Mechanism of the interaction of human platelet profilin with actin.

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.

The actin-binding protein profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C.

Profilin is generally thought to regulate actin polymerization, but the observation that acidic phospholipids dissociate the complex of profilin and actin raised the possibility that profilin might also regulate lipid metabolism. Profilin isolated from platelets binds with high affinity to small clusters of phosphatidylinositol 4,5-bisphosphate (PIP2) molecules in micelles and also in bilayers with other phospholipids. The molar ratio of the complex of profilin with PIP2 is 1:7 in micelles of pure PIP2 and 1:5 in bilayers composed largely of other phospholipids. Profilin competes efficiently with platelet cytosolic phosphoinositide-specific phospholipase C for interaction with the PIP2 substrate and thereby inhibits PIP2 hydrolysis by this enzyme. The cellular concentrations and binding characteristics of these molecules are consistent with profilin being a negative regulator of the phosphoinositide signaling pathway in addition to its established function as an inhibitor of actin polymerization.

Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation.

Epidermal growth factor and platelet-derived growth factor can stimulate the production of the second messenger inositol trisphosphate in responsive cells, but the biochemical pathway for these signaling events has been uncertain because the reactions have not been reconstituted with purified molecules in vitro. A reconstitution is described that requires not only the growth factor, its receptor with tyrosine kinase activity, and the soluble phospholipase C-gamma 1, but also the small soluble actin-binding protein profilin. Profilin binds to the substrate phosphatidylinositol 4,5-bisphosphate and inhibits its hydrolysis by unphosphorylated phospholipase C-gamma 1. Phosphorylation of phospholipase C-gamma 1 by the epidermal growth factor receptor tyrosine kinase overcomes the inhibitory effect of profilin and results in an effective activation of phospholipase C-gamma 1.

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

Regulation of endothelial cell adhesion by profilin.

BACKGROUND: Although profilin is believed to be an essential regulator of the actin cytoskeleton in most cells, its precise role in mammalian cells remains unknown. We have used replication-incompetent adenovirus carrying the human profilin I cDNA as a means rapidly to increase the concentration of profilin in human aortic endothelial cells 12-31-fold above baseline--levels never before achieved in mammalian cells. RESULTS: The concentration of filamentous actin was not detectably affected by profilin overexpression. Actin stress fibers were, however, absent from areas of high profilin content in overexpressing cells, and the bulk of filaments was located at the periphery of the cells. We observed a gradient in the distribution of overexpressed profilin in migrating endothelial cells, with most profilin molecules concentrated near the advancing edge where focal contacts are being formed and focal adhesion proteins are located. Profilin overexpression resulted in increased recruitment of fibronectin receptors to the plasma membrane. Adhesion of endothelial cells to fibronectin was markedly and selectively increased by profilin overexpression. CONCLUSIONS: We conclude that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor.

The Pl(A2) polymorphism of integrin beta(3) enhances outside-in signaling and adhesive functions.

Genetic factors are believed to influence the development of arterial thromboses. Because integrin alpha(IIb)beta(3) plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the Pl(A1) or Pl(A2) polymorphic forms of alpha(IIb)beta(3). Soluble fibrinogen binding was no different between Pl(A1) and Pl(A2) cells, either in a resting state or when alpha(IIb)beta(3) was activated with anti-LIBS6. Pl(A1) and Pl(A2) cells bound equivalently to immobilized fibronectin. In contrast, significantly more Pl(A2) cells bound to immobilized fibrinogen in an alpha(IIb)beta(3)-dependent manner than did Pl(A1) cells. Disruption of the actin cytoskeleton by cytochalasin D abolished the increased binding of Pl(A2) cells. Compared with Pl(A1) cells, Pl(A2) cells exhibited a greater extent of polymerized actin and cell spreading, enhanced tyrosine phosphorylation of pp125(FAK), and greater fibrin clot retraction. These adhesion differences appear to depend on a signaling mechanism sensitive to receptor occupancy. Thus, the Pl(A2) polymorphism altered integrin-mediated functions of adhesion, spreading, actin cytoskeleton rearrangement, and clot retraction.

