Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation.

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro. This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39-deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment.

The endothelium is morphologically and functionally adapted to meet the unique demands of the underlying tissue. At the present time, little is known about the molecular basis of endothelial cell diversity. As one approach to this problem, we have chosen to study the mechanisms that govern differential expression of the endothelial cell-restricted von Willebrand factor (vWF) gene. Transgenic mice were generated with a fragment of the vWF gene containing 2,182 bp of 5' flanking sequence, the first exon and first intron coupled to the LacZ reporter gene. In multiple independent lines of mice, beta-galactosidase expression was detected within endothelial cells in the brain, heart, and skeletal muscle. In isogeneic transplantation models, LacZ expression in host-derived auricular blood vessels was specifically induced by the microenvironment of the heart. In in vitro coculture assays, expression of both the transgene and the endogenous vWF gene in cardiac microvascular endothelial cells (CMEC) was upregulated in the presence of cardiac myocytes. In contrast, endothelial cell levels of thrombomodulin protein and mRNA were unchanged by the addition of ventricular myocytes. Moreover, CMEC expression of vWF was not influenced by the addition of 3T3 fibroblasts or mouse hepatocytes. Taken together, the results suggest that the vWF gene is regulated by vascular bed-specific pathways in response to signals derived from the local microenvironment.

Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2 adrenergic receptor cDNA.

Cardiac pacemaking offers a unique opportunity for direct gene transfer into the heart. An experimental system was developed to assay the effects of transferring the human beta2 adrenergic receptor (beta2AR) under in vitro, ex vivo, and finally in vivo conditions. Constructs encoding either beta2AR or LacZ were used in chronotropy studies with isolated myocytes, and transplanted as well as endogenous murine hearts. Murine embryonic cardiac myocytes were transiently transfected with plasmid constructs. The total percentage of myocytes spontaneously contracting was greater in beta2AR transfected cells, as compared with control cells (67 vs. 42+/-5%). In addition, the percentage of myocytes with chronotropic rates > 60 beats per minute (bpm) was higher in the beta2AR population, as compared with control cells (37 vs. 15+/-5%). The average contractile rate was greater in the beta2AR transfected myocytes at baseline (71+/-14 vs. 50+/-10 bpm; P < 0.001) as well as with the addition of 10(-)3 M isoproterenol (98+/-26 vs. 75+/-18 bpm; P < 0.05). Based on these results, a murine neonatal cardiac transplantation model was used to study the ex vivo effects of targeted expression of beta2AR. The constructs were transfected into the right atrium of transplanted hearts. Injection of the beta2AR construct increased the heart rate by approximately 40% (224+/-37 vs. 161+/-42 bpm; P < 0.005). Finally, the constructs were tested in vivo with injection into the right atrium of the endogenous heart. These results were similar to the ex vivo data with injection of the beta2AR constructs increasing the endogenous heart rates by approximately 40%, as compared with control injected hearts (550+/-42 vs. 390+/-37 bpm; P < 0.05). These studies demonstrate that local targeting of gene expression may be a feasible modality to regulate the cardiac pacemaking activity.

Neonatal plasminogen displays altered cell surface binding and activation kinetics. Correlation with increased glycosylation of the protein.

Plasminogen isolated from 60 full-term newborns differs from adult plasminogen in carbohydrate composition, kinetic activation constants, and cell binding. Amino acid composition and amino-terminal sequence analysis data indicate that the plasminogens of neonates and adults have the same amino acid sequence. Like the adult, the neonate has two glycoforms, but both have significantly more mannose and sialic acid than the adult forms. The difference in the neonatal glycosylation is probably responsible for the altered migration observed by isoelectric focusing. Moreover, the difference in carbohydrate composition appears to be the basis of the decreased functional activity of the neonatal plasminogen. The kcat/Km ratios indicate that the overall activation rates of the two neonatal plasminogen glycoforms are lower compared with the adult glycoforms. In addition, neonatal plasminogen does not bind as well to cellular receptors compared with adult plasminogen. These studies suggest a basis for the decreased fibrinolytic activity observed in neonates.

PDGF mediates cardiac microvascular communication.

The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.

A murine model of myocardial microvascular thrombosis.

Disorders of hemostasis lead to vascular pathology. Endothelium-derived gene products play a critical role in the formation and degradation of fibrin. We sought to characterize the importance of these locally produced factors in the formation of fibrin in the cardiac macrovasculature and microvasculature. This study used mice with modifications of the thrombomodulin (TM) gene, the tissue-type plasminogen activator (tPA) gene, and the urokinase-type plasminogen activator (uPA) gene. The results revealed that tPA played the most important role in local regulation of fibrin deposition in the heart, with lesser contributions by TM and uPA (least significant). Moreover, a synergistic relationship in fibrin formation existed in mice with concomitant modifications of tPA and TM, resulting in myocardial necrosis and depressed cardiac function. The data were fit to a statistical model that may offer a foundation for examination of hemostasis-regulating gene interactions.

