) receptor, to wild-type mice. CONCLUSIONS: Our results demonstrate a role of the P2Y(1) receptor in thrombotic states involving thrombin generation and provide further evidence for the potential relevance of this receptor as a target for antithrombotic drugs.

A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion.

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry.

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.

Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells.

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbalpha in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

Use of von Willebrand diseased kidney as donor in a pig-to-primate model of xenotransplantation.

BACKGROUND: The coagulation process in hyperacute and delayed xenograft rejection is essential and depends upon platelet adhesion and aggregation. The initial binding of platelets to the damaged endothelium is due to the interaction of the platelet receptor glycoprotein Ib with von Willebrand factor (vWF), which is present on activated endothelial cells and bound to the subendothelial matrix. We hypothesized that the use of organs from animals with homozygous von Willebrand disease (vWD), severely deficient in vWF, might prevent the thrombosis encountered in delayed xenograft rejection. METHODS: Ten baboons were treated by extracorporeal immunoadsorption of xenoreactive natural antibodies (XNA) through the donor pig liver to inhibit hyperacute rejection and received heterotopic vWD or control pig kidney xenografts. XNA levels, coagulation, and platelet activation markers were studied, and specimens of rejected kidneys were analyzed histologically. RESULTS: Although XNA depletion was comparable in both groups, neither kidney function nor survival times of control (n=5) or vWD (n=5) porcine kidneys showed any difference. Platelet and coagulation activation was evidenced in both groups after surgery and at rejection time. Immunohistochemical analysis revealed a weak endothelial vWF immunostaining in the rejected vWD kidneys, whereas it was undetectable in the nongrafted vWD kidneys, suggesting the deposition of baboon plasma vWF on the porcine vessels. CONCLUSIONS: The use of vWD organs did not improve the survival time of grafted kidneys in this xenotransplantation model. Further studies on the use of vWD organs, in association with other therapeutic approaches, such as complement inhibition, are nevertheless necessary to evaluate the usefulness of vWF deficiency as an adjunctive therapy to decrease the coagulation process during xenograft rejection.

cADP-ribose formation by blood platelets is not responsible for intracellular calcium mobilization.

Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of D-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets.