Progress in the development of antiplatelet agents: Focus on the targeted molecular pathway from bench to clinic.

Antiplatelet drugs serve as a first-line antithrombotic therapy for the management of acute ischemic events and the prevention of secondary complications in vascular diseases. Numerous antiplatelet therapies have been developed; however, currently available agents are still associated with inadequate efficacy, risk of bleeding, and variability in individual response. Understanding the mechanisms of platelet involvement in thrombosis and the clinical development process of antiplatelet agents is critical for the discovery of novel agents. The functions of platelets in thrombosis are regulated by two major mechanisms: the interaction between surface receptors and their ligands, and the downstream intracellular signaling pathways. Recently, most of the progress made in antiplatelet drug development has been achieved with P2Y receptor antagonists. Additionally, the usage of GP IIb/IIIa receptor antagonists has decreased, because it is associated with a higher risk of bleeding and thrombocytopenia. Agents targeting other platelet surface receptors such as PARs, TP receptor, EP3 receptor, GPIb-IX-V receptor, P-selectin, as well as intracellular signaling factors, such as PI3Kß, have been evaluated in an attempt to develop the next generation of antiplatelet drugs, reduce or eliminate interpatient variability of drug efficacy and significantly lower the risk of drug-induced bleeding. The aim of this review is to describe the pathways of platelet activation in thrombosis, and summarize the development process of antiplatelet agents, as well as the preclinical and clinical evaluations performed on these agents.

Aerobic training improves platelet function in type 2 diabetic patients: role of microRNA-130a and GPIIb.

AIMS: MicroRNAs (miRs) that are mediators of gene expression have been implicated in type 2 diabetes mellitus (T2DM). Platelet hyper-reactivity is one of the most important disorders in T2DM patients. In this study, we explored the effects of aerobic training (AT) on platelet aggregation and Glycoprotein IIb (GPIIb) receptor and miR-130a expression. METHODS: In a quasi-experimental controlled trial, 24 sedentary, eligible female participants with T2DM were selected (age 61.92 ± 3.63) and divided into AT and control (CON) groups based on their peak oxygen consumption (VO2peak). AT protocol was performed three times per week in non-consecutive days on a treadmill with mean intensity (60-75% VO2peak) for 8 weeks, while the control group refrained from any type of exercise training. Two blood samples were taken before and after this period. Real-time PCR was used to determine the expression of platelet GPIIb and miR-130a. Moreover, platelet indices (PLT, MPV, PDW, and PCT), collagen-induced platelet aggregation and glycemic variables were measured. RESULTS: Analyses of data showed that anthropometric variables, VO2peak and glycemic control improved significantly (P < 0.01) after AT. Furthermore, MPV, PDW (P < 0.01), and platelet aggregation (P < 0.001) decreased significantly following AT compared with control group. Platelet GPIIb expression down-regulated significantly (P < 0.05) in AT group but up-regulation of miR-130a expression was not significant between two groups (P > 0.05). CONCLUSIONS: Platelet hyper-reactivity in T2DM females might be decreased not only by glycemic control and amelioration of anthropometric and platelet indices, but also the down-regulation of GPIIb following AT. However, more research is needed to determine the effects of exercise training on platelet miR-130a.

Selective Tropism of Dengue Virus for Human Glycoprotein Ib.

Since the hemorrhage in severe dengue seems to be primarily related to the defect of the platelet, the possibility that dengue virus (DENV) is selectively tropic for one of its surface receptors was investigated. Flow cytometric data of DENV-infected megakaryocytic cell line superficially expressing human glycoprotein Ib (CD42b) and glycoprotein IIb/IIIa (CD41 and CD41a) were analyzed by our custom-written software in MATLAB. In two-dimensional analyses, intracellular DENV was detected in CD42b+, CD41+ and CD41a+ cells. In three-dimensional analyses, the DENV was exclusively detected in CD42b+ cells but not in CD42b- cells regardless of the other expressions. In single-cell virus-protein analyses, the amount of DENV was directly correlated with those of CD42b at the Pearson correlation coefficient of 0.9. Moreover, RT- PCR and apoptosis assays showed that DENV was able to replicate itself and release its new progeny from the infected CD42b+ cells and eventually killed those cells. These results provide evidence for the involvement of CD42b in DENV infection.