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

The possible role of group specific component (Gc) (vitamin D-binding protein) in the clearance of cellular actin entering the circulation was examined with 125I-labeled Gc and actin injected into a rabbit model. Although filamentous F-actin is depolymerized primarily by plasma gelsolin, greater than or equal to 90% 125I-actin injected in either monomeric G- or F-form became complexed eventually with Gc (1:1 molar ratio). Clearance of Gc complexes was much faster (greater than 90% within 5 h) than that of native Gc (t1/2 = 17.2 h). Nephrectomy did not significantly alter the clearance of either Gc or actin. Since Gc complexes are dramatically increased in situations of tissue necrosis such as in fulminant hepatic failure, the current results suggest a crucial role for Gc in sequestration and clearance of released cellular actin.

Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia.

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Redox signaling of the arteriolar myogenic response.

Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P(TM)). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P(TM) elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47(phox) or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H(2)O(2), but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). alpha(1)-Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H(2)O(2) constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H(2)O(2), contribute to the myogenic response of arteriolar VSMCs.

Redox regulation of vascular smooth muscle cell differentiation.

Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (alpha-actin, calponin, and SM1 and SM2 myosin), but not beta-actin. ROS activity, determined using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD; MnTMPyP, 25 micromol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC) decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L). None of the treatments altered beta-actin expression. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2). Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 micromol/L) reduced expression of differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38 MAPK-dependent pathway.

Utility of blood pressure genetic risk score in admixed Hispanic samples.

Hypertension is strongly influenced by genetic factors. Although hypertension prevalence in some Hispanic sub-populations is greater than in non-Hispanic whites, genetic studies on hypertension have focused primarily on samples of European descent. A recent meta-analysis of 200 000 individuals of European descent identified 29 common genetic variants that influence blood pressure, and a genetic risk score derived from the 29 variants has been proposed. We sought to evaluate the utility of this genetic risk score in Hispanics. The sample set consists of 1994 Hispanics from 2 cohorts: the Northern Manhattan Study (primarily Dominican/Puerto Rican) and the Miami Cardiovascular Registry (primarily Cuban/South American). Risk scores for systolic and diastolic blood pressure were computed as a weighted sum of the risk alleles, with the regression coefficients reported in the European meta-analysis used as weights. Association of risk score with blood pressure was tested within each cohort, adjusting for age, age2, sex and body mass index. Results were combined using an inverse-variance meta-analysis. The risk score was significantly associated with blood pressure in our combined sample (P=5.65 × 10-4 for systolic and P=1.65 × 10-3 for diastolic) but the magnitude of the effect sizes varied by degree of European, African and Native American admixture. Further studies among other Hispanic sub-populations are needed to elucidate the role of these 29 variants and identify additional genetic and environmental factors contributing to blood pressure variability in Hispanics.

Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system.

OBJECTIVE: Methylation of the promoter region of the estrogen receptor gene alpha (ER alpha) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. METHODS: We used Southern blot analysis to determine the methylation state of the ER alpha gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. RESULTS: An age related increase in ER alpha gene methylation occurs in the right atrium (range 6 to 19%, R = 0.36, P < 0.05). Significant levels of ER alpha methylation were detected in both veins and arteries. In addition, ER alpha gene methylation appears to be increased in coronary atherosclerotic plaques when compared to normal proximal aorta (10 +/- 2% versus 4 +/- 1%, P < 0.01). In endothelial cells explanted from human aorta and grown in vitro, ER alpha gene methylation remains low. In contrast, cultured aortic smooth muscle cells contain a high level of ER alpha gene methylation (19-99%). CONCLUSIONS: Methylation associated inactivation of the ER alpha gene in vascular tissue may play a role in atherogenesis and aging of the vascular system. This potentially reversible defect may provide a new target for intervention in heart disease.

