[Show abstract][Hide abstract] ABSTRACT: The importance of β(2)-glycoprotein I (β(2)GPI)-specific CD4(+) T cells in the development of pathogenic processes in patients with antiphospholipid syndrome (APS) and APS mouse models is well established. Therefore, our objective is to manipulate the β2GPI specific CD4(+) T cells using tolerogenic dendritic cells (tDCs) to induce tolerance. We aim to evaluate the capability of tDCs to induce antigen-specific tolerance in effector/memory T cells from patients with APS and to elucidate the involved mechanism.
DCs and tDCs were produced from patients with APS peripheral-blood-monocytes, using specific cytokines. β(2)GPI-specific tolerance induction was investigated by coculturing control DC (cDC) or tDC, β(2)GPI-loaded, with autologous effector/memory T cells, evaluating the proliferative response, phenotype, cytokines secretion, viability and regulatory T cells.
Human monocyte-derived DCs treated with interleukin (IL)-10 and transforming growth factor β-1 (10/TGF-DC) induced β(2)GPI-specific-unresponsiveness in effector/memory CD4(+) T cells (46.5% ± 26.0 less proliferation) in 16 of 20 analysed patients with APS, without affecting the proliferative response to an unrelated candidin. In five analysed patients, 10/TGF-DC-stimulated T cells acquired an IL-2(low)interferon γ(low)IL-10(high) cytokine profile, with just a propensity to express higher numbers of Foxp3(+)CTLA-4(+) cells, but with an evident suppressive ability. In four of 10 analysed patients, 10/TGF-DC-stimulated T cell hyporesponsiveness could not be reverted and showed higher percentages of late apoptosis, p<0.02.
The inherent tolerance induction resistance of activated T cells present during the development of autoimmune diseases has delayed the application of tDC as an alternative therapy. This study highlights the 10/TGF-DC feasibility to induce antigen-specific unresponsiveness in autoreactive T cells generated in patients with APS by inducing apoptosis or T cells with regulatory abilities.
Full-text · Article · Sep 2011 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: Recent reports have raised concerns regarding potential risk factors for inhibitor development. In Israel, all haemophilia patients (n = 479) are followed by the National Hemophilia Center. Most children are neonatally exposed to factor concentrate (due to circumcision performed at the age of 8 days). The impact of early exposure and recombinant FVIII products (rFVIII) administration (approved in Israel since 1996) upon inhibitor occurrence in our cohort of haemophilia A (HA) patients was analysed. Two hundred ninety-two consecutive paediatric cases with a first symptomatic onset of HA were enrolled and followed over a median time of 7 years [min-max: 9 months to 17 years]. Study endpoint was inhibitor development against factor VIII. In addition, the treatment regimens applied, i.e. bolus administration or 'continuous infusion' and the family history of inhibitor development were investigated. During the follow-up period 31/292 children (10.6%) developed high titre inhibitors. Inhibitors occurred in 14/43 (32.5%) HA patients neonatally exposed to rFVIII, as compared to 22/249 previously treated with Plasma Derived (PD) products (8.8%). The odds ratio for inhibitor formation in rFVIII treated HA patients was 3.43 (95% CI: 1.36-8.65). Transient inhibitor evolved among 2/43 paediatric HA patients, only among those treated with rFVIII. The risk of inhibitor detection significantly increased among HA children treated by continuous infusion (P = 0.025). Our experience shows that the risk of inhibitor formation may be increased by early exposure to recombinant concentrates. The multiple variables affecting inhibitor incidence deserve further attention by larger prospective studies.
[Show abstract][Hide abstract] ABSTRACT: Severe FXI deficiency is a rare injury-related bleeding disorder. In patients with FXI inhibitors, surgeries may be treated using recombinant activated factor VII; however, treatment safety is a major concern and the best dosing regimen as well as mode of administration is still to be defined. We describe four patients with severe factor XI deficiency and inhibitors to FXI, undergoing eight (four major) surgical procedures treated with continuous infusion of rFVIIa. Following acute MI that evolved after surgery of our first patient, all other patients were treated with low-dose bolus rFVIIa followed by low-dose continuous infusion of rFVIIa. Haemostasis was successfully achieved and no further thrombotic complications occurred. To support our clinical results ex-vivo thromboelastography studies were performed, demonstrating the differences of clot formation and lysis between patients with FXI deficiency and healthy controls and suggesting that low-dose rFVIIa corrects coagulation similarly to high-dose rFVIIa in FXI deficiency. Recombinant FVIIa at low doses may effectively induce haemostasis and seems to be a safe treatment mode in patients with FXI deficiency and inhibitors undergoing surgeries.