Imaging the dynamic platelet-neutrophil response in sterile liver injury and repair in mice.

UNLABELLED: Although platelets have been extensively studied in hemostasis and inflammation, their role is not well understood in sterile liver injury and repair. Using a thermally induced focal liver injury and repair model and multichannel spinning disk confocal microscopy allowed visualization of the dynamic behavior of platelets and neutrophils in this insult. Platelets instantaneously adhered to molecularly altered sinusoidal endothelium adjacent to the afflicted area, paving approximately 200 µm abutting the injury. Platelets remained adherent for at least 4 hours, but dissipated by 8 hours. The early recruitment occurred by GPIIbIIIa (CD41) and the later recruitment was dependent upon both GPIIbIIIa and GPIb (CD42B). Platelets did not occlude the vessels, but rather paved the altered endothelium. Endothelin-induced vasoconstriction by hepatic stellate cells, and not platelet accumulation or coagulation, was responsible for temporarily restricted perfusion around the injury. Neutrophils crawled into the injury from significant distances through the sinusoids. The crawling neutrophils required the platelet-paved endothelium given that very little neutrophil recruitment was noted in thrombocytopenic or CD41-deficient mice. As platelets slowly dissipated, neutrophil recruitment was also halted. Previous work suggested that platelets binding to immobilized neutrophils induced neutrophil extracellular trap (NET) formation in response to infection as well as during thrombosis and other forms of sterile injury. In this model of neutrophils crawling on immobilized platelets, very few NETs were observed and no additional injury was noted. In fact, GPIIbIIIa-deficient mice had delayed repair. CONCLUSION: In a liver model of sterile injury and repair, platelets play a critical role in forming a substratum and pave the way for neutrophils to enter the injured site for subsequent repair.

Proteasome function is required for platelet production.

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

(alpha)IIb Integrin, a novel marker for hemopoietic progenitor cells.

Integrin (alpha)IIb(beta)3 (abbreviated as (alpha)IIb), also known as GPIIb-IIIa or CD41/CD61, is a cell adhesion molecule expressed on cells belonging to the megakaryocytic lineage. Aiming to identify new markers of hemopoietic progenitor cells (HPC), we undertook a developmental study of this molecule since it remains controversial if this integrin is expressed by various progenitors. We reported the expression pattern of two integrins, in both of which the beta3 chain is present, respectively associated with alphaV and alpha IIb in the chick embryo. While at E3.5, the earliest time at which these integrins can be detected, (alpha)V(beta)3 becomes expressed by endothelial cells in the aorta (and only in the aorta), (alpha)IIb(beta)3 becomes detected in the well-defined intra-aortic clusters made up of HPC. The latter were found to be multilineage progenitors when sorted for (alpha)IIb expression and analyzed by means of clonogenic assays. In mice also, (alpha)IIb is expressed in the intra-embryonic site of HPC generation, the intra-arterial clusters in the embryo proper, as well as in sites where HPC migrate. Finally we provided the first evidence in two species that multipotent HPC expressing (alpha)IIb are able to differentiate not only into cells of the erythroid and myeloid lineages but also into lymphocytes. These cell populations actually coexpress (alpha)IIb and c-Kit. These data establish (alpha)IIb as a novel marker for HPC, which appears at very early stages in the embryo. Capitalizing on this finding, other investigators confirmed it and suggested that (alpha)IIb plays a role in regulating hematopoietic development.

GPIIb (CD41) integrin is expressed on mast cells and influences their adhesion properties.

