Effect of Vorapaxar Alone and in Combination with Aspirin on Bleeding Time and Platelet Aggregation in Healthy Adult Subjects.

The effect of the protease-activated receptor-1 (PAR-1) antagonist vorapaxar on human bleeding time is not known. This was a randomized, two-period, open-label trial in healthy men (n = 31) and women (n = 5). In period 1, subjects received 81 mg aspirin q.d. or a vorapaxar regimen achieving steady-state plasma concentrations equivalent to chronic 2.5 mg q.d. doses, for 7 days. In period 2, each group added 7 days of the therapy alternate to that of period 1 without washout. Bleeding time and platelet aggregation using arachidonic acid, ADP, and TRAP agonists were assessed. Bleeding time geometric mean ratio (90% CI) for vorapaxar/baseline was 1.01 (0.88-1.15), aspirin/baseline was 1.32 (1.15-1.51), vorapaxar + aspirin/vorapaxar was 1.47 (1.26-1.70), and vorapaxar + aspirin/aspirin was 1.12 (0.96-1.30). Unlike aspirin, vorapaxar did not prolong bleeding time compared with baseline. Bleeding time following administration of vorapaxar with aspirin was similar to that following aspirin alone.

Characterization and chromosomal localization of JAM-1, a platelet receptor for a stimulatory monoclonal antibody.

We have previously reported the purification and characterization of a 32 kDa platelet surface glycoprotein that is recognized by the stimulatory monoclonal antibody, F11. The cDNA has been cloned and found to encode the human homolog of the murine junctional adhesion molecule, JAM; we therefore named this human homolog JAM-1. Northern blot analysis indicated that JAM-1 mRNA is expressed as multiple species, the predominant transcript being approximately 4.0 kb in size. Genetic mapping analysis using fluorescence in situ hybridization (FISH) showed that it is localized to chromosome 1q21.1-21.3. Recombinant JAM-1, when expressed in Chinese hamster ovary (CHO) cells, localized to the cell membrane with intense staining where two adjacent cells actually made contact with each other, suggesting that, similar to murine JAM, human JAM-1 may also localize at the cell-cell junction. In well-spread cells, JAM-1 co-localized with F-actin at the cell-cell contacts and at the membrane ruffles, but not at the stress fibers. Interestingly, JAM-1 localizes only to the cell-cell junctions formed by two transfected cells and not to the cell-cell junctions formed by a transfected cell with an untransfected cell, suggesting that JAM-1 may facilitate cell adhesion through homophilic binding. In addition, human platelets specifically bind to a monolayer of CHO cells expressing human JAM-1, further supporting homophilic interactions. The results presented here indicate that JAM-1, a receptor for a platelet-activating antibody, is the human homolog of the junctional adhesion molecule. JAM-1 is a single copy gene, which is constitutively expressed on various tissues and cells, and may be involved in cell to cell adhesion through homophilic interaction.

Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain.

The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the alphaIIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calcium- and integrin-binding protein) that appears to be specific for the cytoplasmic domain of alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca2+-binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca2+ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45Ca2+ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein ( approximately 25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3 indicates a specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3.

A new protein kinase C, nPKC eta', and nPKC theta are expressed in human platelets: involvement of nPKC eta' and nPKC theta in signal transduction stimulated by PAF.

We have detected in human platelets two protein kinase C isozymes that have not been reported previously. Using an anti-nPKC theta antibody and Western blotting, we calculated the molecular weight of platelet nPKC theta as 79K. This molecular weight is identical to that described for nPKC theta in skeletal muscle and in COS cells transfected with the nPKC theta-cDNA. Using an anti-nPKC eta antibody, we determined the molecular weight of an immunoreactive protein, which we called nPKC eta', to be 95K. This molecular weight is higher than that of nPKC eta found in lung and skin tissue of 82K and 78K, and it is higher than nPKC eta of COS cells transfected with the nPKC eta-cDNA expression plasmid. Together with previous reports, these findings make the total number of PKC isozymes in human platelets equal to six. These are the PKC isozymes: alpha, beta, delta and zeta, which have been previously described, and eta' and theta which we describe here. To assess the functionality of these new PKC isoforms, we stimulated platelets with PAF. We found a 200% and 175% increase in the levels of membrane-bound nPKC eta' and nPKC theta, respectively, in human platelets stimulated by PAF. A concomitant decrease in the level of these isoforms in the cytoplasm was observed. This PAF-induced translocation was time-dependent, and it reached its peak after a 1 minute incubation of human platelets with PAF for nPKC theta and 30 seconds for nPKC eta'.

Cyclosporine A enhances agonist-induced aggregation of human platelets by stimulating protein phosphorylation.

Use of the immunosuppressant drug cyclosporine A (CSA) has resulted in improved renal graft survival. However, an increased incidence of arterial and venous thrombotic diseases, hemolytic-uremic type syndrome, and findings resembling vasculitis in the kidneys of patients with CSA nephrotoxicity and accelerated atherogenesis have been reported. These disorders may be related to CSA-induced abnormalities in platelet function. We report here that CSA causes increased ADP-stimulated aggregation in isolated platelet suspensions indicating that CSA has a direct effect on platelet function, independent of CSA interactions with plasma factors. Maximal hyperaggregability of ADP-stimulated platelets occurred following a 1 h preincubation period with CSA. Hyperaggregability of platelets due to the presence of CSA was dose-dependent and approached plateau between 200-500 ng/ml CSA. We determined that CSA exerted its effects through a signal transduction pathway involving the phosphorylation of two intracellular proteins, a 40 kD substrate of PKC (p47) and the 20 kD light chain of myosin (p20), a substrate of calcium/calmodulin dependent kinase. Preincubation with CSA resulted in a 200% increase in the phosphorylation of these proteins in platelets stimulated with ADP. We conclude that CSA enhances ADP-induced platelet aggregation and secretion, in part, by potentiating the phosphorylative response of specific intracellular proteins to stimulation by agonists. This process may be responsible for the increased thrombosis and atherogenesis observed in CSA-treated patients.

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.

Activation of human platelets by a stimulatory monoclonal antibody.

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.