[Show abstract][Hide abstract] ABSTRACT: Bolus injection (BI) of sucrose-formulated recombinant factor VIII (rFVIII-FS) is an approved treatment for haemophilia patients undergoing major surgery. Continuous infusion (CI) during surgery has potential benefits by providing steady administration of replacement factor to the patient, avoiding high peaks and low troughs. We tested the stability of rFVIII-FS under CI conditions and conducted a single-centre, open-label, phase III study to evaluate the efficacy and safety of CI using rFVIII-FS in haemophilia A patients undergoing surgery. Patients received bolus rFVIII-FS to achieve >or=80% FVIII levels 30-60 min presurgery, followed by CI of rFVIII-FS at a rate calculated to maintain haemostatic factor levels until days 8-10 post surgery. The rate of infusion was adjusted according to daily calculations derived from the actual clearance. The stability of rFVIII-FS was found to be appropriate for CI for 7 days under the same conditions as clinical settings. Fourteen patients (mean age 37.8 years) receiving on-demand FVIII treatment without a history of inhibitors underwent 15 surgical procedures including joint replacements, synovectomies, multiple tooth extractions, and cholecystectomy. Bleeding was similar to that observed in non-haemophilia patients undergoing similar operations in the same department. Haemostasis during surgery was considered by the attending surgeons as 'excellent' or 'good' in all cases; study investigators rated all 15 cases as 'excellent' overall. There were no adverse events, including inhibitor formation, related to rFVIII-FS. rFVIII-FS was found to be suitable for use in CI in haemophilia A patients undergoing major surgery.
[Show abstract][Hide abstract] ABSTRACT: Non-invasive biomarkers have gained popularity for estimating fibrosis stage. In our hepatitis C-infected haemophilia patients, Fibrotest (FT) correctly identified clinically advanced or minimal liver disease. More accurate tests, like the FibroMeters, have recently been validated. The aim of the study was to improve the estimation of liver fibrosis in hepatitis C-infected haemophiliacs using a combination of biomarkers and FibroMeters. One hundred and thirty-two hepatitis C-infected haemophilia patients (124 male, mean age: 39+/-14 years) were evaluated. The following biomarkers were used: FT, AST-to-platelet ratio index (APRI), Forns index, hyaluronic acid and FibroMeter. We applied a published algorithm suggesting that if FT is in concordance with APRI and/or Forns score, then the FT concurs with liver biopsy for estimation of fibrosis. Concordance of three or more biomarkers was present in 43.2% (57/132) of the patients. This high discordance rate was mainly because of indeterminate scores. Significant fibrosis (F2-F4) was estimated at 34.8% (46/132) and 37.9% (50/132) by the FT and FibroMeter respectively. The discordance rate between the FT and FibroMeter was 16.7% (22/132), (P<0.01 vs. other biomarkers). Using the algorithm, liver histology could be confidently estimated in 69.7% (92/132) of the patients. Concordance between the FT and FibroMeter in those patients who met the terms of the algorithm was 90.2% (83/92). Discordance between biomarkers is significant, and is mainly because of biomarkers with indeterminate results. The concordance rate between FT and FibroMeter is higher compared with the other biomarkers. Practical combination of tests may potentially limit the need of liver biopsy in the majority of haemophilia patients.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Liver biopsy remains the gold standard for the evaluation of fibrosis despite its risks and limitations, especially in haemophilia patients. Recently, non-invasive biomarkers have been used to assess histological features. The most thoroughly evaluated biomarker is the FibroTest (FT) (AUROC 0.80 for fibrosis stages F2F3F4 vs. F0F1).
Aim: To estimate liver fibrosis in haemophilia patients infected with hepatitis C (HCV) using non-invasive biomarkers without liver biopsy.