OBJECTIVES: GPIIb integrin expression has been found on platelets and megakaryocytes, and more recently on immature hematopoietic progenitors. We set out to investigate expression of GPIIb in other hematopoietic cell lineages and, having detected it on mast cells, aimed to determine what possible role it might perform. METHODS: We have made use of cultured human and murine bone marrow mast cells (BMMC) in order to characterize the expression of GPIIb. Further, BMMC cultures from wild type and GPIIb deficient (gpIIb-/-) mice were used for comparison of the adhesive properties mediated by this receptor. Finally, peritoneal mast cells were analyzed from both wild type and (gpIIb-/-) mice. RESULTS: We demonstrate expression of GPIIb on cultured BMMC. Using cells derived from mice homozygous for a null allele of gbIIb we show that the absence of GPIIb has no effect on mast cells with respect to a number of measures of cell growth and differentiation. However, loss of GPIIb on BMMC results in an increase in surface expression of aV integrin, the alternative partner of GPIIIa. CONCLUSION: The results in this study demonstrate that GPIIb is expressed in human and murine mast cells. A function for GPIIb on mast cells is suggested by the altered adhesion of gbIIb-/- BMMC to fibronectin- and vitronectin-coated surfaces. Moreover, comparison of mast cells from the peritoneal cavity of wild type and gbIIb-/- mice indicates that GPIIb could influence the in vivo differentiation or homing of tissue mast cells.

The beta3 subunit of the integrin alphaIIbbeta3 regulates alphaIIb-mediated outside-in signaling.

Bidirectional signaling is an essential feature of alphaIIbbeta3 function. The alphaIIb cytoplasmic domain negatively regulates beta3-mediated inside-out signaling, but little is known about the regulation of alphaIIb-mediated outside-in signaling. We show that alphaIIb-mediated outside-in signaling is enhanced in platelets of a patient lacking the terminal 39 residues of the beta3 cytoplasmic tail. This enhanced signaling was detected as thromboxane A(2) (TxA(2)) production and granule secretion, and required ligand cross-linking of alphaIIbbeta3 and platelet aggregation. This outside-in signaling was specifically inhibited by a palmitoylated version of a beta3 peptide corresponding to cytoplasmic domain residues R724-R734. Unlike the palmitoylated peptide, the nonpalmitoylated beta3 peptide could not cross the platelet membrane and did not inhibit this outside-in signaling. The physiologic relevance of this beta3-mediated negative regulation of alphaIIb outside-in signaling was demonstrated in normal platelets treated with the palmitoylated peptide and a physiologic agonist. Binding of alphaIIbbeta3 complexes to immobilized peptides demonstrated that a peptide corresponding to beta3 residues R724-R734 appears to bind to an alphaIIb cytoplasmic domain peptide containing residues K989-D1002, but not to control peptides. These results demonstrate that alphaIIb-mediated outside-in signaling resulting in TxA(2) production and granule secretion is negatively regulated by a sequence of residues in the membrane distal beta3 cytoplasmic domain sequence RKEFAKFEEER.

A push-pull mechanism for regulating integrin function.

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.

Platelet depletion by anti-CD41 (alphaIIb) mAb injection early but not late in the course of disease protects against Plasmodium berghei pathogenesis by altering the levels of pathogenic cytokines.

Accumulating evidence indicates that platelets play a critical role in the pathogenesis of experimental severe malaria (ESM) elicited by infection with Plasmodium berghei. Mice injected on day 1 of P berghei infection (early) with either anti-CD41 or anti-CD61 monoclonal antibodies (mAbs) exhibited significantly (P<.001) increased survival from ESM compared with infection controls, indicating that platelets function early in the disease. In contrast, groups of mice treated on days 4, 5, and 6 (late) with anti-CD41 mAb exhibited similar mortality as controls. Because platelet depletion by anti-CD41 mAb on day 4 of infection did not protect mice, and platelet adherence occurs on day 6, platelet adherence to endothelium is not required to mediate malarial pathogenesis. Few platelet microparticles were detected in the blood during the course of malaria, but large numbers of erythrocyte vesicles, microparticles, and debris were detected. The protective effect of early anti-CD41 mAb treatment was independent of the number of platelets, platelet microparticles, erythrocyte-platelet conjugates, and erythrocyte vesicles. Mice treated early with anti-CD41 mAb exhibited markedly altered cytokine production on day 4 of P berghei infection (increased interleukin 10 [IL-10], IL-1alpha, IL-6, interferon-gamma [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]; decreased IL-2) but no decline in coagulation factors compared with rat immunoglobulin G (IgG)-treated controls, indicating that platelets regulate the levels of pathogenic cytokines.

The role of the platelet glycoprotein IIb/IIIa in thrombosis and haemostasis.