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis.

Regulation of vascular bed-specific prothrombotic potential.

Hemostasis is the result of interdependent and complex systemic and local endothelial pathways that govern vascular integrity and rheology. A striking feature of hypercoagulable conditions is the focal nature of the resultant thrombotic pathology. Such disorders in hemostasis may be associated with distinct vascular beds, thus implying that the relative combined contribution of individual regulatory pathways may be specific and/or unique to a particular locale in the vasculature. Systemic factors and platelets mediate the formation of fibrin deposition; however, it is the diverse interrelationships in the interaction of these systemic elements with the local endothelial components that dictate vascular bed-specific hemostatic regulation. Indeed, the local activation of coagulation cascades, rather than increases in systemic thrombotic potential, is what leads to fibrin formation in different vascular beds. Hence, the propensity for congenital or acquired disorders to result in local thrombotic pathology is based on the relative contribution of the various hemostatic regulatory pathways in individual vascular beds. The present review highlights the role of local endothelial regulation in the interaction between local and systemic elements that contribute to vascular bed-specific prothrombotic potential.

Lipoprotein (a) in the regulation of fibrinolysis.

Elevated plasma levels of lipoprotein(a) [LP(a)] are associated with increased an risk of developing atherosclerosis. This increased risk may be due to an Lp(a)-mediated depression of fibrinolytic activity. Lp(a) regulates fibrinolysis by controlling the activity of plasminogen activators. Lp(a) is a low density lipoprotein with an apoprotein(a) subunit which has a high degree of homology with the fibrinolytic zymogen plasminogen. The apoprotein(a) subunit contains up to thirty seven copies of a domain homologous to the plasminogen kringle 4 domain, which enables Lp(a) to bind to fibrin. The subunit also has a zymogen domain, but it is not activated by plasminogen activators. Lp(a) inhibits plasminogen activation by competing with plasminogen for access to plasminogen activators bound to vascular surfaces. Lp(a) also competes with the irreversible inhibitor of plasminogen activators, plasminogen activator inhibitor-1. Therefore increases in Lp(a) concentration may decrease fibrinolytic activity by preventing activation of plasminogen, but Lp(a) may also prolong plasminogen activation by preventing the irreversible inhibition of the activators. At elevated levels of Lp(a) the decreased rate of plasmin generation may not be offset by the prolongation in plasminogen activation, and fibrinolysis will be inhibited.

Lipoprotein(a) inhibits plasminogen activation in a template-dependent manner.

Lipoprotein(a) [Lp(a)] is a low density lipoprotein whose plasma levels strongly correlate with the occurrence of atherosclerotic disease. Structural studies have demonstrated that Lp(a) contains two disulphide bonded subunits, one of which has structural similarity to plasminogen. This subunit, designed apo-lipoprotein(a), contains multiple repeat copies of a kringle homologous to kringle-4 of plasminogen, one copy of a kringle-5-like structure and a domain homologous to the catalytic light chain of plasmin. This subunit, however, lacks the site where plasminogen activators cleave plasminogen to generate the active proteinase. Recent studies demonstrate that Lp(a) competes with plasminogen for binding to endothelial cells and macrophages and thus prevents assembly of the fibrinolytic system on cell surfaces. Lp(a) also inhibits activation of plasminogen by streptokinase, urokinase-type plasminogen activator or tissue-type plasminogen activator (t-PA). Inhibition of plasminogen activation by t-PA requires the presence of a template on which activation occurs. This template can be either fibrin or heparin. This review considers the role of Lp(a) as an inhibitor of template-dependent activation of the fibrinolytic system.

Aging-associated changes in vascular activity: a potential link to geriatric cardiovascular disease.

Ischemic cardiovascular disease is a common cause of morbidity and mortality in the United States population over the age of 65. Prior clinical studies have demonstrated that the severity of cardiovascular pathophysiology is increased in older individuals. Both in vitro and in vivo experimental studies have shown that age-associated clinical events parallel changes in vascular function. Aging is associated with systemic as well as cardiac alterations in three basic vascular regulatory functions: vascular tone, hemostasis, and vascular repair/angiogenesis. This article reviews the molecular and cellular events that may contribute to senescent cardiac pathology. Indeed, a better understanding of the biology of aging-associated vascular dysfunction is fundamental for the development of therapeutics targeted for the treatment of cardiovascular disease in older individuals.

p38 mitogen-activated protein kinase targets the production of proinflammatory endothelial microparticles.

