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Biomedical subjects

F A Ofosu

Publications and source records attributed to F A Ofosu.

At least 19 recordsLinked to original sources
PubMed2002

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C).

Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had significantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation.

Adolescent↗
PubMed2002

The blood platelet as a model for regulating blood coagulation on cell surfaces and its consequences.

Platelets actively participate in regulating thrombin production following physical or chemical injury to blood vessels. Injury to blood vessels initiates activation of the large numbers of platelets that appear in the subendothelium where they become exposed to tissue factor and to molecules adhesive for platelets and normally found in the extracellular matrix. The complex of plasma factor VIIa with extravascular tissue factor both initiates and localizes thrombin production on platelets and on extravascular cells. Thrombin production at these sites in turn enhances platelet activation and the subsequent hemostatic plug formation to minimize bleeding. Thrombin production and platelet activation also initiate the process of wound healing requiring thrombin-dependent cell activation and platelet-dependent formation of new blood vessels (angiogenesis). Activated platelets release from their storage granules several proteins and other factors that regulate local thrombin formation and the responses of blood vessel cells to injury to assure hemostasis and effective wound healing. Failure to localize and adequately regulate thrombin production and/or platelet activation can have pathological consequences, including the development and propagation of atherosclerosis and enhancement of tumor development. The primary basis for the pathological consequences of the failure to adequately regulate thrombin production is that the multi-functional thrombin activates several types of cells to initiate their mitogenesis. Mitogenesis precedes many of the undesirable consequences of poorly regulated thrombin production and platelet activation. In addition, activated platelets release a variety of products which influence the functions of several cell types to the extent that inadequate regulation of platelet activation (by excessive thrombin production) could contribute to the pathogenesis of acute and chronic arterial thrombosis and to tumor development. Activated platelets participate in tumor development by releasing several factors that positively (and negatively) regulate blood vessel formation.

Blood Coagulation↗
PubMed2001

Sustained and therapeutic delivery of factor IX in nude haemophilia B mice by encapsulated C2C12 myoblasts: concurrent tumourigenesis.

This study reports the generation of an immunodeficient murine model for haemophilia B, obtained by breeding factor IX-deficient mice with an immunodeficient mouse strain, and use of this mouse model to evaluate the long-term efficacy and safety of a gene therapy strategy for treating haemophilia B. Nude haemophilic mice were implanted with biocompatible microcapsules enclosing recombinant myoblasts secreting human factor IX. The activated partial thromboplastin time (APTT) of plasma of mice thus treated was invariably shortened 3 weeks after microcapsule implantation, and remained shortened for at least 77 days. Shortening of the APTT of the haemophilia mice coincided with the appearance of human factor IX in mice plasmas (up to 600 ng mL(-1) on day 77), and normalization of the tail-bleeding time. Thus, the microencapsulated myoblasts reversed the clinical phenotype of haemophilia B. In contrast, plasmas of immunocompetent haemophilic mice similarly implanted with microcapsules only showed a transient shortening of APTT, and coincident transient delivery of human factor IX antigen. Rapid disappearance of human factor IX from plasmas of immunocompetent mice also coincided with production of antibodies to the human transgene. Significantly, 86% of the nude haemophilia mice developed tumours of myoblast origin. Thus, while this study revealed the feasibility of this gene therapy approach to treat severe haemophilia B, it also highlights the importance of using safer cell lines to prevent tumour development.

Animals↗
PubMed2000

Activation and active site occupation alter conformation in the region of the first epidermal growth factor-like domain of human factor VII.

The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.

Allosteric Site↗
PubMed2000

Statin drugs and dietary isoprenoids as antithrombotic agents.

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

Animals↗
PubMed2000

Human platelet thrombin receptors. Roles in platelet activation.

Platelets are essential participants in hemostasis and thrombosis. Platelets normally circulate in blood as discoid resting cells that become critical constituents of hemostatic plugs or arterial thrombi only after specific receptors on platelet membranes interact with their ligands (agonists) to initiate the reactions that lead to platelet activation. The well-characterized events associated with platelet activation include activation of membrane receptors, shape change, granular secretion, cytoskeletal reassembly, platelet cohesion, and aggregation. The plasma protease alpha-thrombin is the most potent physiologic platelet agonist; this enzyme has other key roles in hemostasis, in the genesis of arterial thrombi, and in embryonic development, inflammation, wound healing, and cell proliferation.

Blood Platelets↗
PubMed2000

Delivery of recombinant product from subcutaneous implants of encapsulated recombinant cells in canines.

