Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression.

OBJECTIVES: Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia--reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. METHODS AND RESULTS: Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg(-1)) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s(-1)) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. CONCLUSION: The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion.

Synthesis of GPIb beta with novel transmembrane and cytoplasmic sequences in a Bernard-Soulier patient resulting in GPIb-defective signaling in CHO cells.

The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.

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.

GPV is a marker of in vivo platelet activation--study in a rat thrombosis model.

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.

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.

Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA--its use to follow platelet storage in transfusion.

Thrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.

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.

Diets enriched in (n-3) fatty acids affect rat coagulation factors dependent on vitamin K.

The effects of dietary lipids on haemostasis were investigated in rats fed high fat diets enriched in saturated fatty acids (SAT), oleic acid (OLEIC), MaxEPA oil (MaxEPA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) and results were compared to those for rats fed standard chow (ST). Coagulant activities of factor IIc and factor VII-Xc were reduced by about 70% in the MaxEPA group and 50% in the EPA and DHA groups relative to the OLEIC, SAT and ST groups. Liver vitamin K levels were five times lower in the experimental groups than in the ST group, which would indicate an effect of high fat diets on vitamin K metabolism. However, only (n-3) fatty acids prolonged the prothrombin time. These components could act at the post-translational modification level of vitamin K-dependent plasma clotting factors. The changes in haemostatic factors found in the MaxEPA group were counteracted by vitamin K supplementation.

One-step assay for the determination of free protein S antigen in plasma using real-time biospecific interaction analysis.

Real-time biospecific interaction analysis based on optical detection by surface plasmon resonance was used to develop an accurate one-step method for the direct measurement of free protein S in human plasma. This assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protein S deficiency. The method relies on the specific capture of free protein S directly from plasma by a monoclonal antibody (mAb), 34G2, immobilized on a sensor chip surface. A calibration curve was established with serial dilutions of standard plasma (working range 5-50%) and a linear relationship was found to exist between the relative response in resonance units (RU) and the concentration of free protein S expressed as percentage plasma dilution (r = 0.99). The specificity of the assay was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissociation of C4b-BP-protein S complexes occurred under the chosen experimental conditions. The technique was reproducible with inter-assay, intra-assay and inter-sensor chip variation coefficients of 1.5-5.4%, 2-3.1% and 4.4-4.9%, respectively, as evaluated in two different plasma samples. Since all tests are automatic and long series of analyses can be performed with the same sensor chip, the method was applied to the determination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent correlation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-treated plasma (r = 0.93).

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.

Antibodies to human adhesion molecules and von Willebrand factor: in vitro cross-species reactivity in the xenotransplantation setting.

Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD3 1, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.

Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

PF4 inhibits thrombin-stimulated MMP-1 and MMP-3 metalloproteinase expression in human vascular endothelial cells.

We investigated whether PF4 could regulate the constitutive and thrombin-stimulated expression of metalloproteinases (MMPs) in endothelial cells (EC). PF4 inhibited the increase in the expression of MMP-1 and MMP-3 promoted by thrombin or the thrombin receptor agonist peptide SFLLRNPNDKYEPF (SFLL..) by 50% but did not modify the constitutive expression of these MMPs. This inhibitory effect was not mediated through a direct interaction of PF4 with thrombin or with the MMPs themselves. The interaction of PF4 with heparan sulfates at the surface of the EC appeared to be implicated in the inhibition mechanism of MMP-1 but not in that of MMP-3. MMP-1 transcription levels remained unchanged after PF4 treatment, whereas the increase in MMP-3 transcription induced by thrombin or SFLL.. was inhibited by approximately 50%. Expression of the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 was not affected by PF4. The present data provide new evidence that the antiangiogenic properties of PF4 involve the inhibition of matrix breakdown and suggest that this property of PF4 could be especially relevant in the context of thrombin-regulated tissue remodelling.

Species specific immunoassays to measure blood platelet and coagulation activation in the rat.

The purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thrombo-plastin (90 to 2400 microliters/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.