BACKGROUND: Endothelial microparticles (EMPs) are irregularly shaped membrane fragments shed into the circulation in patients with vascular diseases, and may themselves act to enhance the endothelial response to inflammation. On the basis of the importance of p38 mitogen-activated protein kinase (MAPK) in endothelial responses to inflammatory stimuli, we sought to define the role of p38 in EMP generation and function. METHODS: Microparticle generation from cultures of human aortic endothelial cells (hAECs) treated with tumor necrosis factor-alpha (TNF-alpha) and p38 inhibition was quantified via multiple modalities. The response of target endothelial cells was assessed by treatment of cells with EMPs generated under various conditions. RESULTS: Inhibition of p38 in hAECs, using pharmacologic agents, resulted in a 50% reduction of TNF-alpha-induced EMPs. Importantly, suppression of microparticles was specific to p38 MAPK pathways. EMPs triggered by TNF-alpha activation induced an approximately four-fold increase in soluble intercellular adhesion molecule-1 (sICAM-1) release from targeted cells. However, inhibition of p38 MAPK in the targeted cell prior to EMP treatment did not alter the sICAM1 response. CONCLUSIONS: Our findings implicate p38 MAPK signaling as significant and selective in the formation and maturation of EMPs. EMPs elicited a proinflammatory response from targeted hAECs that was dependent on the conditions under which EMPs were generated. However, our results imply a unidirectional model in which p38 MAPK is critical at the source of microparticle formation, but not the target cell response to EMPs. These findings indicate a novel mechanism by which p38 inhibition may offer therapeutic benefit in vivo via direct inhibition of EMP formation.

Lipoprotein (a) promotes plasmin inhibition by alpha 2-antiplasmin.

Plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) is regulated by the vascular components fibrin(ogen) fragments, plasminogen and lipoprotein (a). Kinetic analysis demonstrates that CNBr-derived fibrinogen fragments completely protect plasmin from alpha 2AP. Plasminogen and 6-aminohexanoic acid decrease the rate of inhibition by 5- and 10-fold respectively. These studies show that CNBr-derived fibrinogen fragments and 6-aminohexanoic acid bind plasmin kringle(s) with binding constants of 2 micrograms/ml and 120 microM respectively, and that plasminogen binds to alpha 2AP with an affinity of 0.5 nM. The unmodulated inhibition is not effected by the presence of lipoprotein (a), but in the presence of protective CNBr-derived fibrinogen fragments the rate of inhibition is increased by the presence of the lipoprotein. The kinetics demonstrate that lipoprotein (a) binds to CNBr-derived fibrinogen fragments with an affinity of 4 nM, displacing plasmin from the protective surface. In addition, tissue-type plasminogen activator and trypsin inhibition by alpha 2AP is not slowed by the presence of CNBr-derived fibrinogen fragments or plasminogen (Pg), respectively. These kinetics suggest that the initial reversible interaction between plasmin and alpha 2AP is mediated by binding of the inhibitor to the kringle 1 domain of plasmin, with a reversible inhibition constant (Ki) of 5.0 x 10(-10) M. Under conditions where this kringle-inhibitor interaction is blocked, the reversible inhibition still occurs between the plasmin and alpha 2AP, but the initial Ki is increased to 5.0 x 10(-9) M. These data suggest that, in the circulation, plasmin inhibition by alpha 2AP may be down-regulated by fibrin, fibrin(ogen) fragments and Pg, but up-regulated by lipoprotein (a) in the presence of fibrin or fibrin(ogen) fragments. The lipoprotein (a)-mediated promotion of plasmin inhibition may provide an additional mechanism by which the lipoprotein impairs fibrinolysis and promotes atherosclerosis.

Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation.

Heparin sulfate and the less sulfated glycosaminoglycan heparan sulfate enhance human plasminogen (Pg) conversion to plasmin by tissue-type plasminogen activator (t-PA). Kinetic studies indicate that both heparin and heparan increase the kcat of t-PA-mediated Pg activation by 25- and 3.5-fold, respectively. The Km of plasmin formation is unaltered by the presence of either heparin or heparan. Both heparin and heparan stimulate the activity of t-PA by interacting with the finger domain of t-PA, with association constants of 1 microM and 200 nM, respectively. Additionally, the lipoproteins lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) inhibit the heparin enhancement of Pg activation. Lp(a) is a competitive inhibitor and LDL is a mixed inhibitor of t-PA-mediated Pg activation, with inhibition constants of 30 and 70 nM, respectively. The inhibition constants correspond to physiologic concentrations of these lipoproteins. These data suggest that heparin, heparan, and lipoproteins may play an important in vivo role in regulating cell surface associated activation of the fibrinolytic system.