Methylation of the estrogen receptor-alpha gene promoter is selectively increased in proliferating human aortic smooth muscle cells.

OBJECTIVE: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-alpha gene (ERalpha) promoter blocks the expression of ERalpha, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERalpha is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. METHODS: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERalpha in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). RESULTS: We provide evidence that methylation of the ERalpha in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERalpha promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS-M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERalpha and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. CONCLUSIONS: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERalpha, could contribute to the switch in phenotype observed in these cells.

Identification of a major endogenous substrate for phospholipid/Ca2+-dependent kinase in pancreatic acini as Gc (vitamin D-binding protein).

A major 56 kDa substrate for phospholipid/Ca2+-dependent kinase (C-kinase) in pancreatic acinar cells is physicochemically and immunologically indistinguishable from the vitamin D-binding protein, Gc or group-specific component. Cellular Gc was also phosphorylated in intact cells following treatment with carbachol as a physiological stimulus. These findings indicate the potential usefulness of Gc as a defined substrate for further studies of the biological role of C-kinase activity in pancreatic acini and possibly in other cells.

The actin cytoskeleton reorganization induced by Rac1 requires the production of superoxide.

The small GTPase rac1 controls actin redistribution to membrane ruffles in fibroblasts and other cell types, as well as the activation of the NADPH oxidase in phagocytes. We explored the possibility that these two processes could be related. We used a replication-deficient adenoviral vector to overexpress the constitutively active form of rac1, racV12, in human and mouse aortic endothelial cells. We show here that, in addition to membrane ruffle formation, racV12 induced an increase in the total amount of F-actin within endothelial cells. Concurrently, racV12-overexpressing cells produced significantly higher amounts of free radicals, as detected by the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate, than cells infected with a control virus encoding the bacterial beta-galactosidase (Ad-betaGal). To assess the specific role of superoxide in racV12-induced actin reorganization, we co-expressed the human enzyme Cu,Zn-superoxide dismutase (SOD), by means of another adenoviral vector construct. Overexpressed SOD reduced the concentration of superoxide detected in Ad-racV12-transfected cells and reversed the effects of Ad-racV12 on the content of filamentous actin. MnTMPyP, an SOD mimetic, as well as the antioxidant N-acetyl cysteine, had similar effects, in that they reduced not only the free radicals production, but also ruffle formation and the concentration of F-actin within racV12-overexpressing endothelial cells. Our data support the hypothesis that superoxide is one of the important mediators acting downstream of rac1 on the pathway of actin cytoskeleton remodeling in endothelial cells.

Fas-mediated apoptosis in accelerated graft arteriosclerosis.

Pathological conditions have been recognized where vessel destruction is a prominent feature of the pathogenic process. One such condition consists of the chronic rejection of blood vessels in transplanted solid organs. Accelerated graft arteriosclerosis (AGA) is a multifactorial process characterized by the concentric proliferation of smooth muscle cells (SMCs) within the intima of the vessel wall of transplanted organs. Proliferation of SMCs within the intima corresponds to a response of these cells to injury. In situations like restenosis post-angioplasty, the mechanism of injury: the mechanical disruption of the tunica media, is evident. However, in the case of AGA, the mechanism of injury has remained elusive. In this report, we provide evidence that injury to SMCs in AGA vessels requires an intact Fas pathway. The resulting damage to the tunica media and internal elastic lamina, in turn, might trigger the proliferation of intimal smooth muscle cells that appear to be less sensitive to Fas mediated killing, particularly when supported by a favorable context of inflammatory cytokines and growth factors, as it is the case in AGA. This pathogenic process results in a absolute loss of functional blood vessels that is not being compensated by an efficient angiogenic response.

The GPIIIa (beta3 integrin) PlA polymorphism in the early development of coronary atherosclerosis.