Delivering recombinant therapeutic proteins from a universal microencapsulated cell line is an alternate method for gene therapy. It has proved effective in the treatment of several murine models of human genetic diseases. However, in scaling up to large animal models, intraperitoneal Implantations of these microcapsules in canines were associated with excessive Inflammatory response and rapid degradation. We now show that subcutaneous implantation of microencapsulated cells in canines is effective in delivering recombinant product systemically for extended periods, provides a surgically benign site, leads to less inflammatory response, and permits longer-term survival of microcapsules. Allogeneic MDCK cells engineered to secrete human growth hormone (hGH) were microencapsulated in alginate-poly-L-lysine-alginate and implanted subcutaneously. Systemic delivery of hGH was evident within 4 hours and peaked by day 1 after implantation in all dogs. The gradual decline of hGH in the circulation in the first 2 weeks coincided with the development of anti-hGH antibodies by day 11. The high titer persisted for more than 1 month, demonstrating indirectly the persistent delivery of hGH. Microcapsules retrieved from the subcutaneous implant maintained their structure throughout the experiment and were free of host cellular adhesions. The mechanical integrity of the subcutaneously implanted microcapsules also appeared superior to that of the intraperitoneal implant. Hence the subcutaneously implanted microcapsules required minimal surgical intervention and led to a low level of inflammatory response, and the implant survived for at least 1 month, thus demonstrating the feasibility of systemic delivery of recombinant products via subcutaneous implantation in large animals.

Animals↗
PubMed2000

The cleaved peptide of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the surface-connected canalicular system.

The only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PARI) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbalpha, and TM60, directed against the thrombin binding site on GPIbalpha). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbalpha binding sites for von Willebrand factor and thrombin.

Animals↗
PubMed1999

Persistent delivery of factor IX in mice: gene therapy for hemophilia using implantable microcapsules.

Severe hemophilia B is a life-threatening, life long condition caused by absence of or defective coagulation factor IX. Gene therapy could provide an alternative treatment to repeated injection of plasma-derived concentrate or recombinant factor IX. We have previously described the use of implantable microcapsules containing recombinant myoblasts to deliver human factor IX in mice. This study reports the generation of improved myoblast-specific expression vectors. Mouse myoblast clones transfected with the various vectors secreted factor IX in vitro, at rates between 70 and 1000 ng/10(6) cells/day. The recombinant myoblast clones were then encapsulated and implanted into mice. Immunocompetent mice implanted with encapsulated myoblasts had up to 65 ng of factor IX per milliliter in their plasma for up to 14 days, after which antibodies to human factor IX became detectable, and this coincided with decreased factor IX in mouse plasma. In immunodeficient mice, however, factor IX delivery was maintained at a constant level for at least 6 weeks (end of experiment). Interestingly, the highest-secreting myoblast clone in vitro did not deliver the highest level of hFIX in vivo. This discrepancy observed between performance in vitro and in vivo may have important implications for the development of gene therapy protocols based on recombinant cells.

Animals↗
PubMed1998

A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation.

Protease-activated receptor-1 (PAR-1) is a G-protein-linked receptor on platelets and perivascular cells activated by alpha-thrombin and the PAR-1-activating peptide, SFLLRN. alpha-Thrombin activates PAR-1 by cleaving it at R41-S42 to release the 41-residue peptide TR(1-41). Unexpectedly, platelet activation with SFLLRN was also associated with PAR-1 cleavage and the release of TR(1-41). Both PAR-1 cleavage and platelet activation resulting from SFLLRN addition to platelets were markedly inhibited by the serine protease inhibitor 4, 2-(aminoethyl)-benzene sulphonylfluoride.HCl (pefabloc SC) and soybean trypsin inhibitor, but not by inhibitors of calpain, cysteine proteases or metalloproteases. Thus, a trypsin-like platelet protease propagates SFLLRN-dependent PAR-1 cleavage and platelet activation.

Collagen↗
PubMed1998

Factor VII deficiency caused by a structural variant N57D of the first epidermal growth factor domain.

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.

Animals↗
PubMed1998

Thrombin and antithrombotics.

From injury through healing, thrombin has several important functions in blood clotting, subsequent clot lysis, and tissue repair. These include edema, inflammation, cell recruitment, cellular releases, transformations, mitogenesis, and angiogenesis. Thrombin also participates in disease states, such as venous thrombosis, coronary thrombosis, stroke, and pulmonary emboli, among others and is implicated in atherosclerosis, the growth and metastasis of certain cancers, Alzheimer's disease, and perhaps other conditions. Thrombin must be continually generated to sustain normal and pathogenic processes. This is because of a variety of consumptive mechanisms. Unlike other activated factors in thrombotic and fibrinolytic pathways, and because thrombin promotes its own generation (feedback and cellular activation), thrombin is a primary target for therapeutics. Besides recombinant hirudins, Argatroban (Novastan) and Bivalirudin (Hirulog) are promising thrombin-directed inhibitors for antithrombotic intervention.