Heparin oligosaccharides enhance tissue-type plasminogen activator: a correlation between oligosaccharide length and stimulation of plasminogen activation.

The rate of plasminogen (Pg) activation by tissue-type Pg activator (t-PA) is enhanced by heparin-derived oligosaccharides. Kinetic analysis of the effects of heparin oligosaccharides, ranging in size from di- to dodecasaccharides, on Pg activation demonstrates that stimulation of the reaction is dependent on the size of the heparin oligosaccharides. Di- and tetrasaccharides enhance the activation through 2-fold increases in kcat and 4-fold decreases in Km. Hexasaccharide and larger oligosaccharides stimulate the reaction by increasing the kcat by as much as 4-fold, but do not affect the Km. Previous experiments have shown that lipoprotein(a) [Lp(a)] inhibits Pg activation by t-PA, but only in the presence of a template which enhances t-PA activity such as fibrinogen fragments or intact heparin. Similiarly, Lp(a) inhibits the enhancement of t-PA activity by the larger heparin oligosaccharides but has no effect on t-PA activity in the presence of di- and tetrasaccharides. The results of this study when considered with our previous observations (Edelberg & Pizzo, 1990) suggest that the enhancement in Pg activation by the smaller oligosaccharides is mediated exclusively via binding to t-PA while the larger oligosaccharides may interact with both t-PA and Pg. Furthermore, studies of Pg activation in the presence of both heparin oligosaccharides and fibrinogen fragments demonstrate that t-PA is stimulated preferentially by fibrinogen fragments.

Kinetic analysis of the effects of glycosaminoglycans and lipoproteins on urokinase-mediated plasminogen activation.

The glycosaminoglycans (GAGs) heparin, heparan sulphate and chondroitin 6-sulphate stimulate the rate of urokinase activation of human plasminogen. Kinetic analysis of plasminogen activation demonstrates that heparin, heparan sulphate and chondroitin 6-sulphate increased the catalytic rate (Kcat) by 5.3-, 3.5- and 2.5-fold respectively. These stimulatory GAGs had no effect on the affinity of urokinase for plasminogen, since the Km of the reaction is unaltered by the GAGs. The GAGs may enhance the rate of plasminogen activation through an interaction with the catalytic domain of the urokinase, with dissociation constants of approx. 30 nM. Additionally, the lipoproteins, lipoprotein (a) [Lp(a)] and low-density lipoprotein (LDL) inhibit heparin and heparan sulphate stimulation of plasmin formation. Lp(a) is a competitive inhibitor (Kic 20 nM) and LDL is a mixed inhibitor of heparin-enhanced urokinase-mediated plasminogen activation (Kic 24 nM and Kiu 60 nM). These inhibition constants correlate with physiological concentrations of these lipoproteins. These data suggest that these GAGs and lipoproteins may play an important role in vivo in regulating urokinase-mediated plasmin formation.

Further characterization of the cellular plasminogen binding site: evidence that plasminogen 2 and lipoprotein a compete for the same site.

Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types and serve to promote fibrinolysis and local Pg proteolysis. Pg types 1 and 2, separated by chromatography on concanavalin A-Sepharose, were utilized to determine their binding to the monocytoid U937 cell line. Both forms bind in a dose-dependent manner. However, Pg 2 binds to the cellular receptor considerably better than Pg 1 and at equilibrium demonstrates approximately 10-fold greater binding. Lipoprotein a [Lp(a)], which possesses a subunit showing considerable homology to Pg, competes with Pg 2 for the Pg receptor in U937 cells. Moreover, Pg 1 is not able to displace Pg 2 from the receptor. These studies suggest that high levels of Lp(a) may alter the profibrinolytic activity at the cell surface and increase the risks of atherosclerosis and thrombosis. This hypothesis is in accord with the 2-5-fold increased risk of atherosclerosis in patients having high levels of Lp(a).

Lipoprotein a inhibits streptokinase-mediated activation of human plasminogen.

Lipoprotein a [Lp(a)] inhibits human plasminogen (Pg) conversion to plasmin (Pm) by streptokinase- (SK-) mediated activation. Kinetic and binding studies indicate that Lp(a) inhibits Pg activation by competitive and uncompetitive inhibition. Lp(a) competes with Pg for SK and forms a stable complex. Lp(a) does not, however, inhibit Pg activation by the proteolytic SK-Pm complex. The SK-Pg and SK-Pg(act) intermediate complexes are possible targets of the Lp(a) uncompetitive inhibition. The competitive inhibition constant (Kic) is 45 nM or 14 mg/dL, and the uncompetitive inhibition constant (Kiu) is 140 nM or 42 mg/dL, corresponding to physiologic and pathophysiologic Lp(a) concentrations, respectively.