Methods: One hundred and thirty-two haemophilia patients (124 male, mean age 38 ± 14 years) with anti-HCV antibodies were evaluated. These patients were stratified into several groups: patients with features of advanced liver disease – seven, persistently HCV RNA-negative – 21, persistently normal liver function tests (LFTs)– 24, HCV/HIV co-infected – 27. The following biomarkers of fibrosis were used: FT, AST-to-platelet ratio index (APRI), Forns index, age-platelet index and hyaluronic acid. The obtained scores were correlated with the clinical features of the patients.
Results: Estimated by the FT, the distribution of the stage of fibrosis in the 132 patients was F0F1 = 65% (86/132), F2 = 5% (7/132), F3 = 13% (17/132) and F4 = 17% (22/132). Using FT, all patients with clinical suspicion of advanced liver disease were classified as F3F4, whereas patients with persistently HCV RNA-negative were all classified as F0F1. Twenty-one per cent (5/24) of the patients with persistently normal LFTs had fibrosis stage F3F4. The proportion of F3F4 among HCV/HIV co-infected patients was significantly higher than among HCV mono-infected (52% vs. 33%; P = 0.05). Concordance of three or more biomarkers was present in 43% (57/132) of the patients. Liver biopsy could be avoided in 70% (92/132) using a practical assumption that if FT is in concordance with APRI and/or Forns, then we may confidently rely on the biomarker. Concordance rate for patients with presumably advanced or minimal liver disease was excellent (100% and 95% respectively).
Conclusions: In our HCV-infected haemophilia patients, FT correctly identified clinically advanced or minimal liver disease. Discordance among the various biomarkers of fibrosis was considerate; nevertheless, practical combination of FT, APRI, and Forns may predict stage of fibrosis with accuracy, potentially avoiding liver biopsy in the majority of the patients.
[Show abstract][Hide abstract] ABSTRACT: Haemophilia patients who received non-virucidally treated large pool clotting factors before 1987 have a high rate of chronic hepatitis C viral infection (HCV). Some patients are coinfected with HIV. Haemophilia patients and other coagulation disorders were treated at one centre since the beginning of the 1970, and the Israeli National Hemophilia Center (INHC) was officially founded in 1987. To characterize patients with HCV as well as patients with HCV/HIV coinfection at the INHC. Patients with haemophilia and other coagulation disorders positive for HCV antibodies were evaluated between 2001 and 2004. Demographic data, type and severity of coagulation disorder, frequency of coagulation factor usage and treatment with concentrated clotting factors prior to 1987 were recorded. Liver enzymes, viral load, genotype and data supporting advanced liver disease were evaluated. About 179 of 239 haemophilia patients (75%) tested positive for anti-HCV antibodies. Our cohort consisted of 165 patients in whom clinical, biochemical and virological data were available. About 117 patients had active HCV infection with HCV-RNA-positive, and 27 were HCV/HIV coinfected. Twenty-one patients (13%) persistently tested HCV-RNA-negative, hence were considered to clear their HCV infection. There was no former USSR immigrants among HCV/HIV coinfected compared with HCV-infected or HCV-RNA-negative groups (0 vs. 30% and 38%, respectively; P < 0.001). HCV-RNA-negative patients used concentrated coagulation factor less frequently than HCV or HCV/HIV-infected patients (48% vs. 73%; P = 0.023, and 48% vs. 74%; P = 0.043, respectively). The use of concentrated clotting factors before 1987 was significantly more frequent in HCV/HIV than in either HCV-infected or HCV-RNA-negative patients (96% vs. 49% and 48%, respectively; P < 0.001). Compared with HCV/HIV subjects, patients with HCV monoinfection were characterized by a higher proportion of infection with genotype 1 (80% vs. 61%; P = 0.027). The rate of persistently normal liver enzymes in these patients was higher (24% vs. 7%; P = 0.05) than in the HCV/HIV-coinfected patients. Advanced liver disease was significantly more common in patients with HCV/HIV-coinfection than in HCV-monoinfected patients (11% vs. 3%; P = 0.045). The majority of haemophilia patients are infected with HCV. Viral clearance occurred in a minority of these patients. HCV monoinfected and HCV/HIV coinfected differ clinically and prognostically.