Haemostasis is a finely balanced and complex process ideally initiated only in response to disruption of the vascular endothelium as a means of preventing loss of blood from an injured vessel. Deviations from the ideal can lead to serious disease. Firstly, thrombosis, which arises as a consequence of inappropriate platelet-platelet interactions at a region of vessel damaged by atherosclerosis, can lead to occlusion of the affected vessel as in myocardial infarction or stroke. Secondly, loss of the ability of platelets to form aggregates leads to Glanzmann's thrombasthenia (GT) with a tendency to bleed for prolonged periods following injury. Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a major role in the regulation of platelet adhesion and aggregation during haemostasis. Upon platelet activation by an agonist a signalling process is initiated, termed "inside-out" signalling, which gives rise to conformational changes within GPIIb/IIIa. These conformational changes increase the affinity of the receptor for its primary ligand, fibrinogen. Bound fibrinogen then acts as a bridging molecule facilitating the interaction of adjacent platelets. Upon fibrinogen binding GPIIb/IIIa undergoes further conformational changes and through a process termed "outside-in" signalling the receptor signals in to the platelet ultimately resulting in acceleration of the aggregation process. Qualitative or quantitative abnormalities in GPIIb/IIIa give rise to GT, a recessive bleeding disorder, and analysis of affected individuals has provided invaluable insights into the structure/function relationship of this receptor. Due to its critical role in mediating platelet aggregate formation GPIIb/IIIa has become a primary target for the development of antithrombotic agents.

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells.

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.

Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif.

The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets.

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

Mechanisms of platelet retention in the collagen-coated-bead column.

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.

A mutation in the integrin alphaIIb subunit that selectively inhibits alphaIIb beta3 receptor function.

The alpha(IIb) and alpha(v) integrins have been shown to play a significant role in a variety of disease processes. alpha(IIb)beta(3) is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemostasis. Determination of the basis of ligand recognition by alpha(IIb)beta(3) is essential for modulation of platelet function. To identify alpha(IIb) residues involved in alpha(IIb)beta(3) ligand binding function, cells expressing a constitutively active variant of alpha(IIb)beta(3) were randomly mutagenized and selected for loss of alpha(IIb)beta(3) ligand binding function. One mutant isolated in this manner contained a single amino acid substitution at position 96 in alpha(IIb) (Ser9-->Leu). Cells expressing this alpha(IIb) mutant did not bind the ligand mimetic antibody PAC1 or adhere to fibrinogen. In addition, the mutant receptor did not bind to an RGD affinity matrix. Substitution of conserved serine residues at position 1 in beta strand A of all seven repeats of alpha(IIb) similarly inhibited ligand binding to alpha(IIb)beta(3). alpha(IIb)S96 maps to the central cavity of the beta-propeller fold of the alpha(IIb) subunit immediately adjacent to a structurally important sequence at the center of the alphaand beta subunit interface. In contrast, substitution of the analogous residues in alpha(v) or alpha(4) did not disrupt the ligand binding function of alpha(v)beta(3) or alpha(4)beta(1). These data support a potential unique structural or mechanistic role for this residue in alpha(IIb)beta(3) receptor function.

Platelet and osteoclast beta3 integrins are critical for bone metastasis.

Mice with a targeted deletion of beta3 integrin were used to examine the process by which tumor cells metastasize and destroy bone. Injection of B16 melanoma cells into the left cardiac ventricle resulted in osteolytic bone metastasis in 74% of beta3+/+ mice by 14 days. In contrast, only 4% of beta3-/- mice developed bone lesions. Direct intratibial inoculation of tumor resulted in marrow replacement by tumor in beta3-/- mice, but no associated trabecular bone resorption as seen inbeta3+/+ mice. Bone marrow transplantation studies showed that susceptibility to bone metastasis was conferred by a bone marrow-derived cell. To dissect the roles of osteoclast and platelet beta3 integrins in this model of bone metastasis, osteoclast-defective src-/- mice were used. Src-null mice were protected from tumor-associated bone destruction but were not protected from tumor cell metastasis to bone. In contrast, a highly specific platelet aggregation inhibitor of activated alphaIIbbeta3 prevented B16 metastases. These data demonstrate a critical role for platelet alphaIIbbeta3 in tumor entry into bone and suggest a mechanism by which antiplatelet therapy may be beneficial in preventing the metastasis of solid tumors.

Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria.

Some patients with Plasmodium falciparum infections develop cerebral malaria, acute respiratory distress, and shock and ultimately die even though drug therapy has eliminated the parasite from the blood, suggesting that a systemic inflammatory response contributes to malarial pathogenesis. Plasmodium berghei-infected mice are a well-recognized model of severe malaria (experimental severe malaria [ESM]), and infected mice exhibit a systemic inflammatory response. Because platelets are proposed to contribute to ESM and other systemic inflammatory responses, we determined whether platelet adherence contributes to experimental malarial pathogenesis. Indeed, a significant (P < 0.005) increase in the number of rolling and adherent platelets was observed by intravital microscopy in brain venules of P. berghei-infected mice compared with the number in uninfected controls. P-selectin- or ICAM-1-deficient mice exhibit increased survival after P. berghei infection. We observed a significant (P < 0.0001) reduction in the morbidity of mice injected with anti-CD41 (alpha(IIb) or gpIIb) monoclonal antibody on day 1 of P. berghei infection compared with the morbidity of infected controls injected with rat immunoglobulin G. Additionally, platelet rolling and adhesion in brain venules were reduced in P. berghei mice lacking either P-selectin or ICAM-1 or when the platelets were coated with anti-CD41 monoclonal antibody. Unlike other inflammatory conditions, we did not detect platelet-leukocyte interactions during P. berghei malaria. Because (i). leukocyte adhesion is not markedly altered in the absence of P-selectin or ICAM-1 and (ii). CD41 is not an adhesion molecule for parasitized erythrocytes, these findings support the hypothesis that inhibition of platelet adhesion to the brain microvasculature protects against development of malarial pathogenesis.

[Role of platelet membrane glycoprotein II b/III a receptor activation in no-reflow after myocardial reperfusion].

OBJECTIVE: To observe the changes in the activity of platelet glycoprotein (GP) IIb/IIIa receptor following the occurrence of no-reflow after myocardial reperfusion, to explore the measures for clinical management of this condition. METHODS: Nine canine models of no-reflow following myocardial ischemia verified by reperfusion myocardial contrast echocardiography were used in this investigation. The peripheral venous blood (PVB) and sinus venous blood (SVB) were sampled before myocardial reperfusion (after a 3-hour myocardial ischemia) and after a 3-hour reperfusion for determinating GP IIb/IIIa receptor activities by means of enzyme-linked immunosorbent assay (ELISA). RESULTS: The activity of platelet GP IIb/IIIa of PVB, either in 3-hour ischemic group or in 3-hour reperfusion group, was elevated significantly (P < 0.001), suggesting platelet activation. Elevated platelet GP IIb/IIIa receptor activity in SVB occurred only in 3-hour ischemic group, and the 3-hour reperfusion group (P < 0.001) and the no-reflow group had significantly decreased activities, which was more obvious in the latter group (P < 0.01). The findings signified the consumption of the platelet GP IIb/IIIa receptors in the myocardial microcirculation and the presence of platelet aggregation. CONCLUSIONS: Myocardial ischemia may activate the platelets to express GP IIb/IIIa receptors through stress response mechanism and damage the endothelia of the microvasculature to incur adhesion and aggregation of the platelets within the microcirculation, which may be one of the pathogeneses for myocardial no-reflow phenomenon.

The glycoprotein IIb molecule is expressed on early murine hematopoietic progenitors and regulates their numbers in sites of hematopoiesis.

The alpha integrin GPIIb is a marker of hematopoietic progenitors. Using a marking strategy based on Cre-loxP technology to trace the fate of GPIIb-expressing cells, we show that GPIIb is expressed during early definitive embryonic hematopoiesis. However, the marked fetal population is distinct from the hematopoietic cells that predominate in the adult, suggesting that at least two waves of progenitors arise concurrently or consecutively in the fetus. Furthermore, using an inactivated allele of gpIIb, we provide evidence for a functional role of GPIIb on progenitors. We observe an increase in hematopoietic progenitors in the yolk sac, fetal liver, and bone marrow, an effect which may, in part, be explained by loss of binding to fibronectin.