The GPIIIa (beta3 integrin) is an integral part of two glycoprotein receptors - the GP(IIb/IIIa) fibrinogen receptors in platelets and the GP(V/IIIa) vitronectin receptors in endothelium and vascular smooth muscle cells (vSMC). The PlA polymorphism of the gene for GPIIIa (beta3 integrin) has been suggested to play an important role in the progression of coronary artery disease (CAD) and in coronary thrombosis. Whether the action of the PlA polymorphism is due to differences in platelet aggregability or function of the vSMC and endothelial GPIIIa is not known. The association of the PlA polymorphism with the early, non-complicated atherosclerosis and CAD was studied in the Helsinki Sudden Death Study (HSDS) comprising two independent, autopsy series of altogether 700 middle-aged Caucasian Finnish men (33-70 year) suffering sudden out-of-hospital death. The burden of complicated lesions was greater in men with the A2 allele (heterozygotes or homozygotes for A2) (P=0.01) compared with PlA1/A1 homozygotes in the entire series. To further estimate the role of platelet-independent GPIIIa receptors, we excluded all cases with coronary thrombosis and thrombus-overlaid complicated lesions. In this subset of men, fibrous coronary lesions were more frequent (OR 2.9; P<0.01) in the coronary arteries of PlA1/A1 homozygotes compared with men with the PlA2 allele. Moreover, men with the PlA1/A1 genotype also had more stenotic coronary arteries (P<0.05) compared with men with the A2 allele at this early, non-complicated stage of atherosclerosis. The findings of this study suggest that Pl(A1/A1) homozygotes may be prone to early atherosclerosis and more rapid progression of stable CAD whereas carriers of the PlA2 allele are more prone to thrombotic complications. We hypothesize that the PlA polymorphism may account for the early atherosclerosis by affecting the function of endothelial and vSMC GP(V/IIIa) receptors, whereas the PlA polymorphism on platelet GP(IIb/IIIa) receptors may play a major role in coronary thrombosis.

Gender differences in platelet GPIIb-IIIa activation.

Gender differences in the development of thrombotic diseases have been described in numerous clinical settings. Enhanced platelet reactivity in both sexes is associated with the development of vascular thromboses. Because activation of platelet GPIIb-IIIa receptors is a central event in thrombus formation, we examined GPIIb-IIIa function in normal male and female volunteers. Using flow cytometry, we quantitated gender differences in the number of binding sites for FITC-labeled fibrinogen (FITC-FGN) and FITC-labeled PAC-1 antibody (FITC-PAC-1). Washed platelets were incubated with either FITC-FGN or FITC-PAC-1 and activated with either ADP or thrombin receptor activating peptide (TRAP) prior to cytometric acquisition of data. The dissociation constant for FITC-FGN was the same in both sexes (approx. 1.6 x 10(-7)M), however, the number of GPIIb-IIIa receptors per platelet capable of binding fibrinogen was significantly greater in women than men in response to 2 microM ADP (16,319 +/- 1871 vs 9669 +/- 1994, p = 0.02), 20 microM ADP (39,951 +/- 4711 vs 25,948 +/- 4953, p = 0.05) and 50 microM TRAP (39,236 +/- 3965 vs 21,848 +/- 4159, p = 0.007). Similarly, the number of GPIIb-IIIa receptors capable of binding PAC-1 in response to ADP and TRAP was 50% to 80% greater in women than men. Binding experiments using specific anti-GPIIb-IIIa monoclonal antibodies (P2 and 10E5), as well as quantitative Western blotting experiments, showed no gender difference in the total number of GPIIb-IIIa molecules expressed. Analysis of data from female subgroups demonstrated an association of GPIIb-IIIa reactivity with menstrual phase. We conclude that GPIIb-IIIa receptors on platelets from premenopausal women are more "activatable" than those on platelets from young men. Variations in the serum concentrations of estrogens and/or progestins may modulate GPIIb-IIIa function.