[Show abstract][Hide abstract] ABSTRACT: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by mutations in either alpha(IIb)[glycoprotein (GP) IIb] or beta(3) (GPIIIa) genes that lead to a lack or dysfunction of the integrin alpha(IIb)beta(3) which serves as a fibrinogen receptor.
Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in three members of a Cypriot family: a 3-year-old proband, her father and her paternal uncle.
To determine the molecular basis of GT in this family and to characterize possible biochemical and structural defects.
Analysis of the patients' platelets by fluorescence-activated cell sorting demonstrated trace amounts of beta(3), no alpha(IIb) and no alpha(IIb)beta(3) on the membrane. Sequence analysis revealed a novel T607G transversion in exon 5 of the alpha(IIb) gene predicting a Phe171Cys alteration that created a PstI recognition site. All three patients were homozygous for the mutation, the mother and paternal grandparents of the proband were heterozygous, whereas 110 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alpha(IIb) and wild-type beta(3) failed to express alpha(IIb)beta(3) as shown by immunoprecipitation and immunohistochemistry experiments. Structural analysis of the alpha(IIb)beta(3) model, which was based on the crystal structure of alpha(v)beta(3), indicated that Phe171 plays an essential role in the interface between the beta-propeller domain of alpha(IIb) and the betaA domain of beta(3).
A novel Phe171Cys mutation in the alpha(IIb) gene of patients with GT is associated with abrogation of alpha(IIb)beta(3) complex formation.
Full-text · Article · Aug 2004 · Journal of Thrombosis and Haemostasis
[Show abstract][Hide abstract] ABSTRACT: The use of autologous platelet-rich plasma (PRP) as a source for growth factors in bone grafting is a relatively new and promising technique. Early controlled studies indicate that combining PRP with autologous bone grafts significantly enhances the rate of bone formation and maturation. The study consisted of 105 patients who required sinus augmentation with crestal bone height of less than 5 mm in the posterior maxilla. All patients received a composite bone graft that consisted of 30% to 40% autogenous bone harvested from the lateral wall of the maxilla zygomatic-maxillary buttress and the tuberosity and 60% to 70% xenograft. A total of 50 mL of blood was obtained from each patient before the surgical treatment for preparation of 10 mL of PRP. The graft-PRP mixture was activated by human thrombin. All sinus augmentations were carried out simultaneously with dental implants. At 6 months postoperatively, implants were exposed showing no clinical evidence of crestal bone loss around the implants both clinically and radiographically. All implants were clinically osseointegrated and loaded with fixed porcelain fused to metal prosthesis. The use of PRP in augmenting the severely atrophic posterior maxilla has obvious clinical benefits in terms of reducing the healing period of bone maturation, better graft handling, and accelerated soft tissue healing.
No preview · Article · Apr 2004 · Implant Dentistry
[Show abstract][Hide abstract] ABSTRACT: New bone formation requires sufficient number of osteogenic progenitors capable of forming the bone desired. The site of engraftment must be filled with a matrix that facilitates attachment, migration and differentiation of osteoblastic progenitors. It is also necessary that the cells receive stimuli by growth factors that allow them to progress toward a bone phenotype. Another critical step in new tissue formation is the construction of new blood vessels--angiogenesis. Platelets contain growth factors that induce osteoinductive stimuli and accelerate angiogenesis. One strategy for harnessing this benefit is to apply platelet rich plasma (PRP) to bone graft site. The present article review platelets and growth factors physiology. We discuss the interaction between growth factors, thrombin and cells that form bone and blood vessels: osteoblasts, mesenchimal stem cells and endothelial cells. Methods and defaults of PRP preparation and safety issues are presented. The knowledge of platelet physiology and the mechanism by which growth factors effect cell proliferation and differentiation allow the dental surgeon to properly use this treatment modality and to achieve the ultimate goal of durable and effectively functioning bone.