Blood Coagulation↗
PubMed1998

Pharmacological actions of sulodexide.

This report summarizes the results of some of the studies that have evaluated the pharmacokinetic, pharmacodynamic, anticoagulant, and antithrombotic properties of Sulodexide, which consists of a mixture of electrophoretically fast moving heparin (80% of the mass) and dermatan sulfate (the balance), with an average product (Mr) <8000. The low molecular weight (Mr) of the constituents of Sulodexide would predict that the product has the high bioavailability associated with low-Mr heparin and low-Mr dermatan sulfate. Given orally, subcutaneously, or by intravenous injection, Sulodexide exhibits antithrombotic and profibrinolytic properties in several animal models of venous and arterial thrombosis and has relatively high affinity for endothelial (and possibly other) cells. Additionally, in a large multicenter clinical trial involving 3986 patients who had recovered from acute myocardial infarction, oral Sulodexide was associated with a 32% reduction in death and a significant reduction of left ventricular thrombus formation. Compared with heparin, low-Mr heparin, and unfractionated and low-Mr dermatan sulfates, the doses of Sulodexide required for antithrombotic efficacy suggest that the combination of heparin and dermatan sulfate in Sulodexide provides a more effective antithrombotic mechanism than heparin/low-Mr heparins (which catalyze the antiprotease actions of antithrombin III) or dermatan sulfate/low-Mr dermatan sulfate (which catalyze thrombin inhibition by heparin cofactor II).

Animals↗
PubMed1997

Binding of thrombin to the G-protein-linked receptor, and not to glycoprotein Ib, precedes thrombin-mediated platelet activation.

The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as alpha-thrombin-binding sites on platelets remain controversial. alpha-Thrombin has been proposed to bind to both GPIb and the hirudin-like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is alpha-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with >/=0.5 nM alpha-thrombin for 10-60 s at 37 degrees C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 10(11) platelets/liter) after their incubation with 10 nM alpha-thrombin for 10 s. Parallel binding of alpha-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein-linked receptor abrogated alpha-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by </=1.0 nM (but not 10 nM) alpha-thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on alpha-thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein-linked receptor, abrogated alpha-thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported alpha-thrombin-binding site on GPIb inhibited neither alpha-thrombin binding to nor activation of the platelets. Thus, alpha-thrombin binds to and cleaves the G-protein-linked receptor when it activates platelets, and GPIb does not appear to serve as an important binding site when alpha-thrombin activates platelets.

Antibodies, Monoclonal↗
PubMed1996

Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B.

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

Alginates↗
PubMed1996

Plasmin accelerates platelet-dependent prothrombinase formation without activating the platelets.

Patients with acute myocardial infarction who undergo thrombolytic therapy may shortly thereafter present evidence for increased platelet activation and thrombin activity, and recurrent thrombosis. This study investigated whether plasmin activates platelets and prothrombin in recalcified platelet-rich plasma (RPRP) to cause (at least in part) these side-effects of thrombolytic therapy. Plasmin (0.1 and 1.0 CU/ml) addition to RPRP with microM r-tick anticoagulant peptide (the latter a factor Xa inhibitor which abrogates prothrombin activation by prothrombinase at the concentration used) resulted in no change in the concentration of prothrombin fragment 1 + 2, or in the expression of GMP-140, the resting and activated GP IIb-IIIa conformers, and GPIb on platelets. Thus, plasmin neither activates platelets nor prothrombin in RPRP. However, plasmin accelerated platelet activation and secretion, and prothrombin fragment 1 + 2 production in RPRP. When combined with 1 microM r-tick anticoagulant peptide and 1 or 10 mM alpha-thrombin to RPRP, plasmin also increased the number of GMP-140 molecules expressed/platelet without enhancing alpha-thrombin binding to the platelets. Additionally, plasmin accelerated prothrombin activation when it was added to washed platelets resuspended in factor V depleted plasma simultaneously with 10 mM CaCl2, 10 nM alpha-thrombin for 10 s (to activate platelets and platelet factor V), followed by 4 microM hirudin and 1 nM factor Xa. Thus, plasmin potentiates the platelet release reaction in response to alpha-thrombin (probably by increasing the availability of factor V on the platelets) to enhance prothrombin activation in RPRP. These actions of plasmin may contribute to the increased platelet activation and thrombotic side-effects that can occur after thrombolytic therapy.

Blood Platelets↗