No preview · Article · Feb 2004 · Refuʾat ha-peh ṿeha-shinayim (1993)
[Show abstract][Hide abstract] ABSTRACT: Human T lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 infection in patients with B cell-type chronic lymphocytic leukemia (B-CLL) is rare and has been reported only in areas in which HTLV-1 is endemic. In the present study, we detected HTLV-1 proviral DNA by polymerase chain reaction, using tax primers, in peripheral blood lymphocytes from a B-CLL patient, an immigrant to Israel, where HTLV-1 infection is not endemic. F344 rats injected intravenously with peripheral blood lymphocytes obtained from the patient developed HTLV-1 antibodies. Titers of antibody to HTLV-1 in the rat blood were 1:512 by particle agglutination; enzyme-linked immunosorbent assay and Western blotting were also positive. No antibody against HTLV-1 was demonstrated in the animal model after inoculation of either purified B lymphocytes from the B-CLL patient or peripheral blood mononuclear cells from healthy donors. This is one of the few studies showing the presence of HTLV-1 provirus in T lymphocytes of a B-CLL patient who had multiple infections, and died of salmonella sepsis, and the first report of HTLV-1 antibody induction in an animal model by inoculation of lymphocytes obtained from an HTLV-1-infected B-CLL patient.
No preview · Article · Dec 2003 · Medical Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: We describe a possible relationship between inferior vena cava anomalies and extensive thrombosis of the inferior vena cava and the iliac and femoral veins. CONCLUSION: An anomaly of the inferior vena cava should be considered in young patients who present with deep vein thrombosis of the femoral and iliac veins. Coagulation abnormalities, frequently found in these patients, may be a contributory factor.
No preview · Article · Apr 2003 · American Journal of Roentgenology
[Show abstract][Hide abstract] ABSTRACT: Recombinant activated factor VII (rFVIIa, NovoSeven) represents an effective treatment for hemophilia patients with inhibitors, but no consensus as to the best dosing regimen exists. We assessed the efficacy and safety of a rFVIIa 'megadose' (300 micro g kg(-1) bolus) as treatment for bleeds in three young inhibitor patients. Of 114 bleeds, 95 responded to a single dose. Pain relief was faster and therapy duration significantly shorter than with continuous infusion (CI) regimens or standard boluses (90 micro g kg(-1) every 3 h). Rebleeding occurred in 9.6% of cases and 19/114 episodes required a second bolus injection. Although rFVIIa consumption per bleed (median: 300 micro g kg(-1)) was higher than with standard boluses (180-270 micro g kg(-1)), patients found single bolus administration more convenient than recurrent injections or CI. With two exceptions, no complications occurred within 3 h of treatment, despite high FVII:C levels (median: 27.4 U mL(-1); range: 19.8-54 U mL(-1)). Treatment of bleeds with a rFVIIa megadose in young inhibitor patients is effective and well tolerated.
Full-text · Article · Apr 2003 · Journal of Thrombosis and Haemostasis
[Show abstract][Hide abstract] ABSTRACT: To report our experience with recombinant activated factor VII (rFVIIa) to control hemorrhage in trauma patients with profound multifactorial coagulopathy. rFVIIa forms a complex with tissue factor exposed at sites of tissue damage and induces activation of coagulation limited to the site of injury. It is approved for use in hemophilia patients, however, its use in trauma is still controversial due to the theoretical risk of thromboembolic complications.
Nineteen critically ill, multi-transfused patents with trauma (ten blunt and nine penetrating), aged 25 + 17 yr,were treated with rFVIIa after all conventional hemostatic measures had failed. After one to three doses of rFVIIa, hemorrhaging ceased within minutes in 15/19 (78.9%) patients. The total dose of rFVIIa required to control bleeding was 195 +/- 112.7 microg x kg(-1). Shortening of prothrombin time and partial thromboplastin time was observed within 15-30 min from 22.7 +/- 7.9 to 10.4 +/- 2.6 sec and 71 +/- 38.9 to 42.2 +/- 24 sec respectively, (P < 0.05). Transfusion requirements decreased from 30 +/- 18.3 units used within 5.6 +/- 3.4 hr of admission to 2.8 +/- 2.5 within the following 24 hr (P < 0.05). One patient developed clinical deep vein thrombosis. No systemic activation of coagulation was observed clinically. Thirteen patients (68.4%) survived and recovered. Four patents did not respond to rFVIIa treatment and exsanguinated within 24 hr. Two patients died after one week, one from sepsis and one from multi-organ failure.
rFVIIa is a promising adjunctive hemostatic treatment for trauma patients suffering from massive bleeding. Controlled trials are warranted to evaluate the safety and efficacy of this drug.
No preview · Article · Dec 2002 · Canadian Journal of Anaesthesia
[Show abstract][Hide abstract] ABSTRACT: Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.
[Show abstract][Hide abstract] ABSTRACT: The present study was undertaken to evaluate stability, pharmacokinetic profile and efficacy of continuous infusion of 8Y in patients with different types of von Willebrand disease (vWD). Following reconstitution, 8Y levels of von Willebrand factor ristocetin cofactor (vWF:Rco), vWF antigen and factor VIII coagulant activity (FVIII:C) decreased to about 80% of the baseline levels; addition of low molecular weight heparin decreased the level of FVIII:C even further. Reconstituted 8Y was found to be sterile for up to 6 days postreconstitution. Ten vWD patients (four with type 2A, three with type 3, two with type 1 and one with 2N) underwent pharmacokinetic analysis. The recovery of vWF: RCo was significantly lower in patients with type 3 vWD (1.4 +/- 0.05% U(-1) kg(-1)) compared with that of the patients with types 1 (2.3 +/- 0.52% U(-1) kg(-1)) or 2A (2.0 +/- 0.06% U(-1) kg(-1)) vWD (P = 0.015). Type 3 vWD patients exhibited significantly higher vWF:RCo clearance (5.1 +/- 1.1 mL kg(-1) h(-1)) compared with that of patients with type 2A (2.8 +/- 0.7 mL kg(-1) h(-1)) and type 1 (2.6 +/- 1.0 mL kg(-1) h(-1)) vWD (P = 0.028). Accordingly, terminal half-life was lower in patients with type 3 vWD (8.0 +/- 0.6 h(-1)) compared with type 2A (12.7 +/- 5.9 h(-1)) or type 1 (14 +/- 1.2 h(-1)) vWD patients. Multimeric pattern of vWF from patients' plasma was similar to that of 8Y. In two patients treated with 8Y by continuous infusion for prevention or treatment of bleeding haemostasis was achieved. Thus, 8Y is suitable and haemostatically effective for continuous infusion treatment in patients with vWD.
[Show abstract][Hide abstract] ABSTRACT: Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor αIIbβ3 is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified αIIbβ3, specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in αIIbβ3 ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to αIIbβ3 as well as ligation-induced allosteric changes in the conformation of αIIbβ3. We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of αIIbβ3function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin αIIbβ3 on the intact platelet depends totally on their enzymatically catalyzed surface expression.
[Show abstract][Hide abstract] ABSTRACT: Recombinant activated factor VII (rFVIIa) was approved for treatment of hemorrhages in patients with hemophilia who develop inhibitors to factors VIII or IX. Conditions with increased thromboembolic risk, including trauma with or without disseminated intravascular coagulation, were considered a contraindication for the drug. The mechanism of action of rFVIIa suggests enhancement of hemostasis limited to the site of injury without systemic activation of the coagulation cascade. Therefore, use of the drug in trauma patients suffering uncontrolled hemorrhage appears to be rational.
Seven massively bleeding, multitransfused (median, 40 units [range, 25-49 units] of packed cells), coagulopathic trauma patients were treated with rFVIIa (median, 120 microg/kg [range, 120-212 microg/kg]) after failure of conventional measures to achieve hemostasis.
Administration of rFVIIa resulted in cessation of the diffuse bleed, with significant decrease of blood requirements to 2 units (range, 1-2 units) of packed cells (p < 0.05); shortening of prothrombin time and activated partial thromboplastin time from 24 seconds (range, 20-31.8 seconds) to 10.1 seconds (range, 8-12 seconds) (p < 0.005) and 79 seconds (range, 46-110 seconds) to 41 seconds (range, 28-46 seconds) (p < 0.05), respectively; and an increase of FVII level from 0.7 IU/mL (range, 0.7-0.92 IU/mL) to 23.7 IU/mL (range, 18-44 IU/mL) (p < 0.05). Three of the seven patients died of reasons other than bleeding or thromboembolism.
The results of this report suggest that in trauma patients rFVIIa may play a role as an adjunctive hemostatic measure, in addition to surgical hemostatic techniques, and provides the motivation for controlled animal and clinical trials.
Full-text · Article · Sep 2001 · The Journal of trauma